Cold adaptation drives population genomic divergence in the ecological specialist, Drosophila montana.

Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here, we analysed patterns of geographic variation in the cold-adapted species Drosophila montana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and fourth chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and fourth chromosomes, which also showed highest interpopulation FST . This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome-wide effects.

A rare exception to Haldane's rule: Are X chromosomes key to hybrid incompatibilities?

The prevalence of Haldane's rule suggests that sex chromosomes commonly have a key role in reproductive barriers and speciation. However, the majority of research on Haldane's rule has been conducted in species with conventional sex determination systems (XY and ZW) and exceptions to the rule have been understudied. Here we test the role of X-linked incompatibilities in a rare exception to Haldane's rule for female sterility in field cricket sister species (Teleogryllus oceanicus and T. commodus). Both have an XO sex determination system. Using three generations of crosses, we introgressed X chromosomes from each species onto different, mixed genomic backgrounds to test predictions about the fertility and viability of each cross type. We predicted that females with two different species X chromosomes would suffer reduced fertility and viability compared with females with two parental X chromosomes. However, we found no strong support for such X-linked incompatibilities. Our results preclude X-X incompatibilities and instead support an interchromosomal epistatic basis to hybrid female sterility. We discuss the broader implications of these findings, principally whether deviations from Haldane's rule might be more prevalent in species without dimorphic sex chromosomes.

Sexual selection and assortative mating: an experimental test.

Mate choice and mate competition can both influence the evolution of sexual isolation between populations. Assortative mating may arise if traits and preferences diverge in step, and, alternatively, mate competition may counteract mating preferences and decrease assortative mating. Here, we examine potential assortative mating between populations of Drosophila pseudoobscura that have experimentally evolved under either increased ('polyandry') or decreased ('monogamy') sexual selection intensity for 100 generations. These populations have evolved differences in numerous traits, including a male signal and female preference traits. We use a two males: one female design, allowing both mate choice and competition to influence mating outcomes, to test for assortative mating between our populations. Mating latency shows subtle effects of male and female interactions, with females from the monogamous populations appearing reluctant to mate with males from the polyandrous populations. However, males from the polyandrous populations have a significantly higher probability of mating regardless of the female's population. Our results suggest that if populations differ in the intensity of sexual selection, effects on mate competition may overcome mate choice.

The locus of sexual selection: moving sexual selection studies into the post-genomics era.

Sexual selection drives fundamental evolutionary processes such as trait elaboration and speciation. Despite this importance, there are surprisingly few examples of genes unequivocally responsible for variation in sexually selected phenotypes. This lack of information inhibits our ability to predict phenotypic change due to universal behaviours, such as fighting over mates and mate choice. Here, we discuss reasons for this apparent gap and provide recommendations for how it can be overcome by adopting contemporary genomic methods, exploiting underutilized taxa that may be ideal for detecting the effects of sexual selection and adopting appropriate experimental paradigms. Identifying genes that determine variation in sexually selected traits has the potential to improve theoretical models and reveal whether the genetic changes underlying phenotypic novelty utilize common or unique molecular mechanisms. Such a genomic approach to sexual selection will help answer questions in the evolution of sexually selected phenotypes that were first asked by Darwin and can furthermore serve as a model for the application of genomics in all areas of evolutionary biology.

How consistent are the transcriptome changes associated with cold acclimation in two species of the Drosophila virilis group?

For many organisms the ability to cold acclimate with the onset of seasonal cold has major implications for their fitness. In insects, where this ability is widespread, the physiological changes associated with increased cold tolerance have been well studied. Despite this, little work has been done to trace changes in gene expression during cold acclimation that lead to an increase in cold tolerance. We used an RNA-Seq approach to investigate this in two species of the Drosophila virilis group. We found that the majority of genes that are differentially expressed during cold acclimation differ between the two species. Despite this, the biological processes associated with the differentially expressed genes were broadly similar in the two species. These included: metabolism, cell membrane composition, and circadian rhythms, which are largely consistent with previous work on cold acclimation/cold tolerance. In addition, we also found evidence of the involvement of the rhodopsin pathway in cold acclimation, a pathway that has been recently linked to thermotaxis. Interestingly, we found no evidence of differential expression of stress genes implying that long-term cold acclimation and short-term stress response may have a different physiological basis.

The genetic architecture of sexually selected traits in two natural populations of Drosophila montana.

