• The Interactive Fly

    Mechanisms of Evolution

  • Haldane's Paradox - The cost of evolution
  • Haploid selection
  • Horizontal Gene Transfer
  • Hybridization: Genetic exchange between species
  • Karyotype evolution: Genetic divergence through chromosome rearrangement
  • Lifestyle: From Symbiosis to Predation
  • Linkage
  • Metabolism
  • Morphological Evolution
  • Ploidy
  • Polygenic Inheritance
  • Population size
  • Positive Selection
  • Protein Evolution
  • Pseudogenization: loss of functionality of duplicate genes with respect to their parent genes
  • Purifying/Negative Selection
  • Recombination
  • Regulatory Network Evolution
  • Positive/Directional Selection
  • Reproductive traits, reproductive isolation, reproductive barriers, hybrid breakdown, hybrid sterility
  • Sexual Selection and Sperm Competition
  • Social Evolution and Altruism
  • Speciation
  • Trade-offs - cost-benefit relations in evolution
  • Transposable Elements as affectors of molecular evolution
  • Variation

    Extensive local adaptation within the chemosensory system following Drosophila melanogaster's global expansion

    How organisms adapt to new environments is of fundamental biological interest, but poorly understood at the genetic level. Chemosensory systems provide attractive models to address this problem, because they lie between external environmental signals and internal physiological responses. To investigate how selection has shaped the well-characterized chemosensory system of Drosophila melanogaster, this study analysed genome-wide data from five diverse populations. By couching population genomic analyses of chemosensory protein families, including odorant receptors, gustatory receptors, and odorant-binding proteins, within parallel analyses of other large families, it was demonstrated that chemosensory proteins are not outliers for adaptive divergence between species. However, chemosensory families often display the strongest genome-wide signals of recent selection within D. melanogaster. Recent adaptation has operated almost exclusively on standing variation, and patterns of adaptive mutations predict diverse effects on protein function. Finally, evidence is provided that chemosensory proteins have experienced relaxed constraint, and it is argued that this has been important for their rapid adaptation over short timescales (Arguello, 2016).

    A 24 h age difference causes twice as much gene expression divergence as 100 generations of adaptation to a novel environment

    Gene expression profiling is one of the most reliable high-throughput phenotyping methods, allowing researchers to quantify the transcript abundance of expressed genes. Because many biotic and abiotic factors influence gene expression, it is recommended to control them as tightly as possible. This study shows that a 24 h age difference of Drosophila simulans females that were subjected to RNA sequencing (RNA-Seq) five and six days after eclosure resulted in more than 2000 differentially expressed genes. This is twice the number of genes that changed expression during 100 generations of evolution in a novel hot laboratory environment. Importantly, most of the genes differing in expression due to age introduce false positives or negatives if an adaptive gene expression analysis is not controlled for age. These results indicate that tightly controlled experimental conditions, including precise developmental staging, are needed for reliable gene expression analyses, in particular in an evolutionary framework (Hsu, 2019).

    Inverse resource allocation between vision and olfaction across the genus Drosophila

    Divergent populations across different environments are exposed to critical sensory information related to locating a host or mate, as well as avoiding predators and pathogens. These sensory signals generate evolutionary changes in neuroanatomy and behavior; however, few studies have investigated patterns of neural architecture that occur between sensory systems, or that occur within large groups of closely-related organisms. This study examine 62 species within the genus Drosophila and describes an inverse resource allocation between vision and olfaction, which was consistently observe at the periphery, within the brain, as well as during larval development. This sensory variation was noted across the entire genus and appears to represent repeated, independent evolutionary events, where one sensory modality is consistently selected for at the expense of the other. Moreover, evidence is provided of a developmental genetic constraint through the sharing of a single larval structure, the eye-antennal imaginal disc. In addition, the ecological implications of visual or olfactory bias were examined, including the potential impact on host-navigation and courtship (Keesey, 2019).

    Local thermal adaptation detected during multiple life stages across populations of Drosophila melanogaster

    Thermal adaptation is typically detected by examining the tolerance of a few populations to extreme temperatures within a single life stage. However, the extent to which adaptation occurs among many different populations might depend on the tolerance of multiple life stages and the average temperature range that the population experiences. This study examined local adaptation to native temperature conditions in eleven populations of the well-known cosmopolitan fruit fly, Drosophila melanogaster. These populations were sampled from across the global range of D. melanogaster. Traits related to fitness were measured during each life stage to determine if certain stages are more sensitive to changes in temperature than others. D. melanogaster appeared to show local adaptation to native temperatures during the egg, larval, and adult life stages, but not the pupal stage. This suggests that across the entire distribution of D. melanogaster, certain life stages might be locally adapted to native temperatures, while other stages might use phenotypic plasticity or tolerance to a wide range of temperatures experienced in the native environment of this species (Austin, 2019).

    Interspecies comparative analyses reveal distinct carbohydrate-responsive systems among Drosophila species

    During evolution, organisms have acquired variable feeding habits. Some species are nutritional generalists that adapt to various food resources, while others are specialists, feeding on specific resources. However, much remains to be discovered about how generalists adapt to diversified diets. Larvae of the generalists Drosophila melanogaster and D. simulans develop on three diets with different nutrient balances, whereas specialists D. sechellia and D. elegans cannot develop on carbohydrate-rich diets. The generalist D. melanogaster downregulates the expression of diverse metabolic genes systemically by transforming growth factor beta (TGF-beta)/Activin signaling, maintains metabolic homeostasis, and successfully adapts to the diets. In contrast, the specialist D. sechellia expresses those metabolic genes at higher levels and accumulates various metabolites on the carbohydrate-rich diet, culminating in reduced adaptation. Phenotypic similarities and differences strongly suggest that the robust carbohydrate-responsive regulatory systems are evolutionarily retained through genome-environment interactions in the generalists and contribute to their nutritional adaptabilities (Watanabe, 2019).

    Major range loss predicted from lack of heat adaptability in an alpine Drosophila species

    Climate warming is threatening biodiversity worldwide. Climate specialists such as alpine species are especially likely to be vulnerable. Adaptation by rapid evolution is the only long-term option for survival of many species, but the adaptive evolutionary potential of heat resistance has not been assessed in an alpine invertebrate. This study show that the alpine fly Drosophila nigrosparsa cannot readily adapt to heat stress. Heat-exposed flies from a regime with increased ambient temperature and a regime with increased temperature plus artificial selection for heat tolerance were less heat tolerant than the control group. Increased ambient temperature affected negatively both fitness and competitiveness. Ecological niche models predicted the loss of three quarters of the climatically habitable areas of this fly by the end of this century. These findings suggest that, alongside with other climate specialists, species from mountainous regions are highly vulnerable to climate warming and unlikely to adapt through evolutionary genetic changes (Kinzner, 2019).

    Genomic analysis of the four ecologically distinct cactus host populations of Drosophila mojavensis

    Relationships between an organism and its environment can be fundamental in the understanding how populations change over time and species arise. Local ecological conditions can shape variation at multiple levels, among these are the evolutionary history and trajectories of coding genes. This study examines the rate of molecular evolution at protein-coding genes throughout the genome in response to host adaptation in the cactophilic Drosophila mojavensis. These insects are intimately associated with cactus necroses, developing as larvae and feeding as adults in these necrotic tissues. Drosophila mojavensis is composed of four isolated populations across the deserts of western North America and each population has adapted to utilize different cacti that are chemically, nutritionally, and structurally distinct. High coverage Illumina sequencing was performed on three previously unsequenced populations of D. mojavensis. Genomes were assembled using the previously sequenced genome of D. mojavensis from Santa Catalina Island (USA) as a template. Protein coding genes were aligned across all four populations and rates of protein evolution were determined for all loci using a several approaches. Loci that exhibited elevated rates of molecular evolution tend to be shorter, have fewer exons, low expression, be transcriptionally responsive to cactus host use and have fixed expression differences across the four cactus host populations. Fast evolving genes were involved with metabolism, detoxification, chemosensory reception, reproduction and behavior. Results of this study give insight into the process and the genomic consequences of local ecological adaptation (Allan, 2019).

    Microbiome composition shapes rapid genomic adaptation of Drosophila melanogaster

    Population genomic data has revealed patterns of genetic variation associated with adaptation in many taxa. Yet understanding the adaptive process that drives such patterns is challenging; it requires disentangling the ecological agents of selection, determining the relevant timescales over which evolution occurs, and elucidating the genetic architecture of adaptation. Doing so for the adaptation of hosts to their microbiome is of particular interest with growing recognition of the importance and complexity of host-microbe interactions. This study tracked the pace and genomic architecture of adaptation to an experimental microbiome manipulation in replicate populations of Drosophila melanogaster in field mesocosms. Shifts in microbiome composition altered population dynamics and led to divergence between treatments in allele frequencies, with regions showing strong divergence found on all chromosomes. Moreover, at divergent loci previously associated with adaptation across natural populations, the more common allele was found in fly populations experimentally enriched for a certain microbial group was also more common in natural populations with high relative abundance of that microbial group. These results suggest that microbiomes may be an agent of selection that shapes the pattern and process of adaptation and, more broadly, that variation in a single ecological factor within a complex environment can drive rapid, polygenic adaptation over short timescales (Rudman, 2019).

    Decreased temperature sensitivity of Vestigial gene expression in temperate populations of Drosophila melanogaster

    Drosophila melanogaster recently spread from its tropical origin in Africa and became a cosmopolitan species that has adapted to a wide range of different thermal environments, including temperate climates. An important limiting factor of temperate climates has probably been their low and varying temperatures. The transcriptional output of genes can vary across temperatures, which might have been detrimental while settling in temperate environments. The reduction of temperature-sensitive expression of functionally important genes to ensure consistent levels of gene expression might have been relevant while adapting to such environments. This study focuses on the gene vestigial (vg) whose product is a key factor in wing development. Evidence is provided that temperature-sensitivity of vg has been buffered in populations from temperate climates. Temperature-sensitivity of vg gene expression was investigated in six natural populations, including four temperate populations (three from Europe and one from high-altitude Africa), and two tropical populations from the ancestral species range. All temperate populations exhibited a lower degree of temperature-induced expression plasticity than the tropical populations (Voigt, 2019).

    Recurrent collection of Drosophila melanogaster from Wild african environments and genomic insights into species history

    Drosophila melanogaster is thought to originate from sub-Saharan Africa. This study documents the collection of 288 D. melanogaster individuals from multiple African wilderness areas in Zambia, Zimbabwe, and Namibia. The presence of D. melanogaster in these remote woodland environments is consistent with an ancestral range in southern-central Africa, as opposed to equatorial regions. After sequencing the genomes of 17 wilderness-collected flies collected from Kafue National Park in Zambia, reduced genetic diversity relative to town populations, elevated chromosomal inversion frequencies, and strong differences at specific genes including known insecticide targets were found. Combining these genomes with existing data, this study probed the history of this species' geographic expansion. Demographic estimates indicated that expansion from southern-central Africa began approximately 10,000 years ago, with a Saharan crossing soon after, but expansion from the Middle East into Europe did not begin until roughly 1,400 years ago. This improved model of demographic history will provide an important resource for future evolutionary and genomic studies of this key model organism. These findings add context to the history of D. melanogaster, while opening the door for future studies on the biological basis of adaptation to human environments (Sprengelmeyer, 2019).

    A-to-I RNA editing uncovers hidden signals of adaptive genome evolution in animals

    In animals, the most common type of RNA editing is the deamination of adenosines (A) into inosines (I). Because inosines base-pair with cytosines (C), they are interpreted as guanosines (G) by the cellular machinery and genomically encoded G alleles at edited sites mimic the function of edited RNAs. The contribution of this hardwiring effect on genome evolution remains obscure. This study looked for population genomics signatures of adaptive evolution associated with A-to-I RNA edited sites in humans and Drosophila melanogaster. Single nucleotide polymorphisms at edited sites occur 3 (humans) to 15 times (Drosophila) more often than at unedited sites, the nucleotide G is virtually the unique alternative allele at edited sites and G alleles segregate at higher frequency at edited sites than at unedited sites. This study study reveals that a significant fraction of coding synonymous and nonsynonymous as well as silent and intergenic A-to-I RNA editing sites are likely adaptive in the distantly related human and Drosophila lineages (Popitsch, 2020).

    Rapid sex-specific adaptation to high temperature in Drosophila

    The pervasive occurrence of sexual dimorphism demonstrates different adaptive strategies of males and females. While different reproductive strategies of the two sexes are well-characterized, very little is known about differential functional requirements of males and females in their natural habitats. The impact environmental change on the selection response was studied in both sexes. Exposing replicated Drosophila populations to a novel temperature regime, sex-specific changes were demonstrated in gene expression, metabolic and behavioral phenotypes in less than 100 generations. This indicates not only different functional requirements of both sexes in the new environment but also rapid sex-specific adaptation. Supported by computer simulations it is proposed that altered sex-biased gene regulation from standing genetic variation, rather than new mutations, is the driver of rapid sex-specific adaptation. This discovery of environmentally driven divergent functional requirements of males and females has important implications-possibly even for gender aware medical treatments (Hsu, 2020).

    Neuronal function and dopamine signaling evolve at high temperature in Drosophila

    Neuronal activity is temperature-sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet not enough is known about the evolutionary response of neuronal phenotypes in new temperature environments. This study used long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes. A direct relationship was demonstrated between thermal selective pressure and the evolution of neuronally expressed molecular and behavioral phenotypes. Several essential neuronal genes evolve lower expression at high temperatures and higher expression at low temperatures, with dopaminergic neurons standing out by displaying the most consistent expression change across independent replicates. The link between evolved gene expression and behavioral changes was functionally validated by pharmacological intervention in the experimentally evolved D. simulans populations as well as by genetically triggered expression changes of key genes in D. melanogaster. Since natural temperature clines confirm these results for Drosophila and Anopheles populations, it is concluded that neuronal dopamine evolution is a key factor for temperature adaptation (Jaksic, 2020).

    Genome-wide variation and transcriptional changes in diverse developmental processes underlie the rapid evolution of seasonal adaptation

    Many organisms enter a dormant state in their life cycle to deal with predictable changes in environments over the course of a year. The timing of dormancy is therefore a key seasonal adaptation, and it evolves rapidly with changing environments. The hypothesis that differences in the timing of seasonal activity are driven by differences in the rate of development during diapause was tested in Rhagoletis pomonella, a fly specialized to feed on fruits of seasonally limited host plants. Transcriptomes from the central nervous system across a time series during diapause show consistent and progressive changes in transcripts participating in diverse developmental processes, despite a lack of gross morphological change. Moreover, population genomic analyses suggested that many genes of small effect enriched in developmental functional categories underlie variation in dormancy timing and overlap with gene sets associated with development rate in Drosophila melanogaster. These transcriptional data also suggested that a recent evolutionary shift from a seasonally late to a seasonally early host plant drove more rapid development during diapause in the early fly population. Moreover, genetic variants that diverged during the evolutionary shift were also enriched in putative cis regulatory regions of genes differentially expressed during diapause development. Overall, these data suggest polygenic variation in the rate of developmental progression during diapause contributes to the evolution of seasonality in R. pomonella. Patterns that suggest hourglass-like developmental divergence early and late in diapause development are discussed along with an important role for hub genes in the evolution of transcriptional divergence (Dowle, 2020).

    Phenotypic coupling of sleep and starvation resistance evolves in D. melanogaster

    One hypothesis for the function of sleep is that it serves as a mechanism to conserve energy. Recent studies have suggested that increased sleep can be an adaptive mechanism to improve survival under food deprivation in Drosophila melanogaster. To test the generality of this hypothesis, Sleep and its plastic response to starvation was compared in a temperate and tropical population of Drosophila melanogaster. Flies from the temperate population were found to be more starvation resistant, and it was hypothesized that they would engage in behaviors that are considered to conserve energy, including increased sleep and reduced movement. Surprisingly, temperate flies slept less and moved more when they were awake compared to tropical flies, both under fed and starved conditions, therefore sleep did not correlate with population-level differences in starvation resistance. In contrast, total sleep and percent change in sleep when starved were strongly positively correlated with starvation resistance within the tropical population, but not within the temperate population. Thus, unexpectedly complex relationships between starvation and sleep were observed that vary both within and across populations. These observations falsify the simple hypothesis of a straightforward relationship between sleep and energy conservation. The hypothesis that starvation is correlated with metabolic phenotypes was tested by investigating stored lipid and carbohydrate levels; stored metabolites were found to partially contributed towards variation starvation resistance. These findings demonstrate that the function of sleep under starvation can rapidly evolve on short timescales and raise new questions about the physiological correlates of sleep and the extent to which variation in sleep is shaped by natural selection (Sarikaya, 2020).

    Adaptation of circadian neuronal network to photoperiod in high-latitude European Drosophilids
    The genus Drosophila contains over 2,000 species that, stemming from a common ancestor in the Old World Tropics, populate today very different environments. This study found significant differences in the activity pattern of Drosophila species belonging to the holarctic virilis group, i.e., D. ezoana and D. littoralis, collected in Northern Europe, compared to that of the cosmopolitan D. melanogaster, collected close to the equator. These behavioral differences might have been of adaptive significance for colonizing high-latitude habitats and hence adjust to long photoperiods. Most interestingly, the flies' locomotor activity correlates with the neurochemistry of their circadian clock network, which differs between low and high latitude for the expression pattern of the blue light photopigment cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF). In D. melanogaster, CRY and PDF are known to modulate the timing of activity and to maintain robust rhythmicity under constant conditions. The rhythmic behavior of the high-latitude virilis group species could be partially stimulated by mimicking their CRY/PDF expression patterns in a laboratory strain of D. melanogaster. Data suggest that these alterations in the CRY/PDF clock neurochemistry might have allowed the virilis group species to colonize high-latitude environments (Menegazzi, 2017).

    Parallel gene expression evolution in natural and laboratory evolved populations

    Ecological adaptation is frequently inferred by the comparison of natural populations from different environments. Nevertheless, the inference of the selective forces suffers the challenge that many environmental factors covary. With well-controlled environmental conditions, experimental evolution provides a powerful approach to complement the analysis of natural populations. On the other hand, it is apparent that laboratory conditions differ in many ways from natural environments, which raises the question to what extent selection responses in experimental evolution studies can inform about adaptation processes in the wild. This study compared the expression profiles of replicated Drosophila melanogaster populations which have been exposed to two distinct temperature regimes (18/28 °C and 10/20 °C) in the laboratory for more than 80 generations. Using gene-wise differential expression analysis and co-expression network analysis, 541 genes and three co-regulated gene modules were identified that evolved in the same direction in both temperature regimes, and most of these changes probably reflect an adaptation to the space constrain or diurnal temperature fluctuation that is common in both selection regimes. 203 genes and seven modules evolved temperature-specific expression changes. Remarkably, a significant overlap was detected of these temperature-adaptive genes/modules from experimental evolution with temperature-adaptive genes inferred from natural Drosophila populations covering two different temperature clines. It is concluded that well-designed experimental evolution studies are a powerful tool to dissect evolutionary responses (Hsu, 2020).

    The evolution of phenotypic plasticity in response to temperature stress

    Phenotypic plasticity is the ability of a single genotype to produce different phenotypes in response to environmental variation. The importance of phenotypic plasticity in natural populations and its contribution to phenotypic evolution during rapid environmental change is widely debated. This study shows that thermal plasticity of gene expression in natural populations is a key component of its adaptation: evolution to novel thermal environments increases ancestral plasticity rather than mean genetic expression. The evolution of plasticity in gene expression was determined by conducting laboratory natural selection on a Drosophila simulans population in hot and cold environments. After more than 60 generations in the hot environment, 325 genes evolved a change in plasticity relative to the natural ancestral population. Plasticity increased in 75% of these genes, which were strongly enriched for several well-defined functional categories (e.g. chitin metabolism, glycolysis and oxidative phosphorylation). Furthermore, this study showed that plasticity in gene expression of populations exposed to different temperatures is rather similar across species. It is concluded that most of the ancestral plasticity can evolve further in more extreme environments (Mallard, 2020).

    A clinal polymorphism in the insulin signaling transcription factor foxo contributes to life-history adaptation in Drosophila

    A fundamental aim of adaptation genomics is to identify polymorphisms that underpin variation in fitness traits. In D. melanogaster latitudinal life-history clines exist on multiple continents and make an excellent system for dissecting the genetics of adaptation. Previous work has identified numerous clinal SNPs in insulin/insulin-like growth factor signaling (IIS), a pathway known from mutant studies to affect life history. However, the effects of natural variants in this pathway remain poorly understood. This study investigated how two clinal alternative alleles at foxo, a transcriptional effector of IIS, affect fitness components (viability, size, starvation resistance, fat content). This polymorphism from the North American cline was assessed by reconstituting outbred populations, fixed for either the low- or high-latitude allele, from inbred DGRP lines. Since diet and temperature modulate IIS, alleles were phenotyped across two temperatures (18 ° C, 25 ° C) and two diets differing in sugar source and content. Consistent with clinal expectations, the high-latitude allele conferred larger body size and reduced wing loading. Alleles also differed in starvation resistance and expression of InR, a transcriptional target of FOXO. Allelic reaction norms were mostly parallel, with few GxE interactions. Together, these results suggest that variation in IIS makes a major contribution to clinal life-history adaptation ( , ).

    Temperature, rainfall and wind variables underlie environmental adaptation in natural populations of Drosophila melanogaster

    While several studies in a diverse set of species have shed light on the genes underlying adaptation, knowledge on the selective pressures that explain the observed patterns lags behind. Drosophila melanogaster is a valuable organism to study environmental adaptation because this species originated in Southern Africa and has recently expanded worldwide, and also because it has a functionally well-annotated genome. This work aimed to decipher which environmental variables are relevant for adaptation of D. melanogaster natural populations in Europe and North America. 36 whole-genome pool-seq samples of D. melanogaster natural populations were ezamined, collected in 20 European and 11 North American locations. The BayPass software was used to identify SNPs and transposable elements showing signature of adaptive differentiation across populations, as well as significant associations with 59 environmental variables related to temperature, rainfall, evaporation, solar radiation, wind, daylight hours, and soil type. Besides temperature and rainfall, wind related variables are also relevant for D. melanogaster environmental adaptation. Interestingly, 23% to 51% of the genes that showed significant associations with environmental variables were not found overly differentiated across populations. Besides SNPs, ten reference transposable element insertions associated with environmental variables were identified. These results showed that genome-environment association analysis can identify adaptive genetic variants that are undetected by population differentiation analysis while also allowing the identification of candidate environmental drivers of adaptation (Bogaerts-Marquez, 2020).

    Multiple mechanisms drive genomic adaptation to extreme O(2) levels in Drosophila melanogaster

    To detect the genomic mechanisms underlying evolutionary dynamics of adaptation in sexually reproducing organisms, this study analyze multigenerational whole genome sequences of Drosophila melanogaster adapting to extreme O(2) conditions over an experiment conducted for nearly two decades. Methods to analyze time-series genomics data and predict adaptive mechanisms were developed. This study report a remarkable level of synchronicity in both hard and soft selective sweeps in replicate populations as well as the arrival of favorable de novo mutations that constitute a few asynchronized sweeps. Additionally direct experimental observations were made of rare recombination events that combine multiple alleles on to a single, better-adapted haplotype. Based on the analyses of the genes in genomic intervals, this study provides a deeper insight into the mechanisms of genome adaptation that allow complex organisms to survive harsh environments (Iranmehr, 2021).

    The genomic architecture of adaptation to larval malnutrition points to a trade-off with adult starvation resistance in Drosophila

    Periods of nutrient shortage impose strong selection on animal populations. Experimental studies of genetic adaptation to nutrient shortage largely focus on resistance to acute starvation at adult stage; it is not clear how conclusions drawn from these studies extrapolate to other forms of nutritional stress. The genomic signature of adaptation to chronic juvenile malnutrition was studied in six populations of Drosophila melanogaster evolved for 150 generations on an extremely nutrient-poor larval diet. Comparison with control populations evolved on standard food revealed repeatable genomic differentiation between the two set of population, involving >3,000 candidate SNPs forming >100 independently evolving clusters. The candidate genomic regions were enriched in genes implicated in hormone, carbohydrate, and lipid metabolism, including some with known effects on fitness-related life-history traits. Rather than being close to fixation, a substantial fraction of candidate SNPs segregated at intermediate allele frequencies in all malnutrition-adapted populations. This, together with patterns of among-population variation in allele frequencies and estimates of Tajima's D, suggests that the poor diet results in balancing selection on some genomic regions. Candidate genes for tolerance to larval malnutrition showed a high overlap with genes previously implicated in acute starvation resistance. However, adaptation to larval malnutrition in this study was associated with reduced tolerance to acute adult starvation. Thus, rather than reflecting synergy, the shared genomic architecture appears to mediate an evolutionary trade-off between tolerances to these two forms of nutritional stress (Kawecki, 2021).

    Male fertility thermal limits predict vulnerability to climate warming

    Forecasting which species/ecosystems are most vulnerable to climate warming is essential to guide conservation strategies to minimize extinction. Tropical/mid-latitude species are predicted to be most at risk as they live close to their upper critical thermal limits (CTLs). However, these assessments assume that upper CTL estimates, such as CTmax, are accurate predictors of vulnerability and ignore the potential for evolution to ameliorate temperature increases. This study used experimental evolution to assess extinction risk and adaptation in tropical and widespread Drosophila species. Tropical species were found to succumb to extinction before widespread species. Male fertility thermal limits, which are much lower than CTmax, are better predictors of species' current distributions and extinction in the laboratory. Little evidence was found of adaptive responses to warming in any species. These results suggest that species are living closer to their upper thermal limits than currently presumed and evolution/plasticity are unlikely to rescue populations from extinction (van Heerwaarden, 2021).

    Parallel and Population-specific Gene Regulatory Evolution in Cold-Adapted Fly Populations

    hanges in gene regulation at multiple levels may comprise an important share of the molecular changes underlying adaptive evolution in nature. However, few studies have assayed within- and between-population variation in gene regulatory traits at a transcriptomic scale, and therefore inferences about the characteristics of adaptive regulatory changes have been elusive. This study assessed quantitative trait differentiation in gene expression levels and alternative splicing (intron usage) between three closely-related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. The cold-adapted populations were known to show population genetic evidence for parallel evolution at the SNP level, and this study found evidence for parallel expression evolution between them, with stronger parallelism at larval and adult stages than for pupae. A flexible method was implemented to estimate cis- versus trans-encoded contributions to expression or splicing differences at the adult stage. The apparent contributions of cis- versus trans-regulation to adaptive evolution vary substantially among population pairs. While two of three population pairs show a greater enrichment of cis-regulatory differences among adaptation candidates, trans-regulatory differences are more likely to be implicated in parallel expression changes between population pairs. Genes with significant cis-effects are enriched for signals of elevated genetic differentiation between cold- and warm-adapted populations, suggesting that they are potential targets of local adaptation. These findings expand knowledge of adaptive gene regulatory evolution and the ability to make inferences about this important and widespread process (Huang, 2021).

    Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster

    Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, this study examined the genetic basis and evolutionary history of ovarian diapause. Outbred females were exposed to different temperatures and day lengths, ovarian development was characterized for over 2800 flies, and their complete, phased genomes were reconstructed. Diapause, scored at two different developmental cutoffs, was found to be modest heritability, and hundreds of SNPs associated with each of the two phenotypes were identified. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, outdoor mesocosms were used to track diapause under natural conditions. Hybrid swarms reared outdoors were found to evolve increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. These results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space (Erickson, 2020).

    Contingency in the convergent evolution of a regulatory network: Dosage compensation in Drosophila

    The repeatability or predictability of evolution is a central question in evolutionary biology and most often addressed in experimental evolution studies. This study inferred how genetically heterogeneous natural systems acquire the same molecular changes to address how genomic background affects adaptation in natural populations. In particular, advantage was taken of independently formed neo-sex chromosomes in Drosophila species that have evolved dosage compensation by co-opting the dosage-compensation male-specific lethal (MSL) complex to study the mutational paths that have led to the acquisition of hundreds of novel binding sites for the MSL complex in different species. This complex recognizes a conserved 21-bp GA-rich sequence motif that is enriched on the X chromosome, and newly formed X chromosomes recruit the MSL complex by de novo acquisition of this binding motif. Recently formed sex chromosomes were identified in the D. melanica and D. robusta species groups by genome sequencing and generate genomic occupancy maps of the MSL complex to infer the location of novel binding sites. Diverse mutational paths were utilized in each species to evolve hundreds of de novo binding motifs along the neo-X, including expansions of microsatellites and transposable element (TE) insertions. However, the propensity to utilize a particular mutational path differs between independently formed X chromosomes and appears to be contingent on genomic properties of that species, such as simple repeat or TE density. This establishes the 'genomic environment' as an important determinant in predicting the outcome of evolutionary adaptations (Ellison, 2019).

    This study took advantage of naturally occurring variation in sex chromosome karyotype in Drosophila species to study independent replicates of solving the same evolutionary challenge: to dosage compensate newly formed neo-X chromosomes by acquiring hundreds of MSL-binding sites in response to Y degeneration (Ellison, 2019).

    The independent acquisition of dosage compensation in Drosophila allows several important questions in evolutionary biology and gene regulation to be addressed: first, how repeatable is evolution? Evolutionary biologists have long debated the predictability of the evolutionary process. At one extreme, evolution could be highly idiosyncratic and unpredictable, since the survival of the fittest could occur along a great number of forking paths. Alternatively, constraints on evolution may force independent lineages to frequently converge on the same genetic solutions for the same evolutionary challenge. Second, how do regulatory networks evolve? And what is the contribution of TEs to regulatory evolution? Evolutionary innovations and adaptations often require rapid and concerted changes in regulation of gene expression at many loci. TEs constitute the most dynamic part of eukaryotic genomes, and the dispersal of TEs that contain a regulatory element may allow for the same regulatory motif to be recruited at many genomic locations, thereby drawing multiple genes into the same regulatory network. Third, what makes a binding motif functional? The genomes of complex organisms encompass megabases of DNA, and regulatory molecules must distinguish specific targets within this vast landscape. Regulatory factors typically identify their targets through sequence-specific interactions with the underlying DNA, but they typically bind only a fraction of the candidate genomic regions containing their specific target sequence motif. An unresolved mystery in regulatory evolution is what drives the specificity of binding to a subset of genomic regions that all appear to have a sequence that matches the consensus binding motif (Ellison, 2019).

    Several features make dosage compensation in Drosophila a promising system to tackle these questions. The genetic architecture for most adaptations -- especially those involving regulatory changes -- as well as the timing and exact selective forces driving them is generally little understood. In contrast, detailed knowledge is available of the molecular mechanism of dosage compensation in Drosophila. The cis- and trans-acting components of this regulatory network and the regulatory motif for targeting the MSL complex to the X are known. Clear expectations are available of which genomic regions should acquire dosage compensation and about the timing and the evolutionary forces that drive wiring of hundreds of genes into the dosage-compensation network on newly evolved X chromosomes. Specifically, Y degeneration is a general facet of sex chromosome evolution, creating selective pressures to up-regulate X-linked genes in males. Dosage compensation should thus only evolve on neo-X chromosomes whose neo-Y homologs have started to degenerate and should evolve simultaneously or shortly after substantial gene loss has occurred on the neo-Y. Indeed, comparative data in Drosophila support this model of dosage-compensation evolution. Drosophila species with partially eroded neo-Y chromosomes exist that have not yet evolved MSL-mediated dosage compensation, including D. busckii and D. albomicans, lending empirical support to the notion that dosage compensation evolves in response to Y degeneration and not the other way round. Thus, a refined understanding of how, when, why, and where dosage compensation in Drosophila evolves makes this an ideal model system to study the repeatability of evolution and the evolution of regulatory networks (Ellison, 2019).

    Results from evolution experiments indicate that although evolution is not identical in replicate populations, there is an important degree of predictability. Experimentally evolved populations under controlled, identical conditions consistently show parallelism in which mutations in certain genes are repeatedly selected. However, organisms adapting to similar environments are not genetically identical, but their genome instead carries the legacy of their unique evolutionary trajectory, raising the question of how genomic differences affect genetic parallelism (Ellison, 2019).

    Sex chromosome-autosome fusions have independently created neo-sex chromosomes in different Drosophila lineages. This provides everal independent replicates to study how, on the molecular level, evolution has solved the same adaptive challenge: acquiring hundreds of binding sites to recruit the MSL complex to newly formed X chromosomes. This allows quantification of how much variation there is, both within and between species, in the underlying mutational paths to acquire hundreds of MSL-binding sites on neo-X chromosomes and identify genomic contingencies that will influence the repeatability of evolutionary trajectories. Importantly, neo-sex chromosomes of Drosophila are evolutionarily young (between 0.1-15 MY old), which allows, in many cases, inferring of the causative mutations that have resulted in the gain of a regulatory element and decipher the evolutionary processes at work to draw hundreds of genes into a new regulatory network (Ellison, 2019).

    The results suggest that the evolution of MSL-binding sites is highly opportunistic but contingent on genomic background. In particular, each independently evolved neo-X chromosome was found to use a diverse set of mutational pathways to acquire MSL-binding sites on a new neo-X chromosome, ranging from microsatellite expansions to the utilization of presites to TE insertions. However, different lineages differ with regards to the frequency of which mutational paths are most often followed to acquire novel binding sites, and this propensity may depend on the genomic background. In particular, species found with the higher density of simple repeats are more prone to utilize expansions in GA microsatellites to gain a novel MSL-binding site. In contrast, D. robusta has an elevated TE density compared to its sibling species, and it was found that the dispersal of a TE has played an important role in the acquisition of MSL-binding sites on its neo-X chromosome. Thus, this suggests that the genomic background of a species predisposes it to evolve along a particular path, yet the evolutionary process is random and resourceful with regards to utilizing a variety of mutations to create novel MSL-binding sites. However, different phenotypes show drastic differences in their underlying genetic architecture, and the importance of genomic background likely differs among traits and (Ellison, 2019).

    Evolutionary innovations and adaptations often require rapid and concerted changes in regulation of gene expression at many loci. It has been suggested that TEs play a key role in rewiring regulatory networks, since the dispersal of TEs that contain a regulatory element may allow for the same regulatory motif to be recruited at many genomic locations. A handful of recent studies have implicated TEs as drivers of key evolutionary innovations, including placentation in mammals or rewiring the core regulatory network of human embryonic stem cells. While these studies demonstrate that TEs can, in principle, contribute to the creation or rewiring of regulatory networks, they do not address the question of how often regulatory elements evolve by TE insertions versus by other mutations. That is, the importance of TEs in contributing to regulatory evolution is not known. Quantification of the role of TEs would require a priori knowledge of how and when regulatory networks evolve and a detailed molecular understanding of which genes are being drawn into a regulatory network and how. As discussed above, these parameters are well understood for dosage compensation in flies (Ellison, 2019).

    Previous work in D. miranda has shown that a helitron TE was recruited into the dosage-compensation network at two independent time points. The younger 1.5-MY-old neo-X chromosome of D. miranda is in the process of evolving dosage compensation, and dozens of new CESs on this chromosome were created by insertions of the ISX element. The domesticated ISX TE gained a novel MRE motif by a 10-bp deletion in the ISY element, which is a highly abundant TE in the D. miranda genome. The remnants of a related (but different) TE at CES was found on the older neo-X of this species (which formed roughly 13-15 MY ago), but the TE was too eroded to reconstruct its evolutionary history. This study, identified another domesticated TE that was utilized to deliver MSL-binding sites to a newly formed neo-X chromosome, but no significant TE contribution was found for MSL-binding site evolution in two independent neo-X chromosomes (Ellison, 2019).

    The data shed light on the question of when TEs are expected to be important in regulatory evolution. For TEs to contribute to regulatory rewiring, two conditions have to be met: a regulatory element (or a progenitor sequence that can easily mutate into the required binding motif) needs to be present in the TE, and TE needs to be active in the genome (and not yet silenced by the host machinery). TEs undergo a characteristic life cycle in which they invade a new species (or escape the genome defense by mutation) and transpose until they are silenced by the host genome. Once a TE is robustly repressed, it no longer can serve as a vehicle to disperse regulatory elements, so the time window when a particular TE family can be domesticated is probably short and needs to coincide with a necessity to disperse regulatory motifs. A high TE burden does increase that chance, but at a cost: maintaining active TEs in the genome allows a rapid response to evolutionary challenges but also creates a major source of genomic mutation, illegitimate recombination, genomic rearrangements, and genome size inflation (Ellison, 2019).

    The current findings support this view of a TE tradeoff. The ISY element in D. miranda is the most highly abundant transposon in the D. miranda genome and is massively contributing to the degeneration of the neo-Y in this specie. Indeed, the genomic analysis has revealed >20,000 novel insertions of the ISY element on the neo-Y, often within genes. Yet, it contained a sequence that was only one mutational step away from a functional MSL-binding site (that is, a single 10-bp deletion), and domestication of this element allowed for the rapid dispersal of functional binding sites for the MSL complex along the neo-X. The domestication of the TE in D. robusta occurred too long ago for to reconstruct its exact evolutionary history and the potential damage its mobilization may have caused while it was active. However, consistent with a tradeoff that the host genome faces, it was found that D. robusta has a higher TE density than its sister species and also a considerably larger genome size, yet a TE contributed to wiring hundreds of genes into the dosage-compensation network on its neo-X (Ellison, 2019).

    Perhaps surprisingly, in many instances, it was not possible to detect specific mutations that would generate a novel MSL binding motif. Instead, it was found that functional MSL-binding sites are derived from presites containing the GA-rich motif that was already present in an ancestor in which the neo-X is autosomal and in which these sequences do not recruit the MSL complex. The MSL binding motif is only modestly enriched on the X chromosome compared to the autosomes (only approximately 2-fold), and only a small fraction of putative binding sites are actually bound by the MSL complex. The dosage-compensation machinery shares this characteristic with many other sequence-specific binding factors whose predicted target motifs are often in vast excess to the sites actually utilized. It has been speculated that other genomic aspects, such as chromatin context or the 3D organization of the genome, could help to distinguish between utilized and nonutilized copies of a motif. The finding that a large number of sites can acquire the ability to recruit the MSL complex, without any apparent associated changes at the DNA level, supports the view that epigenetic modifications or changes to the 3D architecture of the genome help to ultimately determine which putative binding sites in the genome are actually utilized. In D. melanogaster, the X chromosome has a unique satellite DNA composition, and it was suggested that these repeats play a primary role in determining X identity during dosage compensation. Furthermore, localization of the MSL complex to MREs is dependent on an additional cofactor, the CLAMP protein. CLAMP binds directly to GA-rich MRE sequences and targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome. Recent work has shown that variability in sequence composition within similar GA-rich motifs drive specificity for CLAMP binding, and variation within seemingly similar cis elements may also drive context-specific targeting of the MSL complex. Future investigations of changes in the chromatin level, the repeat content, and the genomic architecture of these newly formed sex chromosomes will help to resolve this outstanding question (Ellison, 2019).

    Allelic polymorphism at foxo contributes to local adaptation in Drosophila melanogaster

    The insulin/insulin-like growth factor signaling pathway has been hypothesized as a major determinant of life-history profiles that vary adaptively in natural populations. In Drosophila melanogaster, multiple components of this pathway vary predictably with latitude; this includes foxo, a conserved gene that regulates insulin signaling and has pleiotropic effects on a variety of fitness-associated traits. It was hypothesized that allelic variation at foxo contributes to genetic variance for size-related traits that vary adaptively with latitude. Patterns of variation were examined among natural populations along a latitudinal transect in the eastern United States; thorax length, wing area, wing loading, and starvation tolerance were found to exhibit significant latitudinal clines for both males and females but that development time does not vary predictably with latitude. Recombinant outbred populations were generated, naturally occurring allelic variation at foxo, which exhibits stronger clinality than expected, were shown to be associated with the same traits that vary with latitude in the natural populations. These results suggest that allelic variation at foxo contributes to adaptive patterns of life-history variation in natural populations of this genetic model (Betancourt, 2021).

    Gene expression clines reveal local adaptation and associated trade-offs at a continental scale

    Local adaptation, where fitness in one environment comes at a cost in another, should lead to spatial variation in trade-offs between life history traits and may be critical for population persistence. Recent studies have sought genomic signals of local adaptation, but often have been limited to laboratory populations representing two environmentally different locations of a species' distribution. This study measured gene expression, as a proxy for fitness, in males of Drosophila subobscura, occupying a 20 degrees latitudinal and 11 ° C thermal range. Uniquely, six populations were sampled, and both common garden and semi-natural responses to identify signals of local adaptation were identified. Contrasting patterns of investment were found: transcripts with expression positively correlated to latitude were enriched for metabolic processes, expressed across all tissues whereas negatively correlated transcripts were enriched for reproductive processes, expressed primarily in testes. When using only the end populations, to compare the results to previous studies, it was found that locally adaptive patterns were obscured. While phenotypic trade-offs between metabolic and reproductive functions across widespread species are well-known, the results identify underlying genetic and tissue responses at a continental scale that may be responsible for this. This may contribute to understanding population persistence under environmental change (Porcelli, 2016).

    Experimental evolution of gene expression and plasticity in alternative selective regimes

    Little is known of how gene expression and its plasticity evolves as populations adapt to different environmental regimes. Expression is expected to evolve adaptively in all populations but only those populations experiencing environmental heterogeneity are expected to show adaptive evolution of plasticity. This study measured the transcriptome in a cadmium-enriched diet and a salt-enriched diet for experimental populations of Drosophila melanogaster that evolved for ~130 generations in one of four selective regimes: two constant regimes maintained in either cadmium or salt diets and two heterogeneous regimes that varied either temporally or spatially between the two diets. For populations evolving in constant regimes, a strong signature of counter-gradient evolution was found; the evolved expression differences between populations adapted to alternative diets is opposite to the plastic response of the ancestral population that is naive to both diets. Based on expression patterns in the ancestral populations, a set of genes was identified for which selection in heterogeneous regimes was predicted to result in increases in plasticity, and the expected pattern was found. In contrast, a set of genes where reduced plasticity was predicted did not follow expectation. Nonetheless, both gene sets showed a pattern consistent with adaptive expression evolution in heterogeneous regimes, highlighting the difference between observing 'optimal' plasticity and improvements in environment-specific expression. Looking across all genes, there is evidence in all regimes of differences in biased allele expression across environments ('allelic plasticity') and this is more common among genes with plasticity in total expression (Huang, 2016).

    Reasons for success: rapid evolution for desiccation resistance and life-history changes in the polyphagous fly Anastrepha ludens

    Species that exhibit broad ranges of distribution may successfully navigate environmental changes by modifying some of their life history traits. Environmental humidity imposes a critical stress that organisms may overcome by increasing their resistance to desiccation. This study used experimental evolution to investigate adaptation to desiccation in the tephritid Anastrepha ludens, a species with high fecundity, late maturation and long lifespan. This study measured morphological, physiological, developmental as well as demographic changes involved in the adaptation to desiccation. Notwithstanding a low heritability (h2 = 0.237), desiccation resistance evolved extremely rapidly and few negative trade-offs were detected. Selected flies exhibited correlated increases in longevity, body size, the amount of body lipids and bulk water content, and in the duration of the pupal stage. Females further delayed sexual maturation, decreased daily fecundity but retained high lifetime reproductive potential. No differences in male mating competitiveness were found. Selected and control lines differed in longevity but not in total female fecundity, demonstrating that A. ludens flies have the capability for fast adaptation to desiccation without loosing their reproductive capability. Thus, it seems that a rapid evolutionary response to desiccation in this polyphagous insect works as a buffer for environmental variation and reduces the strength of selection on reproductive traits (Tejeda, 2016).

    Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster

    Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. The direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. This study investigated flexibility of nutrient catabolism during cold stress in Drosophila artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using 13C-labelled glucose, this study first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers cold-hardy flies were shown to have higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories (Williams, 2016).

    Colder environments did not select for a faster metabolism during experimental evolution of Drosophila melanogaster

    The effect of temperature on the evolution of metabolism has been the subject of debate for a century; however, no consistent patterns have emerged from comparisons of metabolic rate within and among species living at different temperatures. This study used experimental evolution to determine how metabolism evolves in populations of Drosophila melanogaster exposed to one of three selective treatments: a constant 16 ° C, a constant 25 ° C, or temporal fluctuations between 16 and 25 ° C. August Krogh's controversial hypothesis was tested that colder environments select for a faster metabolism. Given that colder environments also experience greater seasonality, the hypothesis was also tested that temporal variation in temperature may be the factor that selects for a faster metabolism. The metabolic rate of flies from each selective treatment was measured at 16, 20.5, and 25 ° C. Although metabolism was faster at higher temperatures, flies from the selective treatments had similar metabolic rates at each measurement temperature. Based on variation among genotypes within populations, heritable variation in metabolism was likely sufficient for adaptation to occur. It is concluded that colder or seasonal environments do not necessarily select for a faster metabolism. Rather, other factors besides temperature likely contribute to patterns of metabolic rate over thermal clines in nature (Alton, 2016).

    Adaptive patterns of phenotypic plasticity in laboratory and field environments in Drosophila melanogaster

    Identifying mechanisms of adaptation to variable environments is essential in developing a comprehensive understanding of evolutionary dynamics in natural populations. Phenotypic plasticity allows for phenotypic change in response to changes in the environment, and as such may play a major role in adaptation to environmental heterogeneity. This study examined the plasticity of stress response in D. melanogaster originating from two distinct geographic regions and ecological habitats. Adults were given a short-term, 5-day exposure to combinations of temperature and photoperiod to elicit a plastic response for three fundamental aspects of stress tolerance that vary adaptively with geography. This was replicated in both the laboratory and in outdoor enclosures in the field. In the laboratory, geographic origin was found to be the primary determinant of the stress response. Temperature and the interaction between temperature and photoperiod were also found to significantly affect stress resistance. In the outdoor enclosures, plasticity was distinct among traits and between geographic regions. These results demonstrate that short-term exposure of adults to ecologically relevant environmental cues results in predictable effects on multiple aspects of fitness. These patterns of plasticity vary among traits and are highly distinct between the two examined geographic regions, consistent with patterns of local adaptation to climate and associated environmental parameters (Mathur, 2016).

