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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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)
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).
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)
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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). 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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).