We investigated the genetic architecture of courtship song and cuticular hydrocarbon traits in two phygenetically distinct populations of Drosophila montana. To study natural variation in these two important traits, we analysed within-population crosses among individuals sampled from the wild. Hence, the genetic variation analysed should represent that available for natural and sexual selection to act upon. In contrast to previous between-population crosses in this species, no major quantitative trait loci (QTLs) were detected, perhaps because the between-population QTLs were due to fixed differences between the populations. Partitioning the trait variation to chromosomes suggested a broadly polygenic genetic architecture of within-population variation, although some chromosomes explained more variation in one population compared with the other. Studies of natural variation provide an important contrast to crosses between species or divergent lines, but our analysis highlights recent concerns that segregating variation within populations for important quantitative ecological traits may largely consist of small effect alleles, difficult to detect with studies of moderate power.

The evolution of novelty in conserved genes; evidence of positive selection in the Drosophila fruitless gene is localised to alternatively spliced exons.

There has been much debate concerning whether cis-regulatory or coding changes are more likely to produce evolutionary innovation or adaptation in gene function, but an additional complication is that some genes can dramatically diverge through alternative splicing, increasing the diversity of gene function within a locus. The fruitless gene is a major transcription factor with a wide range of pleiotropic functions, including a fundamental conserved role in sexual differentiation, species-specific morphology and an important influence on male sexual behaviour. Here, we examine the structure of fruitless in multiple species of Drosophila, and determine the patterns of selective constraint acting across the coding region. We found that the pattern of selection, estimated from the ratio of non-synonymous to synonymous substitutions, varied considerably across the gene, with most regions of the gene evolutionarily conserved but with several regions showing evidence of divergence as a result of positive selection. The regions that showed evidence of positive selection were found to be localised to relatively consistent regions across multiple speciation events, and are associated with alternative splicing. Alternative splicing may thus provide a route to gene diversification in key regulatory loci.

Hybridization and speciation.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near-instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky-Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock-on effects on speciation both within and outside regions of hybridization.

Two distinct genomic regions, harbouring the period and fruitless genes, affect male courtship song in Drosophila montana.

Acoustic signals often have a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated quantitative trait locus for male courtship song carrier frequency (FRE) in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for 10 candidate genes known to affect song production in Drosophila melanogaster. We also extended the analyses to additional song characters (pulse train length (PTL), pulse number (PN), interpulse interval, pulse length (PL) and cycle number (CN)). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (PTL and PN) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (PL, CN and carrier FRE) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.

Multiple quantitative trait loci influence intra-specific variation in genital morphology between phylogenetically distinct lines of Drosophila montana.

The evolution of animal genitalia has gained renewed interest because of their potential roles during sexual selection and early stages of species formation. Although central to understanding the evolutionary process, knowledge of the genetic basis of natural variation in genital morphology is limited to a very few species. Using an outbred cross between phylogenetically distinct lines of Drosophila montana, we characterized quantitative trait loci (QTLs) affecting the size and shape of the distiphallus, a prominent part of the male intromittent organ. Our microsatellite-based linkage analysis shows that intra-specific variation in the distiphallus involves several QTLs of largely additive effect and that a highly significant QTL co-localizes with the same inversion where we have earlier localized a large QTL for a sexually selected courtship song trait. The latter indicates that inversions can play an important role in shaping the evolution of rapidly evolving traits with a potential influence on speciation.

A microsatellite linkage map for Drosophila montana shows large variation in recombination rates, and a courtship song trait maps to an area of low recombination.

Current advances in genetic analysis are opening up our knowledge of the genetics of species differences, but challenges remain, particularly for out-bred natural populations. We constructed a microsatellite-based linkage map for two out-bred lines of Drosophila montana derived from divergent populations by taking advantage of the Drosophila virilis genome and available cytological maps of both species. Although the placement of markers was quite consistent with cytological predictions, the map indicated large heterogeneity in recombination rates along chromosomes. We also performed a quantitative trait locus (QTL) analysis on a courtship song character (carrier frequency), which differs between populations and is subject to strong sexual selection. Linkage mapping yielded two significant QTLs, which explained 3% and 14% of the variation in carrier frequency, respectively. Interestingly, as in other recent studies of traits which can influence speciation, the strongest QTL mapped to a genomic region partly covered by an inversion polymorphism.

Identification of quantitative trait loci function through analysis of multiple cuticular hydrocarbons differing between Drosophila simulans and Drosophila sechellia females.