    Genomic trajectories to desiccation resistance: Convergence and divergence among replicate selected Drosophila lines

    Adaptation to environmental stress is critical for long-term species persistence. With climate change and other anthropogenic stressors compounding natural selective pressures, understanding the nature of adaptation is as important as ever in evolutionary biology. This study investigated this issue in a set of replicated Drosophila lines selected for increased desiccation resistance, a classical physiological trait that has been closely linked to Drosophila species distributions. Pooled whole-genome sequencing was used to compare the genetic basis of their selection responses. While selected SNPs in replicates of the same treatment (desiccation-selection or lab adaptation) tended to change frequency in the same direction, suggesting some commonality in the selection response, candidate SNP and gene lists often differed among replicates. Three of the five desiccation-selection replicates showed significant overlap at the gene and network level. All five replicates showed enrichment for ovary-expressed genes, suggesting maternal effects on the selected trait. Divergence between pairs of replicate lines for desiccation-candidate SNPs was greater than between pairs of control lines. This difference also far exceeded the divergence between pairs of replicate lines for neutral SNPs. Overall, while there was overlap in the direction of allele frequency changes and the network and functional categories affected by desiccation selection, replicates showed unique responses at all levels likely reflecting hitchhiking effects, and highlighting the challenges in identifying candidate genes from these types of experiments when traits are likely to be polygenic (Griffin, 2016).

    Independent natural genetic variation of punishment- versus relief-memory

    A painful event establishes two opponent memories: cues that are associated with pain onset are remembered negatively, whereas cues that coincide with the relief at pain offset acquire positive valence. Such punishment- versus relief-memories are conserved across species, including humans, and the balance between them is critical for adaptive behaviour with respect to pain and trauma. In the fruit fly, Drosophila melanogaster as a study case, this study found that both punishment- and relief-memories display natural variation across wild-derived inbred strains, but they do not covary, suggesting a considerable level of dissociation in their genetic effectors. This provokes the question whether there may be heritable inter-individual differences in the balance between these opponent memories in man, with potential psycho-clinical implications (Appel, 2016).

    The genetic basis of natural variation in Drosophila (Diptera: Drosophilidae) virgin egg retention

    Drosophila melanogaster is able to thrive in harsh northern climates through adaptations in life-history traits and physiological mechanisms that allow for survival through the winter. This examined the genetic basis of natural variation in one such trait, female virgin egg retention, which was previously shown to vary clinally and seasonally. To further understanding of the genetic basis and evolution of virgin egg retention, a genome-wide association study (GWAS) was performed using the previously sequenced Drosophila Genetic Reference Panel (DGRP) mapping population. Twenty-nine single nucleotide polymorphisms (SNPs) associated with virgin egg retention were found, and six available mutant lines, each harboring a mutation in a candidate gene, were examined for effects on egg retention time. Four out of the six mutant lines had defects in egg retention time as compared with the respective controls: mun, T48, Mes-4, and Klp67A Surprisingly, none of these genes has a recognized role in ovulation control, but three of the four genes have known effects on fertility or have high expression in the ovaries. The SNP set associated with egg retention time was enriched for clinal SNPs. The majority of clinal SNPs had alleles associated with longer egg retention present at higher frequencies in higher latitudes. These results support previous studies that show higher frequency of long retention times at higher latitude, providing evidence for the adaptive value of virgin egg-retention (Akhund-Zade, 2016).

    Testing for local adaptation and evolutionary potential along altitudinal gradients in rainforest Drosophila: beyond laboratory estimates

    Predicting how species will respond to the rapid climatic changes predicted this century is an urgent task. Species distribution models (SDMs) use the current relationship between environmental variation and species' abundances to predict the effect of future environmental change on their distributions. However, two common assumptions of SDMs are likely to be violated in many cases: (i) that the relationship of environment with abundance or fitness is constant throughout a species' range and will remain so in future and (ii) that abiotic factors (e.g. temperature, humidity) determine species' distributions. These assumptions were tested by relating field abundance of the rainforest fruit fly Drosophila birchii to ecological change across gradients that include its low and high altitudinal limits. Then, how such ecological variation affects the fitness of 35 D. birchii families transplanted in 591 cages to sites along two altitudinal gradients, was tested to determine whether genetic variation in fitness responses could facilitate future adaptation to environmental change. Overall, field abundance was highest at cooler, high-altitude sites, and declined towards warmer, low-altitude sites. By contrast, cage fitness (productivity) increased towards warmer, lower-altitude sites, suggesting that biotic interactions (absent from cages) drive ecological limits at warmer margins. In addition, the relationship between environmental variation and abundance varied significantly among gradients, indicating divergence in ecological niche across the species' range. However, there was no evidence for local adaptation within gradients, despite greater productivity of high-altitude than low-altitude populations when families were reared under laboratory conditions. Families also responded similarly to transplantation along gradients, providing no evidence for fitness trade-offs that would favour local adaptation. These findings highlight the importance of (i) measuring genetic variation in key traits under ecologically relevant conditions, and (ii) considering the effect of biotic interactions when predicting species' responses to environmental change (O'Brien, 2017)

    Genomics of parallel experimental evolution in Drosophila

    What are the genomic foundations of adaptation in sexual populations? This question was addressed using fitness-character and whole-genome sequence data from 30 Drosophila laboratory populations. These 30 populations are part of a nearly forty-year laboratory radiation featuring three selection regimes, each shared by ten populations for up to 837 generations, with moderately large effective population sizes. Each of three sets of ten populations that shared a selection regime consist of five populations that have long been maintained under that selection regime, paired with five populations that had only recently been subjected to that selection regime. A high degree of evolutionary parallelism in fitness phenotypes was found when most-recent selection regimes are shared, as in previous studies from this laboratory. Genomic parallelism was also found with respect to the frequencies of single-nucleotide polymorphisms, transposable elements, insertions, and structural variants, which was expected. Entirely unexpected was a high degree of parallelism for linkage disequilibrium. The evolutionary genetic changes among these sexual populations are rapid and genomically extensive. This pattern may be due to segregating functional genetic variation that is abundantly maintained genome-wide by selection, variation that responds immediately to changes of selection regime (Graves, 2017).

    Geographical analysis of diapause inducibility in European Drosophila melanogaster populations

    Seasonal overwintering in insects represents an adaptation to stressful environments and in European Drosophila melanogaster females, low temperatures and short photoperiods can induce an ovarian diapause. Diapause may represent a recent (<15Ky) adaptation to the colonisation of temperate Europe by D. melanogaster from tropical sub-Saharan Africa, because African D. melanogaster and the sibling species D. simulans, have been reported to fail to undergo diapause. Over the past few centuries, D. melanogaster have also invaded North America and Australia, and eastern populations on both continents show a predictable latitudinal cline in diapause induction. In Europe however, a new diapause-enhancing timeless allele, ls-tim, is observed at high levels in southern Italy ( approximately 80%), where it appears to have arisen and has spread throughout the continent with a frequency of approximately 20% in Scandinavia. Given the phenotype of ls-tim and its geographical distribution, it was predicted that it would work against any latitudinal cline in diapause induction within Europe. Indeed this study revealed that any latitudinal cline for diapause in Europe is very weak, as predicted by ls-tim frequencies. In contrast, ls-tim frequencies were determined in North America and it was observed that they would be expected to strengthen the latitudinal pattern of diapause. The results reveal how a newly arisen mutation, can, via the stochastic nature of where it initially arose, blur an otherwise adaptive geographical pattern (Pegoraro, 2017)

    Evolution of circadian rhythms in Drosophila melanogaster populations reared in constant light and dark regimes for over 330 generations

    Organisms are believed to have evolved circadian clocks as adaptations to deal with cyclic environmental changes, and therefore it has been hypothesized that evolution in constant environments would lead to regression of such clocks. This study examined whether circadian clocks and the associated properties evolve differently under constant light and constant darkness. Activity-rest, adult emergence and oviposition rhythms were measured of D. melanogaster populations that have been maintained for over 19 years (~330 generations) under three different light regimes - constant light (LL), light-dark cycles of 12:12 h (LD) and constant darkness (DD). While circadian rhythms in all the three behaviors persist in both LL and DD stocks with no differences in circadian period, they differed in certain aspects of the entrained rhythms when compared to controls reared in rhythmic environment (LD). Interestingly, it was also observed that DD stocks have evolved significantly higher robustness or power of free-running activity-rest and adult emergence rhythms compared to LL stocks. Thus, this study, in addition to corroborating previous results of circadian clock evolution in constant light, also highlights that, contrary to the expected regression of circadian clocks, rearing in constant darkness leads to the evolution of more robust circadian clocks which may be attributed to an intrinsic adaptive advantage of circadian clocks and/or pleiotropic functions of clock genes in other traits (Shindey, 2017).

    Transcriptional polymorphism of piRNA regulatory genes underlies the mariner activity in D. simulans testes

    During colonization of new areas, natural populations have to deal with changing environments, and transposable elements (TEs) can be useful "tools" in the adaptation process since they are major contributor to the structural and functional evolution of genomes. In this general context, the activity (copy number, transcriptional and excision rate) of the mariner mos1 element was estimated in 19 natural populations of D. simulans. It is shown (1) that mos1 expression is always higher and more variable in testes than in ovaries; (2) that mos1 activity is higher in colonizing populations compared to the sub-Saharan African ones (ancestral populations); (3) that mos1 variations in transcript levels and copy number are negatively correlated to transcriptional variations of piRNA genes, aubergine and argonaute3. Furthermore, mos1 levels of expression in testes highly contrast with the low expression patterns of ago3. These results strongly suggest that the expression polymorphism of piRNA genes could be responsible for the mos1 variations, first between male and female germlines and second, according to the status of natural populations (colonizing or not). These results provide new perspectives about TEs and piRNA genes co-evolution in Drosophila germlines (Saint-Leandre, 2017).

    A test for gene flow among sympatric and allopatric Hawaiian picture-winged Drosophila

    The Hawaiian Drosophila are one of the most species-rich endemic groups in Hawaii and a spectacular example of adaptive radiation. Drosophila silvestris and D. heteroneura are two closely related picture-winged Drosophila species that occur sympatrically on Hawaii Island and are known to hybridize in nature, yet exhibit highly divergent behavioral and morphological traits driven largely through sexual selection. Their closest-related allopatric species, D. planitibia from Maui, exhibits hybrid male sterility and reduced behavioral reproductive isolation when crossed experimentally with D. silvestris or D. heteroneura. A modified four-taxon test for gene flow was applied to recently obtained genomes of the three Hawaiian Drosophila species. The analysis indicates recent gene flow in sympatry, but also, although less extensive, between allopatric species. This study underscores the prevalence of gene flow, even in taxonomic groups considered classic examples of allopatric speciation on islands. The potential confounding effects of gene flow in phylogenetic and population genetics inference are discussed, as well as the implications for conservation (Kang, 2017).

    Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster

    Identifying the genetic basis for adaptive differences between species requires explicit tests of historical hypotheses concerning the effects of past changes in gene sequence on molecular function, organismal phenotype and fitness. This challenge was addressed by combining ancestral protein reconstruction with biochemical experiments and physiological analysis of transgenic animals that carry ancestral genes. A widely held hypothesis of molecular adaptation was tested in this study-that changes in the alcohol dehydrogenase protein (ADH) along the lineage leading to Drosophila melanogaster increased the catalytic activity of the enzyme and thereby contributed to the ethanol tolerance and adaptation of the species to its ethanol-rich ecological niche. These experiments strongly refute the predictions of the adaptive ADH hypothesis and caution against accepting intuitively appealing accounts of historical molecular adaptation that are based on correlative evidence. The experimental strategy employed can be used to decisively test other adaptive hypotheses and the claims they entail about past biological causality (Siddiq, 2017).

    Adaptive evolution of gene expression in Drosophila

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. This study presents evidence that adaptation dominates the evolution of gene expression levels in flies. 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. The results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, a large group of genes was identifed with sex-specific adaptation of expression, which predominantly occurs in males. This analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis (Nourmohammad, 2017).

    Adaptive evolution of gene expression in Drosophila

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. This study presents evidence that adaptation dominates the evolution of gene expression levels in flies. 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. The results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, a large group of genes was identified with sex-specific adaptation of expression, which predominantly occurs in males. This analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis (Nourmohammad, 2017).

    The inference of adaptation exploits the complex dependence of the expression divergence on the evolutionary distance between species. It reflects two fundamental evolutionary features of quantitative traits. First, such traits generate a divergence pattern with two distinct molecular clocks: at a short evolutionary distance, the divergence is always near the expected value under neutrality; at a longer distance, it depends jointly on stabilizing and directional selection. This feature reconciles seemingly contradictory results of previous studies: analysis of closely related species produces a signal of neutral evolution, whereas evolutionary constraint becomes apparent for more distant species. Second, the phenotypic evolution of gene expression decouples from details of its genetic basis. This explains why overall strong selection on gene expression levels was found even though selection on individual QTLs is often weak. The probabilistic extension of the curren inference scheme, which is based on gene-specific expression divergence, identifies functional gene classes associated with adaptive evolution of regulation (Nourmohammad, 2017).

    The selection model underlying this analysis is a single-peak fitness seascape, which contains components of stabilizing and directional selection on a quantitative trait. These components are well-established notions of quantitative genetics on micro-evolutionary timescales. Each of them can provide a snapshot of the predominant selection pressure in a population. However, the description of selection remains incomplete as a description of selection over macro-evolutionary periods. If selection on a trait is directional at a given evolutionary time, will that selection relax after the trait value has significantly adapted in the direction of selection? If selection is stabilizing, can it be assumed that the optimal trait value will remain invariant in the context of a different species? To address these questions, a conceptual and quantitative synthesis of stabilizing and directional selection is need. The single-peak seascape model arguably provides the simplest such synthesis. It also provides a simple picture of continual adaptation over macro-evolutionary periods: a species follows a moving fitness peak, and this process generates positive fitness flux but no net increase in fitness (Nourmohammad, 2017).

    This method of selection inference can be applied to a spectrum of molecular quantitative traits with a complex genetic basis, provided that comparative data from multiple, sufficiently diverged species are available. Such traits include genome-wide protein levels, protein-DNA binding interactions, and enzymatic activities. For most of these traits, only partial knowledge is available of the underlying genetic loci and their effects on trait and fitness. This method complements QTL studies and opens a way to infer quantitative phenotype-fitness maps at the systems level (Nourmohammad, 2017).

    Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates

    Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. Several hundred differentially expressed genes and dozens of differentially edited sites were identified between flies from each slope; these changes were correlate with genetic differences, and CRISPR mutagenesis was used to validate that an intronic SNP in prominin regulates its editing levels. It was also demonstrated that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences (Yablonovitch, 2017).

    Inter- and intra-specific genomic divergence in Drosophila montana shows evidence for cold adaptation

    The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. This study describes the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species. Branch tests were used to identify genes showing accelerated divergence in contrasts between cold- and warm adapted species, and about 250 genes were identified that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. Evidence was sought of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, and no strong evidence was found for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. Three populations of D. montana were resequenced from its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales (Parker, 2018).

    Effects of evolutionary history on genome wide and phenotypic convergence in Drosophila populations

    Studies combining experimental evolution and next-generation sequencing have found that adaptation in sexually reproducing populations is primarily fueled by standing genetic variation. Consequently, the response to selection is rapid and highly repeatable across replicate populations. Some studies suggest that the response to selection is highly repeatable at both the phenotypic and genomic levels, and that evolutionary history has little impact. Other studies suggest that even when the response to selection is repeatable phenotypically, evolutionary history can have significant impacts at the genomic level. This study tests two hypotheses that may explain this discrepancy. Hypothesis 1: Past intense selection reduces evolutionary repeatability at the genomic and phenotypic levels when conditions change. Hypothesis 2: Previous intense selection does not reduce evolutionary repeatability, but other evolutionary mechanisms may. These hypotheses were tested using D. melanogaster populations that were subjected to 260 generations of intense selection for desiccation resistance and have since been under relaxed selection for the past 230 generations. It was found that, with the exception of longevity and to a lesser extent fecundity, 230 generations of relaxed selection has erased the extreme phenotypic differentiation previously found. No signs were found of genetic fixation, and only limited evidence of genetic differentiation between previously desiccation resistance selected populations and their controls. These findings suggest that evolution in this system is highly repeatable even when populations have been previously subjected to bouts of extreme selection. It is therefore concluded that evolutionary repeatability can overcome past bouts of extreme selection in Drosophila experimental evolution, provided experiments are sufficiently long and populations are not inbred (Phillips, 2018).

    Positive selection at sites of chemosensory genes is associated with the recent divergence and local ecological adaptation in cactophilic Drosophila

    Adaptation to new hosts in phytophagous insects often involves mechanisms of host recognition by genes of sensory pathways. Most often the molecular evolution of sensory genes has been explained in the context of the birth-and-death model. The role of positive selection is less understood, especially associated with host adaptation and specialization. This study aimed to contribute evidence for this latter hypothesis by considering the case of Drosophila mojavensis, a species with an evolutionary history shaped by multiple host shifts in a relatively short time scale, and its generalist sister species, D. arizonae. A phylogenetic and population genetic analysis framework was used to test for positive selection in a subset of four chemoreceptor genes, one gustatory receptor (Gr) and three odorant receptors (Or), for which their expression has been previously associated with host shifts. Strong evidence was found of positive selection at several amino acid sites in all genes investigated, most of which exhibited changes predicted to cause functional effects in these transmembrane proteins. A significant portion of the sites identified as evolving positively were largely found in the cytoplasmic region, although a few were also present in the extracellular domains. The pattern of substitution observed suggests that some of these changes likely had an effect on signal transduction as well as odorant recognition and protein-protein interactions. These findings support the role of positive selection in shaping the pattern of variation at chemosensory receptors, both during the specialization onto one or a few related hosts, but as well as during the evolution and adaptation of generalist species into utilizing several hosts (Dizz, 2018).

    Ultrastructural variation and adaptive evolution of the ovipositor in the endemic Hawaiian Drosophilidae

    Ecological diversification of the endemic Hawaiian Drosophilidae has been accompanied by striking divergence in egg morphology, and ovarian structure and function. To determine how these flies successfully oviposit in a variety of breeding substrates, Scanning Electron Microscopy was used to examine the ultrastructure of the ovipositor of a sample of 65 Drosophila species and five Scaptomyza species of this hyperdiverse monophyletic group. The Drosophila species analyzed included representatives of the fungus-breeding haleakalae group, the leaf-breeding antopocerus and modified tarsus groups, the modified mouthparts species group, the nudidrosophila, and the picture wing clade; the latter sample of 41 species from four species groups included stem- and bark-breeders, as well as tree sap flux-breeders. Ovipositor length was found to vary more than 12-fold among Hawaiian drosophilids, with the longest ovipositors observed in the bark-breeding species and the shortest among the Scaptomyza and fungus-breeders. More noteworthy is the striking variation in overall shape and proportions of the ovipositor, in the shape of the apical region, and in the pattern of sensory structures or ovisensilla. Ultrastructural observations of the pair of long subapical sensilla on the ventral side identify these as taste bristles. Ovipositor form correlates strongly with the oviposition substrate used by the species, being of a distinctive shape and size in each case. It is inferred that the observed morphological divergence in the ovipositor is adaptive and the product of natural selection for successful reproduction in alternate microhabitats (Craddock, 2018).

    Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species

    Insights into the genetic capacities of species to adapt to future climate change can be gained by using comparative genomic and transcriptomic data to reconstruct the genetic changes associated with such adaptations in the past. This study investigated the genetic changes associated with adaptation to arid environments, specifically climatic extremes and new cactus hosts, through such an analysis of five repleta group Drosophila species. Disproportionately high rates of gene gains were found in internal branches in the species' phylogeny where cactus use and subsequently cactus specialisation and high heat and desiccation tolerance evolved. The terminal branch leading to the most heat and desiccation resistant species, Drosophila aldrichi, also shows disproportionately high rates of both gene gains and positive selection. Several Gene Ontology terms related to metabolism were enriched in gene gain events in lineages where cactus use was evolving, while some regulatory and developmental genes were strongly selected in the Drosophila aldrichi branch. Transcriptomic analysis of flies subjected to sublethal heat shocks showed many more downregulation responses to the stress in a heat sensitive versus heat resistant species, confirming the existence of widespread regulatory as well as structural changes in the species' differing adaptations. Gene Ontology terms related to metabolism were enriched in the differentially expressed genes in the resistant species while terms related to stress response were over-represented in the sensitive one. It is concluded that daptations to new cactus hosts and hot desiccating environments were associated with periods of accelerated evolutionary change in diverse biochemistries. The hundreds of genes involved suggest adaptations of this sort would be difficult to achieve in the timeframes projected for anthropogenic climate change (Rane, 2019).

    Phenotypic plasticity facilitates initial colonization of a novel environment

    Phenotypic plasticity can allow organisms to respond to environmental changes by producing better matching phenotypes without any genetic change. Because of this, plasticity is predicted to be a major mechanism by which a population can survive the initial stage of colonizing a novel environment. This prediction was tested by challenging wild Drosophila melanogaster with increasingly extreme larval environments and then examining expression of alcohol dehydrogenase (ADH) and its relationship to larval survival in the first generation of encountering a novel environment. Most families responded in the adaptive direction of increased ADH activity in higher alcohol environments and families with higher plasticity were also more likely to survive in the highest alcohol environment. Thus, plasticity of ADH activity was positively selected in the most extreme environment and was a key trait influencing fitness. Furthermore, there was significant heritability of ADH plasticity that can allow plasticity to evolve in subsequent generations after initial colonization. The adaptive value of plasticity, however, was only evident in the most extreme environment and had little impact on fitness in less extreme environments. The results provide one of the first direct tests of the adaptive role of phenotypic plasticity in colonizing a novel environment (Wang, 2019).

    Genetic redundancy fuels polygenic adaptation in Drosophila

    The genetic architecture of adaptive traits is of key importance to predict evolutionary responses. Most adaptive traits are polygenic-i.e., result from selection on a large number of genetic loci-but most molecularly characterized traits have a simple genetic basis. This discrepancy is best explained by the difficulty in detecting small allele frequency changes (AFCs) across many contributing loci. To resolve this, laboratory natural selection was used to detect signatures for selective sweeps and polygenic adaptation. This study exposed 10 replicates of a Drosophila simulans population to a new temperature regime and uncovered a polygenic architecture of an adaptive trait with high genetic redundancy among beneficial alleles. Convergent responses were observed for several phenotypes-e.g., fitness, metabolic rate, and fat content-and a strong polygenic response (99 selected alleles; mean s = 0.059). However, each of these selected alleles increased in frequency only in a subset of the evolving replicates. Different evolutionary paradigms were discerned based on the heterogeneous genomic patterns among replicates. Redundancy and quantitative trait (QT) paradigms fitted the experimental data better than simulations assuming independent selective sweeps. These results show that natural D. simulans populations harbor a vast reservoir of adaptive variation facilitating rapid evolutionary responses using multiple alternative genetic pathways converging at a new phenotypic optimum. This key property of beneficial alleles requires the modification of testing strategies in natural populations beyond the search for convergence on the molecular level (Barghi, 2019).

    Demographic history of the human commensal Drosophila melanogaster

    The cohabitation of Drosophila melanogaster with humans is nearly ubiquitous. Though it has been well-established that this fly species originated in sub-Saharan Africa, and only recently has spread globally, many details of its swift expansion remain unclear. Elucidating the demographic history of D. melanogaster provides a unique opportunity to investigate how human movement might have impacted patterns of genetic diversity in a commensal species, as well as providing neutral null models for studies aimed at identifying genomic signatures of local adaptation. This study used whole-genome data from five populations (Africa, North America, Europe, Central Asia, and the South Pacific) to carry out demographic inferences, with particular attention to the inclusion of migration and admixture. The importance of these parameters for model fitting is demonstrated, and how previous estimates of divergence times are likely to be significantly underestimated as a result of not including them is shown. Finally, how human movement along early shipping routes might have shaped the present-day population structure of D. melanogaster is discussed (Arguello, 2019).

    A 24 h age difference causes twice as much gene expression divergence as 100 generations of adaptation to a novel environment

    Gene expression profiling is one of the most reliable high-throughput phenotyping methods, allowing researchers to quantify the transcript abundance of expressed genes. Because many biotic and abiotic factors influence gene expression, it is recommended to control them as tightly as possible. This study shows that a 24 h age difference of Drosophila simulans females that were subjected to RNA sequencing (RNA-Seq) five and six days after eclosure resulted in more than 2000 differentially expressed genes. This is twice the number of genes that changed expression during 100 generations of evolution in a novel hot laboratory environment. Importantly, most of the genes differing in expression due to age introduce false positives or negatives if an adaptive gene expression analysis is not controlled for age. These results indicate that tightly controlled experimental conditions, including precise developmental staging, are needed for reliable gene expression analyses, in particular in an evolutionary framework (Hsu, 2019).

    Estimating the timing of multiple admixture pulses during local ancestry inference

    Admixture, the mixing of genetically distinct populations, is increasingly recognized as a fundamental biological process. One major goal of admixture analyses is to estimate the timing of admixture events. Whereas most methods today can only detect the most recent admixture event, this study presents coalescent theory and associated software that can be used to estimate the timing of multiple admixture events in an admixed population. This approach was extensively validated and the conditions under which it can successfully distinguish one from two-pulse admixture models was validated. This approach was applied to real and simulated data of Drosophila melanogaster. Evidence was found of a single very recent pulse of cosmopolitan ancestry contributing to African populations as well as evidence for more ancient admixture among genetically differentiated populations in sub-Saharan Africa. These results suggest this method can quantify complex admixture histories involving genetic material introduced by multiple discrete admixture pulses. The new method facilitates the exploration of admixture and its contribution to adaptation, ecological divergence, and speciation (Medina, 2018).

    The making of a pest: Insights from the evolution of chemosensory receptor families in a pestiferous and invasive fly

    Drosophila suzukii differs from other melanogaster group members in their proclivity for laying eggs in fresh fruit rather than in fermenting fruits. Earlier work has revealed how the olfactory landscape of D. suzukii is dominated by volatiles derived from its unique niche. This study annotated the Olfactory receptors and Gustatory Receptors in D. suzukii and two close relatives, D. biarmipes and D. takahashii, to identify candidate chemoreceptors associated with D. suzukii's unusual niche utilization. A total of 71 Or genes were annotated in D. suzukii, with nine of those being pseudogenes (12.7 %). Alternative splicing of two genes brings the total to 62 genes encoding 66 Ors. Duplications of Or23a and Or67a expanded D. suzukii's Or repertoire, while pseudogenization of Or74a, Or85a, and Or98b reduced the number of functional Ors to roughly the same as other annotated species in the melanogaster group. Seventy-one intact Gr genes and three pseudogenes were annotated in D. suzukii. Alternative splicing in three genes brings the total number of Grs to 81. Signatures of positive selection were identified in two Ors and three Grs at nodes leading to D. suzukii, while three copies in the largest expanded Or lineage, Or67a, also showed signs of positive selection at the external nodes. This analysis of D. suzukii's chemoreceptor repertoires in the context of nine melanogaster group drosophilids, including two of its closest relatives (D. biarmipes and D. takahashii), revealed several candidate receptors associated with the adaptation of D. suzukii to its unique ecological niche (Hickner, 2016).

    A genome-wide scan for genes under balancing selection in Drosophila melanogaster

    In the history of population genetics balancing selection has been considered as an important evolutionary force, yet until today little is known about its abundance and its effect on patterns of genetic diversity. Several well-known examples of balancing selection have been reported from humans, mice, plants, and parasites. However, only very few systematic studies have been carried out to detect genes under balancing selection. This study carried out a genome scan in Drosophila melanogaster to find signatures of balancing selection in a derived (European) and an ancestral (African) population. A total of 34 genomes were scanned, searching for regions of high genetic diversity and an excess of SNPs with intermediate frequency. In total, 183 candidate genes were found: 141 in the European population and 45 in the African one, with only three genes shared between both populations. Most differences between both populations were observed on the X chromosome, though this might be partly due to false positives. Functionally, an overrepresentation of genes involved in neuronal development and circadian rhythm were found. Furthermore, some of the top genes identified are involved in innate immunity. These results revealed evidence of genes under balancing selection in European and African populations. More candidate genes have been found in the European population. They are involved in several different functions (Croze, 2017).

    Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster

    Evolutionary theory predicts that antagonistically selected alleles, such as those with divergent pleiotropic effects in early and late life, may often reach intermediate population frequencies due to balancing selection, an elusive process when sought out empirically. Alternatively, genetic diversity may increase as a result of positive frequency-dependent selection and genetic purging in bottlenecked populations. While experimental evolution systems with directional phenotypic selection typically result in at least local heterozygosity loss, this study reports that selection for increased lifespan in Drosophila melanogaster leads to an extensive genome-wide increase of nucleotide diversity in the selected lines compared to replicate control lines, pronounced in regions with no or low recombination, such as chromosome 4 and centromere neighborhoods. These changes, particularly in coding sequences, are most consistent with the operation of balancing selection and the antagonistic pleiotropy theory of aging and life history traits that tend to be intercorrelated. Genes involved in antioxidant defenses, along with multiple lncRNAs, were among those most affected by balancing selection. Despite the overwhelming genetic diversification and the paucity of selective sweep regions, two genes with functions important for central nervous system and memory, Ptp10D and Ank2, evolved under positive selection in the longevity lines. Overall, the 'evolve-and-resequence' experimental approach proves successful in providing unique insights into the complex evolutionary dynamics of genomic regions responsible for longevity (Michalak, 2017).

    Balancing selection drives the maintenance of genetic variation in Drosophila antimicrobial peptides

    Genes involved in immune defense against pathogens provide some of the most well-known examples of both directional and balancing selection. Antimicrobial peptides (AMPs) are innate immune effector genes, playing a key role in pathogen clearance in many species, including Drosophila. Conflicting lines of evidence have suggested AMPs may be under directional, balancing or purifying selection. This study used both a linear model and control gene-based approach to show that balancing selection is an important force shaping AMP diversity in Drosophila. In D. melanogaster, this is most clearly observed in ancestral African populations. Furthermore, the signature of balancing selection is even more striking once background selection has been accounted for. Balancing selection also acts on AMPs in D. mauritiana, an isolated island endemic separated from D. melanogaster by about 4 million years of evolution. This suggests that balancing selection may be broadly acting to maintain adaptive diversity in Drosophila AMPs, as has been found in other taxa (Chapman, 2019).

    Genetic convergence in the evolution of male-specific color patterns in Drosophila

    Convergent evolution provides a type of natural replication that can be exploited to understand the roles of contingency and constraint in the evolution of phenotypes and the gene networks that control their development. For sex-specific traits, convergence offers the additional opportunity for testing whether the same gene networks follow different evolutionary trends in males versus females. Thus study used an unbiased, systematic mapping approach to compare the genetic basis of evolutionary changes in male-limited pigmentation in several pairs of Drosophila species that represent independent evolutionary transitions. A strong evidence for repeated recruitment of the same genes to specify similar pigmentation in different species was found. At one of these genes, ebony, convergent evolution of sexually dimorphic and monomorphic expression through cis-regulatory changes was observed. However, this functional convergence has a different molecular basis in different species, reflecting both parallel fixation of ancestral alleles and independent origin of distinct mutations with similar functional consequences. These results show that a strong evolutionary constraint at the gene level is compatible with a dominant role of chance at the molecular level (Signor, 2016).

    Recent studies have led to an emerging consensus that convergent phenotypes often reflect evolutionary changes in the same genes (the 'evolutionary hotspot' model). However, most case studies supporting the hotspot model relied heavily on candidate gene approaches, which results in a positive ascertainment bias and some difficulty in interpreting the results. Although genetic mapping is more laborious than candidate gene analysis, it offers several key advantages: it is unbiased in that the loci implicated in other species are no more likely to be discovered than any other genes, it produces direct evidence of a gene's causative role in trait evolution (and, equally importantly, allows other genes to be ruled out), and it provides a quantitative estimate of the relative importance of each gene to the overall genetic architecture of a phenotype. This study applied this approach to the ananassae species subgroup, in which multiple species have independently evolved similar male-specific color patterns. The convergent phenotypes were found ti be controlled by overlapping, but not identical, sets of genes in different evolutionary contrasts. The only gene that was implicated in all three high-resolution mapping crosses and could not be ruled out in the fourth, low-resolution cross (D. m. malerkotliana/D. bipectinata) was ebony. Moreover, ebony is the strongest QTL in all contrasts, contributing 35% to 80% of overall divergence. ebony has previously been implicated in both intraspecific and interspecific differences in pigmentation in several other Drosophila species. Clearly, ebony fits the definition of an evolutionary hotspot. ebony encodes an enzyme, β-alanyl-dopamine synthase, that synthesizes light pigment precursors so that higher ebony expression causes lighter pigmentation. Like other pigmentation genes, it has additional roles in other tissues, such as control of circadian locomotor activity in brain glial cells. Every time ebony has been implicated in the evolution of pigmentation, cis-regulatory rather than coding mutations were involved. Presumably, this pattern reflects the ability of cis-regulatory mutations to overcome pleiotropic constraints by uncoupling gene functions in different cell types (Signor, 2016).

    Repeated involvement of the same gene in multiple phenotypic transitions could potentially result from different evolutionary processes: recurrent de novo mutation, lineage sorting of ancestral variation, or interspecific introgression. Adaptation from standing variation is likely to be faster than awaiting a new mutation because potentially beneficial alleles are available immediately and are likely to be present at higher frequencies than alleles arising de novo. Cases of parallel fixation appear to be fairly common, most notably when a reservoir of newly adaptive alleles is available to colonizing populations. Similarly, introgressive hybridization has been implicated, among other traits, in the evolution of wing color patterns in Heliconius butterflies and high-altitude adaptation in humans. Genome-wide analyses suggest that interspecific introgression may play a more important role in evolution than previously thought. This study found that in D. malerkotliana, D. bipectinata, and D. parabipectinata, at least some of the putative causative SNPs at the ebony locus are shared across species, most likely reflecting ancestral lineage sorting. However, these variants are not found either in D. pseudoananassae or in the ercepeae species complex, suggesting a role for independent ebony mutations in these taxa. The region of the ebony locus associated with the evolution of color patterns in the bipectinata complex evolves so rapidly that it cannot be aligned with other taxa. Outside of the ananassae subgroup, evolutionary changes in ebony expression that lead to divergent pigmentation map to a different, more upstream region of the gene; population-genetic analysis rules out this region in the bipectinata complex. It can be concluded that the widely convergent involvement of ebony in the evolution of color patterns is not due solely to fixation of pre-existing variation, but reflects independent origin of distinct, though functionally similar, cis-regulatory mutations (Signor, 2016).

    Cuticle color depends not just on the level of ebony, but on the balance between ebony, tan, yellow, and potentially other enzymes. For example, tan encodes a β-alanyl-dopamine hydrolase, which reverses the chemical reaction catalyzed by ebony. Although tan is implicated in the evolution of pigmentation in several species, it has been ruled out in many other studies including this one (Table S5). Interestingly, the evolutionary changes that were found to involve tan are never male limited; for example, tan controls a female-limited color polymorphism in D. erecta, and the secondary loss of pigmentation in D. santomea affects both sexes. It is possible that ebony could be favored as the male hotspot due to its dosage sensitivity and chromosomal location. ebony, but not tan or yellow, has a semi-dominant loss-of-function phenotype, suggesting that cis-regulatory mutations in ebony could be more readily visible to directional selection. At the same time, yellow and tan are X-linked, whereas ebony is autosomal, suggesting that it could harbor higher levels of standing genetic variation when not under directional selection. Interestingly, in D. melanogaster, D. americana, and the bipectinata species complex, the role of ebony in phenotypic evolution appears to derive from a combination of pre-existing and de novo mutations. Thus, chromosomal sex, the topology of the regulatory network, the kinetics of the pigment synthesis pathway, and population-genetic factors may all contribute to the evolutionary hotspot status of ebony (Signor, 2016).

    In an accompanying paper (Yassin, 2016), a similarly unbiased and systematic approach was taken to map the genetic basis of natural variation in female-specific abdominal pigmentation in multiple species of the Drosophila montium species subgroup, which is closely related to the ananassae lineage. This variation was found to map to the pdm3 transcription factor in several distantly related species. Moreover, convergent involvement of pdm3 appears to reflect independent mutations in this gene in different species. In contrast, ebony does not contribute to color pattern variation in any of the four montium-subgroup species examined. Why do evolutionary hotspots differ in closely related lineages? Is this a matter of historical contingency or different gene network topology in different clades? Or are different genes within a shared network favored for cis-regulatory evolution in different sexes, resulting in sex-specific evolutionary hotspots? Intriguingly, in the only montium subgroup species in which ebony was implicated in color pattern variation, the pigmentation phenotype is male specific, supporting the latter hypothesis (Signor, 2016).

    One can envision two principal mechanisms for the gain and loss of sex-specific traits. First (the instructive model), the genes responsible for phenotypic changes may be the same genes that are regulated in a dimorphic manner to generate sex-specific phenotypes, as was observed for ebony. Alternatively (the permissive model), the causative genes could be monomorphic, while sexual dimorphism is encoded in parallel to or downstream of these genes. For example, ebony could have been expressed equally between sexes in all species, while a different, 'gatekeeper' gene is sexually dimorphic in all species. In the latter scenario, high levels of ebony expression in both sexes would mask the dimorphic phenotypes promoted by the sex-specific gatekeeper gene, while low ebony levels would uncover the underlying dimorphism. Thus, ebony would be the causative gene responsible for differences between lineages, while the gatekeeper gene is responsible for sexual dimorphism (Signor, 2016).

    These two models suggest very different mechanisms for the evolution of sexual dimorphism. Under the instructive model, gain and loss of sex-specific traits is caused by frequent changes in sex-specific gene regulation. Under the permissive model, the targets of the sex determination pathway can remain static over long evolutionary distances, while the underlying sexual dimorphism is revealed or obscured by sexually monomorphic genetic changes that occur elsewhere in the developmental pathway (Signor, 2016).

    The current results argue for the instructive model: in all evolutionary contrasts, sex-specific pigmentation is associated with sex-specific ebony expression, and sexually monomorphic pigmentation is associated with monomorphic expression. Thus, sex-specific transcriptional regulation of ebony has been gained and/or lost several times within the ananassae species subgroup. A similar pattern has been observed in the expression of desat-F, a hydrocarbon desaturase enzyme involved in the synthesis of cuticular pheromones. It appears that sex-specific gene regulation can be gained and lost quite easily over short evolutionary timescales and that the evolution of sexually dimorphic traits is more likely to follow the instructive model (Signor, 2016).

    Although the study of color pattern evolution in Drosophila has largely been dominated by candidate gene analyses, it has now been enriched by several unbiased, high-resolution genetic mapping studies, including this work. These studies have gone beyond spotlighting individual genes to provide a more holistic picture of the genetic architecture of evolutionary changes and have confirmed the predominance of cis-regulatory mutations in phenotypic evolution. Although no single gene is involved in all cases, the number of players appears to be limited, and most genes have been implicated repeatedly in multiple taxa. Collectively, parallel genetic analyses in multiple species suggest a 'toolkit model' of convergent evolution. For any trait, there are a limited number of genes that can potentially evolve to produce phenotypic changes. Within that toolkit, the relative likelihood of each gene's involvement may depend on its position in the regulatory network that controls the development of that trait, on the historical contingencies specific to each evolving lineage, and, potentially, on sex. Together, these trends result in a pattern where convergent phenotypes have distinct yet overlapping genetic basis in different species (Signor, 2016).

    Patterning the insect eye: From stochastic to deterministic mechanisms

    While most processes in biology are highly deterministic, stochastic mechanisms are sometimes used to increase cellular diversity. In human and Drosophila eyes, photoreceptors sensitive to different wavelengths of light are distributed in stochastic patterns, and one such patterning system has been analyzed in detail in the Drosophila retina. Interestingly, some species in the dipteran family Dolichopodidae (the "long legged" flies, or "Doli") instead exhibit highly orderly deterministic eye patterns. In these species, alternating columns of ommatidia (unit eyes) produce corneal lenses of different colors. Occasional perturbations in some individuals disrupt the regular columns in a way that suggests that patterning occurs via a posterior-to-anterior signaling relay during development, and that specification follows a local, cellular-automaton-like rule. It is hypothesized that the regulatory mechanisms that pattern the eye are largely conserved among flies and that the difference between unordered Drosophila and ordered dolichopodid eyes can be explained in terms of relative strengths of signaling interactions rather than a rewiring of the regulatory network itself. A simple stochastic model is presented that is capable of explaining both the stochastic Drosophila eye and the striped pattern of Dolichopodidae eyes and thereby characterize the least number of underlying developmental rules necessary to produce both stochastic and deterministic patterns. Only small changes to model parameters are needed to also reproduce intermediate, semi-random patterns observed in another Doli species, and quantification of ommatidial distributions in these eyes suggests that their patterning follows similar rules (Ebadi, 2018).

    A Continuum of Evolving De Novo Genes Drives Protein-Coding Novelty in Drosophila

    Orphan genes, lacking detectable homologs in outgroup species, typically represent 10-30% of eukaryotic genomes. This study investigated the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, 6297 orphan genes were found within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7-39.2%) of these genes, this study describes for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, it was shown that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. It was found that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, these results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes (Heames, 2020).

    Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet

    Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. This study found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. The genome of S. flava was sequenced, and this species' odorant receptor repertoire was characterized. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires (Goldman-Huertas, 2005).

    Understanding the origins and consequences of trophic shifts, especially the transition to herbivory, has been a central problem in evolutionary biology. The paleontological record suggests that evolutionary transitions to herbivory have been rare in insects, and the first transitions to herbivory in vertebrates occurred long after the colonization of land. However, species radiations result from herbivorous transitions in insects and vertebrates, suggesting that herbivory is a key innovation. Identifying functional genomic changes associated with the evolutionary transition to herbivory could yield insight into the mechanisms that have driven their success. However, the origins of the most diverse clades of herbivorous insects are ancient and date to the Jurassic or earlier, limiting meaningful genomic comparisons. In contrast, herbivory has evolved more times in Diptera than in any other order. The Drosophilidae is an excellent system to study the evolution of herbivory from a functional genomic perspective because it includes several transitions to herbivory, and the genomic model Drosophila melanogaster (Goldman-Huertas, 2005).

    The transition to herbivory involves adaptations in physiology, morphology, and behavior. The evolution of sensory repertoires could reinforce or even precipitate these adaptations through adaptive loss or relaxation of functional constraint subsequent to a trophic shift. Adaptive loss of chemoreceptors has been rarely shown but occurs in nematodes, although their olfactory systems are distinct from insects. Families of mammalian olfactory receptor proteins have been remodeled during transitions to flight, aquatic lifestyles, and frugivory. Similarly, the evolution of diet specialization in Drosophila species correlates with chemoreceptor gene losses, and hematophagous flies have lost gustatory receptors that detect sweet compounds. More profound changes such as the evolution of new protein families are associated with major evolutionary transitions such as the evolution of flight in insects. Although gene loss is unlikely to be a driving force of innovation, loss-of-function mutations may be exeptations that allow novel behaviors to evolve by disrupting ancestral attractions. If detection of different chemical cues becomes selected in a novel niche, then loss through relaxed constraint may indicate which chemical cues have changed during a trophic shift (Goldman-Huertas, 2005).

    The chemosensory repertoires of many drosophilid species have been functionally annotated. The genus Drosophila includes 23 species with published genome sequences, and D. melanogaster presents the most fully characterized insect olfactory system, allowing potential linkage of receptor remodeling to a mechanistic understanding of behavioral change (Goldman-Huertas, 2005).

    Most drosophilids feed on yeast and other microbes growing on decaying plant tissues. Adult female D. melanogaster and distantly related species innately prefer yeast chemical cues to those produced by the fruit on which they oviposit. D. melanogaster detects volatiles with chemoreceptors of several different protein families, but especially receptors from the odorant receptor (OR) gene family, some of which, such as Or42b, are highly conserved across species. Or42b is necessary for attraction and orientation to vinegar and aliphatic esters. Similar compounds activate Or42b across many Drosophila species, suggesting that volatile cues for yeast, and the associated receptors, are conserved across the Drosophilidae (Goldman-Huertas, 2005).

    The ancestral feeding niche for the genus Scaptomyza (Drosophilidae) is microbe-feeding, but Scaptomyza use decaying leaves and stems rather than the fermenting fruit used by D. melanogaster and other members of the subgenus Sophophora. The close association of Scaptomyza with decaying plant tissues may have precipitated the evolution of herbivory <20 MyBP. Adult females of the species S. flava feed and oviposit on living leaves of many cruciferous plants (Brassicales) including Arabidopsis thaliana. Females puncture leaves with serrated ovipositors to create feeding and oviposition sites, and larvae mine and pupate within the living leaves (Goldman-Huertas, 2005).

    This study used Scaptomyza as a model to test the hypothesis that functional loss of chemosensory genes has played a role in a major ecological transition to herbivory in insects. It was hypothesized that the conserved detection of yeast volatiles would be lost in the herbivorous Scaptomyza lineage. This loss was tested by comparing D. melanogaster and S. flava at behavioral, physiological, and genetic levels. First, it was hypothesized that gravid ovipositing S. flava females would not be attracted to yeast volatiles. Second, it was hypothesized that the olfactory sensory organs of S. flava would have a decreased ability to detect individual yeast volatiles and volatile mixtures. Third, chemoreceptor genes from the OR gene family implicated in detection of yeast volatiles would be lost in the S. flava genome. Finally, it was predicted that chemoreceptor genes potentially mediating detection of plant volatiles would show evidence of positive selection and possibly, neofunctionalization (Goldman-Huertas, 2005).

    Olfaction is used by insects to find resources, mates, and oviposition substrates. This study tested the hypothesis that S. flava is not attracted to yeast volatiles, whereas D. melanogaster is attracted to yeast volatiles. A four-field olfactometer assay was used in which filtered air blown through four corners of a diamond-shaped arena establishes four independent airfields. Two of the four fields were exposed to yeast volatiles from Saccharomyces cerevisiae cultures. The presence of gravid adult females of both species in either yeast or control fields was recorded every 6 s for 10 min. D. melanogaster flies spent 82.4 ± 18.2% SD of the assay time in yeast-volatile fields and more time in yeast-volatile fields than S. flava. S. flava did not spend more time in yeast-volatile fields and divided residence time evenly between yeast and control fields, consistent with a loss of attraction to yeast volatiles in S. flava flies (Goldman-Huertas, 2005).

    Because S. flava flies failed to increase their residence time in olfactometer quadrants exposed to yeast volatiles, it was hypothesized that S. flava antennal olfactory sensory neurons (OSNs) were deficient in their ability to detect yeast volatiles. This hypothesis was tested by conducting electroantennogram (EAG) measurements in adult D. melanogaster and S. flava flies of both sexes 4-20 d after eclosion, exposed to the same yeast volatiles used in the olfactometer assays and to crushed rosette leaves of the host plant of S. flava flies in laboratory colonies (Arabidopsis thaliana accession Col-0). EAG responses are driven by the aggregate depolarization of OSNs in the antennae and scale with the concentration and identity of stimulants. No difference were found between sexes and data for male and female flies were combined. Consistently lower EAG signals were recorded in S. flava flies compared with D. melanogaster, preventing interspecific comparisons of signal amplitude, possibly due to differences in electrical properties of antennae (Goldman-Huertas, 2005).

    The antennae of S. flava were more strongly stimulated by Arabidopsis volatiles than by yeast, whereas the antennae of D. melanogaster were more responsive to volatiles from yeast than those from Arabidopsis. Responses were recorded to a small panel of three volatiles associated with A. thaliana [(Z)-3-hexenol, myrcene, phenethyl isothiocyanate] and two with S. cerevisiae (2-phenylethanol, ethyl acetate). Antennae of both species detected all volatiles compared with a negative control. The antennae of S. flava were most responsive to (Z)-3-hexenol, a volatile produced by damaged leaves of many plant species, and were also highly attuned to phenethyl isothiocyanate, a hydrolyzed product of glucosinolates, which are the major defensive compound in host plants of S. flava. Responses to myrcene and 2-phenylethanol were not in the expected direction, although 2-phenylethanol, as a widespread floral volatile, may remain an important chemical cue for Scaptomyza adults (Goldman-Huertas, 2005).

    Antennae of S. flava were less responsive to yeast and the yeast-associated volatile ethyl acetate than to plant-related volatiles, but these relative comparisons were insufficient to prove that the detection threshold for yeast volatiles had decreased in Scaptomyza. Therefore the sensitivity of S. flava and D. melanogaster flies to this and other short-chain aliphatic esters was tested by exposing females to half-log dilution series of ethyl acetate, ethyl propionate, and isobutyl acetate. Sensitivity was defined as the first concentration increase that generated an increased antennal response. S. flava was insensitive to ethyl acetate at the concentrations tested. S. flava was also less responsive to ethyl propionate and isobutyl acetate compared with D. melanogaster. Scaptomyza is considerably less sensitive to short aliphatic esters, which may account for differences in signal strength in response to plant and yeast volatile mixtures and the lack of attraction to yeast volatiles by S. flava. This unresponsiveness is consistent with the fact that deficits in the production of aliphatic esters in a yeast strain decreased attractiveness to D. melanogaster flies (Goldman-Huertas, 2005).

    The lack of attraction and minimal EAG response to yeast volatiles in S. flava suggested that chemosensory genes have been lost or changed in herbivorous Scaptomyza species. ORs are expressed in the dendrites of OSNs in the antennae and maxillary palps and are the primary receptors by which most neopteran insects detect odors in their environments. The OR family has been functionally annotated in D. melanogaster, and members of subfamily H OR genes in particular are highly conserved and enriched in receptors for aliphatic esters, a group of compounds S. flava detected poorly (Goldman-Huertas, 2005).

    To characterize changes in the OR gene repertoire in S. flava associated with the olfactory phenotypes, the genome of S. flava and annotated OR genes were annotated by using reciprocal tBLASTn searches of previously annotated Drosophila OR protein sequences against this de novo S. flava genome assembly. 65 full-length ORFs for OR genes were found in S. flava. Consistent with previous OR gene-naming conventions, ORs were named after the D. melanogaster ortholog or the most closely related gene, with the exception of OrN1 and OrN2 orthologs, which are not present in D. melanogaster (Goldman-Huertas, 2005).

    Protein translations of S. flava genes were included in a phylogeny of D. melanogaster, Drosophila virilis, Drosophila mojavensis, and Drosophila grimshawi OR protein sequences to assess homology. The latter three species are the closest relatives of Scaptomyza with fully sequenced genomes (Goldman-Huertas, 2005).

    S. flava retains duplicates of Or42a, Or67a, Or74a, Or83c, Or98a, and OrN2 found in other sequenced Drosophila species. Scaptomyza also has duplications not shared with close relatives, although nine of these genes are pseudogenized. The majority of paralogs (56%) were found on the same scaffold in tandem arrays. The functional significance of these gene duplications is not yet clear, but it is suggestive that Or67b, with three copies in S. flava, is in single copy in nearly all sequenced Drosophila. In D. melanogaster, neurons expressing Or67b respond to green leaf volatiles such as (Z)-3-hexenol, to which S. flava also has a robust antennal response (Goldman-Huertas, 2005).

    Only four widely conserved ORs were uniquely lost (Or22a and Or85d) or pseudogenized (Or9a, Or42b) in the Scaptomyza lineage. Syntenic regions flanking OR losses were recovered in the genome assembly. Orthologs of Or9a, Or22a, and Or42b are intact in 23 Drosophila species with genome sequences, and Or85d is missing only in the Drosophila albomicans and Drosophila rhopaloa genome assemblies. As predicted, orthologs of ORs that persist in microbe-feeding Drosophila species and are lost in S. flava, function in yeast-volatile detection. Or42b is highly conserved in sequence among Drosophila species, and the receptor is highly attuned to aliphatic esters at low concentrations. Knockouts of Or42b in adult D. melanogaster result in failure to orient in flight toward aliphatic ester odor plumes , and rescuing these neurons restores attraction to yeast volatiles. Similarly, no sequences similar to Or22a were present in the S. flava assembly, although conserved intergenic regions were found in S. flava that flank Or22a in other Drosophila species. Or22a also detects aliphatic esters and in the specialist species Drosophila erecta and Drosophila sechellia, Or22a detects volatiles produced by host fruit. Both Or22a and Or42b are activated by floral volatiles of Arum palestinum, which mimics yeast fermentation volatiles and attracts a diversity of drosophilids. Finally, Or85d orthologs were not detected in the S. flava genome by BLAST or by inspection of genome regions flanking Or85d in other species. Or85d is expressed in the maxillary palps and in D. melanogaster is responsive to the yeast metabolites 2-heptanol, ethyl acetate, and isoamyl acetate. Or85d is highly sensitive to phenethyl acetate, a common volatile of many yeast species. In D. melanogaster, Or9a is activated by a broad range of ketone-, alcohol-, and carboxylic acid-containing ligands. Some of these ligands, such as acetoin, are common yeast volatiles and strong attractants. The consequences of Or9a pseudogenization will require further study (Goldman-Huertas, 2005).

    A time-calibrated phylogeny of the family Drosophilidae suggests that herbivory evolved in Scaptomyza ca.13.5 million years ago (95% highest posterior density 10.02–17.48 million years ago), overlapping with age ranges inferred from previous analyses. Ancestral state reconstructions were performed in the APE package by using an equal rates model. This analysis indicated that microbe feeding is ancestral in Drosophila and Scaptomyza (99.7% probability) and that herbivory evolved once within the genus Scaptomyza (Goldman-Huertas, 2005).

    It was hypothesized that OR gene losses would coincide with the evolution of herbivory. Degenerate PCR primers were developed from genomes of multiple Scaptomyza and Drosophila species that targeted exonic sequences of Or22a and Or9a, and conserved, flanking, intergenic sequences of Or42b and Or85d (Goldman-Huertas, 2005).

    Gene losses in S. flava were confirmed by PCR screen in three natural populations, with the exception of SflaOr9a-1, which appeared to be present in a functional copy in a population from Arizona. A preliminary genome assembly of Scaptomyza pallida was consistent with PCR screening results for OR loss patterns in this species. The presence/absence of S. flava gene losses was reconstructed along ancestral nodes and found that three of the four OR gene losses in S. flava (Or22a, Or85d, Or42b) coincided with or preceded the evolution of herbivory in Scaptomyza. Losses were shared by herbivorous congeners. Or22a, while lost in S. flava, is intact in the microbe-feeding species Scaptomyza apicata and S. pallida and is also lost in two other herbivorous species, Scaptomyza nigrita and Scaptomyza graminum (Goldman-Huertas, 2005).

    Specialist, microbe-feeding Drosophila species, such as D. sechellia and D. erecta have an accelerated rate of chemoreceptor gene loss, but this pattern could also be due to nearly neutral processes. S. flava feeds almost exclusively on plants within the Brassicales, and it was hypothesized that this species has experienced an accelerated rate of chemosensory gene loss compared with other microbe-feeding Drosophila species. This hypothesis was tested by coding homologous groups of ORs as present or absent in S. flava, D. virilis, D. mojavensis and D. grimshawi (the closest Drosophila relatives of Scaptomyza), and two models of gene loss were inferred in the Brownie software package. No evidence was found for the alternative model of increased rate of loss in Scaptomyza, but it cannot be ruled out that there were insufficient loss events to parameterize the more complex model or that other chemoreceptor gene families have undergone accelerated loss in S. flava. Also, S. flava is oligophagous, feeding on many plant species in the Brassicales, and it is less specialized than D. sechellia and D. erecta (Goldman-Huertas, 2005).

    Because the shift to herbivory in Scaptomyza likely involved many changes in olfactory cues, it was hypothesized that some S. flava OR genes should bear signatures of episodic positive selection, as flies adapted to a novel environment. To test this hypothesis, null and alternative (branch-site) models were inferred in PAML 4.7a where subsets of codons in extant S. flava ORs could evolve under (i) purifying or neutral selection or (ii) purifying, neutral, or positive selection, relative to 12 Drosophila species. A phylogeny-aware alignment program, PRANK, was used to identify regions where indels were probable while minimizing sensitivity to alignment errors. Alignments where more than one taxon had an inferred indel in greater than two regions were trimmed by using Gblocks to remove columns with ambiguous homology(Goldman-Huertas, 2005).

    After correcting for false discovery, two ORs were found in which the branch-site model consistent with episodic positive selection was more likely than the null model. Or88a had the strongest statistical support for the branch-site model. In D. melanogaster, Or88a functions in recognition of male and virgin female conspecifics . Two other branches among the S. flava Or67b paralogs also supported the branch-site model: an ancestral branch preceding a Scaptomyza-specific duplication event and a branch leading to Or67b-3. Homologs of this gene in D. melanogaster encode ORs that respond to the green-leaf volatile (Z)-3-hexenol, one of the most salient ligands found in EAG studies of S. flava. Experimental, functional, and population-based tests are needed to verify whether positive selection has fixed amino acid changes in the Scaptomyza lineage (Goldman-Huertas, 2005).

    It is concluded that trophic transitions in the history of animal life, such as herbivory, may be mediated by genetic changes in chemosensory repertoires. The majority of Drosophilidae feed on microbes, and distantly related drosophilid lineages are attracted by the same yeast-mimicking floral scent produced by A. palestinum. A subset of the ORs stimulated by this scent are highly conserved in other drosophilids, which may be part of a homologous and conserved olfactory circuit used to find fermenting host substrates across the family. It was hypothesized that mutations disrupting the function of OR homologs in this conserved olfactory circuit could mediate the evolution of herbivory or other novel food preferences (Goldman-Huertas, 2005).

    S. flava, an herbivorous drosophilid, has lost orthologs of ORs involved in this generalized yeast olfactory circuit. Consistent with these findings, S. flava did not respond to yeast volatiles in a behavioral assay. Antennae of S. flava were weakly activated by active yeast cultures and short-chain aliphatic esters, key compounds found in yeast volatile blends and known ligands of ORs in D. melanogaster lost in S. flava. However, retention of some ORs implicated in yeast-volatile detection, such as Or92a and Or59b, implies that S. flava may retain the ability to detect some untested yeast compounds (Goldman-Huertas, 2005).

    It is hypothesized that OR genes would be intact in nonherbivorous Scaptomyza and gene losses would coincide with the transition to herbivory. Or22a loss did coincide with the evolution of herbivory, but losses of Or42b and Or85d likely predate the evolution of plant feeding. These more ancient losses of conserved yeast-volatile receptors suggest ancestral Scaptomyza may have already evolved novel olfactory pathways that were later co-opted by herbivorous lineages, and in fact, many Scaptomyza species feed on microbes living within decaying leaves or in leaf mines produced by other insects. Sister groups of many major herbivorous insect lineages also feed on detritus and fungi, suggesting that the transition from microbe feeding to herbivory may be common. The genetic changes that underlie host-finding remain to be identified, but recently duplicated ORs, such as the unique triplication of Or67b in Scaptomyza, are likely candidates for further functional study. Subtle, targeted remodeling of chemoreceptor repertoires may be a general mechanism driving changes in behavior, facilitating trophic shifts and ultimately diversification in animals (Goldman-Huertas, 2005).

    Molecular and functional evolution at the Odorant receptor Or22 locus in Drosophila melanogaster

    Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviours. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. This study shows that several naturally occurring Or22 variants generate major shifts in neuronal response properties. The molecular changes were determined that underpin these response shifts, one of which represents a chimaeric gene variant previously suggested to be under natural selection. In addition it was shown that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. These data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behaviour, it is predicted that Or22 may be a key player in underlying one or more olfactory-driven behaviours of significant adaptive importance (Shaw, 2019).

    Color preference of the spotted wing Drosophila, Drosophila suzukii

    Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a significant invasive pest in soft-skin fruits and berries in Asia, Europe, and North and South America. Many herbivorous insects use multiple cues for host selection, particularly olfactory and visual stimuli. The visual system of closely-related Drosophila melanogaster is well-documented, expressing strong sensitivity to short-wavelength colors (ultraviolet to green) and only limited sensitivity to long-wavelength colors (red to infrared). The results suggest that D. suzukii have limited ability to distinguish red consistent with visual sensitivity range within the melanogaster subgroup. It is proposed that color contrast rather than color appearance may be of greater importance in orientation and attraction. It is proposed that differences in reflectance between light wavelengths important for color opponency are key to color discrimination to provide color contrast between foreground and background, as occurs between fruit and foliage, during host-finding (Little, 2019).

    The loci of behavioral evolution: evidence that Fas2 and tilB underlie differences in pupation site choice behavior between Drosophila melanogaster and D. simulans

    The behaviors of closely related species can be remarkably different, and these differences have important ecological and evolutionary consequences. While the recent boom in genotype-phenotype studies has led to a greater understanding of the genetic architecture and evolution of a variety of traits, studies identifying the genetic basis of behaviors are, comparatively, still lacking. This is likely because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. The Drosophila species complex holds promise for addressing these challenges, as the behaviors of closely related species can be readily assayed in a common environment. This study investigated the genetic basis of an evolved behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, A significant contribution was demonstrated of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deficiencies, the majority of the X chromosome was screened for causal loci, and two regions were identified associated with this X-effect. Gene disruption and RNAi data were collecting supporting a single gene that affects pupation behavior within each region: Fas2 and tilB. Finally, differences in tilB expression were shown to correlate with the differences in pupation site choice behavior between species. This evidence associating two genes with differences in a complex, environmentally sensitive behavior represents the first step towards a functional and evolutionary understanding of this behavioral divergence (Pischedda, 2019).

    Back to the light, coevolution between vision and olfaction in the "Dark-flies" (Drosophila melanogaster)

    Trade-off between vision and olfaction, the fact that investment in one correlates with decreased investment in the other, has been demonstrated by a wealth of comparative studies. However, there is still no empirical evidence suggesting how these two sensory systems coevolve, i.e. simultaneously or alternatively. The "Dark-flies" (Drosophila melanogaster) constitute a unique model to investigate such relation since they have been reared in the dark since 1954, approximately 60 years (~1500 generations). To observe how vision and olfaction evolve, populations of Dark-flies were reared in normal lighting conditions for 1 (DF1G) and 65 (DF65G) generations. The sizes of the visual (optic lobes, OLs) and olfactory (antennal lobes, ALs) primary centres, as well as the rest of the brain, and compared the results with the original and its genetically most similar strain (Oregon flies). Whereas the ALs decreased in size, the OLs (together with the brain) increased in size in the Dark-flies returned back to the light, both in the DF1G and DF65G. These results experimentally show that trade-off between vision and olfaction occurs simultaneously, and suggests that there are possible genetic and epigenetic processes regulating the size of both optic and antennal lobes. Furthermore, although the Dark-flies were able to mate and survive in the dark with a reduced neural investment, individuals being returned to the light seem to have been selected with reinvestment in visual capabilities despite a potential higher energetic cost (Ozer, 2020).

    Olfactory receptor and circuit evolution promote host specialization

    The evolution of animal behaviour is poorly understood. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare. This study develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster that are linked to its extreme specialization on noni fruit (Morinda citrifolia). Using calcium imaging, this study identified olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. This mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. The molecular determinants of this functional change were identified, and their evolutionary origin and behavioural importance were characterized. Circuit tracing was performed in the D. sechellia brain, and receptor adaptations were found to be accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system (Auer, 2020).

    A courtship behavior that makes monandrous females polyandrous

    Females of many animal species mate several times with different males (polyandry), whereas females of some species mate with a single male (monandry) only once. Little is known about the mechanisms by which these different mating systems evolve. Females of Drosophila prolongata mate serially, unlike Drosophila melanogaster females that refuse to remate for several days after their first mating (remating suppression [RS]). Nevertheless, interestingly, nonvirgin D. prolongata females refuse to remate with males that are prohibited from performing their species-specific courtship behavior, leg vibration (LV), suggesting that LV overrides RS making it cryptic in D. prolongata. This study examined how long this cryptic RS persists. Surprisingly, it was sustained for at least 2 weeks, showing that RS is substantially augmented in D. prolongata compared to that of D. melanogaster. The two most closely related species to D. prolongata, Drosophila rhopaloa and Drosophila carrolli, do not perform LV and showed augmented RS, supporting the idea that augmented RS could have evolved before LV was acquired. These results suggested that D. prolongata females are intrinsically monandrous, whereas the newly evolved courtship behavior makes them polyandrous. This is a rare case in which a proximate mechanism of polyandry evolution from monandry is demonstrated (Minekawa, 2020).

    Competitive history shapes rapid evolution in a seasonal climate

    Eco-evolutionary dynamics will play a critical role in determining species' fates as climatic conditions change. Unfortunately, there is little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. The effects of rapid evolution in response to interspecific competition were tested on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster. Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. these populations' ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) were then quantified when exposed to a cooling fall climate. It was found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection (Grainger, 2021).

    Sex ratio and the evolution of aggression in fruit flies

    Aggressive behaviours are among the most striking displayed by animals, and aggression strongly impacts fitness in many species. Aggression varies plastically in response to the social environment, but direct tests of how aggression evolves in response to intra-sexual competition are lacking. This study investigated how aggression in both sexes evolves in response to the competitive environment, using populations of Drosophila melanogaster that were experimentally evolved under female-biased, equal, and male-biased sex ratios. After evolution in a female-biased environment-with less male competition for mates-males fought less often on food patches, although the total frequency and duration of aggressive behaviour did not change. In females, evolution in a female-biased environment-where female competition for resources is higher-resulted in more frequent aggressive interactions among mated females, along with a greater increase in post-mating aggression. These changes in female aggression could not be attributed solely to evolution either in females or in male stimulation of female aggression, suggesting that coevolved interactions between the sexes determine female post-mating aggression. Evidence was found consistent with a positive genetic correlation for aggression between males and females, suggesting a shared genetic basis. This study demonstrates the experimental evolution of a behaviour strongly linked to fitness, and the potential for the social environment to shape the evolution of contest behaviours (Bath, 2021).

    Drosophila adaptation to viral infection through defensive symbiont evolution

    Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. This study shows that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit (Faria, 2016).

    Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila

    While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. This study tested the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. Strong evidence was found for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the alpha-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, was found to escape protection by Spiroplasma, and its ribosomes do not show high levels of depurination. It was also shown that fly ribosomes show little evidence of targeting by RIPs. Finally, the genome of D. neotestacea's defensive Spiroplasma was found to encode a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions (Ballinger, 2017).

    An innovative ovipositor for niche exploitation impacts genital coevolution between sexes in a fruit-damaging Drosophila

    Limited attention has been given to ecological factors influencing the coevolution of male and female genitalia. The innovative ovipositor of Drosophila suzukii, an invading fruit pest, represents an appealing case to document this phenomenon. The serrated saw-like ovipositor is used to pierce the hard skin of ripening fruits that are not used by other fruit flies that prefer soft decaying fruits. This study highlights another function of the ovipositor related to its involvement in genital coupling during copulation. The morphology and coupling of male and female genitalia in this species were compared to its sibling species, Drosophila subpulchrella, and to an outgroup species, Drosophila biarmipes. These comparisons and a surgical manipulation indicated that the shape of male genitalia in D. suzukii has had to be adjusted to ensure tight coupling, despite having to abandon the use of a hook-like structure, paramere, because of the more linearly elongated ovipositor. This phenomenon demonstrates that ecological niche exploitation can directly affect the mechanics of genital coupling and potentially cause incompatibility among divergent forms. This model case provides new insights towards elucidating the importance of the dual functions of ovipositors in other insect species that potentially induce genital coevolution and ecological speciation (Muto, 2018).

    Neural evolution of context-dependent fly song

    It is unclear where in the nervous system evolutionary changes tend to occur. To localize the source of neural evolution that has generated divergent behaviors, a new approach was developed to label and functionally manipulate homologous neurons across Drosophila species. Homologous descending neurons were examined that drive courtship song in two species that sing divergent song types, and relevant evolutionary changes were localized in circuit function downstream of the intrinsic physiology of these descending neurons. This evolutionary change causes different species to produce divergent motor patterns in similar social contexts. Artificial stimulation of these descending neurons drives multiple song types, suggesting that multifunctional properties of song circuits may facilitate rapid evolution of song types (Ding, 2019).

    Evolutionary History of the Hymenoptera

    Hymenoptera (sawflies, wasps, ants, and bees) are one of four mega-diverse insect orders, comprising more than 153,000 described and possibly up to one million undescribed extant species. As parasitoids, predators, and pollinators, Hymenoptera play a fundamental role in virtually all terrestrial ecosystems and are of substantial economic importance. To understand the diversification and key evolutionary transitions of Hymenoptera, most notably from phytophagy to parasitoidism and predation (and vice versa) and from solitary to eusocial life, this study inferred the phylogeny and divergence times of all major lineages of Hymenoptera by analyzing 3,256 protein-coding genes in 173 insect species. These analyses suggest that extant Hymenoptera started to diversify around 281 million years ago (mya). The primarily ectophytophagous sawflies are found to be monophyletic. The species-rich lineages of parasitoid wasps constitute a monophyletic group as well. The little-known, species-poor Trigonaloidea are identified as the sister group of the stinging wasps (Aculeata). Finally, the evolutionary root of bees were located within the apoid wasp family "Crabronidae." These results reveal that the extant sawfly diversity is largely the result of a previously unrecognized major radiation of phytophagous Hymenoptera that did not lead to wood-dwelling and parasitoidism. They also confirm that all primarily parasitoid wasps are descendants of a single endophytic parasitoid ancestor that lived around 247 mya. These findings provide the basis for a natural classification of Hymenoptera and allow for future comparative analyses of Hymenoptera, including their genomes, morphology, venoms, and parasitoid and eusocial life styles (Peters, 2017).

    Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): Evolutionary history of faunal divergence between the old and the new worlds

    The current subgenus Drosophila (the traditional immigrans-tripunctata radiation) includes major elements of temperate drosophilid faunas in the northern hemisphere. Despite previous molecular phylogenetic analyses, the phylogeny of the subgenus Drosophila has not fully been resolved: the resulting trees have more or less varied in topology. One possible factor for such ambiguous results is taxon-sampling that has been biased towards New World species in previous studies. In this study, taxon sampling was balanced between Old and New World species, and phylogenetic relationships among 45 ingroup species selected from ten core species groups of the subgenus Drosophila were analyzed using nucleotide sequences of three nuclear and two mitochondrial genes. Based on the resulting phylogenetic tree, ancestral distributions and divergence times were estimated for each clade to test Throckmorton's hypothesis that there was a primary, early-Oligocene disjunction of tropical faunas and a subsequent mid-Miocene disjunction of temperate faunas between the Old and the New Worlds that occurred in parallel in separate lineages of the Drosophilidae. The results of this study substantially support Throckmorton's hypothesis of ancestral migrations via the Bering Land Bridge mainly from the Old to the New World, and subsequent vicariant divergence of descendants between the two Worlds occurred in parallel among different lineages of the subgenus Drosophila. However, the results also indicate that these events took place multiple times over a wider time range than Throckmorton proposed, from the late Oligocene to the Pliocene (Izumitani, 2016).

    Adaptive protein divergence of BMP ligands takes place under developmental and evolutionary constraints

    The bone morphogenetic protein (BMP) signaling network, comprising evolutionary conserved BMP2/4/Decapentaplegic (Dpp) and Chordin/Short gastrulation (Sog), is widely utilized for dorsal-ventral (DV) patterning during animal development. A similar network is required for posterior crossvein (PCV) formation in the Drosophila pupal wing. Although both transcriptional and post-transcriptional regulation of co-factors in the network appears to give rise to tissue-specific and species-specific properties, their mechanisms are incompletely understood. In Drosophila, BMP5-8 type ligands, Screw (Scw) and /aGlass bottom boat (Gbb), form heterodimers with Dpp for DV patterning and PCV development, respectively. Sequence analysis indicates that the Scw ligand contains two N-glycosylation motifs; one being highly conserved between BMP2/4 and BMP5-8 type ligands, and the other being Scw ligand-specific. The data reveal that N-glycosylation of the Scw ligand boosts BMP signaling both in cell culture and in the embryo. In contrast, N-glycosylation modifications of Gbb or Scw ligands reduce the consistency of PCV development. These results suggest that tolerance for structural changes of BMP5-8 type ligands is dependent on developmental constraints. Furthermore, gain and loss of N-glycosylation motifs in conserved signaling molecules under evolutionary constraints appear to constitute flexible modules to adapt to developmental processes (Tauscher, 2016).

    Genomics of parallel adaptation at two timescales in Drosophila

    Two interesting unanswered questions are the extent to which both the broad patterns and genetic details of adaptive divergence are repeatable across species, and the timescales over which parallel adaptation may be observed. Drosophila melanogaster is a key model system for population and evolutionary genomics. Findings from genetics and genomics suggest that recent adaptation to latitudinal environmental variation (on the timescale of hundreds or thousands of years) associated with Out-of-Africa colonization plays an important role in maintaining biological variation in the species. Additionally, studies of interspecific differences between D. melanogaster and its sister species D. simulans have revealed that a substantial proportion of proteins and amino acid residues exhibit adaptive divergence on a roughly few million years long timescale. This study used population genomic approaches to attack the problem of parallelism between D. melanogaster and a highly diverged conger, D. hydei, on two timescales. D. hydei, a member of the repleta group of Drosophila, is similar to D. melanogaster, in that it too appears to be a recently cosmopolitan species and recent colonizer of high latitude environments. Parallelism was observed both for genes exhibiting latitudinal allele frequency differentiation within species and for genes exhibiting recurrent adaptive protein divergence between species. Greater parallelism was observed for long-term adaptive protein evolution and this parallelism includes not only the specific genes/proteins that exhibit adaptive evolution, but extends even to the magnitudes of the selective effects on interspecific protein differences. Thus, despite the roughly 50 million years of time separating D. melanogaster and D. hydei, and despite their considerably divergent biology, they exhibit substantial parallelism, suggesting the existence of a fundamental predictability of adaptive evolution in the genus (Zhao, 2017).

    Inferring the distribution of selective effects from a time inhomogeneous model

    A Poisson random field model has been developed for estimating the distribution of selective effects of newly arisen nonsynonymous mutations that could be observed as polymorphism or divergence in samples of two related species under the assumption that the two species populations are not at mutation-selection-drift equilibrium. The model is applied to 91 Drosophila genes by comparing levels of polymorphism in an African population of D. melanogaster with divergence to a reference strain of D. simulans. Based on the difference of gene expression level between testes and ovaries, the 91 genes were classified as 33 male-biased, 28 female-biased, and 30 sex-unbiased genes. Under a Bayesian framework, Markov chain Monte Carlo simulations are implemented to the model in which the distribution of selective effects is assumed to be Gaussian with a mean that may differ from one gene to the other to sample key parameters. Based on estimates, the majority of newly-arisen nonsynonymous mutations that could contribute to polymorphism or divergence in Drosophila species are mildly deleterious with a mean scaled selection coefficient of -2.81, while almost 86% of the fixed differences between species are driven by positive selection. There are only 16.6% of the nonsynonymous mutations observed in sex-unbiased genes that are under positive selection in comparison to 30% of male-biased and 46% of female-biased genes that are beneficial. It was also estimated that D. melanogaster and D. simulans may have diverged 1.72 million years ago (Amei, 2019).

    Contrasting patterns of molecular evolution in metazoan germ line genes

    Germ lines are the cell lineages that give rise to the sperm and eggs in animals. The germ lines first arise from primordial germ cells (PGCs) during embryogenesis: these form from either a presumed derived mode of preformed germ plasm (inheritance) or from an ancestral mechanism of inductive cell-cell signalling (induction). Numerous genes involved in germ line specification and development have been identified and functionally studied. However, little is known about the molecular evolutionary dynamics of germ line genes in metazoan model systems. This study examined the molecular evolution of germ line genes within three metazoan model systems. These include the genus Drosophila (N=34 genes, inheritance), the fellow insect Apis (N=30, induction), and their more distant relative Caenorhabditis (N=23, inheritance). Using multiple species and established phylogenies in each genus, this study reports that germ line genes exhibited marked variation in the constraint on protein sequence divergence (dN/dS) and codon usage bias (CUB) within each genus. Importantly, it was found that de novo lineage-specific inheritance (LSI) genes in Drosophila (osk, pgc) and in Caenorhabditis (pie-1, pgl-1), which are essential to germ plasm functions under the derived inheritance mode, displayed rapid protein sequence divergence relative to the other germ line genes within each respective genus. This may reflect the evolution of specialized germ plasm functions and/or low pleiotropy of LSI genes, features not shared with other germ line genes. In addition, it was observed that the relative ranking of dN/dS and of CUB between genera were each more strongly correlated between Drosophila and Caenorhabditis, from different phyla, than between Drosophila and its insect relative Apis, suggesting taxonomic differences in how germ line genes have evolved. Taken together, the present results advance understanding of the evolution of animal germ line genes within three well-known metazoan models. Further, the findings provide insights to the molecular evolution of germ line genes with respect to LSI status, pleiotropy, adaptive evolution as well as PGC-specification mode (Whittle, 2019a).

    Evolution of salivary glue genes in Drosophila species

    At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly. Available genome sequences and PCR-sequencing of regions of interest were used to investigate the glue genes in 20 Drosophila species. A new gene in addition was discovered to the seven glue genes annotated in D. melanogaster. A phase 1 intron was identified at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. High repeat number variation was also detected at the intrapopulation level in D. melanogaster. It is concluded that most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes (Da Lage, 2019).

    Phylogenetic position of the Drosophila fima and dentissima lineages, and the status of the D. melanogaster species group

    The subgenus Sophophora of Drosophila, which includes D. melanogaster, is an important model for the study of molecular evolution, comparative genomics, and evolutionary developmental biology. Numerous phylogenetic studies have examined species relationships in the well-known melanogaster, obscura, willistoni, and saltans species groups, as well as the relationships among these clades. In contrast, other species groups of Sophophora have been relatively neglected and have not been subjected to molecular phylogenetic analysis. This study focuses on the endemic African Drosophila fima and dentissima lineages. Both these clades fall within the broadly defined melanogaster species group, but are otherwise distantly related to each other. The new phylogeny supports pervasive divergent and convergent evolution of male-specific grasping structures (sex combs). The implications of these results are discussed for defining the boundaries of the melanogaster species group, and weigh the relative merits of "splitting" and "lumping" approaches to the taxonomy of this key model system (Kopp, 2019).

    A phylogeny for the Drosophila montium species group: a model clade for comparative analyses

    The Drosophila montium species group is a clade of 94 named species closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. This study used genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of the phylogenetic inferences, three non-overlapping sets of 20 single-copy coding sequences were used, and all 60 genes were analyzed with both Bayesian and maximum likelihood methods. This analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, this analyses also support the monophyly of all seven subgroups proposed within the montium group. Phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. Methodological issues are discussed that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade (Conner, 2020).

    How phenotypic convergence arises in experimental evolution

    Evolutionary convergence is a core issue in the study of adaptive evolution, as well as a highly debated topic at present. Few studies have analyzed this issue using a 'real-time' or evolutionary trajectory approach. Do populations that are initially differentiated converge to a similar adaptive state when experiencing a common novel environment? Drosophila subobscura populations founded from different locations and years showed initial differences and variation in evolutionary rates in several traits during short-term (approximately 20 generations) laboratory adaptation. This study has extended that analysis to 40 more generations to analyze (1) how differences in evolutionary dynamics among populations change between shorter and longer time spans, and (2) whether evolutionary convergence occurs after 60 generations of evolution in a common environment. Substantial variation was found in longer term evolutionary trajectories and differences between short- and longer term evolutionary dynamics. Although pervasive patterns of convergence were observed toward the character values of long-established populations, populations still remain differentiated for several traits at the final generations analyzed. This pattern might involve transient divergence, as is reported in some cases, indicating that more generations should lead to final convergence. These findings highlight the importance of longer term studies for understanding convergent evolution (Simoes, 2019).

    Polymorphism and Divergence of Novel Gene Expression Patterns in Drosophila melanogaster

    Transcriptomes may evolve by multiple mechanisms, including the evolution of novel genes, the evolution of transcript abundance, and the evolution of cell, tissue, or organ expression patterns. This study focused on the last of these mechanisms in an investigation of tissue and organ shifts in gene expression in Drosophila melanogaster. In contrast to most investigations of expression evolution, this study sought to provide a framework for understanding the mechanisms of novel expression patterns on a short population genetic timescale. To do so population samples of D. melanogaster transcriptomes were generated from five tissues: accessory gland, testis, larval salivary gland, female head, and first instar larva. These data were combined with comparable data from two outgroups to characterize gains and losses of expression, both polymorphic and fixed, in D. melanogaster. A large number of gain or loss of expression phenotypes were observed, most of which were polymorphic within D. melanogaster. Several polymorphic, novel expression phenotypes were strongly influenced by segregating cis-acting variants. In support of previous literature on the evolution of novelties functioning in male reproduction, many more novel expression phenotypes were found in the testis and accessory gland than in other tissues. Additionally, genes showing novel expression phenotypes tend to exhibit greater tissue specific expression. Finally, in addition to qualitatively novel expression phenotypes, genes exhibiting major quantitative expression divergence in the D. melanogaster lineage were identified (Cridland, 2020).

    Interspecific introgression reveals a role of male genital morphology during the evolution of reproductive isolation in Drosophila

    Rapid divergence in genital structures among nascent species has been posited to be an early-evolving cause of reproductive isolation, although evidence supporting this idea as a widespread phenomenon remains mixed. Using a collection of interspecific introgression lines between two Drosophila species that diverged ∼240,000 years ago, this study tested the hypothesis that even modest divergence in genital morphology can result in substantial fitness losses. The reproductive consequences were studied of variation in the male epandrial posterior lobes between Drosophila mauritiana and D. sechellia; divergence in posterior lobe morphology has significant fitness costs on several pre-fertilization and post-copulatory reproductive measures. Males with divergent posterior lobe morphology also significantly reduced the life span of their mates. Interestingly, one of the consequences of genital divergence was decreased oviposition and fertilization, which suggests that a sensory bias for posterior lobe morphology could exist in females, and thus posterior lobe morphology may be the target of cryptic female choice in these species. These results provide evidence that divergence in genitalia can in fact give rise to substantial reproductive isolation early during species divergence, and they also reveal novel reproductive functions of the external male genitalia in Drosophila (Frazee, 2021).

    Soft Selective Sweep on Chemosensory Genes Correlates with Ancestral Preference for Toxic Noni in a Specialist Drosophila Population

    Understanding how organisms adapt to environmental changes is a major question in evolution and ecology. In particular, the role of ancestral variation in rapid adaptation remains unclear because its trace on genetic variation, known as soft selective sweep, is often hardly recognizable from genome-wide selection scans. This study investigate the evolution of chemosensory genes in Drosophila yakuba mayottensis, a specialist subspecies on toxic noni (Morinda citrifolia) fruits on the island of Mayotte. Population genomics analyses and behavioral assays were combined to evaluate the level of divergence in chemosensory genes and perception of noni chemicals between specialist and generalist subspecies of D. yakuba. A signal of soft selective sweep was identified on a handful of genes, with the most diverging ones involving a cluster of gustatory receptors expressed in bitter-sensing neurons. These results highlight the potential role of ancestral genetic variation in promoting host plant specialization in herbivorous insects and identify a number of candidate genes underlying behavioral adaptation (Ferreira, 2020).

    Detection of hard and soft selective sweeps from Drosophila melanogaster population genomic data

    Whether hard sweeps or soft sweeps dominate adaptation has been a matter of much debate. Recently, haplotype homozygosity statistics were developed that (i) can detect both hard and soft sweeps with similar power and (ii) can classify the detected sweeps as hard or soft. The application of this method to population genomic data from a natural population of Drosophila melanogaster (DGRP) allowed rediscovery of three known cases of adaptation at the loci Ace, Cyp6g1, and CHKov1 known to be driven by soft sweeps, and additional candidate loci were detected for recent and strong sweeps. Surprisingly, all of the top 50 candidates showed patterns much more consistent with soft rather than hard sweeps. Recently, this work has been criticized by suggesting that all the candidate loci detected by the haplotype statistics, including the positive controls, are unlikely to be sweeps at all and that instead these haplotype patterns can be more easily explained by complex neutral demographic models. This criticism also claimed that these neutral non-sweeps are likely to be hard instead of soft sweeps. This study reanalyze the DGRP data using a range of complex admixture demographic models and reconfirmed the original published results suggesting that the majority of recent and strong sweeps in D. melanogaster are first likely to be true sweeps, and second, that they do appear to be soft. Furthermore, ways to take this work forward are discussed given that most demographic models employed in such analyses are necessarily too simple to capture the full demographic complexity, while more realistic models are unlikely to be inferred correctly because they require a large number of free parameters (Garud, 2021).

    Allometry constrains the evolution of sexual dimorphism in Drosophila across 33 million years of divergence

    Sexual dimorphism is widely viewed as adaptive, reflecting the evolution of males and females towards divergent fitness optima. Its evolution, however, may often be constrained by the shared genetic architecture of the sexes, and by allometry. This study investigated the evolution of sexual size dimorphism, shape dimorphism, and their allometric relationship, in the wings of 82 taxa in the family Drosophilidae which have been diverging for at least 33 million years. Shape dimorphism among species was remarkably similar, with males characterized by longer thinner wings than females. There was, however, quantitative variation among species in both size and shape dimorphism, with evidence that they have adapted to different evolutionary optima in different clades on timescales of about 10 million years. Within species, shape dimorphism was predicted by size, and among species, there was a strong relationship between size dimorphism and shape dimorphism. Allometry constrained the evolution of shape dimorphism for the two most variable traits studied, but dimorphism was evolutionary labile in other traits. The keys for disentangling alternative explanations for dimorphism evolution are studies of natural and sexual selection, together with a deeper understanding of how microevolutionary parameters of evolvability relate to macroevolutionary patterns of divergence (Sztepanacz, 2021).

    Accelerated inbreeding depression suggests synergistic epistasis for deleterious mutations in Drosophila melanogaster

    Synergistic epistasis for deleterious alleles is relevant to the mutation load paradox and the evolution of sex and recombination. Some studies have shown evidence of synergistic epistasis for spontaneous or induced deleterious mutations appearing in mutation-accumulation experiments. However, many newly arising mutations may not actually be segregating in natural populations because of the erasing action of natural selection. A demonstration of synergistic epistasis for naturally segregating alleles can be achieved by means of inbreeding depression studies, as deleterious recessive allelic effects are exposed in inbred lines. This paper reports the results of two independent inbreeding experiments carried out with two different populations of Drosophila melanogaster. The results show a consistent accelerated inbreeding depression for fitness, suggesting synergistic epistasis among deleterious alleles. Computer simulations were performed assuming different possible models of epistasis and mutational parameters for fitness, finding some of them to be compatible with the results observed. These results suggest that synergistic epistasis for deleterious mutations not only occurs among newly arisen spontaneous or induced mutations, but also among segregating alleles in natural populations (Dominguez-Garcia, 2019).

    Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo

    Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. This study used genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na(+),K(+)-ATPase. Surprisingly, frequently observed adaptive substitutions were found at at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. A phylogenetically correlated substitution, A119S, was found that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. These results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, the results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms (Taverner, 2019).

    The ability of Drosophila hybrids to locate food declines with parental divergence

    Hybrids are generally less fit than their parental species, and the mechanisms underlying their fitness reductions can manifest through different traits. For example, hybrids can have physiological, behavioral, or ecological defects, and these defects can generate reproductive isolation between their parental species. However, the rate that mechanisms of postzygotic isolation other than hybrid sterility and inviability evolve has remained largely uninvestigated, despite isolated studies showing that behavioral defects in hybrids are not only possible but might be widespread.This work studied a fundamental animal behavior - the ability of individuals to find food - and tested the rate at which it breaks down in hybrids. The ability of hybrids from 94 pairs of Drosophila species to find food was measured, and this ability was shown to decrease with increasing genetic divergence between the parental species and that male hybrids are more strongly (and negatively) affected than females. These findings quantify the rate that hybrid dysfunction evolves across the diverse radiation of Drosophila and highlights the need for future investigations of the genetic and neurological mechanisms that affect a hybrid's ability to find a suitable substrate on which to feed and breed (Turissini, 2017).

    The Drosophila speciation factor HMR localizes to genomic insulator sites

    Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr (Hybrid male rescue) and Lhr (Lethal hybrid rescue) genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, this study measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, HMR was found to localize to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. These data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species (Gerland, 2017).

    Biodiversity is the result of the emergence and the extinction of species. New species form by pre- and post-zygotic isolation mediated by genetic incompatibility. One of the best characterized examples of hybrid incompatibility is the gene pair Hybrid male rescue (Hmr) and Lethal hybrid rescue (Lhr). Hmr and Lhr cause hybrid incompatibility between the closely related fly species Drosophila melanogaster and D. simulans. Hmr diverged in both Drosophila sibling species under positive selection. HMR and LHR from both species interact physically and localize predominantly to centromeric regions. A reduction of HMR expression results in a misregulation of transposable elements, satellite DNAs and heterochromatic genes. The major difference between HMR and LHR in D. melanogaster and D. simulans is their substantial difference in protein amounts which has been proposed to result in a lethal gain of function in male hybrids. High levels of HMR and LHR in hybrids and overexpression of these proteins in pure species lead to an increased number of binding sites of the complex. Such spreading phenomena based on protein amount have been observed for several chromatin-associated complexes such as the dosage compensation complex, the polycomb complex or components of pericentromeric heterochromatin. In most cases, the precise mechanisms for targeting and spreading are not fully understood. Interestingly, several of the components involved in these processes show signs of adaptive evolution and differ substantially even in very closely related organisms. This observation has spurred a model of a dynamic genome that drives the adaptive evolution of chromatin-associated factors (Gerland, 2017 and references therein).

    Eukaryotic genomes of closely related species differ mostly in the amount and sequence of repetitive DNA. This DNA is often derived from transposable elements, which are highly mutagenic and are therefore under tight transcriptional control by the cellular machinery. During evolution transposons or transposon-derived sequences occasionally adopted structural or novel cis-regulatory functions, thereby contributing to the evolution of new, species-specific, phenotypic traits. Genomic insulators are a particular class of such novel, fast evolving, cis-regulatory elements that show signs of transposon ancestry. A strong expansion of these elements is observed in arthropods, which also experienced a successive gain in the number of insulator binding proteins during evolution. In fact, the Drosophila genome harbours a large variety of insulator proteins such as CTCF, BEAF-32, Su(Hw), Mod(mdg4) and CP190, which all affect nuclear architecture. Different Drosophila species underwent multiple genomic rearrangements and transposon invasions, which presumably resulted in an adaptive response of regulatory DNA binding factors to maintain spatial and temporal gene expression. For example, binding sites for the insulator proteins BEAF-32 and CTCF show a high degree of variability when compared among very closely related species. The gain of new insulator sites is associated with chromosome rearrangements, new born genes and species-specific transcription regulation. Similar to insulator proteins, which tend to cluster in specific nuclear regions, the speciation factor HMR clusters at centromeres or pericentromeric regions in diploid cells but is also detected at distinct euchromatic regions along the chromosome arms in polytene chromosomes. A unifying feature for many of these sites is their close proximity to binding sits of the Heterochromatin Protein 1 (HP1a), a HMR interactor and a well-characterized heterochromatic mark (Gerland, 2017).

    Various studies describe HMR's localization to heterochromatin, but the molecular details on HMR's binding sites and its recruitment to these sites are not well understood. To get new insights into HMR's association to chromatin, this study measured HMR's genome-wide localization by chromatin immunoprecipitation (ChIP) in the D. melanogaster embryonic S2 cell line. This analysis demonstrated an extensive colocalization of HMR with a subset of insulator sites across the genome. HMR's binding to genomic gypsy insulators, which constitute the major group of its binding sites, is dependent on the residing insulator protein complex. In a second group, HMR borders heterochromatin together with the insulator protein BEAF-32. In agreement with previous low-resolution techniques in cell lines and fly tissue, these binding sites are enriched at pericentromeric regions, the cytological region 31 on the 2nd chromosome and the entire 4th chromosome. At most of these sites, HMR associates to the promoters of actively transcribed genes. Interestingly, these genes code for transcripts that have been reported to be downregulated in Hmr mutant larvae and ovaries. Altogether, these data provide evidence for a functional link between HMR and insulator proteins, which potentially results in hybrid incompatibilities due to the adaptive evolution of these genome-organizing complexes (Gerland, 2017).

    HMR localizes to centromeric and pericentromeric regions in D. melanogaster cell lines as well as in mitotically dividing embryonic cells where it has been suggested to act as a repressor of transposable elements. Mutations in Hmr lead to overexpression of satellite DNA and transposable elements in ovaries and larvae. Such a derepression is also observed in hybrid flies, where HMR and LHR levels are higher than the ones in pure species and result in a widespread distribution of the HMR/LHR complex. To better understand the targeting principles that mediate HMR binding within the D. melanogaster genome, it was asked whether it is possible to identify HMR binding sites by applying ChIP-Seq in the D. melanogaster S2 cell line. Combining this approach with RNAi mediated knockdown experiments this study uncovered a strong colocalization of HMR with gypsy insulator binding sites and demonstrated that HMR binding to these sites depends on the presence of the residing insulator protein complex. Notably, HMR associates only with a subset of all Su(Hw) binding sites, but almost all those sites can be classified as gypsy-like sites bound by CP190 and mod(mdg4) in addition to Su(Hw) (Gerland, 2017).

    Besides dispersed binding of HMR at genomic gypsy insulator sites along the chromosome arms, dense clusters of HMR binding sites were observed around the centromere and on the 4th chromosome where it potentially serves to separate HP1a binding domains from highly active genes. This dense clustering of binding sites around the centromere correlates well with the strong colocalization of HMR signals with the centromeric H3 variant CID in immunolocalization experiments. Due to its biochemical interaction and partial colocalization with the heterochromatin protein HP1a in Drosophila embryos, HMR has been suggested to be a bona-fide heterochromatin component. However, in contrast to HP1a, this study detected very distinct HMR binding sites within the genome. When HMR is found close to an HP1a binding domain, it rather borders it than covering the whole domain. The sharp HMR binding signals and the fact that almost all euchromatic HMR binding sites contain putative insulator elements, suggest a role of HMR in separating chromatin domains. A distinct boundary that separates constitutive heterochromatin from the core centromere has also been postulated by Olszak and colleagues who suggest that transition zones between heterochromatin and euchromatin are hotspots for sites of CID misincorporation. Unfortunately, centromeres are notoriously difficult to study by next generation sequencing due to their highly repetitive nature. In addition, the microscopic resolution is not sufficiently high to allow a distinction between a binding to the core centromere chromatin and the chromatin immediately adjacent to it. Therefore, it cannot be ruled that HMR binds large domains at the central region of the Drosophila centromere. However, the fact that the purification of chromatin containing the centromeric H3 variant CID did not identify HMR, suggests that it may very well also form a boundary between pericentromeric heterochromatin and the core centromere. To which extent and by which mechanism HMR fulfils a functional role at these genomic sites remains to be elucidated (Gerland, 2017).

    The genomic sites, where HMR was found bound next to an HP1a domain, are highly enriched for recognition sites of the insulator protein BEAF-32. Interestingly, a depletion of BEAF-32 in S2 cells results in an increased rate of mitotic defects, which is very reminiscent of the phenotype detected when HMR is depleted. Similarly to flies carrying a mutation in the Hmr gene, flies in which BEAF-32 is only contributed maternally have defects in female fertility. BEAF-32's role in maintaining associated promoter regions in an environment that facilitates high transcription levels has been suggested to be functionally relevant for this phenotype. Strikingly, this study found most HMR/BEAF-32 binding sites located between HP1a containing heterochromatin and the transcription start site of a highly active gene. HP1a chromatin might fulfil a repressive function at these genomic regions and HMR might block this repressive impact on the neighbouring gene body. However, no extensive spreading of HP1a or H3K9me3 is seen upon HMR knockdown suggesting that the repressive effect is not directly mediated by HP1a binding or the HMR knock down not efficient enough. As there is evidence that HP1a can also promote gene transcription, HMR may also function as a co-activator for HP1a. Currently, HMR binding next to HP1a containing chromatin is considered as a unifying feature of transcriptionally affected genes but the potential mechanism by which HMR exerts its function is as yet unknown (Gerland, 2017).

    Although HMR depletion has a substantial effect on the transcription of multiple transposons, HMR was found enriched only at the 5' insulator region of the gypsy or gtwin retrotransposons and to similar sites within the genome that are presumably derived from these elements. These sites are occupied by insulator proteins Su(Hw), CP190 and Mod(mdg4) and often display enhancer blocking activity in transgenic assays. Artificial targeting of HMR to DNA placed between an enhancer and a promoter of a reporter gene can block the transcription activity, suggesting that HMR may indeed play a role in setting up endogenous boundary elements. Similar to what is known for Su(Hw), HMR binding to this class of binding sites is dependent on the presence of the structural protein CP190, which has a key function in the stabilization of insulator protein complexes. However, as no strong physical interaction is observed between CP190 and HMR, the loss of HMR binding upon a reduction of CP190 levels may also be the result of increased nucleosome occupancy. Such increase in Histone H3 binding cannot be observed upon HMR removal suggesting that HMR acts downstream of CP190. Interestingly, CP190 loss impairs HMR binding to gypsy-like insulator sites but has weak effect on HMR binding to sites containing BEAF-32 recognition motifs. Notably, in contrast to BEAF-32, CP190 is not required for oogenesis, suggesting that the lack of HMR binding to the class 1 sites may be responsible for the female sterility phenotype observed in Hmr mutant flies (Gerland, 2017).

    How can the current findings be integrated with the lethal phenotype of increased HMR/LHR levels in male hybrids? It is tempting to speculate that multiple additional binding sites that are observed in hybrids and on polytene chromosomes of fly strains over-expressing HMR constitute boundary regions. An increased binding to such boundaries, which have been shown to cluster and form aggregates in vivo, may trigger a massive change in nuclear architecture. In turn, this could indirectly activate multiple transposable elements similar to what is observed when centromere clustering is disturbed. Such a disturbed nuclear architecture may then trigger the activation of a cell cycle checkpoint which has been previously suggested to be a major cause of hybrid lethality (Gerland, 2017).

    Altogether, these data provide a novel link between HMR and cis-regulatory elements bound by insulator proteins. It is speculated that divergent evolution of such genomic elements and their corresponding binding factors in sibling species is triggering hybrid incompatibilities (Gerland, 2017).

    Persistent postmating, prezygotic reproductive isolation between populations

    Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate-female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. This study addressed these outstanding questions using multiple populations of Drosophila montana. A recurrent pattern of PMPZ isolation is shown, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. No evidence was found of variability in fertilization outcomes attributable to different female x male genotype interactions, and in combination with other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype x genotype interactions. These results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate-female reproductive tract interactions within species that may cause this PMPZ isolation (Garlovsky, 2018).

    The hpRNA/RNAi pathway is essential to resolve intragenomic conflict in the Drosophila male germline

    Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. This study reveals that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, D. simulans null mutants of Dcr-2 and AGO2 were generated. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, these data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species (Lin, 2018).

    RNA interference (RNAi) has long been recognized as a versatile experimental technique, but its endogenous biological utilities have been less tangible. This topic is in principle more accessible in invertebrates, several of which express diverse endogenous siRNAs (endo-siRNAs) via dedicated RNAi machinery that is distinct from the related miRNA pathway. However, while RNAi mutants in nematodes and flies are compromised at defending viruses and affected by certain extreme environmental perturbations, RNAi mutants generally exhibit few overt phenotypes under non-sensitized conditions (Lin, 2018).

    The biological logic of the Drosophila hairpin RNA (hpRNA) pathway has been described, in which inverted repeat transcripts preferentially generate endo-siRNAs in the testis and repress specific highly complementary mRNAs. Although all known hpRNAs are recently evolved, clear evidence is observed for siRNA:target co-evolution, indicating adaptive properties of this regulatory network. While ovaries detectably express hpRNAs and endo-siRNAs, RNAi mutants have relatively little consequence in females. Instead, genetic ablation of RNAi causes spermatogenesis defects and male subfertility. Nevertheless, Drosophila melanogaster RNAi mutant males are fertile, suggesting this species can formally cope without siRNAs, at least within the laboratory setting (Lin, 2018).

    In searching for other manifestations of the hpRNA pathway, the Winters sex-ratio (SR) system of Drosophila simulans was investigated. This meiotic drive system is absent from D. melanogaster and was born within D. simulans subclade species that diverged ~240,000 years ago. Despite its recent de novo appearance, Winters SR factors have profound activities. The Distorter on X (Dox) promotes X chromosome transmission by suppressing Y-bearing sperm, a patently undesirable 'wild-type' gene activity that must be silenced in order to maintain the D. simulans species. An antidote is encoded by autosomal Not much yang (Nmy), to which an inverted repeat with a sequence similarity to Dox was mapped. Signatures consistent with positive selection on Winters factors have been detected within D. simulans populations, indicating the system is actively evolving under an 'arms-race' scenario. While the relationship of Dox and Nmy was evocative of homology-dependent silencing, there is currently no evidence (1) for molecular species constituting the active output of Nmy, (2) that Nmy directly or indirectly regulates the expression of Dox, (3) whether Winters factors are truly distinct from other SR systems, or (4) that the RNAi pathway participates in SR control (Lin, 2018).

    This study provides first molecular evidence that hpRNA-siRNAs are functional mediators of son protection in the Winters SR system. Moreover, this study reveals that a second, previously uncloned SR suppressor in this species, known as the Durham SR system, involves a previously unknown hpRNA-siRNA locus termed Tmy. Although defined as genetically separable SR systems, this study also shows that Nmy and Tmy are paralogous and have partially overlapping capacity to suppress both Dox as well as its progenitor locus MDox. To demonstrate a connection to the RNAi pathway, CRISPR/Cas9 was used to engineer dcr-2 and ago2 null mutants in this non-model fruit fly species. Remarkably, these exhibit profound, testis-specific phenotypes that are much more severe than their well-studied D. melanogaster counterparts, in that they are completely male sterile due to profound defects in spermatogenesis progression and harbor massive synergistic derepression of Dox and MDox transcripts, consistent with loss of collaborative suppression by Nmy and Tmy hpRNAs (Lin, 2018).

    Altogether, these data demonstrate unanticipated complexity of sex-distorting factors and hpRNA-suppressing loci in D. simulans, all of which are rapidly evolving and none of which exist in D. melanogaster. Thus, RNAi is a key pathway that resolves intragenomic conflict that ensures species survival and fulfills roles in adaptive gonadal gene regulation that are more commonly attributed to the piRNA pathway. This highlights the need to explore a wider range of species to more fully appreciate the evolving functions of germline small RNA pathways (Lin, 2018).

    This study provides critical linkages among the RNAi pathway, hpRNA biogenesis and function, and suppression of SR bias. The evolutionary behavior of SR systems conforms to a 'Red Queen' effect, in which a seemingly static outcome (equal transmission of X and Y sperm) actually involves intense, opposing and rapidly evolving genetic programs. These studies provide striking evidence that endogenous RNAi is a central molecular pathway that resolves SR distortion and may potentially have an impact on hybrid sterility. Molecular and genetic evidence are provided of a potential hierarchy, in that Dox is a prime direct target of Nmy based on the observation that it supplies the majority of targeting siRNAs. Nevertheless, Tmy provides a secondary defense: since Tmy exhibits extensive complementarity to Dox that is non-overlapping with Nmy, Tmy siRNAs maintain targeting to Dox even in nmy mutants. Tmy can directly repress Dox in sensor assays, and most importantly, RNAi mutants exhibit strongly elevated Dox transcripts in testis, consistent with co-targeting endogenous action of both hpRNAs on Dox. Moreover, these principles are shown to be true for MDox, strongly implying this locus as a functional distorter. Overall, this study reveals that multiple evolutionary nascent hpRNAs (Nmy/Tmy) are individually required for species preservation via suppression of Winters and Durham SR (Dox/MDox) distorters in D. simulans (Lin, 2018).

    In the future, further dissection of the genetic contributions of the individual hpRNAs and distorters will shed light on their relative contributions to Winters and Durham SR systems and the extent to which they are distinct systems or partially overlapping as indicated by our studies. This will be a challenge in a non-model fly that lacks the genetic tools available in D. melanogaster but will be important to understand how these newly emerging factors are endowed with such powerful activities. For example, compelling hypotheses to test include whether specific deletions of the Tmy hairpin alone can recapitulate SR bias, whether Tmy exhibits derepression of other distorter factors, and whether nmy/tmy double mutant might exhibit only SR or may prove to recapitulate sterility found in RNAi mutants. Thus far, Nmy and Tmy loci have proven recalcitrant to repeated attempts for CRISPR/Cas9 targeting, and it is not clear whether something about their repeat structure affects this endeavor. Moreover, the close linkage of these loci will be a challenge for any efforts to generate recombinants. Still, it will be worthwhile to pursue the generation of new genetic tools (Lin, 2018).

    Remarkably, there is a third SR distortion system in D. simulans ('Paris'). While it is genetically complex, it was recently shown to depend on HP1D2, a recently evolved paralog of the piRNA factor Rhino. Thus, there are apparently molecular linkages of SR systems with small RNA systems, on both driving and suppressing sides. Although the mechanism of Paris SR remains to be determined, HP1D2 protein localizes to the heterochromatic Y chromosome, which provides a connection to the observation that driving Paris alleles prevent segregation of Y chromatids during meiosis II. On the other hand, the defect in Winters SR appears to be post-meiotic, indicating mechanistic diversity in how depletion of male sperm might be achieved by de novo genes (Lin, 2018).

    On the other hand, the sister species D. melanogaster appears to lack SR systems, testament to the extremely rapid rise and fall of SR systems during evolution. The adaptive properties of hpRNAs make them ideal genetic elements to tame such selfish meiotic drive elements, which are theorized to be under constant cycles of emergence, suppression, and disappearance. There is mounting evidence that genetic systems that manifest in SR defects are often associated with sterility. The findings of this study support the notion that the success or failure to resolve intragenomic conflicts using RNAi would be intimately connected to speciation (Lin, 2018).

    While these roles were unexpected in light of the fact that metazoan RNAi biology has been so challenging to appreciate, the situation would have been different had a species only slightly diverged from D. melanogaster initially been selected as a genetic model. This parallels the inference that RNAi might have been recognized earlier had certain budding yeasts other than Saccharomyces cerevisiae been studied earlier. Indeed, functional studies across a broader phylogeny will be necessary to appreciate the evolving requirements of small RNA regulation, beyond standard model organisms. The availability of the D. simulans RNAi mutants of this study opens the door to molecular identification of novel selfish genetic elements that induce SR bias and/or hybrid sterility, in both the Winters and Durham systems (Lin, 2018).

    Strength of sexual and postmating prezygotic barriers varies between sympatric populations with different histories and species abundances

    The impact of different reproductive barriers on species or population isolation may vary in different stages of speciation depending on evolutionary forces acting within species and through species' interactions. Genetic incompatibilities between interacting species are expected to reinforce prezygotic barriers in sympatric populations and lead to cascade reinforcement between conspecific populations living within and outside the areas of sympatry. These predictions were tested and this work studied whether and how the strength and target of reinforcement between Drosophila montana and Drosophila flavomontana vary between sympatric populations with different histories and species abundances. All barriers between D. montana females and D. flavomontana males were nearly complete, while in the reciprocal cross strong postzygotic isolation was accompanied by prezygotic barriers whose strength varied according to population composition. Sexual isolation between D. flavomontana females and D. montana males was increased in long-established sympatric populations, where D. flavomontana is abundant, while postmating prezygotic (PMPZ) barriers were stronger in populations where this species is a new invader and still rare and where female discrimination against heterospecific males was lower. Strengthening of sexual and PMPZ barriers in this cross also induced cascade reinforcement of respective barriers between D. flavomontana populations, which is a classic signature of reinforcement process (Poikela, 2019).

    Reproductive capacity evolves in response to ecology through common changes in cell number in Hawaiian Drosophila

    Lifetime reproductive capacity is a critical fitness component. In insects, female reproductive capacity is largely determined by the number of ovarioles, the egg-producing subunits of the ovary. Recent work has provided insights into ovariole number regulation in Drosophila melanogaster. However, whether mechanisms discovered under laboratory conditions explain evolutionary variation in natural populations is an outstanding question. This study investigated potential effects of ecology on the developmental processes underlying ovariole number evolution among Hawaiian Drosophila, a large adaptive radiation wherein the highest and lowest ovariole numbers of the family have evolved within 25 million years. Previous studies proposed that ovariole number correlated with oviposition substrate but sampled largely one clade of these flies and were limited by a provisional phylogeny and the available comparative methods. This hypothesis was tested by applying phylogenetic modeling to an expanded sampling of ovariole numbers and substrate types and shows support for these predictions across all major groups of Hawaiian Drosophila, wherein ovariole number variation is best explained by adaptation to specific substrates. Furthermore, oviposition substrate evolution is linked to changes in the allometric relationship between body size and ovariole number. Finally, evidence is provided that the major changes in ovarian cell number that regulate D. melanogaster ovariole number also regulate ovariole number in Hawaiian drosophilids. Thus, evidence is provided that this remarkable adaptive radiation is linked to evolutionary changes in a key reproductive trait regulated at least partly by variation in the same developmental parameters that operate in the model species D. melanogaster (Sarikaya, 2019).

    A common suite of cellular abnormalities and spermatogenetic errors in sterile hybrid males in Drosophila

    When two species interbreed, the resulting hybrid offspring are often sterile, with the heterogametic (e.g. XY) hybrid usually being more severely affected. The prevailing theory for this pattern of sterility evokes divergent changes in separate lineages having maladaptive interactions when placed together in a hybrid individual, with recessive factors on the sex chromosome interacting with dominant factors on the autosomes. The effect of these interactions on gametogenesis should not be uniform across species pairs unless genetic divergence follows the same paths in different lineages or if a specific stage of gametogenesis is more susceptible to detrimental genetic interactions. A detailed cellular characterization of hybrid male sterility was performed across three recently diverged species pairs of Drosophila. Across all three pairs, sterile hybrid sperm are alive but exhibit rapid nuclear de-condensation with age, with active, but non-differentiated, mitochondria. Surprisingly, all three sets of interspecies hybrids produce half of the number of sperm per round of spermatogenesis, with each sperm cell containing two tails. Non-disjunction failures during meiosis I were identified as the likely cause. Thus, errors during meiosis I may be a general phenomenon underlying Drosophila male sterility, indicating either a heightened sensitivity of this spermatogenic stage to failure, or a basis to sterility other than the prevailing model (Kanippayoor, 2020).

    Parallel sequencing of Wolbachia wCer2 from donor and novel hosts reveals multiple incompatibility factors and genome stability after host transfers

    The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit flies by embryonic microinjections. wCer2 genome data was obtained from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host, however only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster) and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes and one Type IV cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species (Morrow, 2020).

    Reproductive isolation caused by azoospermia in sterile male hybrids of Drosophila

    Recently diverged populations in the early stages of speciation offer an opportunity to understand mechanisms of isolation and their relative contributions. Drosophila willistoni is a tropical species with broad distribution from Argentina to the southern United States, including the Caribbean islands. A postzygotic barrier between northern populations (North America, Central America, and the northern Caribbean islands) and southern populations (South American and the southern Caribbean islands) has been recently documented and used to propose the existence of two different subspecies. This study has identified premating isolation between populations regardless of their subspecies status. No evidence of postmating prezygotic isolation was found, and this study proceeded to characterize hybrid male sterility between the subspecies. Sterile male hybrids transfer an ejaculate that is devoid of sperm but causes elongation and expansion of the female uterus. In sterile male hybrids, bulging of the seminal vesicle appears to impede the movement of the sperm toward the sperm pump, where sperm normally mixes with accessory gland products. The results highlight a unique form of hybrid male sterility in Drosophila that is driven by a mechanical impediment to transfer sperm rather than by an abnormality of the sperm itself. Interestingly, this form of sterility is reminiscent of a form of infertility (azoospermia) that is caused by lack of sperm in the semen due to blockages that impede the sperm from reaching the ejaculate (Davis, 2020).

    A morphological trait involved in reproductive isolation between Drosophila sister species is sensitive to temperature

    Male genitalia are usually extremely divergent between closely related species, but relatively constant within one species. This study examined the effect of temperature on the shape of the ventral branches, a male genital structure involved in reproductive isolation, in the sister species Drosophila santomea and Drosophila yakuba. A semi-automatic measurement machine learning pipeline was designed that can reliably identify curvatures and landmarks based on manually digitized contours of the ventral branches. With this method, it was observed that temperature does not affect ventral branches in D. yakuba but that in D. santomea ventral branches tend to morph into a D. yakuba-like shape at lower temperature. Male genitalia structures involved in reproductive isolation can be relatively variable within one species and can resemble the shape of closely related species' genitalia through plasticity to temperature. These results suggest that reproductive isolation mechanisms can be dependent on the environmental context (Peluffo, 2021).

    Identification of misexpressed genetic elements in hybrids between Drosophila-related species

    Crosses between close species can lead to genomic disorders, often considered to be the cause of hybrid incompatibility, one of the initial steps in the speciation process. How these incompatibilities are established and what are their causes remain unclear. To understand the initiation of hybrid incompatibility, reciprocal crosses were performed between two species of Drosophila (D. mojavensis and D. arizonae) that diverged less than 1 Mya. A genome-wide transcriptomic analysis was performed on ovaries from parental lines and on hybrids from reciprocal crosses. Using an innovative procedure of co-assembling transcriptomes, it was shown that parental lines differ in the expression of their genes and transposable elements. Reciprocal hybrids presented specific gene categories and few transposable element families misexpressed relative to the parental lines. Because TEs are mainly silenced by piwi-interacting RNAs (piRNAs), it was hypothesized that in hybrids the deregulation of specific TE families is due to the absence of such small RNAs. Small RNA sequencing confirmed the hypothesis, and it is therefore proposed that TEs can indeed be major players of genome differentiation and be implicated in the first steps of genomic incompatibilities through small RNA regulation (Lopez-Maestre, 2017).

    Introduction of a male-harming mitochondrial haplotype via 'Trojan Females' achieves population suppression in fruit flies

    Pests are a global threat to biodiversity, ecosystem function, and human health. Pest control approaches are thus numerous, but their implementation costly, damaging to non-target species, and ineffective at low population densities. The Trojan Female Technique (TFT) is a prospective self-perpetuating control technique that is species-specific and predicted to be effective at low densities. The goal of the TFT is to harness naturally-occurring mutations in the mitochondrial genome that impair male fertility while having no effect on females. This study provides proof-of-concept for the TFT, by showing that introduction of a male fertility-impairing mtDNA haplotype into replicated populations of Drosophila melanogaster causes numerical population suppression, with the magnitude of effect positively correlated with its frequency at trial inception. Further development of the TFT could lead to establishing a control strategy that overcomes limitations of conventional approaches, with broad applicability to invertebrate and vertebrate species, to control environmental and economic pests (Wolff, 2017).

    Male mate choice via cuticular hydrocarbon pheromones drives reproductive isolation between Drosophila species

    Mate discrimination is a key mechanism restricting gene flow between species. While studied extensively with respect to female mate choice, mechanisms of male mate choice between species are far less studied. Thus, there is little knowledge of the relative frequency, importance, or overall contribution of male mate discrimination to reproductive isolation. This study estimated the relative contributions of male and female choice to reproductive isolation between Drosophila simulans and D. sechellia, and showed that male mate discrimination accounts for the majority of the current isolation between these species. It was further demonstrate that males discriminate based on female cuticular hydrocarbon pheromones, and collect evidence supporting the hypothesis that male mate discrimination may alleviate the costs associated with heterospecific courtship and mating. These findings highlight the potentially significant contribution of male mate choice to the formation of reproductive isolating barriers, and thus the speciation process (Shahandeh, 2017).

    Genes underlying species differences in CHC production between Drosophila melanogaster and D. simulans

    Surface chemical compounds are key components of survival and reproduction in many species. Cuticular hydrocarbons (CHCs) are chemical compounds produced by all insects that are used for both desiccation resistance and chemical communication, including communication related to mating. In the species pair of Drosophila melanogaster and D. simulans, female CHCs stimulate conspecific males to mate and repel heterospecific males. While CHCs are a critical contributor to both reproductive success within a species and isolation between species, few genes underlying species variation in CHC profiles are known. This study used genetic mapping of the 3rd chromosome to test a suite of candidate genes for interspecies variation in CHCs. Candidate gene CG5946 was found to be involved in species differences in the production of 7,11-heptacosadiene and 7-tricosene between D. melanogaster and D. simulans. This is therefore a new candidate locus contributing to species-specific variation in the CHC profile. In the process of mapping genes for CHCs, 29 candidate genes for the reduced survival or inviability of interspecies hybrids were identified (Ward, 2020).

    The rate of evolution of postmating-prezygotic reproductive isolation in Drosophila

    Reproductive isolation is an intrinsic aspect of species formation. For that reason, the identification of the precise isolating traits, and the rates at which they evolve, is crucial to understanding how species originate and persist. Previous work has measured the rates of evolution of prezygotic and postzygotic barriers to gene flow, yet no systematic analysis has studied the rates of evolution of postmating-prezygotic (PMPZ) barriers. This study measured the magnitude of two barriers to gene flow that act after mating occurs but before fertilization. The magnitude of a premating barrier (female mating rate in nonchoice experiments) and two postzygotic barriers (hybrid inviability and hybrid sterility) was also measured for all pairwise crosses of all nine known extant species within the melanogaster subgroup. The results indicate that PMPZ isolation evolves faster than hybrid inviability but slower than premating isolation. Next, postzygotic isolation was partitioned into different components; as expected, hybrid sterility evolves faster than hybrid inviability. These results lend support for the hypothesis that, in Drosophila, reproductive isolation mechanisms (RIMs) that act early in reproduction (or in development) tend to evolve faster than those that act later in the reproductive cycle. Finally, whether there was evidence for reinforcing selection at any RIM was tested. No evidence was found for generalized evolution of reproductive isolation via reinforcement which indicates that there is no pervasive evidence of this evolutionary process. These results indicate that PMPZ RIMs might have important evolutionary consequences in initiating speciation and in the persistence of new species (Turissini, 2017).

    Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

    Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape Karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, this study investigated an autosome-Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10-15 Mya. Single molecule real-time sequencing reads were used to assemble the D. pseudoobscura dot chromosome, including the Y-to-dot translocation. It was found that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ∼78 Kb and is not repeat-dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y-to-dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, a consistent reduction in intron sizes across the Y-to-dot region was not found. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y-to-dot translocation (Chang, 2017).

    Linked genetic variation and not genome structure causes widespread differential expression associated with chromosomal inversions

    Chromosomal inversions are widely thought to be favored by natural selection because they suppress recombination between alleles that have higher fitness on the same genetic background or in similar environments. Nonetheless, few selected alleles have been characterized at the molecular level. Gene expression profiling provides a powerful way to identify functionally important variation associated with inversions and suggests candidate phenotypes. This study characterized differential expression patterns associated with two chromosomal inversions found in natural Drosophila melanogaster populations. To isolate the impacts of genome structure, synthetic chromosomal inversions were engineered on controlled genetic backgrounds with breakpoints that closely match each natural inversion. Synthetic inversions have negligible effects on gene expression. Nonetheless, natural inversions have broad-reaching regulatory impacts in cis and trans Furthermore, differentially expressed genes associated with both natural inversions are enriched for loci associated with immune response to bacterial pathogens. The results support the idea that inversions in D. melanogaster experience natural selection to maintain associations between functionally related alleles to produce complex phenotypic outcomes (Said, 2018).

    An investigation of Y chromosome incorporations in 400 species of Drosophila and related genera

    Y chromosomes are widely believed to evolve from a normal autosome through a process of massive gene loss (with preservation of some male genes), shaped by sex-antagonistic selection and complemented by occasional gains of male-related genes. The net result of these processes is a male-specialized chromosome. This might be expected to be an irreversible process, but it was found in 2005 that the Drosophila pseudoobscura Y chromosome was incorporated into an autosome. Y chromosome incorporations have important consequences: a formerly male-restricted chromosome reverts to autosomal inheritance, and the species may shift from an XY/XX to X0/XX sex-chromosome system. In order to assess the frequency and causes of this phenomenon Y chromosome incorporations was sought in 400 species from Drosophila and related genera. One additional large scale event of Y chromosome incorporation was found, affecting the whole montium subgroup (40 species in our sample); overall 13% of the sampled species (52/400) have Y incorporations. While previous data indicated that after the Y incorporation the ancestral Y disappeared as a free chromosome, the much larger data set analyzed here indicates that a copy of the Y survived as a free chromosome both in montium and pseudoobscura species, and that the current Y of the pseudoobscura lineage results from a fusion between this free Y and the neoY. The 400 species sample also showed that the previously suggested causal connection between X-autosome fusions and Y incorporations is, at best, weak: the new case of Y incorporation (montium) does not have X-autosome fusion, whereas nine independent cases of X-autosome fusions were not followed by Y incorporations. Y incorporation is an underappreciated mechanism affecting Y chromosome evolution; these results show that at least in Drosophila it plays a relevant role and highlight the need of similar studies in other groups (Dupim, 2018).

    The molecular characterization of fixed inversions breakpoints unveils the ancestral character of the Drosophila guanche chromosomal arrangements

    Cytological studies revealed that the number of chromosomes and their organization varies across species. The increasing availability of whole genome sequences of multiple species across specific phylogenies has confirmed and greatly extended these cytological observations. In the Drosophila genus, the ancestral karyotype consists of five rod-like acrocentric chromosomes (Muller elements A to E) and one dot-like chromosome (element F), each exhibiting a generally conserved gene content. Chromosomal fusions and paracentric inversions are thus the major contributors, respectively, to chromosome number variation among species and to gene order variation within chromosomal element. The subobscura cluster of Drosophila consists in three species that retain the genus ancestral karyotype and differ by a reduced number of fixed inversions. This study used cytological information and the D. guanche genome sequence to identify and molecularly characterize the breakpoints of inversions that became fixed since the D. guanche-D. subobscura split. The results have led to a proposal of a modified version of the D. guanche cytological map of its X chromosome, and to establish that (i) most inversions became fixed in the D. subobscura lineage and (ii) the order in which the four X chromosome overlapping inversions occurred and became fixed (Orengo. 2019).

    Structural variants exhibit widespread allelic heterogeneity and shape variation in complex traits

    It has been hypothesized that individually-rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. This study identified more than 20,000 euchromatic SVs from 14 Drosophila melanogaster genome assemblies, of which ~40% are invisible to high specificity short-read genotyping approaches. SVs are common, with 31.5% of diploid individuals harboring a SV in genes larger than 5kb, and 24% harboring multiple SVs in genes larger than 10kb. SV minor allele frequencies are rarer than amino acid polymorphisms, suggesting that SVs are more deleterious. A number of functionally important genes were shown to harbor previously hidden structural variants likely to affect complex phenotypes. Furthermore, SVs are overrepresented in candidate genes associated with quantitative trait loci mapped using the Drosophila Synthetic Population Resource. It is concluded that SVs are ubiquitous, frequently constitute a heterogeneous allelic series, and can act as rare alleles of large effect (Chakraborty, 2019).

    Complex evolutionary history of the Y chromosome in flies of the Drosophila obscura species group

    The Drosophila obscura species group shows dramatic variation in karyotype, including transitions among sex chromosomes. Members of the affinis and pseudoobscura subgroups contain a neo-X chromosome (a fusion of the X with an autosome), and it was shown that ancestral Y genes have become autosomal in species harboring the neo-X. Detailed analysis of species in the pseudoobscura subgroup revealed that ancestral Y genes became autosomal through a translocation to the small dot chromosome. This study shows that the Y-dot translocation is restricted to the pseudoobscura subgroup, and translocation of ancestral Y genes in the affinis subgroup likely followed a different route. Most ancestral Y genes appear to have translocated to unique autosomal or X-linked locations in different taxa of the affinis subgroup, and a dynamic model of sex chromosome formation and turnover in the obscura species group is proposed. The results suggest that Y genes can find unique paths to escape unfavorable genomic environments that form after sex chromosome-autosome fusions (Bracewell, 2020).

    Fine-scale position effects shape the distribution of inversion breakpoints in Drosophila melanogaster

    Chromosomal inversions are among the primary drivers of genome structure evolution in a wide range of natural populations. While there is an impressive array of theory and empirical analyses that have identified conditions under which inversions can be positively selected, comparatively little data is available on the fitness impacts of these genome structural rearrangements themselves. Because inversion breakpoints can disrupt functional elements and alter chromatin domains, the precise positioning of an inversion's breakpoints can strongly affect its fitness. This study compared the fine-scale distribution of low frequency inversion breakpoints with those of high frequency inversions and inversions that have gone to fixation between Drosophila species. A number of differences were identified among frequency classes that may influence inversion fitness. In particular, breakpoints that are proximal to insulator elements, generate large tandem duplications, and minimize impacts on gene coding spans are more prevalent in high frequency and fixed inversions than in rare inversions. The data suggest that natural selection acts to preserve both genes and larger cis-regulatory networks in the occurrence and spread of rearrangements. These factors may act to limit the availability of high fitness arrangements when suppressed recombination is favorable (McBroome, 2020).

    A hidden markov model approach for simultaneously estimating local ancestry and admixture time using next generation sequence data in samples of arbitrary ploidy

    Admixture-the mixing of genomes from divergent populations-is increasingly appreciated as a central process in evolution. This study introduced a novel hidden Markov model for estimating local ancestry that models the read pileup data, rather than genotypes, is generalized to arbitrary ploidy, and can estimate the time since admixture during local ancestry inference. This method can simultaneously estimate the time since admixture and local ancestry with good accuracy, and it performs well on samples of high ploidy-i.e. 100 or more chromosomes. As this method is very general, it will be useful for local ancestry inference in a wider variety of populations than what previously has been possible. The method was applied to pooled sequencing data derived from populations of Drosophila melanogaster on an ancestry cline on the east coast of North America. Regions of local recombination rates were found to be negatively correlated with the proportion of African ancestry, suggesting that selection against foreign ancestry is the least efficient in low recombination regions. Finally it was shown that clinal outlier loci are enriched for genes associated with gene regulatory functions, consistent with a role of regulatory evolution in ecological adaptation of admixed D. melanogaster populations. These results illustrate the potential of local ancestry inference for elucidating fundamental evolutionary processes (Corbett-Detig, 2017).

    Sex and tissue-specific evolution of developmental plasticity in Drosophila melanogaster

    Developmental plasticity influences the size of adult tissues in insects. Tissues can have unique responses to environmental perturbation during development; however, the prevalence of within species evolution of tissue-specific developmental plasticity remains unclear. To address this, the effects of temperature and nutrition were studied on wing and femur size in D. melanogaster populations from a temperate and tropical region. Wings were more sensitive to temperature, while wings and femurs were equally responsive to nutrition in both populations and sexes. The temperate population was larger under all conditions, except for femurs of starved females. In line with this, greater femur size plasticity was observed in response to starvation in temperate females, leading to differences in sexual dimorphism between populations such that the slope of the reaction norm of sexual dimorphism in the tropical population was double that of the temperate population. Lastly, a significant trend was observed for steeper slopes of reaction norms in temperate than in tropical females, but not in males. These findings highlight that plasticity divergence between populations can evolve heterogeneously across sexes and tissues and that nutritional plasticity can alter sexual dimorphism in D. melanogaster (Sarikaya, 2021).

    Symbiont strain is the main determinant of variation in Wolbachia-mediated protection against viruses across Drosophila species

    Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host-symbiont associations in a range of Drosophila species, this study found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, the symbiont genome was found to have a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programs using Wolbachia to prevent mosquitoes from transmitting disease (Martinez, 2017).

    Inter- and intra-species variation in genome-wide gene expression of Drosophila in response to parasitoid wasp attack

    A study of inter- and intra-species variation in resistance to parasitoid attack used RNA-seq after parasitization in lines experimentally selected for increased resistance. A core set of genes was found that are consistently up-regulated after parasitoid attack. Another set showed no up-regulation or expression in D. sechellia, the species unable to raise an immune response against parasitoids. This set consists largely of genes that are lineage-restricted to the melanogaster subgroup. Artificially selected lines did not show significant differences in gene expression with respect to non-selected lines, but several genes showed differential exon usage. This study has shown substantial similarities, but also notable differences, in the transcriptional responses to parasitoid attack among four closely related Drosophila species. In contrast, within D. melanogaster, the responses were remarkably similar. It was confirmed that in the short-term, selection does not act on a pre-activation of the immune response. Instead it may target alternative mechanisms such as differential exon usage. In the long-term, support was found for the hypothesis that the ability to immunologically resist parasitoid attack is contingent on new genes that are restricted to the melanogaster subgroup (Salazar-Jaramillo, 2017).

    Host-pathogen coevolution increases genetic variation in susceptibility to infection

    It is common to find considerable genetic variation in susceptibility to infection in natural populations. This study has investigated whether natural selection increases this variation by testing whether host populations show more genetic variation in susceptibility to pathogens that they naturally encounter than novel pathogens. In a large cross-infection experiment involving four species of Drosophila and four host-specific viruses, greater genetic variation was always found in susceptibility to viruses that had coevolved with their host. The genetic architecture of resistance was examined in one host species, finding that there are more major-effect genetic variants in coevolved host-pathogen interactions. It is concluded that selection by pathogens has increased genetic variation in host susceptibility, and much of this effect is caused by the occurrence of major-effect resistance polymorphisms within populations (Duxbury, 2019).

    Geographic variation in the spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), based on mitochondrial DNA sequences

    The spotted-wing drosophila (SWD), Drosophila suzukii, is an economically damaging pest that was originally native to a few Asian countries, including Korea, but is now found in North America and Europe. Portions of the mitochondrial (mt) COI and ND4 genes were sequenced from a total of 195 individuals collected mainly from Korea. GenBank-registered COI sequences were combined with the COI data to assess the worldwide diversity, divergence, and relatedness of SWD haplotypes. A total of 139 haplotypes were obtained from the concatenated COI and ND4 sequences. Most haplotypes were confined to single localities, but 12 of them were found in more than two localities, and one haplotype (SWDCN61) was found from Korea to Canada. A dataset combining GenBank sequences with the current data identified a total of 94 worldwide COI haplotypes with a maximum sequence divergence (MSD) of 5.433% (32 bp). Although most haplotypes were found in only a single country, a few haplotypes were found commonly in China, Korea, and Japan; these occurred at a higher frequency and were often involved in introductions. A rough estimate of genetic diversity in each country showed higher diversity in ancestral distributional ranges, but the invasion over Asian countries seems to have been substantial because haplotype diversity was only 2.35 to 3.97-fold lower in the U.S.A, Canada, and Italy than that in the populations' ancestral ranges (Choi, 2017).

    Rapidly evolving Toll-3/4 genes encode male-specific Toll-like receptors in Drosophila

    Animal Toll-like receptors (TLRs) have evolved through a pattern of duplication and divergence. Whereas mammalian TLRs directly recognize microbial ligands, Drosophila Tolls bind endogenous ligands downstream of both developmental and immune signaling cascades. This study found that most Toll genes in Drosophila evolve slowly with little gene turnover (gains/losses), consistent with their important roles in development and indirect roles in microbial recognition. In contrast, the Toll-3/4 genes were found to have experienced an unusually rapid rate of gene gains and losses, resulting in lineage-specific Toll-3/4s and vastly different gene repertoires among Drosophila species, from zero copies (e.g., D. mojavensis) to nineteen copies (e.g., D. willistoni). In D. willistoni, strong evidence was found for positive selection in Toll-3/4 genes, localized specifically to an extracellular region predicted to overlap with the binding site of Spatzle, the only known ligand of insect Tolls. However, because Spatzle genes are not experiencing similar selective pressures, it was hypothesize that Toll-3/4s may be rapidly evolving because they bind to a different ligand, akin to TLRs outside of insects. Unlike other Toll genes in D. melanogaster, Toll-3 and Toll-4 have apparently escaped from essential developmental roles. It is proposed that the Toll-3/4 genes represent an exceptionally rapidly evolving lineage of Drosophila Toll genes, which play an unusual, as-yet-undiscovered role in the male germline (Levin, 2017).

    Many ways to make darker flies: Intra- and interspecific variation in Drosophila body pigmentation components

    Body pigmentation is an evolutionarily diversified and ecologically relevant trait with substantial variation within and between species, and important roles in animal survival and reproduction. Insect pigmentation, in particular, provides some of the most compelling examples of adaptive evolution, including its ecological significance and genetic bases. Pigmentation includes multiple aspects of color and color pattern that may vary more or less independently, and can be under different selective pressures. This study decomposed Drosophila thorax and abdominal pigmentation, a valuable eco-evo-devo model, into distinct measurable traits related to color and color pattern. Intra- and interspecific variation for those traits was investigated, and its different sources were assessed. For each body part, overall darkness was measured, as well as four other pigmentation properties distinguishing between background color and color of the darker pattern elements that decorate each body part. By focusing on two standard D. melanogaster laboratory populations, this study showed that pigmentation components vary and covary in distinct manners depending on sex, genetic background, and temperature during development. Studying three natural populations of D. melanogaster along a latitudinal cline and five other Drosophila species, it was shown that evolution of lighter or darker bodies can be achieved by changing distinct component traits. The results paint a much more complex picture of body pigmentation variation than previous studies could uncover, including patterns of sexual dimorphism, thermal plasticity, and interspecific diversity. These findings underscore the value of detailed quantitative phenotyping and analysis of different sources of variation for a better understanding of phenotypic variation and diversification, and the ecological pressures and genetic mechanisms underlying them (Lafuente, 2021).

    A fitness trade-off between seasons causes multigenerational cycles in phenotype and population size

    Although seasonality is widespread and can cause fluctuations in the intensity and direction of natural selection, there is little information about the consequences of seasonal fitness trade-offs for population dynamics. This study exposed populations of Drosophila melanogaster to repeated seasonal changes in resources across 58 generations and used experimental and mathematical approaches to investigate how viability selection on body size in the non-breeding season could affect demography. Opposing seasonal episodes of natural selection on body size were shown to interact with both direct and delayed density dependence to cause populations to undergo predictable multigenerational density cycles. These results provide evidence that seasonality can set the conditions for life-history trade-offs and density dependence, which can, in turn, interact to cause multigenerational population cycles (Betini, 2017).

    Manipulation of feeding regime alters sexual dimorphism for lifespan and reduces sexual conflict in Drosophila melanogaster

    Sexual dimorphism for lifespan (SDL) is widespread, but poorly understood. A leading hypothesis, which was tested in this study, is that strong SDL can reduce sexual conflict by allowing each sex to maximize its sex-specific fitness. Replicated experimental evolution lines of the fruit fly, Drosophila melanogaster, were used that had been maintained for over 360 generations on either unpredictable 'Random' or predictable 'Regular' feeding regimes. This evolutionary manipulation of feeding regime led to robust, enhanced SDL in Random over control, Regular lines. Enhanced SDL was associated with a significant increase in the fitness of focal males, tested with wild-type (WT) females. This was due to sex-specific changes to male life history, manifested as increased early reproductive output and reduced survival. In contrast, focal female fitness, tested with WT males, did not differ across regimes. Hence increased SDL was associated with a reduction in sexual conflict, which increased male fitness and maintained fitness in females. Differences in SDL were not associated with developmental time or developmental survival. Overall, the results showed that the expression of enhanced SDL, resulting from experimental evolution of feeding regimes, was associated with male-specific changes in life history, leading to increased fitness and reduced sexual conflict (Duxbury, 2017).

    Fear creates an Allee effect: experimental evidence from seasonal populations

    Allee effects, a decline in individual fitness at low population size or density, driven by predation can play a strong role in the decline of small populations but are conventionally thought to occur when generalist predators target specific prey (i.e. type II functional response). However, aside from direct consumption, fear of predators could also increase vigilance and reduce time spent foraging as population size decreases, as has been observed in wild mammals living in social groups. To investigate the role of fear on fitness in relation to population density in a species with limited sociality, varying densities of Drosophila melanogaster were exposed to mantid predators either during an experimental breeding season or non-breeding season. The presence of mantids in either season decreased the reproductive performance of individuals but only at low breeding densities, providing evidence for an Allee effect. The experimental results were used to parametrize a mathematical model to examine the population consequences of fear at low densities. Fear tended to destabilize population dynamics and increase the risk of extinction up to sevenfold. The study provides unique experimental evidence that the indirect effects of the presence of predators can cause an Allee effect and has important consequences for understanding of the dynamics of small populations (Elliott, 2017).

    Pleiotropic effects of DDT resistance on male size and behaviour

    Understanding the evolution and spread of insecticide resistance requires knowing the relative fitness of resistant organisms. In the absence of insecticides, resistance is predicted to be costly. The Drosophila melanogaster DDT resistance allele (DDT-R) is associated with a male mating cost. This could be because resistant males are generally smaller, but DDT-R may also alter courtship behaviours. This study tested for body size and courtship effects of DDT-R on mating success in competitive and non-competitive mating trials respectively. Relative aggression was assessed in resistant and susceptible males because aggression can also influence mating success. While the effect of DDT-R on male size partly contributed to reduced mating success, resistant males also had lower rates of courtship and were less aggressive than susceptible males. These differences contribute to the observed DDT-R mating costs. Additionally, these pleiotropic effects of DDT-R are consistent with the history and spread of resistance alleles in nature (Rostant, 2017).

    Perceptive costs of reproduction drive ageing and physiology in male Drosophila

    Costs of reproduction are thought to result from natural selection optimizing organismal fitness within putative physiological constraints. Phenotypic and population genetic studies of reproductive costs are plentiful across taxa, but an understanding of their mechanistic basis would provide important insight into the diversity in life-history traits, including reproductive effort and ageing. This study dissected the causes and consequences of specific costs of reproduction in male Drosophila melanogaster. Key survival and physiological costs of reproduction arise from perception of the opposite sex, and they are reversed by the act of mating. In the absence of pheromone perception, males are free from reproductive costs on longevity, stress resistance and fat storage. The costs of perception and the benefits of mating are both mediated by evolutionarily conserved neuropeptidergic signalling molecules, as well as the transcription factor dFoxo. These results provide a molecular framework in which certain costs of reproduction arise as a result of self-imposed 'decisions' in response to perceptive neural circuits, which then orchestrate the control of life-history traits independently of physical or energetic effects associated with mating itself (Harvanek, 2017).

    One prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster

    Wolbachia are maternally inherited, intracellular bacteria at the forefront of vector control efforts to curb arbovirus transmission. In international field trials, the cytoplasmic incompatibility (CI) drive system of wMel Wolbachia is deployed to replace target vector populations, whereby a Wolbachia-induced modification of the sperm genome kills embryos. However, Wolbachia in the embryo rescue the sperm genome impairment, and therefore CI results in a strong fitness advantage for infected females that transmit the bacteria to offspring. The two genes responsible for the wMel-induced sperm modification of CI, cifA and cifB, were recently identified in the eukaryotic association module of prophage WO, but the genetic basis of rescue is unresolved. This study used transgenic and cytological approaches to demonstrate that maternal cifA expression independently rescues CI and nullifies embryonic death caused by wMel Wolbachia in Drosophila melanogaster. Discovery of cifA as the rescue gene and previously one of two CI induction genes establishes a "Two-by-One" model that underpins the genetic basis of CI. Results highlight the central role of prophage WO in shaping Wolbachia phenotypes that are significant to arthropod evolution and vector control (Shropshire, 2018).

    Mitochondrial DNA fitness depends on nuclear genetic background in Drosophila

    Mitochondrial DNA (mtDNA) has been one of the most extensively studied molecules in ecological, evolutionary and clinical genetics. In its early application in evolutionary genetics, mtDNA was assumed to be a selectively neutral marker conferring negligible fitness consequences for its host. However, this dogma has been overturned in recent years due to now extensive evidence for non-neutral evolutionary dynamics. Since mtDNA proteins physically interact with nuclear proteins to provide the mitochondrial machinery for aerobic ATP production, among other cell functions, co-variation of the respective genes is predicted to affect organismal fitness. To test this hypothesis, an mtDNA-nuclear DNA introgression model was used in Drosophila melanogaster to test the fitness of genotypes in perturbation-reperturbation population cages and in a non-competitive assay for female fecundity. Genotypes consisted of both conspecific and heterospecific mtDNA-nDNA constructs, with either D. melanogaster or D. simulans mtDNAs on two alternative D. melanogaster nuclear backgrounds, to investigate mitonuclear genetic interactions (G x G effects). Considerable variation was found between nuclear genetic backgrounds on the selection of mtDNA haplotypes. In addition, there was variation in the selection on mtDNAs pre- and post- reperturbation, demonstrating overall poor repeatability of selection. There was a strong influence of nuclear background on non-competitive fecundity across all the mtDNA species types (Mossman, 2019).

    Differential impacts of yeasts on feeding behavior and development in larval Drosophila suzukii (Diptera:Drosophilidae)

    Larval Drosophila encounter and feed on a diverse microbial community within fruit. In particular, free-living yeast microbes provide a source of dietary protein critical for development. However, successional changes to the fruit microbial community may alter host quality through impacts on relative protein content or yeast community composition. For many species of Drosophila, fitness benefits from yeast feeding vary between individual yeast species, indicating differences in yeast nutritional quality. To better understand these associations, this study evaluated how five species of yeast impacted feeding preference and development in larval Drosophila suzukii. Larvae exhibited a strong attraction to the yeast Hanseniaspora uvarum in pairwise yeast feeding assays. However, larvae also performed most poorly on diets containing H. uvarum, a mismatch in preference and performance that suggests differences in yeast nutritional quality are not the primary factor driving larval feeding behavior. Together, these results demonstrate that yeast plays a critical role in D. suzukii's ecology and that larvae may have developed specific yeast associations. Further inquiry, including systematic comparisons of Drosophila larval yeast associations more broadly, will be necessary to understand patterns of microbial resource use in larvae of D. suzukii and other frugivorous species (Lewis, 2019).

    Fitness effects but no temperature-mediated balancing selection at the polymorphic Adh gene of Drosophila melanogaster

    Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to be maintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. This study use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. No evidence was found of a temperature-mediated trade-off: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. These findings tentatively suggest that environment-specific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation (Siddiq, 2019).

    Fitness consequences of the selfish supergene Segregation Distorter

    Segregation distorters are selfish genetic elements that subvert Mendelian inheritance, for example by destroying gametes that do not carry the distorter. Simple theoretical models predict that distorter alleles will either spread to fixation, or stabilise at some high intermediate frequency. However, many distorters have substantially lower allele frequencies than predicted by simple models, suggesting that key sources of selection remain to be discovered. This study measured the fitness of Drosophila melanogaster adults and juveniles carrying zero, one, or two copies of three different variants of the naturally-occurring supergene Segregation Distorter (SD), in order to investigate why SD alleles remain relatively rare within populations despite being preferentially inherited. First, it was shown that the three SD variants differ in the severity and dominance of the fitness costs they impose on individuals carrying them. Second, SD-carrying parents produced less fit offspring in some crosses, independent of of spring genotype, indicating that SD alleles can have non-genetic, transgenerational costs in addition to their direct costs. Third, it was found that SD carriers sometimes produce a biased offspring sex ratio, perhaps due to off-target effects of SD on the sex chromosomes (Wong, 2019).

    Mother's curse and indirect genetic effects: Do males matter to mitochondrial genome evolution?

    Maternal inheritance of mitochondrial DNA (mtDNA) was originally thought to prevent any response to selection on male phenotypic variation attributable to mtDNA, resulting in a male-biased mtDNA mutation load ("mother's curse"). However, the theory underpinning this claim implicitly assumes that a male's mtDNA has no effect on the fitness of females he comes into contact with. If such "mitochondrially encoded indirect genetics effects" (mtIGEs) do in fact exist, and there is relatedness between the mitochondrial genomes of interacting males and females, male mtDNA-encoded traits can undergo adaptation after all. This possibility was tested using strains of Drosophila melanogaster that differ in their mtDNA. The experiments indicate that female fitness is influenced by the mtDNA carried by males that the females encounter, which could plausibly allow the mitochondrial genome to evolve via kin selection. It is argued that mtIGEs are probably common, and that this might ameliorate or exacerbate mother's curse (Keaney, 2019).

    Distinct nutritional and endocrine regulation of prothoracic gland activities underlies divergent life history strategies in Manduca sexta and Drosophila melanogaster

    Life history trade-offs lead to various strategies that maximize fitness, but the developmental mechanisms underlying these alternative strategies continue to be poorly understood. In insects, trade-offs exist between size and developmental time. Recent studies in the fruit fly Drosophila melanogaster have suggested that the steroidogenic prothoracic glands play a key role in determining the timing of metamorphosis. In this study, the nutrient-dependent growth and transcriptional activation of prothoracic glands were studied in D. melanogaster and the tobacco hornworm Manduca sexta. In both species, minimum viable weight (MVW) was associated with activation of ecdysteroid biosynthesis genes and growth of prothoracic gland cells. However, the timing of MVW attainment in M. sexta is delayed by the presence of the sesquiterpenoid hormone, juvenile hormone (JH), whereas in D. melanogaster it is not. Moreover, in D. melanogaster, the transcriptional regulation of ecdysteroidogenesis becomes nutrient-independent at the MVW/critical weight (CW) checkpoint. In contrast, in M. sexta, starvation consistently reduced transcriptional activation of ecdysteroid biosynthesis genes even after CW attainment, indicating that the nature of CW differs fundamentally between the two species. In D. melanogaster, the prothoracic glands dictate the timing of metamorphosis even in the absence of nutritional inputs, whereas in M. sexta, prothoracic gland activity is tightly coupled to the nutritional status of the body, thereby delaying the onset of metamorphosis before CW attainment. It is proposed that selection for survival under unpredictable nutritional availability leads to the evolution of increased modularity in both morphological and endocrine traits (Xu, 2020).

    Insect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, it was reasoned that the cost of insecticide resistance could be modulated by ambient temperature. Experimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = - 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = - 0.031). This unexpected observation is attributed to the advantage of the reduced enzymatic activity of resistance mutations in hot environments. This study shows that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. It is suggested that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change (Langmuller, 2020).

    Repeated duplication of Argonaute2 is associated with strong selection and testis specialization in Drosophila

    Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. This study reconstruct the history of Ago2 duplications across the Drosophila obscura group, and patterns of gene expression were used to infer new functional specialization. Some duplications were shown to be old, shared by the entire species group, and losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura. While the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogues have independently specialized to testis-specific expression. Using population genetic analyses, it was shown that most testis-specific paralogues have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogues. It is speculated that the repeated evolution of testis-specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. This study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in non-model species (Lewis, 2016).

    Insights into DDT Resistance from the Drosophila melanogaster Genetic Reference Panel

    Insecticide resistance is considered a classic model of microevolution, where a strong selective agent is applied to a large natural population, resulting in a change in frequency of alleles that confer resistance. While many insecticide resistance variants have been characterized at the gene level, they are typically single genes of large effect identified in highly resistant pest species. In contrast, multiple variants have been implicated in DDT resistance in Drosophila melanogaster, however only the Cyp6g1 locus has previously been shown to be relevant to field populations. This study used genome-wide association studies to identify DDT-associated polygenes and used selective sweep analyses to assess their adaptive significance. Two candidate DDT resistance loci were identified and verified. A largely uncharacterized gene, CG10737, has a function in muscles that ameliorates the effects of DDT, while a putative detoxifying P450, Cyp6w1, shows compelling evidence of positive selection (Schmidt, 2017).

    Structural variants and selective sweep foci contribute to insecticide resistance in the Drosophila genetic reference panel

    Patterns of nucleotide polymorphism within populations of Drosophila melanogaster suggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweeps to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. This study screened the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome-wide association studies map survival on malathion to the two of the largest sweeps in the D. melanogaster genome; Ace and Cyp6g1. Malathion survivorship also correlates with lines which have high levels of Cyp12d1, Jheh1 and Jheh2 transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived allele seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes: Cyp6g1 exhibits copy number variation and transposable element insertions, Cyp12d1 is tandemly duplicated, the Jheh loci are associated with a Bari1 transposable element insertion, and a Cyp6a17 deletion is associated with susceptibility (Battlay, 2018).

    Directional selection reduces developmental canalization against genetic and environmental perturbations in Drosophila wings

    Natural selection may enhance or weaken the robustness of phenotypes against genetic or environmental perturbations. However, important aspects of the relationship between adaptive evolution and canalization remain unclear. Recent work showed that the evolution of larger wing size in a high altitude natural population of Drosophila melanogaster was accompanied by decanalized wing development--specifically a loss of robustness to genetic perturbation. But that study did not address environmental robustness, and it compared populations that may have numerous biological differences. This study performed artificial selection on this same trait in D. melanogaster (larger wing length) and directly test whether this directional selection resulted in decanalization. In general, size-selected replicates show greater frequencies of wing defects than control replicates both after mutagenesis (genetic perturbation) and when subjected to high temperature stress (environmental perturbation), although the increase in defect frequency varies importantly among replicates. These results support the hypothesis that directional selection may result in the loss of both genetic and environmental robustness-offering a rare window into the relationship between adaptation and canalization (Groth, 2018).

    Genetic trade-offs between male reproductive traits in Drosophila melanogaster

    In Drosophila melanogaster, males engage in both extensive pre- and post-copulatory competition for the opportunity to mate with females and subsequently sire offspring. The selection pressure for increased male reproductive success has resulted in the evolution of a wide diversity of sexual traits. However, despite strong selection, individuals often exhibit considerable phenotypic variation in the expression of these traits, and it is unclear if any of this variation is owing to underlying genetic trade-offs. Using hemiclonal flies this study examined how male reproductive success covaries with their ability to induce long-term stimulation of oogenesis and oviposition in their mates, and how this relationship may change over time. It was found that males from hemiclone lines with phenotypes that were more successful in a short-term reproductive 'scramble' environment were less effective at stimulating long-term fecundity in females. Furthermore, it was observed that males from hemiclone lines which showed the most improvement over a longer reproductive interaction period also tended to stimulate higher long-term fecundity in females. Together, these results indicate the presence of genetic trade-offs between different male reproductive traits and offer insights into the maintenance of their variation (Filice, 2018).

    Genomic analysis of European Drosophila melanogaster populations reveals longitudinal structure, continent-wide selection, and previously unknown DNA viruses

    Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatio-temporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. This study addresses these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and the first continent-wide genomic analysis of genetic variation was performed in European Drosophila melanogaster. These analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. Variation among populations in the composition of the fly microbiome was characterized, and five new DNA viruses were identified in these samples (Kapun, 2020).

    Natural selection on sleep duration in Drosophila melanogaster

    Sleep is ubiquitous across animal species, but why it persists is not well understood. This study observed natural selection act on Drosophila sleep by relaxing bi-directional artificial selection for extreme sleep duration for 62 generations. When artificial selection was suspended, sleep increased in populations previously selected for short sleep. Likewise, sleep decreased in populations previously selected for long sleep when artificial selection was relaxed. The corresponding changes were measured in the allele frequencies of genomic variants responding to artificial selection. The allele frequencies of these variants reversed course in response to relaxed selection, and for short sleepers, the changes exceeded allele frequency changes that would be expected under random genetic drift. These observations suggest that the variants are causal polymorphisms for sleep duration responding to natural selection pressure. These polymorphisms may therefore pinpoint the most important regions of the genome maintaining variation in sleep duration (Souto-Maior, 2020).

    The genetic architecture of temperature adaptation is shaped by population ancestry and not by selection regime

    Understanding the genetic architecture of temperature adaptation is key for characterizing and predicting the effect of climate change on natural populations. One particularly promising approach is Evolve and Resequence, which combines advantages of experimental evolution such as time series, replicate populations, and controlled environmental conditions, with whole genome sequencing. Recent analysis of replicate populations from two different Drosophila simulans founder populations, which were adapting to the same novel hot environment, uncovered very different architectures-either many selection targets with large heterogeneity among replicates or fewer selection targets with a consistent response among replicates. This study exposed the founder population from Portugal to a cold temperature regime. Although almost no selection targets are shared between the hot and cold selection regime, the adaptive architecture was similar. A moderate number was detected of targets under strong selection (19 selection targets, mean selection coefficient = 0.072) and parallel responses in the cold evolved replicates. This similarity across different environments indicates that the adaptive architecture depends more on the ancestry of the founder population than the specific selection regime. These observations will have broad implications for the correct interpretation of the genomic responses to a changing climate in natural populations (Otte, 2021).

    X chromosome drive in a widespread Palearctic woodland fly, Drosophila testacea

    Selfish genes that bias their own transmission during meiosis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes have important evolutionary and ecological consequences, and can be found in a broad range of taxa including plants, mammals, and insects. This study reports a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. Males carrying the driving X (SR males) sire 80-100% female offspring, and possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. The majority of sons produced by SR males are sterile and appear to lack a Y chromosome, suggesting that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. By screening wild-caught flies using progeny sex ratios and a diagnostic marker, it was demonstrated that the driving X is present in wild populations at a frequency of ~10% and that suppressors of drive are segregating in the same population. The testacea species group appears to be a hotspot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves (Keais, 2017).

    A Pooled Sequencing Approach Identifies a Candidate Meiotic Driver in Drosophila

    Meiotic drive occurs when a selfish element increases its transmission frequency above the Mendelian ratio by hijacking the asymmetric divisions of female meiosis. New methods to reliably detect meiotic drive are therefore needed, particularly for discovering moderate-strength drivers that are likely to be more prevalent in natural populations than strong drivers. This study reports an efficient method that uses sequencing of large pools of backcross (BC1) progeny to test for deviations from Mendelian segregation genome-wide of single-nucleotide polymorphisms (SNPs) that distinguish the parental strains. Meiotic drive can be detected by a characteristic pattern of decay in distortion of SNP frequencies, caused by recombination unlinking the driver from distal loci. Control crosses allow allele-frequency distortion caused by meiotic drive to be distinguished from distortion resulting from developmental effects. This approach was used to test whether chromosomes with extreme telomere-length differences segregate at Mendelian ratios, as telomeric regions are a potential hotspot for meiotic drive due to their roles in meiotic segregation and multiple observations of high rates of telomere sequence evolution. Using four different pairings of long and short telomere strains, this study found no evidence that extreme telomere-length variation causes meiotic drive in Drosophila. However, one candidate meiotic driver was identified in a centromere-linked region that shows an ~8% increase in transmission frequency, corresponding to a ~54:46 segregation ratio. These results show that candidate meiotic drivers of moderate strength can be readily detected and localized in pools of F1 progeny (Wei, 2017).

    Efficient allelic-drive in Drosophila

    Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. This study generalizes this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. Two configurations for allelic-drive were tested: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. A phenomenon referred to as lethal-mosaicism was investigated that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process is referred to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox (Guichard, 2019).

    Sex-Ratio meiotic drive shapes the evolution of the Y chromosome in Drosophila simulans

    The recent emergence and spread of X-linked segregation distorters - called "Paris" system - in the worldwide species Drosophila simulans has elicited the selection of drive-resistant Y chromosomes. This study investigated the evolutionary history of 386 Y chromosomes originating from 29 population samples collected over a period of twenty years, showing a wide continuum of phenotypes when tested against the Paris distorters, from high sensitivity to complete resistance (males sire approximately 95% to approximately 40% female progeny). Analyzing around 13 kb of Y-linked gene sequences in a representative subset of nine Y chromosomes, only three polymorphic sites resulting in three haplotypes were found. Remarkably, one of the haplotypes is associated with resistance. This haplotype is fixed in all samples from Sub-Saharan Africa, the region of origin of the drivers. Exceptionally, with the spread of the drivers in Egypt and Morocco, it was possible to record the replacement of the sensitive lineage by the resistant haplotype in real time, within only a few years. In addition, in situ hybridization, using satellite DNA probes, was performed on a subset of 21 Y chromosomes from six locations. In contrast to the low molecular polymorphism, this revealed extensive structural variation suggestive of rapid evolution, either neutral or adaptive. Moreover, the results show that intragenomic conflicts can drive astonishingly rapid replacement of Y chromosomes and suggest that the emergence of Paris segregation distorters in East Africa occurred less than half a century ago (Helleu, 2019).

    Chromosomal rearrangements as a source of new gene formation in Drosophila yakuba

    The origins of new genes are among the most fundamental questions in evolutionary biology. Understanding of the ways that new genetic material appears and how that genetic material shapes population variation remains incomplete. De novo genes and duplicate genes are a key source of new genetic material on which selection acts. To better understand the origins of these new gene sequences, this study explored the ways that structural variation might alter expression patterns and form novel transcripts. Evidence is provided that chromosomal rearrangements are a source of novel genetic variation that facilitates the formation of de novo exons in Drosophila. 51 cases were found of de novo exon formation created by chromosomal rearrangements in 14 strains of D. yakuba. These new genes inherit transcription start signals and open reading frames when the 5' end of existing genes are combined with previously untranscribed regions. Such new genes would appear with novel peptide sequences, without the necessity for secondary transitions from non-coding RNA to protein. This mechanism of new peptide formations contrasts with canonical theory of de novo gene progression requiring non-coding intermediaries that must acquire new mutations prior to loss via pseudogenization. Hence, these mutations offer a means to de novo gene creation and protein sequence formation in a single mutational step, answering a long standing open question concerning new gene formation. Gene expression changes were identified for 134 existing genes, indicating that these mutations can alter gene regulation. Population variability for chromosomal rearrangements is considerable, with 2368 rearrangements observed across 14 inbred lines. More rearrangements were identified on the X chromosome than any of the autosomes, suggesting the X is more susceptible to chromosome alterations. Together, these results suggest that chromosomal rearrangements are a source of variation in populations that is likely to be important to explain genetic and therefore phenotypic diversity (Stewart, 2019).

    Fitness consequences of a non-recombining sex-ratio drive chromosome can explain its prevalence in the wild

    Understanding the pleiotropic consequences of gene drive systems on host fitness is essential to predict their spread through a host population. This paper reports a study sex-ratio (SR) X-chromosome drive in the fly Drosophila recens, where SR causes the death of Y-bearing sperm in male carriers. SR males only sire daughters, which all carry SR, thus giving the chromosome a transmission advantage. The prevalence of the SR chromosome appears stable, suggesting pleiotropic costs. It was previously shown that females homozygous for SR are sterile, and this study tests for additional fitness costs of SR. Females heterozygous for SR were found to have reduced fecundity, and male SR carriers were found to have reduced fertility in conditions of sperm competition. Fitness estimates were used to parametrize theoretical models of SR drive, and sthe decrease in fecundity and sperm competition performance were shown to account for the observed prevalence of SR in natural populations. In addition, it was found that the expected equilibrium frequency of the SR chromosome is particularly sensitive to the degree of multiple mating and performance in sperm competition. Together, these data suggest that the mating system of the organism should be carefully considered during the development of gene drive systems (Dyer, 2019).

    A toxin-antidote CRISPR gene drive system for regional population modification

    Engineered gene drives based on a homing mechanism could rapidly spread genetic alterations through a population. However, such drives face a major obstacle in the form of resistance against the drive. In addition, they are expected to be highly invasive. This study introduce the Toxin-Antidote Recessive Embryo (TARE) drive. It functions by disrupting a target gene, forming recessive lethal alleles, while rescuing drive-carrying individuals with a recoded version of the target. Modeling shows that such drives will have threshold-dependent invasion dynamics, spreading only when introduced above a fitness-dependent frequency. A TARE drive is demonstrated in Drosophila with 88-95% transmission by female heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. These results suggest that TARE drives constitute promising candidates for the development of effective, flexible, and regionally confinable drives for population modification (Champer, 2020).

    A Protamine Knockdown Mimics the Function of Sd in Drosophila melanogaster

    Segregation Distorter (SD) is an autosomal meiotic drive system found worldwide in natural populations of Drosophila melanogaster This gene complex induces the preferential and nearly exclusive transmission of the SD chromosome in SD/SD(+) males. This selfish propagation occurs through the interplay of the Sd locus, its enhancers and the Rsp(s) locus during spermatid development. The key distorter locus, Sd, encodes a truncated but enzymatically active RanGAP (RanGTPase-activating protein), a key nuclear transport factor in the Ran signaling pathway. When encoded by Sd, RanGAP is mislocalized to the nucleus interior, which then traps Ran inside the nucleus and disrupts nuclear import. As a result of this aberrant nuclear transport, a process known as the histone-to-protamine transition that is required for proper spermatid condensation fails to occur in SD/SD (+) males. In this process, sperm-specific protamine proteins enter the spermatid nucleus and replace the formerly chromatin-complexed histones. Previous work has shown that mutations affecting nuclear import and export can enhance distortion in an SD background, thus verifying that a defect in nuclear transport is responsible for the unequal transmission of chromosomes. This study shows that specifically reducing protamines induces distortion in an SD background, verifying that protamines are transported via the RanGAP/GEF pathway and indicating that E(SD) plays a significant and unique role in the process of distortion (Gingell, 2020).

    Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements

    Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. This study shows that resilience can be achieved through cycles of population modification with "Cleave and Rescue" (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvR (n+1), carries a Rescue transgene from an earlier element, ClvR (n) ClvR (n+1) should spread within a population of ClvR (n), while also bringing about a decrease in its frequency. To test this hypothesis, it was first shown that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. It was then shown that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control (Oberhofer, 2020).

    Extensive Recombination Suppression and Epistatic Selection Causes Chromosome-Wide Differentiation of a Selfish Sex Chromosome in Drosophila pseudoobscura

    Sex-Ratio (SR) chromosomes are selfish X-chromosomes that distort Mendelian segregation and are commonly associated with inversions. These chromosomal rearrangements suppress recombination with Standard (ST) X-chromosomes and are hypothesized to maintain multiple alleles important for drive in a single large haplotype. A multifaceted study was conducted of the multiply inverted Drosophila pseudoobscura SR chromosome to understand the evolutionary history, genetic architecture, and present-day dynamics that shape this enigmatic selfish chromosome. The D. pseudoobscura SRchromosome has three non-overlapping inversions: basal, medial, and terminal. 23 of 29 Mb of the D. pseudoobscura XR chromosome arm is highly differentiated between the Standard (ST) and SR arrangements, including a 6.6Mb collinear region between the medial and terminal inversions. Although crossing-over is heavily suppressed on this chromosome arm, it is not completely eliminated, with measured rates indicating recombination suppression alone cannot explain patterns of differentiation or the near-perfect association of the three SR chromosome inversions in nature. The ancient basal and medial inversions of the SR chromosome were demonstrated contain genes sufficient to cause weak drive. In contrast, the younger terminal inversion cannot drive by itself, but contains at least one modifier gene necessary for full manifestation of strong sex chromosome drive. By parameterizing population genetic models for chromosome-wide linkage disequilibrium with the experimental results, it is inferred that strong selection acts to maintain the near-perfect association of SR chromosome inversions in present day populations. It is concluded the combined action of suppressed recombination and strong, ongoing, epistatic selection shape the D. pseudoobscura SR arrangement into a highly differentiated chromosome (Fuller, 2020).

    TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants
    A major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as 'Peto's Paradox'. This study showed that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 (see Drosophila p53) and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore several of the TP53 retrogenes (TP53RTGs) were shown to be transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto's paradox in Proboscideans (Sulak, 2016).

    Optimal scaling of critical size for metamorphosis in the genus Drosophila

    Juveniles must reach a critical body size to become a mature adult. Molecular determinants of critical size have been studied, but the evolutionary importance of critical size is still unclear. Using nine fly species, this study showed that interspecific variation in organism size can be explained solely by species-specific critical size. The observed variation in critical size quantitatively agrees with the interspecific scaling relationship predicted by the life history model, which hypothesizes that critical size mediates an energy allocation switch between juvenile and adult tissues. The mechanism underlying critical size scaling is explained by an inverse relationship between growth duration and growth rate, which cancels out their contributions to the final size. Finally, evolutionary changes in growth duration can be traced back to the scaling of ecdysteroid hormone dynamics. It is concluded that critical size adaptively optimizes energy allocation, and has a central role in organism size determination (Hironaka, 2019).

    Sex-dependent and sex-independent regulatory systems of size variation in natural populations

    Size of organs/organisms is a polygenic trait. Many of the growth-regulatory genes constitute conserved growth signaling pathways. However, how these multiple genes are orchestrated at the systems level to attain the natural variation in size including sexual size dimorphism is mostly unknown. This study has taken a multi-layered systems omics approach to study size variation in the Drosophila wing. Expression levels of many critical growth regulators such as Wnt and TGFbeta pathway components were shown to significantly differ between sexes but not between lines exhibiting size differences within each sex, suggesting a primary role of these regulators in sexual size dimorphism. Only a few growth genes including a receptor of steroid hormone ecdysone exhibit association with between-line size differences. In contrast, between-line size variation was found to be largely regulated by genes with a diverse range of cellular functions, most of which have never been implicated in growth. In addition, it was shown that expression quantitative trait loci (eQTLs) linked to these novel growth regulators accurately predict population-wide, between-line wing size variation. In summary, this study unveils differential gene regulatory systems that control wing size variation between and within sexes (Okada, 2019).

    Gene duplications circumvent trade-offs in enzyme function: Insect adaptation to toxic host plants

    Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase alpha-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster was expressed in vitro and four distinct combinations were introduced of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. These results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier (Dalla, 2016).

    Rapid functional and sequence differentiation of a tandemly-repeated species-specific multigene family in Drosophila

    Gene clusters of recently duplicated genes are hotbeds for evolutionary change. However, understanding of how mutational mechanisms and evolutionary forces shape the structural and functional evolution of these clusters is hindered by the high sequence identity among the copies, which typically results in their inaccurate representation in genome assemblies. The presumed testis-specific, chimeric gene Sdic originated and tandemly expanded in Drosophila melanogaster, contributing to increased male-male xion. Using various types of massively parallel sequencing data, the organization, sequence evolution, and functional attributes of the different Sdic copies were examined. By leveraging long-read sequencing data, both copy number and order differences were uncovered from the currently accepted annotation for the Sdic region. Despite evidence for pervasive gene conversion affecting the Sdic copies, signatures of two episodes of diversifying selection were uncovered, that have contributed to the evolution of a variety of C-termini and miRNA binding site compositions. Expression analyses involving RNA-seq datasets from 59 different biological conditions revealed distinctive expression breadths among the copies, with three copies being transcribed in females, opening the possibility to a sexually antagonistic effect. Phenotypic assays using Sdic knock-out strains indicated that should this antagonistic effect exist, it does not compromise female fertility. These results strongly suggest that the genome consolidation of the Sdic gene cluster is more the result of a quick exploration of different paths of molecular tinkering by different copies than a mere dosage increase, which could be a recurrent evolutionary outcome in the presence of persistent sexual selection (Clifton, 2016).

    Transcriptional interference promotes rapid expression divergence of Drosophila nested genes

    Nested genes are the most common form of protein-coding overlap in eukaryotic genomes. Previous studies have shown that nested genes accumulate rapidly over evolutionary time, typically via the insertion of short young duplicate genes into long introns. However, the evolutionary relationship between nested genes remains unclear. This study compare RNA-seq expression profiles of nested, proximal intra-chromosomal, intermediate intra-chromosomal, distant intra-chromosomal, and inter-chromosomal gene pairs in two Drosophila species. Expression profiles of nested genes were found to be more divergent than those of any other class of genes, supporting the hypothesis that concurrent expression of nested genes is deleterious due to transcriptional interference. Further analysis reveals that expression profiles of derived nested genes are more divergent than those of their ancestral un-nested orthologs, which are more divergent than those of un-nested genes with similar genomic features. Thus, gene expression divergence between nested genes is likely caused by selection against nesting of genes with insufficiently divergent expression profiles, as well as by continued expression divergence after nesting. Moreover, expression divergence and sequence evolutionary rates are elevated in young nested genes and reduced in old nested genes, indicating that a burst of rapid evolution occurs after nesting. Together, these findings suggest that similarity between expression profiles of nested genes is deleterious due to transcriptional interference, and that natural selection addresses this problem both by eradicating highly deleterious nestings and by enabling rapid expression divergence of surviving nested genes, thereby quickly limiting or abolishing transcriptional interference (Assis, 2016).

    The goddard and saturn genes are essential for Drosophila male fertility and may have arisen de novo

    New genes arise through a variety of mechanisms, including the duplication of existing genes and the de novo birth of genes from non-coding DNA sequences. While there are numerous examples of duplicated genes with important functional roles, the functions of de novo genes remain largely unexplored. Many newly evolved genes are expressed in the male reproductive tract, suggesting that these evolutionary innovations may provide advantages to males experiencing sexual selection. Using testis-specific RNA interference, 11 putative de novo genes in Drosophila melanogaster for effects on male fertility, and two, goddard and saturn, were identified that are essential for spermatogenesis and sperm function. goddard knockdown males fail to produce mature sperm, while saturn knockdown males produce fewer sperm that function inefficiently once transferred to females. Consistent with a de novo origin, both genes are identifiable only in Drosophila and are predicted to encode proteins with no sequence similarity to any annotated protein. However, since high levels of divergence prevented the unambiguous identification of the non-coding sequences from which each gene arose, saturn and saturn are considered to be putative de novo genes. Within Drosophila, both genes have been lost in certain lineages, but show conserved, male-specific patterns of expression in the species in which they are found. Goddard is consistently found in single-copy and evolves under purifying selection. In contrast, saturn has diversified through gene duplication and positive selection. These data suggest that de novo genes can evolve essential roles in male reproduction (Gubala, 2017).

    Recurrent gene duplication leads to diverse repertoires of centromeric histones in Drosophila species

    Despite their essential role in the process of chromosome segregation in most eukaryotes, centromeric histones show remarkable evolutionary lability. Not only have they been lost in multiple insect lineages, but they have also undergone gene duplication in multiple plant lineages. Based on detailed study of a handful of model organisms including Drosophila melanogaster, centromeric histone duplication is considered to be rare in animals. Using a detailed phylogenomic study, this study found that Cid, the centromeric histone gene, has undergone at least four independent gene duplications during Drosophila evolution. Duplicate Cid genes were found in D. eugracilis (Cid2), in the montium species subgroup (Cid3, Cid4) and in the entire Drosophila subgenus (Cid5). Cid3, Cid4, Cid5 all localize to centromeres in their respective species. Some Cid duplicates are primarily expressed in the male germline. With rare exceptions, Cid duplicates have been strictly retained after birth, suggesting that they perform non-redundant centromeric functions, independent from the ancestral Cid. Indeed, each duplicate encodes a distinct N-terminal tail, which may provide the basis for distinct protein-protein interactions. Finally, it was shown some Cid duplicates evolve under positive selection whereas others do not. Taken together, these results support the hypothesis that Drosophila Cid duplicates have subfunctionalized. Thus, these gene duplications provide an unprecedented opportunity to dissect the multiple roles of centromeric histones (Kursel, 2017).

    Tandem duplications lead to novel expression patterns through exon shuffling in Drosophila yakuba

    One common hypothesis to explain the impacts of tandem duplications is that whole gene duplications commonly produce additive changes in gene expression due to copy number changes. This study used genome wide RNA-seq data from a population sample of Drosophila yakuba to test this 'gene dosage' hypothesis. Little evidence was observed of expression changes in response to whole transcript duplication capturing 5' and 3' UTRs. Among whole gene duplications, evidence was observed that dosage sharing across copies is likely to be common. The lack of expression changes after whole gene duplication suggests that the majority of genes are subject to tight regulatory control and therefore not sensitive to changes in gene copy number. Rather, changes were observed in expression level due to both shuffling of regulatory elements and the creation of chimeric structures via tandem duplication. Additionally, 30 de novo gene structures were observed arising from tandem duplications, 23 of which form with expression in the testes. Thus, the value of tandem duplications is likely to be more intricate than simple changes in gene dosage. The common regulatory effects from chimeric gene formation after tandem duplication may explain their contribution to genome evolution (Rogers, 2017).

    Diverse cis-regulatory mechanisms contribute to expression evolution of tandem gene duplicates

    Pairs of duplicated genes generally display a combination of conserved expression patterns inherited from their unduplicated ancestor and newly acquired domains. However, how the cis-regulatory architecture of duplicated loci evolves to produce these expression patterns is poorly understood. This study directly examined the gene-regulatory evolution of two tandem duplicates, the Drosophila Ly6 genes CG9336 and CG9338, which arose at the base of the drosophilids between 40 and 60 million years ago. Comparing the expression patterns of the two paralogs in four Drosophila species with that of the unduplicated ortholog in the tephritid Ceratitis capitata, they were shown to diverge from each other as well as from the unduplicated ortholog. Moreover, the expression divergence appears to have occurred close to the duplication event and also more recently in a lineage-specific manner. The comparison of the tissue-specific cis-regulatory modules (CRMs) controlling the paralog expression in the four Drosophila species indicates that diverse cis-regulatory mechanisms, including the novel tissue-specific enhancers, differential inactivation, and enhancer sharing, contributed to the expression evolution. This analysis also reveals a surprisingly variable cis-regulatory architecture, in which the CRMs driving conserved expression domains change in number, location, and specificity. Altogether, this study provides a detailed historical account that uncovers a highly dynamic picture of how the paralog expression patterns and their underlying cis-regulatory landscape evolve. It is argued that these findings will encourage studying cis-regulatory evolution at the whole-locus level in order to understand how interactions between enhancers and other regulatory levels shape the evolution of gene expression (Baudouin-Gonzalez, 2017).

    Adaptation of gene loci to heterochromatin in the course of Drosophila evolution is associated with insulator proteins

    Pericentromeric heterochromatin is generally composed of repetitive DNA forming a transcriptionally repressive environment. Dozens of genes were embedded into pericentromeric heterochromatin during evolution of Drosophilidae lineage while retaining activity. However, factors that contribute to insusceptibility of gene loci to transcriptional silencing remain unknown. This study finds that the promoter region of genes that can be embedded in both euchromatin and heterochromatin exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of certain chromatin remodeling factors, including insulator proteins. Using ChIP-seq data, this study demonstrates that evolutionary gene relocation between euchromatin and pericentric heterochromatin occurred with preservation of sites of insulation of BEAF-32 in evolutionarily distant species, i.e. D. melanogaster and D. virilis. Moreover, promoters of virtually all protein-coding genes located in heterochromatin in D. melanogaster are enriched with insulator proteins BEAF-32, GAF and dCTCF. Applying RNA-seq of a BEAF-32 mutant, this study shows that the impairment of BEAF-32 function has a complex effect on gene expression in D. melanogaster, affecting even those genes that lack BEAF-32 association in their promoters. It is proposed that conserved intrinsic properties of genes, such as sites of insulation near the promoter regions, may contribute to adaptation of genes to the heterochromatic environment and, hence, facilitate the evolutionary relocation of genes loci between euchromatin and heterochromatin (Funikov, 2020).

    Evolved Differences in cis and trans Regulation Between the Maternal and Zygotic mRNA Complements in the Drosophila Embryo

    How gene expression can evolve depends on the mechanisms driving gene expression. Gene expression is controlled in different ways in different developmental stages; this study asked whether different developmental stages show different patterns of regulatory evolution. To explore the mode of regulatory evolution, this study used the early stages of embryonic development controlled by two different genomes, that of the mother and that of the zygote. During embryogenesis in all animals, initial developmental processes are driven entirely by maternally provided gene products deposited into the oocyte. The zygotic genome is activated later, when developmental control is handed off from maternal gene products to the zygote during the maternal-to-zygotic transition. Using hybrid crosses between sister species of Drosophila (D. simulans, D. sechellia, and D. mauritiana) and transcriptomics, this study finds that the regulation of maternal transcript deposition and zygotic transcription evolve through different mechanisms. Patterns of transcript level inheritance in hybrids, relative to parental species, were found to differ between maternal and zygotic transcripts, and maternal transcript levels are more likely to be conserved. Changes in transcript levels occur predominantly through differences in trans regulation for maternal genes, while changes in zygotic transcription occur through a combination of both cis and trans regulatory changes. Differences in the underlying regulatory landscape in the mother and the zygote are likely the primary determinants for how maternal and zygotic transcripts evolve (Cartwright, 2020).

    Widespread cis- and trans-regulatory evolution underlies the origin, diversification, and loss of a sexually dimorphic fruit fly pigmentation trait

    Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait-building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis-regulatory elements (CREs) that drive their expression (cis-evolution) or within genes for CRE-interacting transcription factors (trans-evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. This study investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. These CREs were shown to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male-specific pigmentation. This study evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. The transgene host frequently determines CRE activity, implicating trans-evolution as a significant factor for this trait's diversity. The gain of dimorphic Bab transcription factor expression as a trans-change contributing to the dimorphic trait was evaluated. The findings suggest an amenability to change for the landscape of trans-regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs (Hughes, 2021).

    Inter-embryo gene expression variability recapitulates the hourglass pattern of evo-devo

    The evolution of embryological development has long been characterized by deep conservation. In animal development, the phylotypic stage in mid-embryogenesis is more conserved than either early or late stages among species within the same phylum. Hypotheses to explain this hourglass pattern have focused on purifying the selection of gene regulation. This paper proposes an alternative-genes are regulated in different ways at different stages and have different intrinsic capacities to respond to perturbations on gene expression. To eliminate the influence of natural selection, the expression variability of isogenetic single embryo transcriptomes was quantified throughout fly Drosophila melanogaster embryogenesis. The expression variability is lower at the phylotypic stage, supporting that the underlying regulatory architecture in this stage is more robust to stochastic variation on gene expression. Evidence is presented that the phylotypic stage is also robust to genetic variations on gene expression. Moreover, chromatin regulation appears to play a key role in the variation and evolution of gene expression. It is suggested that a phylum-level pattern of embryonic conservation can be explained by the intrinsic difference of gene regulatory mechanisms in different stages (Liu, 2020).

    Hybrid Incompatibilities and Transgressive Gene Expression Between Two Closely Related Subspecies of Drosophila

    Genome-wide assays of expression between species and their hybrids have identified genes that become either over- or underexpressed relative to the parental species (i.e., transgressive). Transgressive expression in hybrids is of interest because it highlights possible changes in gene regulation linked to hybrid dysfunction. Previous studies in Drosophila that used long-diverged species pairs with complete or nearly complete isolation (i.e., full sterility and partial inviability of hybrids) and high-levels of genome misregulation have found correlations between expression and coding sequence divergence. The work highlighted the possible effects of directional selection driving sequence divergence and transgressive expression. Whether the same is true for taxa at early stages of divergence that have only achieved partial isolation remains untested. This study reanalyzed previously published genome expression data and available genome sequence reads from a pair of partially isolated subspecies of Drosophila to compare expression and sequence divergence. A significant correlation was found in rates of expression and sequence evolution, but no support for directional selection driving transgressive expression in hybrids. Most transgressive genes in hybrids show no differential expression between parental subspecies. SNP data was used to explore the role of stabilizing selection through compensatory mutations. This study also examined possible misregulation through cascade effects that could be driven by interacting gene networks or co-option of off-target cis-regulatory elements (Go, 2020).

    Rapid Evolution of Autosomal Binding Sites of the Dosage Compensation Complex in Drosophila melanogaster and Its Association With Transcription Divergence

    How pleiotropy influences evolution of protein sequence remains unclear. The male-specific lethal (MSL) complex in Drosophila mediates dosage compensation by 2-fold upregulation of the X chromosome in males. Nevertheless, several MSL proteins also bind autosomes and likely perform functions not related to dosage compensation. The evolution of MOF, MSL1, and MSL2 binding sites was studied in Drosophila melanogaster and its close relative Drosophila simulans. Pervasive expansion of the MSL binding sites were found in D. melanogaster, particularly on autosomes. The majority of these newly-bound regions are unlikely to function in dosage compensation and associated with an increase in expression divergence between D. melanogaster and D. simulans. While dosage-compensation related sites show clear signatures of adaptive evolution, these signatures are even more marked among autosomal regions. This study points to an intriguing avenue of investigation of pleiotropy as a mechanism promoting rapid protein sequence evolution (Dai, 2021).

    The hourglass model of evolutionary conservation during embryogenesis extends to developmental enhancers with signatures of positive selection

    Inter-species comparisons of both morphology and gene expression within a phylum have revealed a period in the middle of embryogenesis with more similarity between species compared to earlier and later time-points. This "developmental hourglass" pattern has been observed in many phyla, yet the evolutionary constraints on gene expression, and underlying mechanisms of how this is regulated, remains elusive. Moreover, the role of positive selection on gene regulation in the more diverged earlier and later stages of embryogenesis remains unknown. Using DNase-seq to identify regulatory regions in two distant Drosophila species (D. melanogaster and D. virilis), this study assessed the evolutionary conservation and adaptive evolution of enhancers throughout multiple stages of embryogenesis. This revealed a higher proportion of conserved enhancers at the phylotypic period, providing a regulatory basis for the hourglass expression pattern. Using an in silico mutagenesis approach, signatures of positive selection on developmental enhancers were detected at early and late stages of embryogenesis, with a depletion at the phylotypic period, suggesting positive selection as one evolutionary mechanism underlying the hourglass pattern of animal evolution (Liu, 2021).

    Genes relocated between Drosophila chromosome arms evolve under relaxed selective constraints relative to non-relocated genes

    Gene duplication creates a second copy of a gene either in tandem to the ancestral locus or dispersed to another chromosomal location. Gene relocations may be as common as canonical dispersed duplications in which both the ancestral and derived copies are retained. Relocated genes appear to be under more selective constraints than the derived copies of canonical duplications, and they are possibly as conserved as single-copy non-relocated genes. To test this hypothesis, comparative genomics, population genetics, gene expression, and functional analyses were combined to assess the selection pressures acting on relocated, duplicated, and non-relocated single-copy genes in Drosophila genomes. Relocated genes were found to evolve faster than single-copy non-relocated genes, and there is no evidence that this faster evolution is driven by positive selection. In addition, relocated genes are less essential for viability and male fertility than single-copy non-relocated genes, suggesting that relocated genes evolve fast because of relaxed selective constraints. However, relocated genes evolve slower than the derived copies of canonical dispersed duplicated genes. We therefore conclude that relocated genes are under more selective constraints than canonical duplicates, but are not as conserved as single-copy non-relocated genes (Hart, 2018).

    Origin, composition, and structure of the supernumerary B chromosome of Drosophila melanogaster

    The number of chromosomes carried by an individual species is one of its defining characteristics. Some species, however, can also carry supernumerary chromosomes referred to as B chromosomes. B chromosomes were recently identified in a laboratory stock of Drosophila melanogaster enabling them to be subjected to extensive molecular analysis. The B chromosomes by pulsed-field gel electrophoresis and determined their composition through next-generation sequencing. Although these B chromosomes carry no known euchromatic sequence, they are rich in transposable elements and long arrays of short nucleotide repeats, the most abundant being the uncharacterized AAGAT satellite repeat. Fluorescent in-situ hybridization on metaphase chromosome spreads revealed this repeat is located on Chromosome 4, strongly suggesting the origin of the B chromosomes is Chromosome 4. Cytological and quantitative comparisons of signal intensity between Chromosome 4 and the B chromosomes supports the hypothesis that the structure of the B chromosome is an isochromosome. Also, the identification is reported of a new B chromosome variant in a related laboratory stock. This B chromosome has a similar repeat signature as the original but is smaller and much less prevalent. Additional stocks with similar genotypes were examined and B chromosomes were found, but these stocks lacked the AAGAT satellite repeat. This molecular characterization of D. melanogaster B chromosomes is the first step towards understanding how supernumerary chromosomes arise from essential chromosomes and what may be necessary for their stable inheritance (Hanlon, 2018).

    Functional interplay between ribosomal protein paralogues in the eRpL22 family in Drosophila melanogaster

    Duplicated ribosomal protein (RP) genes in the Drosophila melanogaster eRpL22 family encode structurally-divergent and differentially-expressed rRNA-binding RPs. eRpL22 is expressed ubiquitously and eRpL22-like expression is tissue-restricted with highest levels in the adult male germline. Paralogue functional equivalence was explored using the GAL4-UAS system for paralogue knockdown or overexpression and a conditional eRpL22-like knockout in a heat- shock flippase/FRT line. Ubiquitous eRpL22 knockdown with Actin-GAL4 resulted in embryonic lethality, confirming eRpL22 essentiality. eRpL22-like knockdown (60%) was insufficient to cause lethality; yet, conditional eRpL22-like knockout at one hour following egg deposition caused lethality within each developmental stage. Therefore, each paralogue is essential. Variation in timing of heat-shock-induced eRpL22-like knockout highlighted early embryogenesis as the critical period where eRpL22-like expression (not compensated for by eRpL22) is required for normal development of several organ systems, including testis development and subsequent sperm production. To determine if eRpL22-like can substitute for eRpL22, Actin-GAL4 for ubiquitous eRpL22 knockdown and eRpL22-like-FLAG (or FLAG-eRpL22: control) overexpression. Emergence of adults demonstrated that ubiquitous eRpL22-like-FLAG or FLAG-eRpL22 expression eliminates embryonic lethality resulting from eRpL22 depletion. Adults rescued by eRpL22-like-FLAG (but not by FLAG-eRpL22) overexpression had reduced fertility and longevity. It is concluded that eRpL22 paralogue roles are not completely interchangeable and include functionally-diverse roles in development and spermatogenesis (Mageeney, 2018).

    The molecular basis for the neofunctionalization of the juvenile hormone esterase duplication in Drosophila

    The Drosophila melanogaster enzymes juvenile hormone esterase (DmJHE) and its duplicate, DmJHEdup, present ideal examples for studying the structural changes involved in the neofunctionalization of enzyme duplicates. DmJHE is a hormone esterase with precise regulation and highly specific activity for its substrate, juvenile hormone. DmJHEdup is an odorant degrading esterase (ODE) responsible for processing various kairomones in antennae. Phylogenetic analysis shows that the JHE lineage predates the hemi/holometabolan split. DmJHE has sufficient substrate promiscuity and activity against odorant esters for a duplicate to evolve a general ODE function against a range of mid-long chain food esters, as is shown in DmJHEdup. Both JHEs showed very similar active sites despite low sequence identity (30%). Both ODEs differed drastically from the JHEs and each other, explaining their complementary substrate ranges. A small number of amino acid changes are identified that may have been involved in the early stages of the neofunctionalization of DmJHEdup. These results provide key insights into the process of neofunctionalization and the structural changes that can be involved (Hopkins, 2019).

    Birth-and-death evolution of the fatty acyl-CoA reductase (FAR) gene family and diversification of cuticular hydrocarbon synthesis in Drosophila

    The birth-and-death evolutionary model proposes that some members of a multigene family are phylogenetically stable and persist as a single copy over time whereas other members are phylogenetically unstable and undergo frequent duplication and loss. Functional studies suggest that stable genes are likely to encode essential functions, while rapidly evolving genes reflect phenotypic differences in traits that diverge rapidly among species. One such class of rapidly diverging traits are insect cuticular hydrocarbons (CHCs), which play dual roles in chemical communications as short-range recognition pheromones as well as protecting the insect from desiccation. Insect CHCs diverge rapidly between related species leading to ecological adaptation and/or reproductive isolation. Because the CHC and essential fatty acid biosynthetic pathways share common genes, it was hypothesized that genes involved in the synthesis of CHCs would be evolutionary unstable, while those involved in fatty acid-associated essential functions would be evolutionary stable. To test this hypothesis, the evolutionary history was investigated of the fatty acyl-CoA reductases (FARs) gene family that encodes enzymes in CHC synthesis. A unique dataset was compiled of 200 FAR proteins across 12 Drosophila species. A broad diversity in FAR content was uncovered that is generated by gene duplications, subsequent gene losses, and alternative splicing. FARs expressed in oenocytes and presumably involved in CHC synthesis are more unstable than FARs from other tissues. Taken together, this study provides empirical evidence that a comparative approach investigating the birth-and-death evolution of gene families can identify candidate genes involved in rapidly diverging traits between species (Finet, 2019).

    Recurrent gene co-amplification on Drosophila X and Y chromosomes

    Y chromosomes often contain amplified genes which can increase dosage of male fertility genes and counteract degeneration via gene conversion. This study identified genes with increased copy number on both X and Y chromosomes in various species of Drosophila, a pattern that has previously been associated with sex chromosome drive involving the Slx and Sly gene families in mice. Recurrent X/Y co-amplification appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila. This study also demonstrates that convergent acquisition and amplification of testis expressed gene families are common on Drosophila sex chromosomes, and especially on recently formed ones, and one putative novel X/Y co-amplification system was carefully characterized. Co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D. obscura group, where this normally autosomal gene pair is sex-linked due to a sex chromosome-autosome fusion. Several evolutionary scenarios were explored that would explain this pattern of co-amplification. Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that, like Slx/Sly in mice, these genes may be remnants of a cryptic sex chromosome drive system, however additional transgenic experiments will be necessary to validate this model. Regardless of whether sex chromosome drive is responsible for this co-amplification, the findings suggest that recurrent gene duplications between X and Y sex chromosomes could have a widespread effect on genomic and evolutionary patterns, including the epigenetic regulation of sex chromosomes, the distribution of sex-biased genes, and the evolution of hybrid sterility (Ellison, 2019).

    A burst of genetic innovation in Drosophila actin-related proteins for testis-specific function

    Many cytoskeletal proteins perform fundamental biological processes and are evolutionarily ancient. For example, the superfamily of actin-related proteins (Arps) specialized early in eukaryotic evolution for diverse cellular roles in the cytoplasm and the nucleus. Despite its strict conservation across eukaryotes, this study found that the Arp superfamily has undergone dramatic lineage-specific diversification in Drosophila. Our phylogenomic analyses reveal four independent Arp gene duplications that occurred in the common ancestor of the obscura group of Drosophila and have been mostly preserved in this lineage. All four obscura-specific Arp paralogs are predominantly expressed in the male germline and have evolved under positive selection. We focus the analyses on the divergent Arp2D paralog, which arose via a retroduplication event from Arp2, a component of the Arp2/3 complex that polymerizes branched actin networks. Computational modeling analyses suggests that Arp2D can replace Arp2 in the Arp2/3 complex and bind actin monomers. Together with the signature of positive selection, these findings suggest that Arp2D may augment Arp2's functions in the male germline. Indeed, it was found that Arp2D is expressed during and following male meiosis, where it localizes to distinct locations such as actin cones-specialized cytoskeletal structures that separate bundled spermatids into individual mature sperm. It is hypothesized that this unprecedented burst of genetic innovation in cytoskeletal proteins may have been driven by the evolution of sperm heteromorphism in the obscura group of Drosophila (Schroeder, 2019).

    Expansion of imaginal disc growth factor gene family in diptera reflects the evolution of novel functions

    Imaginal disc growth factors (IDGFs) are a small protein family found in insects. They are related to chitinases and implicated in multiple functions, including cell growth stimulation, antimicrobial activity, insect hemolymph clotting, and maintenance of the extracellular matrix. A number of new IDGFs have been found in several insect species and their detailed phylogenetic analysis provides a good basis for further functional studies. To achieve this goal, Idgf cDNAs were sequenced from several lepidopteran and trichopteran species and the data was supplemented with sequences retrieved from public databases. A comparison of Idgf genes in different species showed that Diptera typically contain several Idgf paralogs with a simple exon-intron structure (2-3 exons), whereas lepidopteran Idgfs appear as a single copy per genome and contain a higher number of exons (around 9). These results show that, while lepidopteran Idgfs, having single orthologs, are characterized by low divergence and stronger purifying selection over most of the molecule, the duplicated Idgf genes in Diptera, Idgf1 and Idgf4, exhibit signs of positive selection. This characterization of IDGF evolution provides the first information on the changes that formed these important molecules (Zurovcova, 2019).

    Ab initio construction and evolutionary analysis of protein-coding gene families with partially homologous relationships: Closely related Drosophila genomes as a case study

    How have genes evolved within a well-known genome phylogeny? Many protein-coding genes should have evolved as a whole at the gene level, and some should have evolved partly through fragments at the subgene level. To comprehensively explore such complex homologous relationships and better understand gene family evolution, in this study, with de novo-identified modules, the subgene units which could consecutively cover proteins within a set of closely related species, a new phylogeny-based approach was applied that considers evolutionary models with partial homology to classify all protein-coding genes in nine Drosophila genomes. Compared with two other popular methods for gene family construction, this approach improved practical gene family classifications with a more reasonable view of homology and provided a much more complete landscape of gene family evolution at the gene and subgene levels. This study found that most expanded gene families might have evolved mainly through module rearrangements rather than gene duplications and mainly generated single-module genes through partial gene duplication, suggesting that there might be pervasive subgene rearrangement in the evolution of protein-coding gene families. The use of a phylogeny-based approach with partial homology to classify and analyze protein-coding gene families may provide a more comprehensive landscape depicting how genes evolve within a well-known genome phylogeny (Han, 2020).

    Run or die in the evolution of new microRNAs - Testing the Red Queen hypothesis on de novo new genes

    The Red Queen hypothesis depicts evolution as the continual struggle to adapt. According to this hypothesis, new genes, especially those originating from non-genic sequences (i.e., de novo genes), are eliminated unless they evolve continually in adaptation to a changing environment. This study analyzed two Drosophila de novo miRNAs that are expressed in a testis-specific manner with very high rates of evolution in their DNA sequence. These miRNAs were knocked out in two sibling species, and their contributions to different fitness components were investigated. It was observed that the fitness contributions of miR-975 in D. simulans seem positive, in contrast to its neutral contributions in D. melanogaster, while miR-983 appears to have negative contributions in both species, as the fitness of the knockout mutant increases. As predicted by the Red Queen hypothesis, the fitness difference of these de novo miRNAs indicates their different fates (Zhao, 2020).

    Structural and functional characterization of a putative de novo gene in Drosophila

    Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, this study elucidated the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. This study shows that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, this study showed that Goddard protein contains a large central α-helix, but is otherwise partially disordered. Similar results were found for Goddard's orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard's structure appears to have been maintained with only minor changes over millions of years (Lange, 2021).

    Functional Diversification, Redundancy and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster

    Large multigene families, such as the insect odorant binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. This study reports the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. CRISPR technology was used to excise the entire cluster while introducing a PhiC31 re-integration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. Whole transcriptome sequencing was performed and sexually dimorphic changes in transcript abundances ("transcriptional niches") associated with each gene-edited genotype were assessed. Using this approach, it was possible to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. The effects were analyzed of gene editing of this cluster on organismal phenotypes, and a significant skewing was found of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families (Johnstun, 2021).

    Genomic analyses of new genes and their phenotypic effects reveal rapid evolution of essential functions in Drosophila development

    It is a conventionally held dogma that the genetic basis underlying development is conserved in a long evolutionary time scale. Ample experiments based on mutational, biochemical, functional, and complementary knockdown/knockout approaches have revealed the unexpectedly important role of recently evolved new genes in the development of Drosophila. The recent progress in the genome-wide experimental testing of gene effects and improvements in the computational identification of new genes (< 40 million years ago, Mya) open the door to investigate the evolution of gene essentiality with a phylogenetically high resolution. These advancements also raised interesting issues in techniques and concepts related to phenotypic effect analyses of genes, particularly of those that recently originated. This study reports analyses of these issues, including reproducibility and efficiency of knockdown experiment and difference between RNAi libraries in the knockdown efficiency and testing of phenotypic effects. This study reports a large data from knockdowns of 11,354 genes (~75% of the Drosophila melanogaster total genes), including 702 new genes (~66% of the species total new genes that aged < 40 Mya), revealing a similarly high proportion (~32.2%) of essential genes that originated in various Sophophora subgenus lineages and distant ancestors beyond the Drosophila genus. The transcriptional compensation effect from CRISPR knockout were detected for highly similar duplicate copies. Knockout of a few young genes detected analogous essentiality in various functions in development. Taken together, this experimental and computational analyses provide valuable data for detection of phenotypic effects of genes in general and further strong evidence for the concept that new genes in Drosophila quickly evolved essential functions in viability during development (Xia, 2021).

    Demographic analyses of a new sample of haploid genomes from a Swedish population of Drosophila melanogaster

    European and African natural populations of Drosophila melanogaster have been the focus of several studies aiming at inferring demographic and adaptive processes based on genetic variation data. However, in these analyses little attention has been given to gene flow between African and European samples. This paper presents a dataset consisting of 14 fully sequenced haploid genomes sampled from a natural population from the northern species range (Umea, Sweden). This new data was co-analyzed with an African population to compare the likelihood of several competing demographic scenarios for European and African populations and shows that gene flow improves the fit of demographic models to data (Kapopoulou, 2020).

    Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA

    Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. This study measured underreplication in 132 species across the Drosophila genus and leveraged these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented in this study estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila (Hjelmen, 2020). >

    Low levels of genetic differentiation with isolation by geography and environment in populations of Drosophila melanogaster from across China

    The fruit fly, Drosophila melanogaster, is a model species in evolutionary studies. However, population processes of this species in East Asia are poorly studied. This study examined the population genetic structure of D. melanogaster across China. There were 14 mitochondrial haplotypes with 10 unique ones out of 23 known from around the globe. Pairwise F(ST) values estimated from 15 novel microsatellites ranged from 0 to 0.11, with geographically isolated populations showing the highest level of genetic uniqueness. STRUCTURE analysis identified high levels of admixture at both the individual and population levels. Mantel tests indicated a strong association between genetic distance and geographical distance as well as environmental distance. Full redundancy analysis (RDA) showed that independent effects of environmental conditions and geography accounted for 62.10% and 31.58% of the total explained genetic variance, respectively. When geographic variables were constrained in a partial RDA analysis, the environmental variables bio2 (mean diurnal air temperature range), bio13 (precipitation of the wettest month), and bio15 (precipitation seasonality) were correlated with genetic distance. This study suggests that demographic history, geographical isolation, and environmental factors have together shaped the population genetic structure of D. melanogaster after its introduction into China (Yue, 2021).

    Rapid Global Spread of wRi-like Wolbachia across Multiple Drosophila

    Maternally transmitted Wolbachia, Spiroplasma, and Cardinium bacteria are common in insects, but their interspecific spread is poorly understood. Because Wolbachia cannot survive outside host cells, spread between distantly related host species requires horizontal transfers that are presumably rare. This study documents spread of wRi-like Wolbachia among eight highly diverged Drosophila hosts (10-50 million years) over only about 14,000 years (5,000-27,000). Comparing 110 wRi-like genomes, it was found </=0.02% divergence from the wRi variant that spread rapidly through California populations of D. simulans. The hosts include both globally invasive species (D. simulans, D. suzukii, and D. ananassae) and narrowly distributed Australian endemics (D. anomalata and D. pandora). Phylogenetic analyses that include mtDNA genomes indicate introgressive transfer of wRi-like Wolbachia between closely related species D. ananassae, D. anomalata, and D. pandora but no horizontal transmission within species. These analyses suggest D. ananassae as the Wolbachia source for the recent wRi invasion of D. simulans and D. suzukii as the source of Wolbachia in its sister species D. subpulchrella. Although six of these wRi-like variants cause strong cytoplasmic incompatibility, two cause no detectable reproductive effects, indicating that pervasive mutualistic effects complement the reproductive manipulations for which Wolbachia are best known. "Super spreader" variants like wRi may be particularly useful for controlling insect pests and vector-borne diseases with Wolbachia transinfections (Turelli, 2018).

    Fine scale mapping of genomic introgressions within the Drosophila yakuba clade

    The process of speciation involves populations diverging over time until they are genetically and reproductively isolated. Hybridization between nascent species was long thought to directly oppose speciation. A natural place to look for individuals with admixed ancestry (indicative of introgression) is in regions where species co-occur. In west Africa, D. santomea and D. yakuba hybridize on the island of Sao Tome, while D. yakuba and D. teissieri hybridize on the nearby island of Bioko. This report quantifies the genomic extent of introgression between the three species of the Drosophila yakuba clade (D. yakuba, D. santomea), D. teissieri). The genomes of 86 individuals were sequenced from all three species. A new statistical framework was developed and applied, using a hidden Markov approach, to identify introgression. Introgression was found to have occurred between both species pairs but most introgressed segments are small (on the order of a few kilobases). After ruling out the retention of ancestral polymorphism as an explanation for these similar regions, this study found that the sizes of introgressed haplotypes indicate that genetic exchange is not recent (>1,000 generations ago). It was additionally shown that in both cases, introgression was rarer on X chromosomes than on autosomes which is consistent with sex chromosomes playing a large role in reproductive isolation. Even though the two species pairs have stable contemporary hybrid zones, providing the opportunity for ongoing gene flow, the results indicate that genetic exchange between these species is currently rare (Turissini, 2017).

    A Maladaptive Combination of Traits Contributes to the Maintenance of a Drosophila Hybrid Zone

    Drosophila teissieri and D. yakuba diverged approximately 3 mya and are thought to share a large, ancestral, African range. These species now co-occur in parts of continental Africa and in west Africa on the island of Bioko. While D. yakuba is a human commensal, D. teissieri seems to be associated with Parinari fruits, restricting its range to forests. Genome data indicate introgression, despite no evidence of contemporary hybridization. This study reports the discovery of D. yakuba-D. teissieri hybrids at the interface of secondary forests and disturbed, open habitats on Bioko. Hybrids are the F1 progeny of D. yakuba females and D. teissieri males. At high temperatures like those found on Bioko, D. teissieri females are generally less receptive to mating, and in combination with temperature effects on egg lay and egg-to-adult viability, this decreases the potential for gene flow between female D. teissieri and male D. yakuba relative to the reciprocal cross. Field and laboratory experiments demonstrate that F1 hybrids have a maladaptive combination of D. yakuba behavior and D. teissieri physiology, generating additional barriers to gene flow. Nevertheless, analysis of introgressed and non-introgressed regions of the genome indicate that, while rare, gene flow is relatively recent. These observations identify precise intrinsic and extrinsic factors that, along with hybrid male sterility, limit gene flow and maintain these species. These data contribute to a growing body of literature that suggests the Gulf of Guinea may be a hotspot for hybridization (Cooper, 2018).

    Rapid and predictable evolution of admixed populations between two Drosophila species pairs

    The consequences of hybridization are varied, ranging from the origin of new lineages, introgression of some genes between species, to the extinction of one of the hybridizing species. This study generated replicate admixed populations between two pairs of sister species of Drosophila: D. simulans and D. mauritiana; and D. yakuba and D. santomea. Each pair consisted of a continental species and an island endemic. The admixed populations were maintained by random mating in discrete generations for over 20 generations. Morphological, behavioral, and fitness-related traits from each replicate population were assessed periodically, and genomic DNA was sequenced from the populations at generation 20. For both pairs of species, species-specific traits and their genomes regressed to those of the continental species. A few alleles from the island species persisted, but they tended to be proportionally rare among all sites in the genome and were rarely fixed within the populations. This paucity of alleles from the island species was particularly pronounced on the X-chromosome. These results indicate that nearly all foreign genes were quickly eliminated after hybridization and that selection against the minor species genome might be similar across experimental replicates (Matute, 2019).

    Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation

    Despite holding a central role in fertilization, reproductive traits often show elevated rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within populations is predicted to cause mis-signalling between the male ejaculate and the female during and after mating resulting in postmating prezygotic (PMPZ) isolation between populations. Crosses between Drosophila montana populations show PMPZ isolation in the form of reduced fertilization success in both noncompetitive and competitive contexts. This study tested whether male ejaculate proteins produced in the accessory glands or ejaculatory bulb differ between populations using liquid chromatography tandem mass spectrometry. More than 150 differentially abundant proteins were found between populations that may contribute to PMPZ isolation, including a number of proteases, peptidases and several orthologues of Drosophila melanogaster Sfps known to mediate fertilization success. Males from the population that elicit the stronger PMPZ isolation after mating with foreign females typically produced greater quantities of Sfps. The accessory glands and ejaculatory bulb show enrichment for different gene ontology (GO) terms and the ejaculatory bulb contributes more differentially abundant proteins. Proteins with a predicted secretory signal evolve faster than nonsecretory proteins. Finally, advantage was taken of quantitative proteomics data for three Drosophila species to determine shared and unique GO enrichments of Sfps between taxa and which potentially mediate PMPZ isolation. This study provides the first high-throughput quantitative proteomic evidence showing divergence of reproductive proteins between populations that exhibit PMPZ isolation (Garlovsky, 2020).

    Comparative genomic analyses provide new insights into the evolutionary dynamics of heterochromatin in Drosophila

    The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, this study has identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. The results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, a new scenario is suggested whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin (Caizzi, 2016).

    Genomics of natural populations: How differentially expressed genes shape the evolution of chromosomal inversions in Drosophila pseudoobscura

    The third chromosome of Drosophila pseudoobscura is a model system to test hypotheses about how rearrangements are established in populations because its third chromosome is polymorphic for > 30 gene arrangements that were generated by a series of overlapping inversion mutations. Circumstantial evidence has suggested that these gene arrangements are selected. Despite the expected homogenizing effects of extensive gene flow, the frequencies of arrangements form gradients or clines in nature, which have been stable since the system was first described more than 80 years ago. Furthermore, multiple arrangements exist at appreciable frequencies across several ecological niches providing the opportunity for heterokaryotypes to form. This study tested whether genes are differentially expressed among chromosome arrangements in first instar larvae, adult females and males. In addition, it was asked whether transcriptional patterns in heterokaryotypes are dominant, semidominant, overdominant, or underdominant. Evidence was found for a significant abundance of differentially expressed genes across the inverted regions of the third chromosome, including an enrichment of genes involved in sensory perception for males. The majority of loci show additivity in heterokaryotypes. These results suggest that multiple genes have expression differences among arrangements that were either captured by the original inversion mutation or accumulated after it reached polymorphic frequencies, providing a potential source of genetic variation for selection to act upon. These data suggest that the inversions are favored because of their indirect effect of recombination suppression that has held different combinations of differentially expressed genes together in the various gene arrangement backgrounds (Fuller, 2016).

    The origin of chromosomal inversions as a source of segmental duplications in the Sophophora subgenus of Drosophila

    Chromosomal inversions can contribute to the adaptation of organisms to their environment by capturing particular advantageous allelic combinations of a set of genes included in the inverted fragment and also by advantageous functional changes due to the inversion process itself that might affect not only the expression of flanking genes but also their dose and structure. Of the two mechanisms originating inversions -ectopic recombination, and staggered double-strand breaks and subsequent repair- only the latter confers the inversion the potential to have dosage effects and/or to generate advantageous chimeric genes. In Drosophila subobscura, there is ample evidence for the adaptive character of its chromosomal polymorphism, with an important contribution of some warm-climate arrangements such as E1+2+9+12. This study has characterized the breakpoints of inversion E12 and established that it originated through the staggered-break mechanism like four of the five inversions of D. subobscura previously studied. This mechanism that also predominates in the D. melanogaster lineage might be prevalent in the Sophophora subgenus and contribute to the adaptive character of the polymorphic and fixed inversions of its species. Finally, the D. subobscura inversion breakpoint regions were shown to have generally been disrupted by additional structural changes that occurred at different time scales (Puerma, 2016).

    Sex-specific evolution of relative leg size in Drosophila prolongata results from changes in the intersegmental coordination of tissue growth

    Evolution of relative organ size is the most prolific source of morphological diversity, yet the underlying molecular mechanisms that modify growth control are largely unknown. Models where organ proportions have undergone recent evolutionary changes hold the greatest promise for understanding this process. Uniquely among Drosophila species, D. prolongata displays a dramatic, male-specific increase in the size of its forelegs relative to other legs. By comparing leg development between males and females of D. prolongata and its closest relative D. carrolli, this study shows that the exaggerated male forelegs are produced by a sex- and segment-specific increase in mitosis during the final larval instar. Intersegmental compensatory control, where smaller leg primordia grow at a faster rate, is observed in both species and sexes. However, the equlibrium growth rates that determine the final relative proportion between the first and second legs have shifted in male D. prolongata compared both to conspecific females and to D. carrolli. It is suggested that the observed developmental changes that produce new adult proportions reflect an interplay between conserved growth coordination mechanisms and evolving organ-specific growth targets (Luecke, 2019).

    Evolution of sexual size dimorphism in the wing musculature of Drosophila

    Male courtship songs in Drosophila are exceedingly diverse across species. While much of this variation is understood to have evolved from changes in the central nervous system, evolutionary transitions in the wing muscles that control the song may have also contributed to song diversity. Focusing on a group of four wing muscles that are known to influence courtship song in Drosophila melanogaster, this study investigated the evolutionary history of wing muscle anatomy of males and females from 19 Drosophila species. Three of the wing muscles have evolved sexual dimorphisms in size multiple independent times, whereas one has remained monomorphic in the phylogeny. These data suggest that evolutionary changes in wing muscle anatomy may have contributed to species variation in sexually dimorphic wing-based behaviors, such as courtship song. Moreover, wing muscles appear to differ in their propensity to evolve size dimorphisms, which may reflect variation in the functional constraints acting upon different wing muscles (Tracy, 2020).

    Life history evolution and cellular mechanisms associated with increased size in high-altitude Drosophila

    Understanding the physiological and genetic basis of growth and body size variation has wide-ranging implications, from cancer and metabolic disease to the genetics of complex traits. This study examined the evolution of body and wing size in high-altitude Drosophila melanogaster from Ethiopia, flies with larger size than any previously known population. Specifically, attempts were made to identify life history characteristics and cellular mechanisms that may have facilitated size evolution. The large-bodied Ethiopian flies laid significantly fewer but larger eggs relative to lowland, smaller-bodied Zambian flies. The highland flies were found to achieve larger size in a similar developmental period, potentially aided by a reproductive strategy favoring greater provisioning of fewer offspring. At the cellular level, cell proliferation was a strong contributor to wing size evolution, but both thorax and wing size increases involved important changes in cell size. Nuclear size measurements were consistent with elevated somatic ploidy as an important mechanism of body size evolution. The significance of these results for the genetic basis of evolutionary changes in body and wing size in Ethiopian D. melanogaster is discussed (Lack, 2016).

    Identification of Evolutionarily Conserved Nuclear Matrix Proteins and Their Prokaryotic Origins

    Compared to prokaryotic cells, a typical eukaryotic cell is much more complex along with its endomembrane system and membrane-bound organelles. Although the endosymbiosis theories convincingly explain the evolution of membrane-bound organelles such as mitochondria and chloroplasts, very little is understood about the evolutionary origins of the nucleus, the defining feature of eukaryotes. Most studies on nuclear evolution have not been able to take into consideration the underlying structural framework of the nucleus, attributed to the nuclear matrix (NuMat), a ribonucleoproteinaceous structure. This can largely be attributed to the lack of annotation of its core components. Since NuMat has been shown to provide a structural platform for facilitating a variety of nuclear functions such as replication, transcription, and splicing, it is important to identify its protein components to better understand these processes. In this study, this issue was addressed using the developing embryos of Drosophila melanogaster and Danio rerio; 362 core NuMat proteins were identified that are conserved between the two organisms. These results were compared with publicly available Mus musculus NuMat dataset and Homo sapiens cellular localization dataset to define the core homologous NuMat proteins consisting of 252 proteins. Of them, 86 protein groups have originated from pre-existing proteins in prokaryotes. While 36 were conserved across all eukaryotic supergroups, 14 new proteins evolved before the evolution of the last eukaryotic common ancestor and together, these 50 proteins out of the 252 core conserved NuMat proteins are conserved across all eukaryotes, indicating their indispensable nature for nuclear function for over 1.5 billion years of eukaryotic history. This analysis paves the way to understand the evolution of the complex internal nuclear architecture and its functions (Sureka, 2021).

    Structural basis of diversity and homodimerization specificity of zinc-finger-associated domains in Drosophila

    In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. This study shows that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. A crystal structure was obtained of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, this study demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. It is speculated that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization (Bonchuk, 2021).

    Accelerated pseudogenization on the neo-X chromosome in Drosophila miranda

    Y chromosomes often degenerate via the accumulation of pseudogenes and transposable elements. By contrast, little is known about X-chromosome degeneration. This study compared the pseudogenization process between genes on the neo-sex chromosomes in Drosophila miranda and their autosomal orthologues in closely related species. The pseudogenization rate on the neo-X is much lower than the rate on the neo-Y, but appears to be higher than the rate on the orthologous autosome in D. pseudoobscura. Genes under less functional constraint and/or genes with male-biased expression tend to become pseudogenes on the neo-X, indicating the accumulation of slightly deleterious mutations and the feminization of the neo-X. A weak trend was found that the genes with female-benefit/male-detriment effects identified in D. melanogaster are pseudogenized on the neo-X, implying the masculinization of the neo-X. These observations suggest that both X and Y chromosomes can degenerate due to a complex suite of evolutionary forces (Nozawa, 2016).

    The landscape of A-to-I RNA editome is shaped by both positive and purifying selection

    The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. This study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, presents a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system (Yu, 2016).

    Weak polygenic selection drives the rapid adaptation of the chemosensory system: lessons from the upstream regions of the major gene families

    The animal chemosensory system is involved in essential biological processes, most of them mediated by proteins encoded in multigene families. These multigene families have been fundamental for the adaptation to new environments, significantly contributing to phenotypic variation. This adaptive potential contrasts, however, with the lack of studies at their upstream regions, especially taking into account the evidence linking their transcriptional changes to certain phenotypic effects. This study explicitly characterised the contribution of the upstream sequences of the major chemosensory gene families to rapid adaptive processes. For that, the genome sequences of 158 lines were analyzed from a population of Drosophila melanogaster that recently colonised North America, and functional and transcriptional data available for this species were integrated. Both, strong negative and strong positive selection were found to shape transcriptional evolution at the genome-wide level. The chemosensory upstream regions, however, exhibit a distinctive adaptive landscape, including multiple mutations of small beneficial effect and a reduced number of cis-regulatory elements. Together, these results suggest that the promiscuous and partially redundant transcription and function of the chemosensory genes provide evolutionarily opportunities for rapid adaptive episodes through weak polygenic selection (Librado, 2016).

    Evolutionary dynamics of abundant stop codon readthrough

    This study leveraged comparative genomic evidence across 21 Anopheles mosquitoes to systematically annotate translational stop codon readthrough genes in the malaria vector Anopheles gambiae, and to provide the first study of abundant readthrough evolution, by comparison with 20 Drosophila species. Evolutionary signatures were identified of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and of 51 additional Drosophila melanogaster stop codons. Most differences between the readthrough repertoires of the two species arose from readthrough gain or loss in existing genes, rather than birth of new genes or gene death; that readthrough-associated RNA structures are sometimes gained or lost while readthrough persists; that readthrough is more likely to be lost at TAA and TAG stop codons; and that readthrough is under continued purifying evolutionary selection in mosquito, based on population genetic evidence. Readthrough-associated gene properties were determined that predate readthrough, and differences were identified in the characteristic properties of readthrough genes between clades. More than 600 functional readthrough stop codons were identified in mosquito and 900 in fruit fly, provide evidence of readthrough control of peroxisomal targeting, and the phylogenetic extent of abundant readthrough as following divergence from centipede was refined (Jungreis, 2016).

    Molecular population genetics of the Polycomb genes in Drosophila subobscura
    Polycomb group (PcG) proteins are important regulatory factors that modulate the chromatin state. They form protein complexes that repress gene expression by the introduction of posttranslational histone modifications. The study of PcG proteins divergence in Drosophila revealed signals of coevolution among them and an acceleration of the nonsynonymous evolutionary rate in the lineage ancestral to the obscura group species, mainly in subunits of the Pcl-PRC2 complex. This study examined the nucleotide polymorphism of PcG genes in a natural population of D. subobscura to detect whether natural selection has also modulated the evolution of these important regulatory genes in a more recent time scale. Results show that most genes are under the action of purifying selection and present a level and pattern of polymorphism consistent with predictions of the neutral model, the exceptions being Su(z)12 and Pho. MK tests indicate an accumulation of adaptive changes in the SU(Z)12 protein during the divergence of D. subobscura and D. guanche. In contrast, the HKA test shows a deficit of polymorphism at Pho. The most likely explanation for this reduced variation is the location of this gene in the dot-like chromosome and would indicate that this chromosome also has null or very low recombination in D. subobscura, as reported in D. melanogaster (Calvo-Martin, 2017).

    Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network
    This study applied a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, both analytical and computational descriptions are provided of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). The sign of SNP influence (activation or repression) may change in time and space. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using this model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, signatures are seen of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, the patterns of polymorphism in the data were analyzed at two phenotypic levels: the strengths of binding and expression. Combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern (Gursky, 2017).

    Negative frequency dependent selection contributes to the maintenance of a global polymorphism in mitochondrial DNA

    Understanding the forces that maintain diversity across a range of scales is at the very heart of biology. Frequency-dependent processes are generally recognized as the most central process for the maintenance of ecological diversity. The same is, however, not generally true for genetic diversity. Negative frequency dependent selection, where rare genotypes have an advantage, is often regarded as a relatively weak force in maintaining genetic variation in life history traits because recombination disassociates alleles across many genes. Yet, many regions of the genome show low rates of recombination and genetic variation in such regions (i.e., supergenes) may in theory be upheld by frequency dependent selection. This group studied what is essentially a ubiquitous life history supergene (i.e., mitochondrial DNA) in the fruit fly Drosophila subobscura, showing sympatric polymorphism with two main mtDNA genotypes co-occurring in populations world-wide. Using an experimental evolution approach involving manipulations of genotype starting frequencies, this study shows that negative frequency dependent selection indeed acts to maintain genetic variation in this region. Moreover, the strength of selection was affected by food resource conditions. This work provides novel experimental support for the view that balancing selection through negative frequency dependency acts to maintain genetic variation in life history genes. It is suggested that the emergence of negative frequency dependent selection on mtDNA is symptomatic of the fundamental link between ecological processes related to resource use and the maintenance of genetic variation (Kurbalija Novicic, 2020).

    Towards an Evolutionarily Appropriate Null Model: Jointly Inferring Demography and Purifying Selection

    The question of the relative evolutionary roles of adaptive and non-adaptive processes has been a central debate in population genetics for nearly a century. While advances have been made in the theoretical development of the underlying models, and statistical methods for estimating their parameters from large-scale genomic data, a framework for an appropriate null model remains elusive. A model incorporating evolutionary processes known known to be in constant operation - genetic drift (as modulated by the demographic history of the population) and purifying selection - is lacking. Without such a null model, the role of adaptive processes in shaping within- and between-population variation may not be accurately assessed. This study investigated how population size changes and the strength of purifying selection affect patterns of variation at neutral sites near functional genomic components. A novel statistical framework is proposed for jointly inferring the contribution of the relevant selective and demographic parameters. By means of extensive performance analyses, the utility of the approach was quantified, the most important statistics for parameter estimation were identified, and the results with existing methods were compared. Finally, genome-wide population-level data was re-analyzed from a Zambian population of Drosophila melanogaster, and it was found that the population has experienced a much slower rate of population growth than was inferred when the effects of purifying selection were neglected. This approach represents an appropriate null model, against which the effects of positive selection can be assessed (Johri, 2020).

    Molecular evolution and the decline of purifying selection with age

    Life history theory predicts that the intensity of selection declines with age, and this trend should impact how genes expressed at different ages evolve. This study finds consistent relationships between a gene's age of expression and patterns of molecular evolution in two mammals (the human Homo sapiens and the mouse Mus musculus) and two insects (the malaria mosquito Anopheles gambiae and the fruit fly Drosophila melanogaster). When expressed later in life, genes fix nonsynonymous mutations more frequently, are more polymorphic for nonsynonymous mutations, and have shorter evolutionary lifespans, relative to those expressed early. The latter pattern is explained by a simple evolutionary model. Further, early-expressed genes tend to be enriched in similar gene ontology terms across species, while late-expressed genes show no such consistency. In humans, late-expressed genes are more likely to be linked to cancer and to segregate for dominant disease-causing mutations. Last, the effective strength of selection (N(e) s) decreases and the fraction of beneficial mutations increases with a gene's age of expression. These results are consistent with the diminishing efficacy of purifying selection with age, as proposed by Medawar's classic hypothesis for the evolution of senescence, and provide links between life history theory and molecular evolution (Cheng, 2021).

    Ancestral male recombination in Drosophila albomicans produced geographically restricted neo-Y chromosome haplotypes varying in age and onset of decay

    Male Drosophila typically have achiasmatic meiosis, and fusions between autosomes and the Y chromosome have repeatedly created non-recombining neo-Y chromosomes that degenerate. Intriguingly, Drosophila nasuta males recombine, but their close relative D. albomicans reverted back to achiasmy after evolving neo-sex chromosomes. This study used genome-wide polymorphism data to reconstruct the complex evolutionary history of neo-sex chromosomes in D. albomicans and examine the effect of recombination and its cessation on the initiation of neo-Y decay. Population and phylogenomic analyses reveal three distinct neo-Y types that are geographically restricted. Due to ancestral recombination with the neo-X, overall nucleotide diversity on the neo-Y is similar to the neo-X but severely reduced within neo-Y types. Consistently, the neo-Y chromosomes fail to form a monophyletic clade in sliding window trees outside of the region proximal to the fusion. Based on tree topology changes, the recombination breakpoints were infered that produced haplotypes specific to each neo-Y type. Recombination became suppressed at different time points for the different neo-Y haplotypes. Haplotype age correlates with onset of neo-Y decay, and older neo-Y haplotypes show more fixed gene disruption via frameshift indels and down-regulation of neo-Y alleles. Genes are downregulated independently on the different neo-Ys, but are depleted of testes-expressed genes across all haplotypes. This indicates that genes important for male function are initially shielded from degeneration. Thesee results offer a time course of the early progression of Y chromosome evolution, showing how the suppression of recombination, through the reversal to achiasmy in D. albomicans males, initiates the process of degeneration (Wei, 2019).

    Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster

    Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. This study tested for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. Winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. The observed differences are ascribed to indirect selection on recombination caused by directional selection on desiccation tolerance. These findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations) (Aggarwal, 2021).

    Structure of the Transcriptional Regulatory Network Correlates with Regulatory Divergence in Drosophila

    Transcriptional control of gene expression is regulated by biochemical interactions between cis-regulatory DNA sequences and trans-acting factors that form complex regulatory networks. Genetic changes affecting both cis- and trans-acting sequences in these networks have been shown to alter patterns of gene expression as well as higher-order organismal phenotypes. This study investigated how the structure of these regulatory networks relates to patterns of polymorphism and divergence in gene expression. To do this, a transcriptional regulatory network inferred for Drosophila melanogaster was compared to differences in gene regulation observed between two strains of D. melanogaster as well as between two pairs of closely related species: Drosophila sechellia and Drosophila simulans, and D. simulans and D. melanogaster. The number of transcription factors predicted to directly regulate a gene ("in-degree") was negatively correlated with divergence in both gene expression (mRNA abundance) and cis-regulation. This observation suggests that the number of transcription factors directly regulating a gene's expression affects the conservation of cis-regulation and gene expression over evolutionary time. The hypothesis that transcription factors regulating more target genes (higher "out-degree") are less likely to evolve changes in their cis-regulation and expression (presumably due to increased pleiotropy) was also tested, but little support was found for this predicted relationship. Taken together, these data show how the architecture of regulatory networks can influence regulatory evolution (Yang, 2017).

    Cis- and trans-regulatory effects on gene expression in a natural population of Drosophila melanogaster

    Cis- and trans-regulatory mutations are important contributors to transcriptome evolution. Quantifying their relative contributions to intraspecific variation in gene expression is essential for understanding the population genetic processes that underlie evolutionary changes in gene expression. This study has examined this issue by quantifying genome-wide allele specific expression (ASE) variation using a crossing scheme that produces F1 hybrids between 18 different Drosophila melanogaster strains sampled from the Drosophila Genetic Reference Panel (DGRP) and a reference strain from another population. Head and body samples from F1 adult females were subjected to RNA-seq and the subsequent ASE quantification. Cis- and trans-regulatory effects on expression variation were estimated from these data. A higher proportion of genes showed significant cis-regulatory variation (~28%) than those showed significant trans-regulatory variation (~9%). The sizes of cis-regulatory effects on expression variation were 1.98 and 1.88 times larger than trans-regulatory effects in heads and bodies, respectively. A generalized linear model analysis revealed that both cis- and trans-regulated expression variation was strongly associated with nonsynonymous nucleotide diversity and tissue specificity. Interestingly, trans-regulated variation showed a negative correlation with local recombination rate. Also, analysis on proximal transposon element (TE) insertions suggested that they affect transcription levels of ovary-expressed genes more pronouncedly than genes not expressed in the ovary, possibly due to defense mechanisms against TE mobility in the germline. Collectively, this detailed quantification of ASE variations from a natural population has revealed a number of new relationships between genomic factors and the effects of cis- and trans-regulatory factors on expression variation (Osada, 2017).

    Pigmentation pattern and developmental constraints: flight muscle attachment sites delimit the thoracic trident of Drosophila melanogaster

    In their seminal paper published in 1979, Gould and Lewontin argued that some traits arise as by-products of the development of other structures and not for direct utility in themselves. This study shows that this applies to the trident, a pigmentation pattern observed on the thorax of Drosophila melanogaster. Using reporter constructs, it was shown that the expression domain of several genes encoding pigmentation enzymes follows the trident shape. This domain is complementary to the expression pattern of stripe (sr), which encodes an essential transcription factor specifying flight muscle attachment sites. sr limits the expression of these pigmentation enzyme genes to the trident by repressing them in its own expression domain, i.e. at the flight muscle attachment sites. Evidence is given that repression of not only yellow but also other pigmentation genes, notably tan, is involved in the trident shape. The flight muscle attachment sites and sr expression patterns are remarkably conserved in dipterans reflecting the essential role of sr. The data suggest that the trident is a by-product of flight muscle attachment site patterning that arose when sr was co-opted for the regulation of pigmentation enzyme coding genes (Gibert, 2018).

    A major role for noncoding regulatory mutations in the evolution of enzyme activity

    The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. To determine the types of genetic changes underlying the quantitative evolution of protein activity, this study dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, this study found that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, both regulatory and coding substitutions were found to contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution (Loehlin, 2019).

    Holding it together: rapid evolution and positive selection in the synaptonemal complex of Drosophila

    The synaptonemal complex (SC) is a highly conserved meiotic structure that functions to pair homologs and facilitate meiotic recombination in most eukaryotes. Five Drosophila SC proteins have been identified and localized within the complex: C(3)G, C(2)M, CONA, ORD, and the newly identified Corolla. The SC is required for meiotic recombination in Drosophila and absence of these proteins leads to reduced crossing over and chromosomal nondisjunction. The proteins display little apparent sequence conservation outside the genus. To identify factors that explain this lack of apparent conservation, a molecular evolutionary analysis of these genes was performed across the Drosophila genus. For the five SC components, gene sequence similarity declines rapidly with increasing phylogenetic distance and only ORD and C(2)M are identifiable outside of the Drosophila genus. SC gene sequences have a higher dN/dS (omega) rate ratio than the genome wide average and this can in part be explained by the action of positive selection in almost every SC component. Omega estimates for the five SC components are in accordance with their physical position within the SC. Components interacting with chromatin evolve slowest and components comprising the central elements evolve the most rapidly. Thus, the Drosophila SC is proposed to be evolving rapidly due to two combined effects: 1) a high rate of evolution can be partly explained by low purifying selection on protein components whose function is to simply hold chromosomes together, 2) positive selection in the SC is driven by its sex-specificity combined with its role in facilitating both recombination and centromere clustering in the face of recurrent bouts of drive in female meiosis (Hemmer, 2016).

    Genomic and transcriptomic associations identify a new insecticide resistance phenotype for the selective sweep at the Cyp6g1 locus of Drosophila melanogaster

    Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel and genome-wide association. Recently released full transcriptome data was used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genomewide association studies to allow systems genetics analyses of phenotypes. Both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. Four novel candidate genes associated with azinphos-methyl resistance were found, all of which are involved in either regulation of fat storage or nervous system development. In Cyp6g1, a demonstrable resistance locus was found, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap was found between peaks of a genome-wide association study and a genome-wide selective sweep analysis (Battlay, 2015).

    Rapid divergence and convergence of life-history in experimentally evolved Drosophila melanogaster

    selection experiments are alluring in their simplicity, power, and ability to inform about how evolution works. A longstanding challenge facing evolution experiments with metazoans is that significant generational turnover takes a long time. This study presents data from a unique system of experimentally evolved laboratory populations of Drosophila melanogaster that have experienced three distinct life-history selection regimes. The goal of the study was to determine how quickly populations of a certain selection regime diverge phenotypically from their ancestors, and how quickly they converge with independently derived populations that share a selection regime. Results indicate that phenotypic divergence from an ancestral population occurs rapidly, within dozens of generations, regardless of that population's evolutionary history. Similarly, populations sharing a selection treatment converge on common phenotypes in this same time frame, regardless of selection pressures those populations may have experienced in the past. These patterns of convergence and divergence were found to emerge much faster than expected, suggesting that intermediate evolutionary history has transient effects in this system. These results are applicable to other experimental evolution projects, and suggest that many relevant questions can be sufficiently tested on shorter timescales than previously thought (Burke, 2016).

    Survey of global genetic diversity within the Drosophila immune system

    Numerous studies across a wide range of taxa have demonstrated that immune genes are routinely among the most rapidly evolving genes in the genome. This observation, however, does not address what proportion of immune genes undergo strong selection during adaptation to novel environments. This study determined the extent of very recent divergence in genes with immune function across five populations of Drosophila melanogaster;t immune genes do not show an overall trend of recent rapid adaptation. The population-based approach uses a set of carefully matched control genes to account for the effects of demography and local recombination rate, allowing identification of whether specific immune functions are putative targets of strong selection. Evidence was found that viral defense genes are rapidly evolving in Drosophila at multiple time scales. Local adaptation to bacteria and fungi is less extreme and primarily occurs through changes in recognition and effector genes rather than large-scale changes to the regulation of the immune response. Surprisingly, genes in the Toll pathway, which show a high rate of adaptive substitutions between the D. melanogaster and D. simulans lineages, show little population differentiation. Quantifying the flies for resistance to a generalist Gram-positive bacterial pathogen, it was found that this genetic pattern of low population differentiation was recapitulated at the phenotypic level. In sum, these results highlight the complexity of immune evolution and suggest that Drosophila immune genes do not follow a uniform trajectory of strong directional selection as flies encounter new environments (Early, 2016).

    Genome-wide analysis of long-term evolutionary domestication in Drosophila melanogaster

    Experimental evolutionary genomics now allows biologists to test fundamental theories concerning the genetic basis of adaptation. This laboratory conducted one of the longest laboratory evolution experiments with any sexually-reproducing metazoan, Drosophila melanogaster. Next-generation resequencing data from this experiment was conducted to examine genome-wide patterns of genetic variation over an evolutionary time-scale that approaches 1,000 generations. Measures of variation within and differentiation between populations were compared to simulations based on a variety of evolutionary scenarios. This analysis yielded no clear evidence of hard selective sweeps, whereby natural selection acts to increase the frequency of a newly-arising mutation in a population until it becomes fixed. Evidence was found for selection acting on standing genetic variation, as independent replicate populations exhibit similar population-genetic dynamics, without obvious fixation of candidate alleles under selection. A hidden-Markov model test for selection also found widespread evidence for selection. More genetic variation was found genome-wide, and less differentiation was found between replicate populations genome-wide, than arose in any of the simulated evolutionary scenarios (Phillips, 2016).

    Variation in the intensity of selection on codon bias over time causes contrasting patterns of base composition evolution in Drosophila

    Four-fold degenerate coding sites form a major component of the genome, and are often used to make inferences about selection and demography, so that understanding their evolution is important. Despite previous efforts, many questions regarding the causes of base composition changes at these sites in Drosophila remain unanswered. To shed further light on this issue, a new whole-genome polymorphism dataset was obtained from D. simulans. Samples were analyzed from the putatively ancestral range of D. simulans, as well as an existing polymorphism dataset from an African population of D. melanogaster. By using D. yakuba as an outgroup, clear evidence was found for selection on 4-fold sites along both lineages over a substantial period, with the intensity of selection increasing with GC content. Based on an explicit model of base composition evolution, it is suggested that the observed AT-biased substitution pattern in both lineages is probably due to an ancestral reduction in selection intensity, and is unlikely to be the result of an increase in mutational bias towards AT alone. By using two polymorphism-based methods for estimating selection coefficients over different timescales, it was shown that the selection intensity on codon usage has been rather stable in D. simulans in the recent past, but the long-term estimates in D. melanogaster are much higher than the short-term ones, indicating a continuing decline in selection intensity, to such an extent that the short-term estimates suggest that selection is only active in the most GC-rich parts of the genome. Finally, evidence is provided for complex evolutionary patterns in the putatively neutral short introns, which cannot be explained by the standard GC-biased gene conversion model. These results reveal a dynamic picture of base composition evolution (Jackson, 2017).

    A variable genetic architecture of melanic evolution in Drosophila melanogaster

    Unraveling the genetic architecture of adaptive phenotypic divergence is a fundamental quest in evolutionary biology. In Drosophila melanogaster, high-altitude melanism has evolved in separate mountain ranges in sub-Saharan Africa, potentially as an adaptation to UV intensity. The genetic basis of this melanism was investigated in three populations using a new bulk segregant analysis mapping method. Nineteen distinct QTL regions were identified from 9 mapping crosses, with several QTL peaks overlapping between two or all populations, and yet different crosses involving the same melanic population commonly yielded distinct QTLs. The strongest QTLs often overlapped well-known pigmentation genes, but wide signals of genetic differentiation (FST) was typically not found between lightly and darkly pigmented populations at these genes. Instead, small numbers of highly differentiated SNPs were found at the probable causative genes. A simulation analysis showed that these patterns of polymorphism were consistent with selection on standing genetic variation. Overall, these results suggest that even for potentially simpler traits like pigmentation, the complexity of adaptive trait evolution poses important challenges for QTL mapping and population genetic analysis (Bastide, 2016).

    Promoter shape varies across populations and affects promoter evolution and expression noise

    Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. This study used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape). The results identify promoter shape as a molecular trait that can evolve independently of promoter strength. Broad promoters typically harbor shape-associated variants, with signatures of adaptive selection. Single-cell measurements demonstrate that variants modulating promoter shape often increase expression noise, whereas heteroallelic interactions with other promoter variants alleviate these effects. These results uncover new functional properties of natural promoters and suggest the minimization of expression noise as an important factor in promoter evolution (Schor, 2016).

    Predictable phenotypic, but not karyotypic, evolution of populations with contrasting initial history

    The relative impact of selection, chance and history will determine the predictability of evolution. There is a lack of empirical research on this subject, particularly in sexual organisms. This study used experimental evolution to test the predictability of evolution. The real-time evolution was analyzed of Drosophila subobscura populations derived from contrasting European latitudes placed in a novel laboratory environment. Each natural population was sampled twice within a three-year interval. Evolutionary responses at both phenotypic (life-history, morphological and physiological traits) and karyotypic levels were studied for around 30 generations of laboratory culture. The results show (1) repeatable historical effects between years in the initial state, at both phenotypic and karyotypic levels; (2) predictable phenotypic evolution with general convergence except for body size; and (3) unpredictable karyotypic evolution. It is concluded that the predictability of evolution is contingent on the trait and level of organization, highlighting the importance of studying multiple biological levels with respect to evolutionary patterns (Simoes, 2017).

    Naturally-segregating variation at Ugt86Dd contributes to nicotine resistance in Drosophila melanogaster

    Identifying the sequence polymorphisms underlying complex trait variation is a key goal of genetics research, since knowing the precise causative molecular events allows insight into the pathways governing trait variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative sequence polymorphisms. Previous studies mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource, and this study use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNAi of this gene reduced resistance. A segregating 22-bp frameshift deletion in Ugt86Dd, and accounting for the InDel during mapping largely eliminates the QTL, implying the event explains the bulk of the effect of the mapped locus. CRISPR/Cas9 editing a relatively resistant genotype to generate lesions in Ugt86Dd that recapitulate the naturally-occurring putative loss-of-function allele leads to a large reduction in resistance. Despite this major effect of the deletion, the allele appears to be very rare in wild-caught populations, and likely explains only a small fraction of the natural variation for the trait. Nonetheless, this putatively causative coding InDel can be a launchpad for future mechanistic exploration of xenobiotic detoxification (Highfill, 2017).

    The genetic basis of susceptibility to low-dose paraquat and variation between the sexes in D. melanogaster

    Toxicant resistance is a complex trait, affected both by genetics and the environment. Like most complex traits, it can exhibit sexual dimorphism, yet sex is often overlooked as a factor in studies of toxicant resistance. Paraquat, one such toxicant, is a commonly used herbicide and is known to produce mitochondrial oxidative stress, decrease dopaminergic neurons and dopamine (DA) levels, and decrease motor ability. The purpose of this study was to map the genes contributing to low-dose paraquat susceptibility in Drosophila melanogaster, and to determine if susceptibility differs between the sexes. One hundred of the Drosophila Genetic Reference Panel (DGRP) lines were scored for susceptibility via climbing ability and used in a genome wide association study (GWAS). Variation in seventeen genes in females and thirty-five genes in males associated with paraquat susceptibility. Only two candidate genes overlapped between the sexes despite a significant positive correlation between male and female susceptibilities. Many associated polymorphisms had significant interactions with sex, with most having conditionally neutral effects. Conditional neutrality between the sexes likely stems from sex-biased expression which may result from partial resolution of sexual conflict. Candidate genes were verified with RNAi knockdowns, gene expression analyses, and DA quantification. Several of these genes are novel associations with paraquat susceptibility. This research highlights the importance of assessing both sexes when studying toxicant susceptibility (Lovejoy, 2021).

    Multiple P450s and variation in neuronal genes underpins the response to the insecticide Imidacloprid in a population of Drosophila melanogaster

    Insecticide resistance is an economically important example of evolution in response to intense selection pressure. In this study, the genetics of resistance to the neonicotinoid insecticide imidacloprid is explored using the Drosophila Genetic Reference Panel, a collection of inbred Drosophila melanogaster genotypes derived from a single population in North Carolina. Imidacloprid resistance varied substantially among genotypes, and more resistant genotypes tended to show increased capacity to metabolize and excrete imidacloprid. Variation in resistance level was then associated with genomic and transcriptomic variation, implicating several candidate genes involved in central nervous system function and the cytochrome P450s Cyp6g1 and Cyp6g2. CRISPR-Cas9 mediated removal of Cyp6g1 suggested that it contributed to imidacloprid resistance only in backgrounds where it was already highly expressed. Cyp6g2, previously implicated in juvenile hormone synthesis via expression in the ring gland, was shown to be expressed in metabolically relevant tissues of resistant genotypes. Cyp6g2 overexpression was shown to both metabolize imidacloprid and confer resistance. These data collectively suggest that imidacloprid resistance is influenced by a variety of previously known and unknown genetic factors (Denecke, 2017).

    Nearly neutral evolution across the Drosophila melanogaster genome

    Under the nearly neutral theory of molecular evolution, the proportion of effectively neutral mutations is expected to depend upon the effective population size (Ne). This study investigate whether this is the case across the genome of Drosophila melanogaster using polymorphism data from North American and African lines. This study shows that the ratio of the number of nonsynonymous and synonymous polymorphisms is negatively correlated to the number of synonymous polymorphisms, even when the nonindependence is accounted for. The relationship is such that the proportion of effectively neutral nonsynonymous mutations increases by approximately 45% as Ne is halved. However, it was also shown that this relationship is steeper than expected from an independent estimate of the distribution of fitness effects from the site frequency spectrum. A number of potential explanations for this was investigated, and it was shown, using simulation, that this is consistent with a model of genetic hitchhiking: Genetic hitchhiking depresses diversity at neutral and weakly selected sites, but has little effect on the diversity of strongly selected sites (Castellano, 2018).

    Pleiotropy modulates the efficacy of selection in Drosophila melanogaster

    Pleiotropy is the well-established idea that a single mutation affects multiple phenotypes. If a mutation has opposite effects on fitness when expressed in different contexts, then genetic conflict arises. Pleiotropic conflict is expected to reduce the efficacy of selection by limiting the fixation of beneficial mutations through adaptation, and the removal of deleterious mutations through purifying selection. While this has been widely discussed, in particular in the context of a putative "gender load", it has yet to be systematically quantified. This work empirically estimates to which extent different pleiotropic regimes impede the efficacy of selection in Drosophila melanogaster. Whole-genome polymorphism data from a single African population and divergence data from D. simulans were used to estimate the fraction of adaptive fixations (alpha), the rate of adaptation (omegaA) and the direction of selection (DoS). After controlling for confounding covariates, it was found that the different pleiotropic regimes have a relatively small, but significant, effect on selection efficacy. Specifically, the results suggest that pleiotropic sexual antagonism may restrict the efficacy of selection, but that this conflict can be resolved by limiting the expression of genes to the sex where they are beneficial. Intermediate levels of pleiotropy across tissues and life stages can also lead to maladaptation in D. melanogaster, due to inefficient purifying selection combined with low frequency of mutations that confer a selective advantage. Thus, this study highlights the need to consider the efficacy of selection in the context of antagonistic pleiotropy, and of genetic conflict in general (Fraisse, 2018).

    Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly

    Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life-history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. This study assesses the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. Response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets, however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age-at-reproduction selection regime. Given that multiple selection pressures are prevalent in nature, these findings suggest that trade-offs should be considered not only among traits within an organism, but also among adaptive responses to different - sometimes conflicting - selection pressures, including across life stages (May, 2019).

    Stage-specific genotype-by-environment interactions for cold and heat hardiness in Drosophila melanogaster

    Environments often vary across a life cycle, imposing fluctuating natural selection across development. Such fluctuating selection can favor different phenotypes in different life stages, but stage-specific evolutionary responses will depend on genetic variance, covariance, and their interaction across development and across environments. Thus, quantifying how genetic architecture varies with plastic responses to the environment and across development is vital to predict whether stage-specific adaptation will occur in nature. Additionally, the interaction of genetic variation and environmental plasticity (GxE) may be stage-specific, leading to a three-way interaction between genotype, environment, and development or GxDxE. To test for these patterns, larvae and adults of Drosophila melanogaster isogenic lines derived from a natural population were exposed to extreme heat and cold stress after developmental acclimation to cool (18 degrees C) and warm (25 degrees C) conditions, and genetic variance for thermal hardiness was measured. Significant GxE was observed that was specific to larvae and adults for cold and heat hardiness (GxDxE), but no significant genetic correlation across development for either trait at either acclimation temperature. However, cross-development phenotypic correlations for acclimation responses suggest that plasticity itself may be developmentally constrained, though rigorously testing this hypothesis requires more experimentation. These results illustrate the potential for stage-specific adaptation within a complex life cycle and demonstrate the importance of measuring traits at appropriate developmental stages and environmental conditions when predicting evolutionary responses to changing climates (Freda, 2019).

    Cross-sex genetic covariances limit the evolvability of wing-shape within and among species of Drosophila

    The independent evolution of males and females is potentially constrained by both sexes inheriting the same alleles from their parents. This genetic constraint can limit the evolvability of complex traits; however, there are few studies of multivariate evolution that incorporate cross-sex genetic covariances in their predictions. Drosophila wing-shape has emerged as a model high-dimensional phenotype; wing-shape is highly evolvable in contemporary populations, and yet perplexingly stable across phylogenetic timescales. This study shows that cross-sex covariances in D. melanogaster, given by the B-matrix, may considerably bias wing-shape evolution. Using random skewers, this study shows that B would constrain the response to antagonistic selection by 90%, on average, but would double the response to concordant selection. Both cross-sex within-trait and cross-sex cross-trait covariances determined the predicted response to antagonistic selection, but only cross-sex within-trait covariances facilitated the predicted response to concordant selection. Similar patterns were observed in the direction of extant sexual dimorphism in D. melanogaster, and in directions of most and least dimorphic variation across the Drosophila phylogeny. These results highlight the importance of considering between-sex genetic covariances when making predictions about evolution on both macro- and micro- evolutionary timescales, and may provide one more explanatory piece in the puzzle of stasis (Sztepanacz, 2019).

    Quantifying selection on standard metabolic rate and body mass in Drosophila melanogaster

    Standard metabolic rate (SMR), defined as the minimal energy expenditure required for self-maintenance, is a key physiological trait. Few studies have estimated its relationship with fitness, most notably in insects. This is presumably due to the difficulty of measuring SMR in a large number of very small individuals. Using high-throughput flow-through respirometry and a Drosophila melanogaster laboratory population adapted to a life-cycle that facilitates fitness measures, SMR, body mass, and fitness were quantified in 515 female and 522 male adults. A novel multivariate approach was used to estimate linear and non-linear selection differentials and gradients from the variance-covariance matrix of fitness, SMR, and body mass, allowing traits specific covariates to be accommodated within a single model. In males, linear selection differentials for mass and SMR were positive and individually significant. Selection gradients were also positive but, despite substantial sample sizes, were non-significant due to increased uncertainty given strong SMR-mass collinearity. In females, only nonlinear selection was detected and it appeared to act primarily on body size, although the individual gradients were again non-significant. Selection did not differ significantly between sexes although differences in the fitness surfaces suggest sex-specific selection as an important topic for further study (Videlier, 2020).

    Sexual antagonism, temporally fluctuating selection, and variable dominance affect a regulatory polymorphism in Drosophila melanogaster

    Understanding how genetic variation is maintained within species is a major goal of evolutionary genetics that can shed light on the preservation of biodiversity. This study examined the maintenance of a regulatory single-nucleotide polymorphism (SNP) of the X-linked Drosophila melanogaster gene fezzik. The derived variant at this site is at intermediate frequency in many worldwide populations, but absent in populations from the ancestral species range in sub-Saharan Africa. Wild-caught individuals from a single European population were collected and genotyped biannually over a period of five years, revealing an overall difference in allele frequency between the sexes and a consistent change in allele frequency across seasons in females but not in males. Modelling based on the observed allele and genotype frequencies suggested that both sexually antagonistic and temporally fluctuating selection may help maintain variation at this site. The derived variant is predicted to be female-beneficial and mostly recessive; however, there was uncertainty surrounding the dominance estimates, and long-term modelling projections suggest that it is more likely to be dominant. By examining gene expression phenotypes, it was found that phenotypic dominance was variable and dependent upon developmental stage and genetic background, suggesting that dominance may be variable at this locus. It was further determined that fezzik expression and genotype are associated with starvation resistance in a sex-dependent manner, suggesting a potential phenotypic target of selection. By characterizing the mechanisms of selection acting on this SNP, the results improve understanding of how selection maintains genetic and phenotypic variation in natura

    Selection on inversion breakpoints favors proximity to pairing sensitive sites in Drosophila melanogaster

    Chromosomal inversions are widespread among taxa, and have been implicated in a number of biological processes including adaptation, sex chromosome evolution, and segregation distortion. Consistent with selection favoring linkage between loci, it is well established that length is a selected trait of inversions. However, the factors that affect the distribution of inversion breakpoints remain poorly understood. 'Sensitive sites' have been mapped on all euchromatic chromosome arms in Drosophila melanogaster, and may be a source of natural selection on inversion breakpoint positions. Briefly, sensitive sites are genomic regions wherein proximal structural rearrangements result in large reductions in local recombination rates in heterozygotes. This study shows that breakpoints of common inversions are significantly more likely to lie within a cytological band containing a sensitive site than are breakpoints of rare inversions. Furthermore, common inversions for which neither breakpoint intersects a sensitive site are significantly longer than rare inversions, but common inversions whose breakpoints intersect a sensitive site show no evidence for increased length. These results are interpreted to mean that selection favors inversions whose breakpoints disrupt synteny near to sensitive sites, possibly because these inversions suppress recombination in large genomic regions. This is the first evidence consistent with positive selection acting on inversion breakpoint positions (Corbett-Detig, 2016).

    A genomic map of the effects of linked selection in Drosophila

    Natural selection at one site shapes patterns of genetic variation at linked sites. Quantifying the effects of 'linked selection' on levels of genetic diversity is key to making reliable inference about demography, building a null model in scans for targets of adaptation, and learning about the dynamics of natural selection. This study introduced the first method that jointly infers parameters of distinct modes of linked selection, notably background selection and selective sweeps, from genome-wide diversity data, functional annotations and genetic maps. The central idea is to calculate the probability that a neutral site is polymorphic given local annotations, substitution patterns, and recombination rates. Information is then combined across sites and samples using composite likelihood in order to estimate genome-wide parameters of distinct modes of selection. In addition to parameter estimation, this approach yields a map of the expected neutral diversity levels along the genome. To illustrate the utility of this approach, it was applied to genome-wide resequencing data from 125 lines in Drosophila melanogaster and diversity levels was reliably predicted at the 1Mb scale. The results corroborate estimates of a high fraction of beneficial substitutions in proteins and untranslated regions (UTR). They allow distinguishing between the contribution of sweeps and other modes of selection around amino acid substitutions and uncovered evidence for pervasive sweeps in untranslated regions (UTRs). This inference further suggests a substantial effect of other modes of linked selection and of adaptation in particular. More generally, it was demonstrated that linked selection has had a larger effect in reducing diversity levels and increasing their variance in D. melanogaster than previously appreciated (Elyashiv, 2016).

    Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster

    Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. This study used single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. The optimal de novo assembly methods and parameter combinations were determined that were required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. It was determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. Evidence was found for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin (Khost, 2017).

    High rate of translocation-based gene birth on the Drosophila Y chromosome

    The Y chromosome is a unique genetic environment defined by a lack of recombination and male-limited inheritance. The Drosophila Y chromosome has been gradually acquiring genes from the rest of the genome, with only seven Y-linked genes being gained over the past 63 million years (0.12 gene gains per million years). Using a next-generation sequencing (NGS)-powered genomic scan, this study shows that gene transfers to the Y chromosome are much more common than previously suspected: at least 25 have arisen across three Drosophila species over the past 5.4 million years (1.67 per million years for each lineage). The gene transfer rate is significantly lower in Drosophila melanogaster than in the Drosophila simulans clade, primarily due to Y-linked retrotranspositions being significantly more common in the latter. Despite all Y-linked gene transfers being evolutionarily recent (<1 million years old), only three showed evidence for purifying selection (omega </= 0.14). Thus, although the resulting Y-linked functional gene acquisition rate (0.25 new genes per million years) is double the longer-term estimate, the fate of most new Y-linked genes is defined by rapid degeneration and pseudogenization. These results show that Y-linked gene traffic, and the molecular mechanisms governing these transfers, can diverge rapidly between species, revealing the Drosophila Y chromosome to be more dynamic than previously appreciated. This analytical method provides a powerful means to identify Y-linked gene transfers and will help illuminate the evolutionary dynamics of the Y chromosome in Drosophila and other species (Tobler, 2017).

    MicroRNA clusters integrate evolutionary constraints on expression and target affinities: the miR-6/5/4/286/3/309 cluster in Drosophila

    A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. This study investigated a microRNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considered together, the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs (Qu, 2020).

    Drosophila as a useful model for understanding the evolutionary physiology of obesity resistance and metabolic thrift

    Evolved metabolic thriftiness in humans is a proposed contributor to the obesity epidemic. Insect models have been shown to evolve both 'metabolic thrift' in response to rearing on high-protein diets that promote leanness, and 'obesity resistance' when reared on fattening high-carbohydrate, low-protein foods. Despite the hypothesis that human obesity is caused by evolved metabolic thrift, genetic contributions to this physiological trait remain elusive. A pilot study was conducted to determine whether thrift and obesity resistance can arise under laboratory based 'quasi-natural selection' in the genetic model organism Drosophila melanogaster. Both these traits were found to evolve within 16 generations. Contrary to predictions from the 'thrifty genotype/phenotype' hypothesis, this study found that when animals from a metabolic thrift inducing high-protein environment are mismatched to fattening high-carbohydrate foods, they did not become 'obese'. Rather, they accumulate less triglyceride than control animals, not more. It is speculated that this may arise through as yet un-quantified parental effects - potentially epigenetic. This study establishes that D. melanogaster could be a useful model for elucidating the role of the trans- and inter-generational effects of diet on the genetics of metabolic traits in higher animals (Gray, 2021).

    The pdm3 locus is a hotspot for recurrent evolution of female-limited color dimorphism in Drosophila

    Sex-limited polymorphisms are an intriguing form of sexual dimorphism that offer unique opportunities to reconstruct the evolutionary changes that decouple male and female traits encoded by a shared genome. This study investigated the genetic basis of a Mendelian female-limited color dimorphism (FLCD) that segregates in natural populations of more than 20 species of the Drosophila montium subgroup. In these species, females have alternative abdominal color morphs, light and dark, whereas males have only one color morph in each species. A comprehensive molecular phylogeny of the montium subgroup supports multiple origins of FLCD. Despite this, FLCD mapped to the same locus in four distantly related species-the transcription factor POU domain motif 3 (pdm3), which acts as a repressor of abdominal pigmentation in D. melanogaster. In D. serrata, FLCD maps to a structural variant in the first intron of pdm3; however, this variant is not found in the three other species-D. kikkawai, D. leontia, and D. burlai-and sequence analysis strongly suggests the pdm3 alleles responsible for FLCD originated independently at least three times. It is proposed that cis-regulatory changes in pdm3 form sexually dimorphic and monomorphic alleles that segregate within species and are preserved, at least in one species, by structural variation. Surprisingly, pdm3 has not been implicated in the evolution of sex-specific pigmentation outside the montium subgroup, suggesting that the genetic paths to sexual dimorphism may be constrained within a clade but variable across clades (Yassin, 2016).

    Mapping QTL contributing to variation in posterior lobe morphology between strains of Drosophila melanogaster

    Closely-related, and otherwise morphologically similar insect species frequently show striking divergence in the shape and/or size of male genital structures, a phenomenon thought to be driven by sexual selection. Comparative interspecific studies can help elucidate the evolutionary forces acting on genital structures to drive this rapid differentiation. However, genetic dissection of sexual trait divergence between species is frequently hampered by the difficulty generating interspecific recombinants. Intraspecific variation can be leveraged to investigate the genetics of rapidly-evolving sexual traits; this study carried out out a genetic analysis of variation in the posterior lobe within D. melanogaster. The lobe is a male-specific process emerging from the genital arch of D. melanogaster and three closely-related species, is essential for copulation, and shows radical divergence in form across species. There is also abundant variation within species in the shape and size of the lobe, and while this variation is considerably more subtle than that seen among species, it nonetheless provides the raw material for QTL mapping. An advanced intercross population was created from a pair of phenotypically-different inbred strains, and after phenotyping and genotyping-by-sequencing the recombinants, several QTL contributing to various measures of lobe morphology were mapped. The additional generations of crossing over in the mapping population led to QTL intervals that are smaller than is typical for an F2 mapping design. The intervals that were mapped overlap with a pair of lobe QTL that was previously identified in an independent mapping cross, potentially suggesting a level of shared genetic control of trait variation. The QTL additionally implicate a suite of genes that have been shown to contribute to the development of the posterior lobe. These loci are strong candidates to harbor naturally-segregating sites contributing to phenotypic variation within D. melanogaster, and may also be those contributing to divergence in lobe morphology between species (Hackett, 2006).

    Sex-specific selection and sex-biased gene expression in humans and flies

    Sexual dimorphism results from sex-biased gene expression, which evolves when selection acts differently on males and females. While there is an intimate connection between sex-biased gene expression and sex-specific selection, few empirical studies have studied this relationship directly. This study compare the two on a genome-wide scale in humans and flies. A distinctive "Twin Peaks" pattern in humans that relates the strength of sex-specific selection, quantified by genetic divergence was yound between male and female adults at autosomal loci, to the degree of sex-biased expression. Genes with intermediate degrees of sex-biased expression show evidence of ongoing sex-specific selection, while genes with either little or completely sex-biased expression do not. This pattern apparently results from differential viability selection in males and females acting in the current generation. The Twin Peaks pattern is also found in Drosophila using a different measure of sex-specific selection acting on fertility. A simple model was developed that successfully recapitulates the Twin Peaks. These results suggest that many genes with intermediate sex-biased expression experience ongoing sex-specific selection in humans and flies (Cheng, 2016).

    Sexual selection shapes development and maturation rates in Drosophila

    Explanations for the evolution of delayed maturity usually invoke trade-offs mediated by growth, but processes of reproductive maturation continue long after growth has ceased. This study tested whether sexual selection shapes the rate of posteclosion maturation in the fruit fly Drosophila melanogaster. Populations maintained for more than 100 generations under a short generation time and polygamous mating system evolved faster posteclosion maturation and faster egg-to-adult development of males, when compared to populations kept under short generations and randomized monogamy that eliminated sexual selection. An independent assay demonstrated that more mature males have higher fitness under polygamy, but this advantage disappears under monogamy. In contrast, for females greater maturity was equally advantageous under polygamy and monogamy. Furthermore, monogamous populations evolved faster development and maturation of females relative to polygamous populations, with no detectable trade-offs with adult size or egg-to-adult survival. These results suggest that a major aspect of male maturation involves developing traits that increase success in sexual competition, whereas female maturation is not limited by investment in traits involved in mate choice or defense against male antagonism. Moreover, rates of juvenile development and adult maturation can readily evolve in opposite directions in the two sexes, possibly implicating polymorphisms with sexually antagonistic pleiotropy (Hollis, 2016).

    Sex-biased transcriptome divergence along a latitudinal gradient

    Sex-dependent gene expression is likely an important genomic mechanism that allows sex-specific adaptation to environmental changes. Among Drosophila species, sex-biased genes display remarkably consistent evolutionary patterns; male-biased genes evolve faster than unbiased genes in both coding sequence and expression level, suggesting sex differences in selection through time. However, comparatively little is known of the evolutionary process shaping sex-biased expression within species. Latitudinal clines offer an opportunity to examine how changes in key ecological parameters also influence sex-specific selection and the evolution of sex-biased gene expression. This study assayed male and female gene expression in Drosophila serrata along a latitudinal gradient in eastern Australia spanning most of its endemic distribution. Analysis of 11,631 genes across eight populations revealed strong sex differences in the frequency, mode and strength of divergence. Divergence was far stronger in males than females and while latitudinal clines were evident in both sexes, male divergence was often population specific, suggesting responses to localized selection pressures that do not covary predictably with latitude. While divergence was enriched for male-biased genes, there was no overrepresentation of X-linked genes in males. By contrast, X-linked divergence was elevated in females, especially for female-biased genes. Many genes that diverged in D. serrata have homologs also showing latitudinal divergence in Drosophila simulans and Drosophila melanogaster on other continents, likely indicating parallel adaptation in these distantly related species. These results suggest that sex differences in selection play an important role in shaping the evolution of gene expression over macro- and micro-ecological spatial scales (Allen, 2017).

    The large X-effect on secondary sexual characters and the genetics of variation in sex comb tooth number in Drosophila subobscura

    This study examined the genetics of a secondary sexual trait, male sex comb size in Drosophila subobscura, to evaluate the amount of variation attributable to the X-chromosome. This species bears unusually large sex combs for its species group, and therefore, this trait may be a good candidate for having been affected by natural or sexual selection. Significant heritable variation was observed in number of teeth of the distal sex comb across strains. While reciprocal F1 crosses seemed to implicate a disproportionate X-chromosome effect, further examination in the F2 progeny showed that transgressive autosomal effects inflated the estimate of variation associated with the X-chromosome in the F1. Instead, the X-chromosome appears to confer the smallest contribution of all major chromosomes to the observed phenotypic variation. Further, effects on copulation latency or duration associated with the observed phenotypic variation were not observed. Overall, this study presents an examination of the genetics underlying segregating phenotypic variation within species and illustrates two common pitfalls associated with some past studies of the genetic basis of secondary sexual traits (Mittleman, 2017).

    Direct benefits of choosing a high fitness mate can offset the indirect costs associated with intralocus sexual conflict

    Intralocus sexual conflict generates a cost to mate choice: high fitness partners transmit genetic variation that confers lower fitness to offspring of the opposite sex. Earlier work in the fruit fly, Drosophila melanogaster, revealed that these indirect genetic costs were sufficient to reverse potential "good genes" benefits of sexual selection. However, mate choice can also confer direct fitness benefits by inducing larger numbers of progeny. This study considers whether direct benefits through enhanced fertility could offset the costs associated with intralocus sexual conflict in D. melanogaster. Using hemiclonal analysis, it was found that females mated to high fitness males produced 11% more offspring compared to those mated to low fitness males, and high fitness females produced 37% more offspring than low fitness females. These direct benefits more than offset the reduction in offspring fitness caused by intralocus sexual conflict, creating a net fitness benefit for each sex to pairing with a high fitness partner. These findings highlight the need to consider both direct and indirect effects when investigating the fitness impacts of mate choice. Direct fitness benefits may shelter sexually antagonistic alleles from selection, suggesting a novel mechanism for the maintenance of fitness variation (Pischedda, 2017).

    Evolutionary dynamics of male reproductive genes in the Drosophila virilis subgroup

    Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and are important targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. This study utilized short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. The majority of male reproductive transcripts are testes-biased, accounting for ~15% of all annotated genes. Ejaculatory bulb-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melnaogaster ejaculatory bulb protein, Peb-me, which is involved in mating-plug formation. In addition, 71 candidate SFPs were identified, and this gene set was show to have the highest rate of nonsynonymous codon substitution relative to testes- and ejaculatory bulb-biased genes. Furthermore, orthologs were identified of 35 D. melanogaster SFPs that have conserved accessory gland expression in the virilis group. Finally, several of the SFPs that have the highest rate of nonsynonymous codon substitution were shown to reside on chromosomal regions that contribute to paternal gametic incompatibility between species. These results show that SFPs rapidly diversify in the virilis group, and suggest that they likely play a role in PCSS and/or gametic isolation (Ahmed-Braimah, 2017).

    Reproductive isolation through experimental manipulation of sexually antagonistic coevolution in Drosophila melanogaster

    Promiscuity can drive the evolution of sexual conflict before and after mating occurs. Post mating, the male ejaculate can selfishly manipulate female physiology, leading to a chemical arms race between the sexes. Theory suggests that drift and sexually antagonistic coevolution can cause allopatric populations to evolve different chemical interactions between the sexes, thereby leading to postmating reproductive barriers and speciation. There is, however, little empirical evidence supporting this form of speciation. This theory was tested by creating an experimental evolutionary model of Drosophila melanogaster populations undergoing different levels of interlocus sexual conflict. Allopatric populations under elevated sexual conflict were shown to exhibit assortative mating, indicating premating reproductive isolation. Further, these allopatric populations also show reduced copulation duration and sperm defense ability when mating happens between individuals across populations compared to that within the same population, indicating postmating prezygotic isolation. Sexual conflict can cause reproductive isolation in allopatric populations through the coevolution of chemical (postmating prezygotic) as well as behavioural (premating) interactions between the sexes. Thus it study provides the first comprehensive evidence of postmating (as well as premating) reproductive isolation due to sexual conflict (Syed, 2017).

    Male relatedness and familiarity are required to modulate male-induced harm to females in Drosophila

    Males compete over mating and fertilization, and often harm females in the process. Inclusive fitness theory predicts that increasing relatedness within groups of males may relax competition and discourage male harm of females as males gain indirect benefits. Recent studies in Drosophila melanogaster are consistent with these predictions, and have found that within-group male relatedness increases female fitness, though others have found no effects. Importantly, these studies did not fully disentangle male genetic relatedness from larval familiarity, so the extent to which modulation of harm to females is explained by male familiarity remains unclear. This study performed a fully factorial design, isolating the effects of male relatedness and larval familiarity on female harm. While no differences were found in male courtship or aggression, there was a significant interaction between male genetic relatedness and familiarity on female reproduction and survival. Relatedness among males increased female lifespan, reproductive lifespan and overall reproductive success, but only when males were familiar. By showing that both male relatedness and larval familiarity are required to modulate female harm, these findings reconcile previous studies, shedding light on the potential role of indirect fitness effects on sexual conflict and the mechanisms underpinning kin recognition in fly populations (Le Page, 2017).

    A species-specific multigene family mediates differential sperm displacement in Drosophila melanogaster

    Sperm competition is a postcopulatory selection mechanism in species in which females mate with multiple males. Despite its evolutionary relevance in shaping male traits, the genetic mechanisms underlying sperm competition are poorly understood. A recently originated multigene family specific to Drosophila melanogaster, Sdic, is important for the outcome of sperm competition in doubly mated females, although the mechanistic nature of this phenotype remained unresolved. This study compared doubly mated females, second mated to either Sdic knockout or nonknockout males, and directly visualize sperm dynamics in the female reproductive tract. A less effective removal of first-to-mate male's sperm within the female's sperm storage organs is consistent with a reduced sperm competitive ability of the Sdic knockout males. These results highlight the role young genes can play in driving the evolution of sperm competition (Jayaswal, 2018).

    Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

    Competition between males creates potential for pre- and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. This study evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. In a setting where males competed directly with a rival male for access to a female and fertilization of her ova, polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, no difference was found in male sperm defense competitiveness between the different selection regimes. Instead, monogamous males were found to be inferior in precopulatory competition, which indicates that in this system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. These findings are discussed in the context of findings from previous experimental evolution studies in Drosophila and other invertebrate species (Wensing, 2017).

    Evolving doublesex expression correlates with the origin and diversification of male sexual ornaments in the Drosophila immigrans species group

    Male ornaments and other sex-specific traits present some of the most dramatic examples of evolutionary innovations. Comparative studies of similar but independently evolved traits are particularly important for identifying repeated patterns in the evolution of these traits. Male-specific modifications of the front legs have evolved repeatedly in Drosophilidae and other Diptera. The best understood of these novel structures is the sex comb of Drosophila melanogaster and its close relatives. This study examined the evolution of another male foreleg modification, the sex brush, found in the distantly related Drosophila immigrans species group. Similar to the sex comb, it was found that the origin of the sex brush correlates with novel, spatially restricted expression of the doublesex (dsx) transcription factor, the primary effector of the Drosophila sex determination pathway. The diversity of Dsx expression patterns in the immigrans species group closely reflects the differences in the presence, position, and size of the sex brush. Together with previous work on sex comb evolution, these observations suggest that tissue-specific activation of dsx expression may be a common mechanism responsible for the evolution of sexual dimorphism and particularly for the origin of novel male-specific ornaments (Rice, 2018).

    Evolutionary divergence in competitive mating success through female mating bias for good genes

    Despite heritable variation for univariate sexually selected traits, recent analyses exploring multivariate traits find evidence consistent with the lek paradox in showing no genetic variation available to choosy females, and therefore no genetic benefits of choice. This study used the preferences of Drosophila melanogaster females to exert bidirectional selection on competitive male mating success to test for the presence and nature of genetic variation underlying this multivariate trait. Male mating success diverged between selection regimens, and flies from success-selected lines had a smaller burden of deleterious, recessive mutations that affect egg-to-adult viability, were better sperm competitors (sperm offence), and did not demonstrate reduced desiccation resistance or components of female fitness (traits thought to trade off with attractiveness) relative to flies from failure-selected populations. Mating success remained subject to inbreeding depression in success-selected lines, suggesting that variation in mating success remains, thanks to numerous genes of small effect. Together, these results provide unique evidence for the evolutionary divergence in male mating success, demonstrating that genetic variation is not exhausted along the axis of precopulatory sexual selection and that female mating biases align with the avoidance of bad genes (Dugand, 2018).

    Evolution of sex-biased gene expression and dosage compensation in the eye and brain of Heliconius butterflies

    Differences in behavior and life history traits between females and males are the basis of divergent selective pressures between sexes. It has been suggested that a way for the two sexes to deal with different life history requirements is through sex-biased gene expression. A comparative sex-biased gene expression analysis was performed of the combined eye and brain transcriptome from five Heliconius species, H. charithonia, H. sara, H. erato, H. melpomene and H. doris, representing five of the main clades from the Heliconius phylogeny. The degree of sexual dimorphism in gene expression is not conserved across Heliconius. Most of the sex-biased genes identified in each species are not sex-biased in any other, suggesting that sexual selection might have driven sexually dimorphic gene expression. Only three genes shared sex-biased expression across multiple species: ultraviolet opsin UVRh1 and orthologs of Drosophila Kruppel-homolog 1 and CG9492. It was also observed that in some species female-biased genes have higher evolutionary rates, but in others, male-biased genes show the fastest rates when compared with unbiased genes, suggesting that selective forces driving sex-biased gene evolution in Heliconius act in a sex- and species-specific manner. Furthermore, dosage compensation was found in all the Heliconius tested, providing additional evidence for the conservation of dosage compensation across Lepidoptera. Finally, sex-biased genes are significantly enriched on the Z, a pattern that could be a result of sexually antagonistic selection (Catalan, 2018).

    Evolution of a central neural circuit underlies Drosophila mate preferences

    Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. This study compared pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. Males of both species were shown to detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. These results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution (Seeholzer, 2018).

    The sensory periphery has been proposed to be the most evolutionarily labile element of the nervous system, as changes in the expression or tuning of sensory receptors can allow for the emergence of species-specific behaviours without necessitating potentially more complex developmental rewiring of central pathways in the brain. By contrast, the current results suggest that species-specific behavioural responses to 7,11-HD arise through functional alterations in how pheromone signals are propagated through a structurally conserved central circuit that consists of parallel excitatory and feedforward inhibitory branches. By reweighting the balance of excitatory vAB3 and inhibitory mAL signalling to P1 neurons, 7,11-HD is transformed from an excitatory signal that promotes courtship in D. melanogaster males into an inhibitory signal that suppresses courtship in D. simulans males. Although the analysis highlights the importance of this pheromone pathway in shaping species-specific mate preferences, the possibility cannot be excluded that additional inputs to P1 neurons or other targets of mAL and vAB3 neurons also contribute to divergent courtship decisions. Nevertheless, the data suggest that the branched architecture of pheromone-processing pathways serves as a substrate for the evolution of mate preferences, pointing to the potential existence of favourable sites within neural circuits to instantiate adaptive behavioural changes, analogous to how specific nodes within developmental regulatory networks contribute to morphological diversity (Seeholzer, 2018).

    The conserved tuning of Ppk23+ neurons suggests that D. simulans males dedicate this sensory pathway to the detection of a female pheromones, rather than sensing the chemical cues carried by conspecific females. Consistent with this idea, although P1 neurons are sufficient to elicit courtship in D. simulans males, they are not excited by the taste of a D. simulans female. Given that D. simulans cuticular pheromones are sexually monomorphic and offer ambiguous signals for mate recognition, males probably rely on additional sensory inputs for their arousal. Indeed, the fervent courtship exhibited by D. simulans ppk23 mutants towards females of different species demonstrates that D. simulans males can be aroused in the absence of any species-specific excitatory cue. Dienes, such as 7,11-HD, actively suppress this arousal, presumably through recruitment of strong mAL-mediated inhibition via Ppk23+ pathways. Together, these observations reinforce the notion that pheromone communication in Drosophila serves to focus a male's desire, such that flies lacking cuticular pheromones can be inherently attractive and appropriate mate choices are honed by specific inhibitory chemical cues (Seeholzer, 2018).

    Peripheral adaptations are likely to play an important role in the evolution of novel chemical sensitivities. In this case, however, preserving the sensory periphery while varying central circuits provides a mechanism to alter the behavioural valence of a single chemical cue. As D. melanogaster and D. simulans diverged, their reproductive isolation was probably strengthened by the ability of both species to detect the same pheromone but assign it a different meaning through these central circuit modifications. Notably, in D. melanogaster, P1 neuron excitability is regulated by the social history of a male. This suggests that both experience-dependent and evolutionary adaptations may act on the same neural substrate to modify sensory integration and mate choices, similar to how phenotypic plasticity may facilitate morphological evolution35. Therefore, functional reweighting of sensory inputs at flexible nodes in the nervous system, shaped by evolutionary selection or individual experience, may allow for alternative behavioural responses to the same sensory signal (Seeholzer, 2018).

    Predicting the evolution of sexual dimorphism in gene expression

    Sexual dimorphism in gene expression is likely to be the underlying source of dimorphism in a variety of traits. Many analyses implicitly make the assumption that dimorphism only evolves when selection favors different phenotypes in the two sexes, although theory makes clear that it can also evolve as an indirect response to other kinds of selection. Furthermore, previous analyses consider the evolution of a single transcript or trait at a time, ignoring the genetic covariance with other transcripts and traits. This study first shows which aspects of the genetic-variance covariance matrix, G, affect dimorphism when these assumptions about selection are relaxed. Gene expression data from Drosophila melanogaster were analyzed with these predictions in mind. Dimorphism of gene expression for individual transcripts shows the signature of both direct selection for dimorphism and indirect responses to selection. To account for the effect of measurement error on evolutionary predictions, a G matrix was estimated for eight linear combinations of expression traits. Sex-specific genetic variances in female- and male-biased transcription, as well as one relatively unbiased combination, were quite unequal, ensuring that most forms of selection on these traits will have large effects on dimorphism. Predictions of response to selection based on the whole G matrix showed that sexually concordant and antagonistic selection are equally capable of changing sexual dimorphism. In addition, the indirect responses of dimorphism due to cross-trait covariances were quite substantial. The assumption that sexual dimorphism in transcription is an adaptation could be incorrect in many specific cases (Houle, 2021).

    The old and the new: discovery proteomics identifies putative novel seminal fluid proteins in Drosophila

    Seminal fluid proteins (SFPs), the non-sperm component of male ejaculates produced by male accessory glands, are viewed as central mediators of reproductive fitness. SFPs effect both male and female post-mating functions and show molecular signatures of rapid adaptive evolution. While SFP identification was historically challenging, advances in label-free quantitative proteomics expands the scope of studying other systems to further advance the field. This study applied label-free quantitative proteomics to identify the accessory gland proteome and secretome in Drosophila pseudoobscura, a close relative of D. melanogaster, and use the dataset to identify both known and putative novel SFPs. Using this approach 163 putative SFPs were identified, 32% of which overlapped with previously identified D. melanogaster SFPs. SFPs with known extracellular annotation evolve more rapidly than other proteins produced by or contained within the accessory gland. These results will further the understanding of the evolution of SFPs and the underlying male accessory gland proteins that mediate reproductive fitness of the sexes (Karr, 2019).

    Males from populations with higher competitive mating success produce sons with lower fitness

    Female mate choice can result in direct benefits to the female or indirect benefits through her offspring. Females can increase their fitness by mating with males whose genes encode increased survivorship and reproductive output. Alternatively, male investment in enhanced mating success may come at the cost of reduced investment in offspring fitness. This study measures male mating success in a mating arena that allows for male-male, male-female and female-female interactions in Drosophila melanogaster. Isofemale line population measurements were used to correlate male mating success with sperm competitive ability, the number of offspring produced and the indirect benefits of the number of offspring produced by daughters and sons. This study finds that males from populations that gain more copulations do not increase female fitness through increased offspring production, nor do these males fare better in sperm competition. Instead, it was found that these populations have a reduced reproductive output of sons, indicating a potential reproductive trade-off between male mating success and offspring quality (Nguyen, 2019).

    Conspecific sperm precedence is reinforced, but postcopulatory sexual selection weakened, in sympatric populations of Drosophila

    Sexual selection can accelerate speciation by driving the evolution of reproductive isolation, but forces driving speciation could also reciprocally feedback on sexual selection. This might be particularly important in the context of 'reinforcement', where selection acts directly to increase prezygotic barriers to reduce the cost of heterospecific matings. Using assays of sperm competition within and between two sister species, a signature of reinforcement is shown where these species interact: populations of Drosophila pseudoobscura that co-occur with sister species D. persimilis have an elevated ability to outcompete heterospecific sperm, consistent with selection for increased postcopulatory isolation. These D. pseudoobscura populations have decreased sperm competitive ability against conspecifics, reducing the opportunity for sexual selection within these populations. These findings demonstrate that direct selection to increase reproductive isolation against other species can compromise the efficacy of sexual selection within species, a collateral effect of reproductive traits responding to heterospecific interactions (Castillo, 2019).

    Selection shapes turnover and magnitude of sex-biased expression in Drosophila gonads

    Sex-biased gene expression is thought to drive the phenotypic differences in males and females in metazoans. Research on sex organs in Drosophila, employing original approaches and multiple-species contrasts, provides a means to gain insights into factors shaping the turnover and magnitude (fold-bias) of sex-biased expression. Using recent RNA-seq data, sex-biased gonadal expression in 10,740 protein coding sequences were examined in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. ananassae (5 to 44 My divergence). Using an approach wherein genes were identified with lineage-specific transitions (LSTs) in sex-biased status (amongst testis-biased, ovary-biased and unbiased; thus, six transition types) standardized to the number of genes with the ancestral state (S-LSTs), and those with clade-wide expression bias status, several key findings were revealed. First, the six categorical types of S-LSTs in sex-bias showed disparate rates of turnover, consistent with differential selection pressures. Second, the turnover in sex-biased status was largely unrelated to cross-tissue expression breadth, suggesting pleiotropy does not restrict evolution of sex-biased expression. Third, the fold-sex-biased expression, for both testis-biased and ovary-biased genes, evolved directionally over time toward higher values, a crucial finding that could be interpreted as a selective advantage of greater sex-bias, and sexual antagonism. Fourth, in terms of protein divergence, genes with LSTs to testis-biased expression exhibited weak signals of elevated rates of evolution (than ovary-biased) in as little as 5 My, which strengthened over time. Moreover, genes with clade-wide testis-specific expression (44 My), a status not observed for any ovary-biased genes, exhibited striking acceleration of protein divergence, which was linked to low pleiotropy. By studying LSTs and clade-wide sex-biased gonadal expression in a multi-species clade of Drosophila, this study describes evidence that interspecies turnover and magnitude of sex-biased expression have been influenced by selection. Further, whilst pleiotropy was not connected to turnover in sex-biased gonadal expression, it likely explains protein sequence divergence (Whittle, 2019b).

    Cis- and trans-acting variants contribute to survivorship in a naive Drosophila melanogaster population exposed to ryanoid insecticides

    Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster. This study used a naive population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which was validated with linkage mapping and transgenic manipulation of gene expression. It is proposed that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naive to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals (Green, 2019).

    Genome-wide sexually antagonistic variants reveal long-standing constraints on sexual dimorphism in fruit flies

    The evolution of sexual dimorphism is constrained by a shared genome, leading to 'sexual antagonism', in which different alleles at given loci are favoured by selection in males and females. Despite its wide taxonomic incidence, little is known about the identity, genomic location, and evolutionary dynamics of antagonistic genetic variants. To address these deficits, this study used sex-specific fitness data from 202 fully sequenced hemiclonal Drosophila melanogaster fly lines to perform a genome-wide association study (GWAS) of sexual antagonism. Approximately 230 chromosomal clusters of candidate antagonistic single nucleotide polymorphisms (SNPs) were identified. In contradiction to classic theory, no clear evidence was found that the X chromosome is a hot spot for sexually antagonistic variation. Characterising antagonistic SNPs functionally, a large excess of missense variants were found, but there was very little enrichment in terms of gene function. The evolutionary persistence of antagonistic variants was also assessed by examining extant polymorphism in wild D. melanogaster populations and closely related species. Remarkably, antagonistic variants are associated with multiple signatures of balancing selection across the D. melanogaster distribution range and in their sister species D. simulans, indicating widespread and evolutionarily persistent (about 1 million years) genomic constraints on the evolution of sexual dimorphism. Based on these results, it is proposed that antagonistic variation accumulates because of constraints on the resolution of sexual conflict over protein coding sequences, thus contributing to the long-term maintenance of heritable fitness variation (Ruzicka, 2019).

    Testing for local adaptation in adult male and female fitness among populations evolved under different mate competition regimes

    Mating/fertilization success and fecundity are influenced by sexual interactions among individuals, the nature and frequency of which can vary among different environments. The extent of local adaptation for such adult fitness components is poorly understood. This study allowed 63 populations of Drosophila melanogaster to independently evolve in one of three mating environments that alter sexual interactions: one involved enforced monogamy, while the other two permitted polygamy in either structurally simple standard fly vials or in larger "cages" with added complexity. Adult male and female reproductive fitness were measured after 16 and 28 generations, respectively, via full reciprocal transplants. In males, reciprocal local adaptation was observed between the monogamy and simple polygamy treatments, consistent with the evolution of reproductively competitive males under polygamy that perform poorly under monogamy because they harm their only mate. However, males evolved in the complex polygamy treatment performed similarly or better than all other males in all mating environments, consistent with previous results showing higher genetic quality in this treatment. Differences in female fitness were more muted, suggesting selection on females was less divergent across the mating treatments and echoing a common pattern of greater phenotypic and expression divergence in males than females (Testing for local adaptation in adult male and female fitness among populations evolved under different mate competition regimes (Yun, 2019).

    Sexual selection drives the evolution of male wing interference patterns

    The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. This study used multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection (Hawkes, 2019).

    Evolution of mechanisms that control mating in Drosophila males

    Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, this study found that D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Together, this study have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.

    tartan underlies the evolution of Drosophila male genital morphology

    Male genital structures are among the most rapidly evolving morphological traits and are often the only features that can distinguish closely related species. This process is thought to be driven by sexual selection and may reinforce species separation. However, while the genetic bases of many phenotypic differences have been identified, knowledge about the genes underlying evolutionary differences in male genital organs and organ size more generally is still lacking. The claspers (surstyli) are periphallic structures that play an important role in copulation in insects. This study shows that divergence in clasper size and bristle number between Drosophila mauritiana and Drosophila simulans is caused by evolutionary changes in tartan (trn), which encodes a transmembrane leucine-rich repeat domain protein that mediates cell-cell interactions and affinity. There are no fixed amino acid differences in trn between D. mauritiana and D. simulans, but differences in the expression of this gene in developing genitalia suggest that cis-regulatory changes in trn underlie the evolution of clasper morphology in these species. Finally, analyses of reciprocal hemizygotes that are genetically identical, except for the species from which the functional allele of trn originates, determined that the trn allele of D. mauritiana specifies larger claspers with more bristles than the allele of D. simulans. Therefore, this study has identified a gene underlying evolutionary change in the size of a male genital organ, which will help to better understand not only the rapid diversification of these structures, but also the regulation and evolution of organ size more broadly (Hagen, 2019).

    Unravelling the genetic basis for the rapid diversification of male genitalia between Drosophila species

    In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, there is only a limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. This study used new D. simulans/D. mauritiana introgression lines on chromosome 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. RNA-seq was carried out on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed testing of the function of expressed positional candidates during genital development in D. melanogaster. Several new genes were found to be involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, it was discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, these results provide new insights into the regulation of genital development and how this has evolved between species (Hagen, 2020).

    No evidence of positive assortative mating for genetic quality in fruit flies

    In sexual populations, the effectiveness of selection will depend on how gametes combine with respect to genetic quality. If gametes with deleterious alleles are likely to combine with one another, deleterious genetic variation can be more easily purged by selection. Assortative mating, where there is a positive correlation between parents in a phenotype of interest such as body size, is often observed in nature, but does not necessarily reveal how gametes ultimately combine with respect to genetic quality itself. This study manipulated genetic quality in fruit fly populations using an inbreeding scheme designed to provide an unbiased measure of mating patterns. While inbred flies had substantially reduced reproductive success, their gametes did not combine with those of other inbred flies more often than expected by chance, indicating a lack of positive assortative mating. Instead, a negative correlation was detected in genetic quality between parents, i.e. disassortative mating, which diminished with age. This pattern is expected to reduce the genetic variance for fitness, diminishing the effectiveness of selection. How mechanisms of sexual selection could produce a pattern of disassortative mating is discussed. This study highlights that sexual selection has the potential to either increase or decrease genetic load (Sharp, 2019).

    Mechanisms of resistance to pathogens and parasites are thought to be costly and thus to lead to evolutionary trade-offs between resistance and life-history traits expressed in the absence of the infective agents. On the other hand, sexually selected traits are often proposed to indicate "good genes" for resistance, which implies a positive genetic correlation between resistance and success in sexual selection. This study shows that experimental evolution of improved resistance to the intestinal pathogen Pseudomonas entomophila in Drosophila melanogaster was associated with a reduction in male sexual success. Males from four resistant populations achieved lower paternity than males from four susceptible control populations in competition with males from a competitor strain, indicating an evolutionary cost of resistance in terms of mating success and/or sperm competition. In contrast, no costs were found in larval viability, larval competitive ability and population productivity assayed under nutritional limitation; together with earlier studies this suggests that the costs of P. entomophila resistance for nonsexual fitness components are negligible. Thus, rather than indicating heritable pathogen resistance, sexually selected traits expressed in the absence of pathogens may be sensitive to costs of resistance, even if no such costs are detected in other fitness traits (Kawecki, 2019).

    Evolution of sexually dimorphic pheromone profiles coincides with increased number of male-specific chemosensory organs in Drosophila prolongata

    Binary communication systems that involve sex-specific signaling and sex-specific signal perception play a key role in sexual selection and in the evolution of sexually dimorphic traits. The driving forces and genetic changes underlying such traits can be investigated in systems where sex-specific signaling and perception have emerged recently and show evidence of potential coevolution. A promising model is found in Drosophila prolongata, which exhibits a species-specific increase in the number of male chemosensory bristles. This transition is shown to coincides with recent evolutionary changes in cuticular hydrocarbon (CHC) profiles. Long-chain CHCs that are sexually monomorphic in the closest relatives of D. prolongata (D. rhopaloa, D. carrolli, D. kurseongensis, and D. fuyamai) are strongly male-biased in this species. An intraspecific female-limited polymorphism, where some females have male-like CHC profiles, was also identifid. Both the origin of sexually dimorphic CHC profiles and the female-limited polymorphism in D. prolongata involve changes in the relative amounts of three mono-alkene homologs, 9-tricosene, 9-pentacosene, and 9-heptacosene, all of which share a common biosynthetic origin and point to a potentially simple genetic change underlying these traits. These results suggest that pheromone synthesis may have coevolved with chemosensory perception and open the way for reconstructing the origin of sexual dimorphism in this communication system (Luo, 2019).

    Sexual Selection Does Not Increase the Rate of Compensatory Adaptation to a Mutation Influencing a Secondary Sexual Trait in Drosophila melanogaster

    Theoretical work predicts that sexual selection can enhance natural selection, increasing the rate of adaptation to new environments and helping purge harmful mutations. While some experiments support these predictions, remarkably little work has addressed the role of sexual selection on compensatory adaptation-populations' ability to compensate for the costs of deleterious alleles that are already present. This study tested whether sexual selection, as well as the degree of standing genetic variation, affect the rate of compensatory evolution via phenotypic suppression in experimental populations of Drosophila melanogaster. These populations were fixed for a spontaneous mutation causing mild abnormalities in the male sex comb, a structure important for mating success.This mutation was mapped to an ~85 kb region on the X chromosome containing three candidate genes, showed that the mutation is deleterious, and that its phenotypic expression and penetrance vary by genetic background. Experimental evolution was then performed, including a treatment where opportunity for mate choice was limited by experimentally enforced monogamy. Although evolved populations did show some phenotypic suppression of the morphological abnormalities in the sex comb, the amount of suppression did not depend on the opportunity for sexual selection. Sexual selection, therefore, may not always enhance natural selection; instead, the interaction between these two forces may depend on additional factors (Chandler, 2020).

    Co-evolving wing spots and mating displays are genetically separable traits in Drosophila

    The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, this study investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and Drosophila gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), an ~440 kb region on the X chromosome was identified that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed that the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior (Massey, 2020).

    Feminization of complex traits in Drosophila melanogaster via female-limited X chromosome evolution

    A handful of studies have investigated sexually antagonistic constraints on achieving sex-specific fitness optima, although exclusively through male-genome-limited evolution experiments. This article has established a female-limited X chromosome evolution experiment, where an X chromosome balancer was used to enforce the inheritance of the X through the matriline, thus removing exposure to male selective constraints. This approach eliminates the effects of sexually antagonistic selection on the X chromosome, permitting evolution toward a single sex-specific optimum. After multiple generations of selection, strong evidence was found that body size and development time had moved toward a female-specific optimum, whereas reproductive fitness and locomotion activity remained unchanged. The changes in body size and development time are consistent with previous results, and suggest that the X chromosome is enriched for sexually antagonistic genetic variation controlling these particular traits. The lack of change in reproductive fitness and locomotion activity could be due to a number of mutually nonexclusive explanations, including a lack of sexually antagonistic variance on the X chromosome for those traits or confounding effects of the use of the balancer chromosome. This study is the first to employ female-genome-limited selection and adds to the understanding of the complexity of sexually antagonistic genetic variation (Lund-Hansen, 2020)

    Positive genetic covariance between male sexual ornamentation and fertilizing capacity

    Postcopulatory sexual selection results from variation in competitive fertilization success among males and comprises powerful evolutionary forces that operate after the onset of mating. Theoretical advances in the field of sexual selection addressing the buildup and coevolutionary consequences of genetic coupling motivate the hypothesis that indirect postcopulatory sexual selection may promote evolution of male secondary sexual traits-those traits traditionally ascribed to mate choice and male fighting. A crucial prediction of this hypothesis is genetic covariance between trait expression and competitive fertilization success, which has been predicted to arise, for example, when traits subject to pre- and postcopulatory sexual selection are under positive correlational selection. This study imposed bidirectional artificial selection on male ornament (sex comb) size in Drosophila bipectinata and demonstrated increased competitive fertilization success as a correlated evolutionary response to increasing ornament size. Transcriptional analyses revealed that levels of specific seminal fluid proteins repeatedly shifted in response to this selection, suggesting that properties of the ejaculate, rather than the enlarged sex comb itself, contributed fertilizing capacity. Ultraprecise laser surgery was used to reduce ornament size of high-line males and found that their fertilizing superiority persisted despite the size reduction, reinforcing the transcriptional results. The data support the existence of positive genetic covariance between a male secondary sexual trait and competitive fertilization success, and suggest the possibility that indirect postcopulatory sexual selection may, under certain conditions, magnify net selection on ornamental trait expression (Polak, 2021).

    Nonadaptive molecular evolution of seminal fluid proteins in Drosophila

    Seminal fluid proteins (SFPs) are a group of reproductive proteins that are among the most evolutionarily divergent known. As SFPs can impact male and female fitness, these proteins have been proposed to evolve under postcopulatory sexual selection (PCSS). However, the fast change of the SFPs can also result from nonadaptive evolution, and the extent to which selective constraints prevent SFPs rapid evolution remains unknown. Using intra- and interspecific sequence information, along with genomics and functional data, this study examine the molecular evolution of approximately 300 SFPs in Drosophila. It was found that 50-57% of the SFP genes, depending on the population examined, are evolving under relaxed selection. Only 7-12% showed evidence of positive selection, with no evidence supporting other forms of PCSS, and 35-37% of the SFP genes were selectively constrained. Further, despite associations of positive selection with gene location on the X chromosome and protease activity, the analysis of additional genomic and functional features revealed their lack of influence on SFPs evolving under positive selection. These results highlight a lack of sufficient evidence to claim that most SFPs are driven to evolve rapidly by PCSS while identifying genomic and functional attributes that influence different modes of SFPs evolution (Patlar, 2021).

    Repeated evidence that the accelerated evolution of sperm is associated with their fertilization function

    Spermatozoa are the most morphologically diverse cell type, leading to the widespread assumption that they evolve rapidly. However, there is no direct evidence that sperm evolve faster than other male traits. Such a test requires comparing male traits that operate in the same selective environment, ideally produced from the same tissue, yet vary in function. This study examined rates of phenotypic evolution in sperm morphology using two insect groups where males produce fertile and non-fertile sperm types (Drosophila species from the obscura group and a subset of Lepidoptera species), where these constraints are solved. Moreover, in Drosophila, the relationship between rates of sperm evolution and the link with the putative selective pressures of fertilization function and postcopulatory sexual selection exerted by female reproductive organs were tested. Repeated evolutionary patterns were found across these insect groups-lengths of fertile sperm evolve faster than non-fertile sperm. In Drosophila, fertile sperm length evolved faster than body size, but at the same rate as female reproductive organ length. Rates of evolution of different sperm components were also compared, showing that head length evolves faster in fertile sperm while flagellum length evolves faster in non-fertile sperm. This study provides direct evidence that sperm length evolves more rapidly in fertile sperm, probably because of their functional role in securing male fertility and in response to selection imposed by female reproductive organs (Fitzpatrick, 2020).

    Divergence of responses to variable socio-sexual environments in laboratory populations of Drosophila melanogaster evolving under altered operational sex ratios

    Post-copulatory sexual selection (PSS) is an important selective force that determines fitness in polyandrous species. PSS can be intense in some cases and can drive the evolution of remarkable ejaculate properties. In males, investment in ejaculate plays an important role in the outcome of PSS. Thus, males are expected to adaptively tailor their ejaculate according to the perceived competition in their vicinity. Plastic responses in ejaculate investment to variation in intrasexual competition are disparate and widespread in males. This study investigated the evolution of plasticity in reproductive traits using Drosophila melanogaster populations evolving for more than 150 generations under male- or female-biased sex ratios. When exposed to different numbers of competitors early in their life, males from these two regimes responded differently in terms of their copulation duration and sperm competitive ability. In addition, the effect of this early life experience wore off at different rates in males of male-biased and female-biased regimes with increasing time from the removal of competitive cues. Furthermore, this study finds that males change their reproductive strategies depending upon the identity of rival males. Together, these results provide evidence of the evolution of male reproductive investment that depends on socio-sexual cues experienced early in life (Maggu, 2020).

    The Drosophila seminal proteome and its role in postcopulatory sexual selection

    Postcopulatory sexual selection (PCSS), comprised of sperm competition and cryptic female choice, has emerged as a widespread evolutionary force among polyandrous animals. There is abundant evidence that PCSS can shape the evolution of sperm. However, sperm are not the whole story: they are accompanied by seminal fluid substances that play many roles, including influencing PCSS. Foremost among seminal fluid models is Drosophila melanogaster, which displays ubiquitous polyandry, and exhibits intraspecific variation in a number of seminal fluid proteins (Sfps) that appear to modulate paternity share. This study first consolidated current information on the identities of D. melanogaster Sfps. Comparing between D. melanogaster and human seminal proteomes, evidence is found of similarities between many protein classes and individual proteins, including some D. melanogaster Sfp genes linked to PCSS, suggesting evolutionary conservation of broad-scale functions. Experimental evidence for the functions of D. melanogaster Sfps in PCSS and sexual conflict is reviewed. Gaps are identified in current knowledge and areas for future research, including an enhanced identification of PCSS-related Sfps, their interactions with rival sperm and with females, the role of qualitative changes in Sfps and mechanisms of ejaculate tailoring. This article is part of the theme issue 'Fifty years of sperm competition' (Wigby, 2020).

    Female-limited X-chromosome evolution effects on male pre- and post-copulatory success

    Intralocus sexual conflict arises when the expression of shared alleles at a single locus generates opposite fitness effects in each sex (i.e. sexually antagonistic alleles), preventing each sex from reaching its sex-specific optimum. Despite its importance to reproductive success, the relative contribution of intralocus sexual conflict to male pre- and post-copulatory success is not well-understood. This study used a female-limited X-chromosome (FLX) evolution experiment in Drosophila melanogaster to limit the inheritance of the X-chromosome to the matriline, eliminating possible counter-selection in males and allowing the X-chromosome to accumulate female-benefit alleles. After more than 100 generations of FLX evolution, the effect of the evolved X-chromosome on male attractiveness and sperm competitiveness was studied. A non-significant increase was found in attractiveness and decrease was found in sperm offence ability in males expressing the evolved X-chromosomes, but a significant increase in their ability to avoid displacement by other males' sperm. This is consistent with a trade-off between these traits, perhaps mediated by differences in body size, causing a small net reduction in overall male fitness in the FLX lines. These results indicate that the X-chromosome in D. melanogaster is subject to selection via intralocus sexual conflict in males (Manat, 2021).

    Sexually antagonistic coevolution between the sex chromosomes of Drosophila melanogaster

    Antagonistic interactions between the sexes are important drivers of evolutionary divergence. Interlocus sexual conflict is generally described as a conflict between alleles at two interacting loci whose identity and genomic location are arbitrary, but with opposite fitness effects in each sex. This study builds on previous theory by suggesting that when loci under interlocus sexual conflict are located on the sex chromosomes it can lead to cycles of antagonistic coevolution between them and therefore between the sexes. This hypothesis was tested by performing experimental crosses using Drosophila melanogaster where the sex chromosomes was reciprocally exchanged between five allopatric wild-type populations in a round-robin design. Disrupting putatively coevolved sex chromosome pairs resulted in increased male reproductive success in 16 of 20 experimental populations (10 of which were individually significant), but also resulted in lower offspring egg-to-adult viability that affected both male and female fitness. After 25 generations of experimental evolution these sexually antagonistic fitness effects appeared to be resolved. To formalize the hypothesis, population genetic models were developed of antagonistic coevolution using fitness expressions based on the empirical results. The model predictions support the conclusion that antagonistic coevolution between the sex chromosomes is plausible under the fitness effects observed in these experiments. Together, these results lend both empirical and theoretical support to the idea that cycles of antagonistic coevolution can occur between sex chromosomes and illustrate how this process, in combination with autosomal coadaptation, may drive genetic and phenotypic divergence between populations (Lund-Hansen, 2021).

    Individual and synergistic effects of male external genital traits in sexual selection

    Male genital traits exhibit extraordinary interspecific phenotypic variation. This remarkable and general evolutionary trend is widely considered to be the result of sexual selection. However, a good understanding of whether or how individual genital traits function in different competitive arenas (episodes of sexual selection), or how different genital traits may interact to influence competitive outcomes, is still not available. This study used an experimental approach based on high-precision laser phenotypic engineering to address these outstanding questions, focusing on three distinct sets of micron-scale external (nonintromittent) genital spines in male Drosophila kikkawai Burla (Diptera: Drosophilidae). Elimination of the large pair of spines on the male secondary claspers sharply reduced male ability to copulate, yet elimination of the other sets of spines on the primary and secondary claspers had no significant effects on copulation probability. Intriguingly, both the large spines on the secondary claspers and the cluster of spines on the primary claspers were found to independently promote male competitive fertilization success. Moreover, when large and small secondary clasper spines were simultaneously shortened in individual males, these males suffered greater reductions in fertilization success relative to males whose traits were altered individually, providing evidence for synergistic effects of external genital traits on fertilization success. Overall, the results are significant in demonstrating that a given genital trait (the large spines on the secondary claspers) can function in different episodes of sexual selection, and distinct genital traits may interact in sexual selection. The results offer an important contribution to evolutionary biology by demonstrating an understudied selective mechanism, operating via subtle trait interactions in a post-insemination context, by which genital traits may be co-evolving (Rodriguez-Exposito, 2019).

    Phylogenomic Insights into the Evolution of Stinging Wasps and the Origins of Ants and Bees

    The stinging wasps (Hymenoptera: Aculeata) are an extremely diverse lineage of hymenopteran insects, encompassing over 70,000 described species and a diversity of life history traits, including ectoparasitism, cleptoparasitism, predation, pollen feeding (bees [Anthophila] and Masarinae), and eusociality (social vespid wasps, ants, and some bees). The most well-studied lineages of Aculeata are the ants, which are ecologically dominant in most terrestrial ecosystems, and the bees, the most important lineage of angiosperm-pollinating insects. Establishing the phylogenetic affinities of ants and bees helps in understanding and reconstruction of patterns of social evolution as well as leading to full appreciation of the biological implications of the switch from carnivory to pollen feeding (pollenivory). Despite recent advancements in aculeate phylogeny, considerable uncertainty remains regarding higher-level relationships within Aculeata, including the phylogenetic affinities of ants and bees. Ultraconserved element (UCE) phylogenomics was used to resolve relationships among stinging-wasp families, gathering sequence data from >800 UCE loci and 187 samples, including 30 out of 31 aculeate families. The 187-taxon dataset was analyzed using multiple analytical approaches, and several alternative taxon sets were analyzed. Alternative hypotheses for the phylogenetic positions of ants and bees were tested. The results present a highly supported phylogeny of the stinging wasps. Most importantly, it was found unequivocal evidence that ants are the sister group to bees+apoid wasps (Apoidea) and that bees are nested within a paraphyletic Crabronidae. It was also demonstrated that taxon choice can fundamentally impact tree topology and clade support in phylogenomic inference (Branstetter, 2017).

    Interactions between the developmental and adult social environments mediate group dynamics and offspring traits in Drosophila melanogaster

    Developmental conditions can strongly influence adult phenotypes and social interactions, which in turn affect key evolutionary processes such as sexual selection and sexual conflict. While the implications of social interactions in phenotypically mixed populations at the individual level are increasingly well known, how these effects influence the fate of groups remains poorly understood, which limits understanding of the broader ecological implications. To address this problem this study manipulated adult phenotypes and social composition in Drosophila melanogaster - by experimentally manipulating the larval density of the group-members - and measured a range of group-level outcomes across the lifespan of groups. Adult groups composed of exclusively low larval-density individuals showed high courtship levels, and low early reproductive rates, group growth rates, offspring mass and offspring eclosion success, relative to high larval-density or mixed larval-density groups. Furthermore, high larval-density groups had lower survival. Offspring mass increased with time, but at a reduced rate in groups when male group members (but not females) were from a mixture of larval-densities; peak reproductive rates were also earlier in these groups. These results suggest that that variation in developmental conditions experienced by adult group members can modify the reproductive output of groups (Morimoto, 2017).

    Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies

    For social animals, the genotypes of group members affect the social environment, and thus individual behavior, often indirectly. This study used genome-wide association studies (GWAS) to determine the influence of individual vs. group genotypes on aggression in honey bees. Aggression in honey bees arises from the coordinated actions of colony members, primarily nonreproductive "soldier" bees, and thus, experiences evolutionary selection at the colony level. This study shows that individual behavior is influenced by colony environment, which in turn, is shaped by allele frequency within colonies. Using a population with a range of aggression, individual whole genomes were sequenced and for genotype-behavior associations were looked for within colonies in a common environment. There were no significant correlations between individual aggression and specific alleles. By contrast, strong correlations were found between colony aggression and the frequencies of specific alleles within colonies, despite a small number of colonies. Associations at the colony level were highly significant and were very similar among both soldiers and foragers, but they covaried with one another. One strongly significant association peak, containing an ortholog of the Drosophila sensory gene dpr4 (see Dips and Dprs) on linkage group (chromosome) 7, showed strong signals of both selection and admixture during the evolution of gentleness in a honey bee population. Links were thus found between colony genetics and group behavior and also, molecular evidence was found for group-level selection, acting at the colony level. It is concluded that group genetics dominates individual genetics in determining the fatal decision of honey bees to sting (Avalos, 2020).

    Population differences in olfaction accompany host shift in Drosophila mojavensis
    Evolutionary shifts in plant-herbivore interactions provide a model for understanding the link among the evolution of behaviour, ecological specialization and incipient speciation. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. This study shows that changes in host plant use between distinct D. mojavensis populations are accompanied by changes in the olfactory system. Specifically, differences were observed in olfactory receptor neuron specificity and sensitivity, as well as changes in sensillar subtype abundance, between populations. Additionally, RNA-seq analyses reveal differential gene expression between populations for members of the odorant receptor gene family. Hence, alterations in host preference are associated with changes in development, regulation and function at the olfactory periphery (Crowley-Gall, 2016).

    Shedding light on the grey zone of speciation along a continuum of genomic divergence

    Speciation results from the progressive accumulation of mutations that decrease the probability of mating between parental populations or reduce the fitness of hybrids-the so-called species barriers. Of primary importance is the prevalence of gene flow between diverging entities, which is central in most species concepts and has been widely discussed in recent years. This study explored the continuum of speciation thanks to a comparative analysis of genomic data from 61 pairs of populations/species of animals with variable levels of divergence. The intermediate "grey zone" of speciation, in which taxonomy is often controversial, spans from 0.5% to 2% of net synonymous Divergence, irrespective of species life history traits or ecology. Thanks to appropriate modeling of among-locus variation in genetic drift and introgression rate, this study clarifies the status of the majority of ambiguous cases and uncovers a number of cryptic species. This analysis also reveals the high incidence in animals of semi-isolated species (when some but not all loci are affected by barriers to gene flow) and highlights the intrinsic difficulty, both statistical and conceptual, of delineating species in the grey zone of speciation (Roux, 2016).

    Genetic differentiation and adaptive evolution at reproductive loci in incipient Drosophila species

    Accessory gland proteins (Acps; see Drosophila Sex Peptide and 0vulin) are part of the seminal fluid of male Drosophila flies. Some Acps have exceptionally high evolutionary rates and evolve under positive selection. Proper interactions between Acps and female reproductive molecules are essential for fertilization. These observations lead to suggestions that fast evolving Acps could be involved in speciation by promoting reproductive incompatibilities between emerging species. To test this hypothesis, population genetics data were used for three sibling species: D. mayaguana, D. parisiena, and D. straubae. The latter two species are morphologically very similar and show only incipient reproductive isolation. This system allowed examination of Acp evolution at different time frames in respect to speciation and reproductive isolation. Comparing data of 14 Acp loci with data obtained for other genomic regions, it was found that some Acps show extraordinarily high levels of divergence between D. mayaguana and its two sister species D. parisiena, and D. straubae. This divergence was likely driven by adaptive evolution at several loci. No fixed nucleotide differences were found between D. parisiena and D. straubae, however. Nevertheless, some Acp loci did show significant differentiation between these species associated with signs of positive selection; these loci may be involved in this early phase of the speciation process (Almeida, 2016).

    Early events in speciation: Cryptic species of Drosophila aldrichi

    Understanding the earliest events in speciation remains a major challenge in evolutionary biology. Thus identifying species whose populations are beginning to diverge can provide useful systems to study the process of speciation. Drosophila aldrichi, a cactophilic fruit fly species with a broad distribution in North America, has long been assumed to be a single species owing to its morphological uniformity. While previous reports either of genetic divergence or reproductive isolation among different D. aldrichi strains have hinted at the existence of cryptic species, the evolutionary relationships of this species across its range have not been thoroughly investigated. This study shows that D. aldrichi actually is paraphyletic with respect to its closest relative, Drosophila wheeleri, and that divergent D. aldrichi lineages show complete hybrid male sterility when crossed. The data support the interpretation that there are at least two species of D. aldrichi, making these flies particularly attractive for studies of speciation in an ecological and geographical context (Castro Vargas, 2017).

    Genetic divergence and the number of hybridizing species affect the path to homoploid hybrid speciation

    Hybridization is often maladaptive and in some instances has led to the loss of biodiversity. However, hybridization can also promote speciation, such as during homoploid hybrid speciation, thereby generating biodiversity. Despite examples of homoploid hybrid species, the importance of hybridization as a speciation mechanism is still widely debated, and a general understanding is lacking of the conditions most likely to generate homoploid hybrid species. This study shows that the level of genetic divergence between hybridizing species has a large effect on the probability that their hybrids evolve reproductive isolation. Populations of hybrids formed by parental species with intermediate levels of divergence were more likely to mate assortatively, and discriminate against their parental species, than those generated from weakly or strongly diverged parental species. Reproductive isolation was also found between hybrid populations, suggesting differential sorting of parental traits across populations. Finally, hybrid populations derived from three species were more likely to evolve reproductive isolation than those derived from two species, supporting arguments that hybridization-supplied genetic diversity can lead to the evolution of novel "adaptive systems" and promote speciation. These results illustrate when hybridization and admixture is expected to promote hybrid speciation. Whether homoploid hybrid speciation is a common speciation mechanism in general remains an outstanding empirical question (Comeault, 2018).

    Tissue-specific cis-regulatory divergence implicates eloF in inhibiting interspecies mating in Drosophila

    Reproductive isolation is a key component of speciation. In many insects, a major driver of this isolation is cuticular hydrocarbon pheromones, which help to identify potential intraspecific mates. When the distributions of related species overlap, there may be strong selection on mate choice for intraspecific partners because interspecific hybridization carries significant fitness costs. Drosophila has been a key model for the investigation of reproductive isolation; although both male and female mate choices have been extensively investigated, the genes underlying species recognition remain largely unknown. To explore the molecular mechanisms underlying Drosophila speciation, tissue-specific cis-regulatory divergence was identifed using RNA sequencing (RNA-seq) in D. simulans x D. sechellia hybrids. By focusing on cis-regulatory changes specific to female oenocytes, the tissue that produces cuticular hydrocarbons, this study identified a small number of candidate genes were identified. One of these, the fatty acid elongase eloF, broadly affects the hydrocarbons present on D. sechellia and D. melanogaster females, as well as the propensity of D. simulans males to mate with them. Therefore, cis-regulatory changes in eloF may be a major driver in the sexual isolation of D. simulans from multiple other species. The RNA-seq approach proved to be far more efficient than quantitative trait locus (QTL) mapping in identifying candidate genes; the same framework can be used to pinpoint candidate drivers of cis-regulatory divergence in traits differing between any interfertile species (Combs, 2018).

    Gene flow mediates the role of sex chromosome meiotic drive during complex speciation

    During speciation, sex chromosomes often accumulate interspecific genetic incompatibilities faster than the rest of the genome. The drive theory posits that sex chromosomes are susceptible to recurrent bouts of meiotic drive and suppression, causing the evolutionary build-up of divergent cryptic sex-linked drive systems and, incidentally, genetic incompatibilities. To assess the role of drive during speciation, this study combined high-resolution genetic mapping of X-linked hybrid male sterility with population genomics analyses of divergence and recent gene flow between the fruitfly species, Drosophila mauritiana and D. simulans. The findings reveal a high density of genetic incompatibilities and a corresponding dearth of gene flow on the X chromosome. Surprisingly, a known drive element recently migrated between species and, rather than contributing to interspecific divergence, caused a strong reduction in local sequence divergence, undermining the evolution of hybrid sterility. Gene flow can therefore mediate the effects of selfish genetic elements during speciation (Meiklejohn, 2018).

    Aggression and courtship differences found in Drosophila melanogaster from two different microclimates at Evolution Canyon, Israel

    Aggression and courtship behavior were examined of wild Drosophila melanogaster flies isolated from two contrasting microclimates found at Evolution Canyon in Mt. Carmel, Israel: an African-like dry tropical Slope (AS) and a European-like humid temperate Slope (ES), separated by 250 meters. Intraslope aggression between same sex fly pairings collected from the same slope was measured and compared. Both male and female flies displayed similar fighting abilities from both slopes. ES males, however, from the humid biome, showed a tendency to lunge more per aggressive encounter, compared with AS males from the dry biome. Interslope aggression was tested by pairing flies from opposite slopes. ES males displayed higher numbers of lunges, and won more fights against their AS opponents. Enhanced courtship performances were observed in ES compared to AS males. The fighting and courtship superiority seen in ES males could reinforce fitness and pre-mating reproductive isolation mechanisms that underlie incipient sympatric speciation. This may support an evolutionary advantage of adaptively divergent fruit fly aggression phenotypes from different environments (Palavicino-Maggio, 2019).

    Experimental introgression to evaluate the impact of sex specific traits on Drosophila melanogaster incipient speciation

    Sex specific traits are involved in speciation but it is difficult to determine whether their variation initiates or reinforces sexual isolation. In some insects, speciation depends of the rapid change of expression in desaturase genes coding for sex pheromones. Two closely related desaturase genes are involved in Drosophila melanogaster pheromonal communication: desat1 affects both the production and the reception of sex pheromones while desat2 is involved in their production in flies of Zimbabwe populations. There is a strong asymmetric sexual isolation between Zimbabwe populations and all other "Cosmopolitan" populations: Zimbabwe females rarely copulate with Cosmopolitan males whereas Zimbabwe males readily copulate with all females. All populations express desat1 but only Zimbabwe strains show high desat2 expression. To evaluate the impact of sex pheromones, female receptivity and desat expression on the incipient speciation process between Zimbabwe and Cosmopolitan populations, the Zimbabwe genome was introgressed into a Cosmopolitan genome labeled with the white mutation, using a multi-generation procedure. The association between these sex-specific traits was determined during the procedure. The production of pheromones was largely dissociated between the sexes. The copulation frequency (but not latency) was highly correlated with the female-but not with the male-principal pheromones. Two stable white lines were finally obtained showing Zimbabwe-like sex pheromones, copulation discrimination and desat expression. This study indicates that the variation of sex pheromones and mating discrimination depend of distinct-yet overlapping-sets of genes in each sex suggesting that their cumulated effects participate to reinforce the speciation process (Cortot, 2019).

    The evolution of male and female mating preferences in Drosophila speciation

    The relative importance of male and female mating preferences in causing sexual isolation between species remains a major unresolved question in speciation. Despite previous work showing that male courtship bias and/or female copulation bias for conspecifics occur in many taxa, the present study is one of the first large-scale works to study their relative divergence. To achieve this, data from the literature and present experiments were used across 66 Drosophila species pairs. The results revealed that male and female mate preferences are both ubiquitous in Drosophila but evolved largely independently, suggesting different underlying evolutionary and genetic mechanisms. Moreover, their relative divergence strongly depended on the geographical relationship of species. Between allopatric species, male courtship and female copulation preferences diverged at very similar rates, evolving approximately linearly with time of divergence. In sharp contrast, between sympatric species pairs, female preferences diverged much more rapidly than male preferences and were the only drivers of enhanced sexual isolation in sympatry and Reproductive Character Displacement (RCD). Not only does this result suggest that females are primarily responsible for such processes as reinforcement, but it also implies that evolved female preferences may reduce selection for further divergence of male courtship preferences in sympatry (Yukilevich, 2019).

    Engineering multiple species-like genetic incompatibilities in insects

    Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. This study demonstrates a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, it was demonstrated that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms (Maselk, 2020).

    Pigmentation and fitness trade-offs through the lens of artificial selection.

    Pigmentation is a classic phenotype that varies widely and adaptively in nature both within and among taxa. Genes underlying pigmentation phenotype are highly pleiotropic, creating the potential for functional trade-offs. However, the basic tenets of this trade-off hypothesis with respect to life-history traits have not been directly addressed. In natural populations of Drosophila melanogaster, the degree of melanin pigmentation covaries with fecundity and several other fitness traits. To examine correlations and potential trade-offs associated with variation in pigmentation, replicate outbred populations were selected for extreme pigmentation phenotypes. Replicate populations responded rapidly to the selection regime and after 100 generations of artificial selection were phenotyped for pigmentation as well as the two basic fitness parameters of fecundity and longevity. The data demonstrate that selection on pigmentation resulted in a significant shift in both fecundity and longevity profiles. Selection for dark pigmentation resulted in greater fecundity and no pronounced change in longevity, whereas selection for light pigmentation decreased longevity but did not affect fecundity. These results indicate the pleiotropic nature of alleles underlying pigmentation phenotype and elucidate possible trade-offs between pigmentation and fitness traits that may shape patterns of phenotypic variation in natural populations (Rajpurohit, 2017).

    Evolutionary history of LTR-retrotransposons among 20 Drosophila species

    The presence of transposable elements (TEs) in genomes is known to explain in part the variations of genome sizes among eukaryotes. Even among closely related species, the variation of TE amount may be striking, as for example between the two sibling species, Drosophila melanogaster and D. simulans. However, not much is known concerning the TE content and dynamics among other Drosophila species. The sequencing of several Drosophila genomes, covering the two subgenus Sophophora and Drosophila, revealed a large variation of the repeat content among these species but no much information is known concerning their precise TE content. The identification of some consensus sequences of TEs from the various sequenced Drosophila species allowed to get an idea concerning their variety in term of diversity of superfamilies but the used classification remains very elusive and ambiguous. This study focused on LTR-retrotransposons because they represent the most widely represented class of TEs in the Drosophila genomes, describing the phylogenetic relationship of each LTR-retrotransposon family described in 20 Drosophila species, computing their proportion in their respective genomes and identifying several new cases of horizontal transfers. CAll these results give a clearer view on the evolutionary history of LTR retrotransposons among Drosophila that seems to be mainly driven by vertical transmissions although the implications of horizontal transfers, losses and intra-specific diversification are clearly also at play (Bargues, 2017).

    Retrotransposons are the major contributors to the expansion of the Drosophila ananassae Muller F element

    The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (~5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in Drosophila ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, the genome sequence was improved and the genes in a 1.4 Mb region of the D. ananassae F element, and a 1.7 Mb region from the D element were annotated for comparison. Transposons (particularly LTR and LINE retrotransposons) were found to be major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F element genes exhibit distinct characteristics compared to D element genes (e.g., larger coding spans, larger introns, more coding exons, lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F element genes in both species are enriched in H3K4me2 while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves understanding of how transposons can affect genome size and how genes can function within highly repetitive domains (Leung, 2017).

    Dissecting the satellite DNA landscape in three cactophilic Drosophila sequenced genomes

    Eukayote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. This study used high throughput sequencing data from three cactophilic Drosophila species, D. buzzatii, D. seriema and D. mojavensis, to access and study their whole satDNA landscape. Five satDNAs were identified, three previously described (pBuM, DBC-150 and CDSTR198) and two novel ones (CDSTR138 and CDSTR130). Only pBuM is shared among all three species. The satDNA repeat length falls within only two classes, between 130-200bp or between 340-390bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Interspersion were revealed between pBuM and CDSTR130 in the microchromosomes of D. mojavensis. Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNAseq data, transcription activity was found for pBuM (in D. mojavensis) and CDSTR198 (in D. buzzatii) in all five analyzed developmental stages, most notably in pupae and adult males. These data revealed that cactophilic Drosophila present the lowest amount of satDNAs (1.9% to 2.9%) within the Drosophila genus reported so far (de Lima, 2017).

    Pervasive epigenetic effects of Drosophila euchromatic transposable elements impact their evolution

    Transposable elements (TEs) are widespread genomic parasites, and their evolution has remained a critical question in evolutionary genomics. This paper describes a study of the relatively unexplored epigenetic impacts of TEs and provides the first genome-wide quantification of such effects in D. melanogaster and D. simulans. Surprisingly, the spread of repressive epigenetic marks (histone H3K9me2) to nearby DNA occurs at >50% of euchromatic TEs, and can extend up to 20 kb. This results in differential epigenetic states of genic alleles and, in turn, selection against TEs. Interestingly, the lower TE content in D. simulans compared to D. melanogaster correlates with stronger epigenetic effects of TEs and higher levels of host genetic factors known to promote epigenetic silencing. This study demonstrates that the epigenetic effects of euchromatic TEs, and host genetic factors modulating such effects, play a critical role in the evolution of TEs both within and between species (Lee, 2017).

    Genomic analysis of P elements in natural populations of Drosophila melanogaster

    The Drosophila melanogaster P transposable element provides one of the best cases of horizontal transfer of a mobile DNA sequence in eukaryotes. Invasion of natural populations by the P element has led to a syndrome of phenotypes known as P-M hybrid dysgenesis that emerges when strains differing in their P element composition mate and produce offspring. This study compared estimates of genomic P element content with gonadal dysgenesis phenotypes for isofemale strains obtained from three worldwide populations of D. melanogaster to illuminate the molecular basis of natural variation in cytotype status. P element abundance estimated from genome sequences of isofemale strains is shown to be highly correlated across different bioinformatics approaches, but abundance estimates are sensitive to method and filtering strategies as well as incomplete inbreeding of isofemale strains. P element content was found to vary significantly across populations, with strains from a North American population having fewer P elements but a higher proportion of full-length elements than strains from populations sampled in Europe or Africa. Despite these geographic differences in P element abundance and structure, neither the number of P elements nor the ratio of full-length to internally-truncated copies is strongly correlated with the degree of gonadal dysgenesis exhibited by an isofemale strain. Thus, variation in P element abundance and structure across different populations does not necessarily lead to corresponding geographic differences in gonadal dysgenesis phenotypes. Finally, it was confirmed that population differences in the abundance and structure of P elements that are observed from isofemale lines can also be observed in pool-seq samples from the same populations. This work supports the view that genomic P element content alone is not sufficient to explain variation in gonadal dysgenesis across strains of D. melanogaster, and informs future efforts to decode the genomic basis of geographic and temporal differences in P element induced phenotypes (Bergman, 2017).

    Phenotypic plasticity through transcriptional regulation of the evolutionary hotspot gene tan in Drosophila melanogaster

    Phenotypic plasticity is the ability of a given genotype to produce different phenotypes in response to distinct environmental conditions. Phenotypic plasticity can be adaptive. Furthermore, it is thought to facilitate evolution. Although phenotypic plasticity is a widespread phenomenon, its molecular mechanisms are only beginning to be unravelled. Environmental conditions can affect gene expression through modification of chromatin structure, mainly via histone modifications, nucleosome remodelling or DNA methylation, suggesting that phenotypic plasticity might partly be due to chromatin plasticity. As a model of phenotypic plasticity, abdominal pigmentation was studied of Drosophila melanogaster females, which is temperature sensitive. Abdominal pigmentation is indeed darker in females grown at 18 degrees C than at 29 degrees C. This phenomenon is thought to be adaptive as the dark pigmentation produced at lower temperature increases body temperature. This study showed that temperature modulates the expression of tan (t), a pigmentation gene involved in melanin production. t is expressed 7 times more at 18 ° C than at 29 &176; C in female abdominal epidermis. Genetic experiments show that modulation of t expression by temperature is essential for female abdominal pigmentation plasticity. Temperature modulates the activity of an enhancer of t without modifying compaction of its chromatin or level of the active histone mark H3K27ac. By contrast, the active mark H3K4me3 on the t promoter is strongly modulated by temperature. The H3K4 methyl-transferase involved in this process is likely Trithorax, since it regulates t expression and the H3K4me3 level on the t promoter and also participates in female pigmentation and its plasticity. Interestingly, t was previously shown to be involved in inter-individual variation of female abdominal pigmentation in Drosophila melanogaster, and in abdominal pigmentation divergence between Drosophila species. Sensitivity of t expression to environmental conditions might therefore give more substrate for selection, explaining why this gene has frequently been involved in evolution of pigmentation (Gibert, 2016).

    On the long-term stability of clines in some metabolic genes in Drosophila melanogaster

    Very little information exists for long-term changes in genetic variation in natural populations. This study took the unique opportunity to compare a set of data for SNPs in 15 metabolic genes from eastern US collections of Drosophila melanogaster that span a large latitudinal range and represent two collections separated by 12 to 13 years. This was expanded to a 22-year interval for the Adh gene and approximately 30 years for the G6pd and Pgd genes. During these intervals, five genes showed a statistically significant change in average SNP allele frequency corrected for latitude. While much remains unchanged, five genes were seen that wx latitudinal clines have been lost or gained and two where the slope significantly changes. The long-term frequency shift towards a southern favored Adh S allele reported in Australia populations is not observed in the eastern US over a period of 21 years. There is no general pattern of southern-favored or northern-favored alleles increasing in frequency across the genes. This observation points to the fluid nature of some allelic variation over this time period and the action of selective responses or migration that may be more regional than uniformly imposed across the cline (Cogni, 2017).

    Genomic variation predicts adaptive evolutionary responses better than population bottleneck history

    The relationship between population size, inbreeding, loss of genetic variation and evolutionary potential of fitness traits is still unresolved, and large-scale empirical studies testing theoretical expectations are surprisingly scarce. This study presents a highly replicated experimental evolution setup with 120 lines of Drosophila melanogaster having experienced inbreeding caused by low population size for a variable number of generations. Genetic variation in inbred lines and in outbred control lines was assessed by genotyping-by-sequencing (GBS) of pooled samples consisting of 15 males per line. All lines were reared on a novel stressful medium for 10 generations during which body mass, productivity, and extinctions were scored in each generation. In addition, egg-to-adult viability was investigated in the benign and the stressful environments before and after rearing at the stressful conditions for 10 generations. Strong positive correlations were found between levels of genetic variation and evolutionary response in all investigated traits, and showed that genomic variation was more informative in predicting evolutionary responses than population history reflected by expected inbreeding levels. It was also found that lines with lower genetic diversity were at greater risk of extinction. For viability, the results suggested a trade-off in the costs of adapting to the stressful environments when tested in a benign environment. This work presents convincing support for long-standing evolutionary theory, and it provides novel insights into the association between genetic variation and evolutionary capacity in a gradient of diversity rather than dichotomous inbred/outbred groups (Orsted, 2019).


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