The genetics of sexual isolation, behavioral differences between species that prevent mating, is understood poorly. Pheromonal differences between species can influence sexual isolation in many animals and in some cases a single locus can cause large functional changes in pheromonal mating signals. Drosophila cuticular hydrocarbons (CHCs) can function as pheromones and consequently affect mate recognition. In a previous study of the two major CHCs in females that affect mating discrimination between Drosophila simulans and D. sechellia, quantitative trait loci (QTL) were identified on the X and third chromosome, and a few candidate genes were potentially implicated. Here we specifically test candidate genes for CHC biosynthesis and determine the genetic architecture of four additional CHCs that differ in abundance between D. simulans and D. sechellia females. The same QTL, and new ones, were found for additional CHCs. By examining all these CHCs and exploring their covariance, we were able to ascribe putative function to the major QTL. Although desaturases have received considerable attention for their role in CHC biosynthesis, evidence here implies that elongases may be just as important. Sex determination genes do not seem to have a role in this species difference although D. sechellia is sexually dimorphic in CHCs, whereas D. simulans is not. Epistatic interactions, only detected for CHCs limited to D. sechellia, imply that complex interactions among loci may also be having a role in these compounds that affect mating isolation.

Characterization of female preference functions for Drosophila montana courtship song and a test of the temperature coupling hypothesis.

Female mate preferences are a major cause of diversity and elaboration in male sexual traits. Here we characterize the shape of female preference functions for pulse length and carrier frequency of the courtship song of Drosophila montana by fitting both parametric and nonparametric functions to the incidence of female receptive gestures to synthetic song. Preference functions for both traits are strongly directional. That for pulse length is linear and favors short pulses, whereas that for carrier frequency is stabilizing in shape, but would exert directional preferences favoring males with high carrier frequency. The preference for carrier frequency has probably evolved under sexual selection, but reasons for the preference for short pulses are less apparent. We also examine the effect of ambient temperature on the carrier frequency of male song and on the preference function for carrier frequency. For many similar acoustic communication systems, temperature coupling, a compensatory effect of temperature on preference functions, is thought to maintain coordination between preferences and signals. However, although the carrier frequency of D. montana song is highly dependent on environmental temperature, there is no temperature coupling of the female preference function. We suggest that temperature coupling may often arise due to a common effect of temperature on song and preference, rather than be an advantageous characteristic whose function is to maintain coordination in temperature-affected communication systems.

The inheritance of female preference functions in a mate recognition system.

Mate recognition systems (MRSs) play a major role in sexual selection and speciation, yet few studies have analysed both male and female components in detail. Here, female preference functions have been characterized for the tettigoniid bushcricket Ephippiger ephippiger, and the inheritance of male song and female preference functions followed in crosses between subspecies. Songs are disproportionately determined by sex-linked genes. However, there is no evidence for a role of maternally derived sex-linked genes in female preference or of maternal effects. At the genetic level, there is a mismatch between peak preferences and male song, consistent with an evolutionary history of persistent directional preferences. Such a pattern of inheritance could contribute to the process of speciation via the evolution of new MRSs.

Postglacial intra-lacustrine divergence of Icelandic threespine stickleback morphs in three neovolcanic lakes.

The geographical context of divergence and local adaptation of lacustrine fish is controversial. Despite recent theoretical support for sympatric and parapatric divergence, empirical studies providing unequivocal support for this remain scant. An important component of such a case would be where multiple lakes have different morphs and a range of markers, both mitochondrial and nuclear, show monophyly within lakes. Here we describe such a situation for threespine sticklebacks in three lakes in Iceland. By analysing the variation at nuclear and mitochondrial markers in several freshwater and marine populations as well as three pairs of intra-lacustrine morphs we infer their phylogenetic relationships and colonization pattern. There were high levels of microsatellite variation in all populations and no evidence was found for either repeated colonization of marine fish or colonization from distinct glacial refugia. Intra-lacustrine threespine stickleback morphs in all three lakes show significant genetic divergence probably indicating restricted gene flow.

Beyond the point of no return? A comparison of genetic diversity in captive and wild populations of two nearly extinct species of Goodeid fish reveals that one is inbred in the wild.

The relative importance of genetic and non-genetic factors in extinction liability has been extensively debated. Here, we examine the levels of genetic variability at 13 (seven informative) loci in wild and captive populations of two endangered species of Mexican Goodeid fish, Ameca splendens and Zoogoneticus tequila. Allelic diversity was higher in the wild populations, and F(IS) lower. Values of theta (=4Nemu) were estimated using a coalescent approach. These implied that the effective population size of all captive populations of A. splendens were smaller than that of the wild population; qualitatively similar results were obtained using an analytical method based on within-population gene identity disequilibrium. However, the wild population of Z. tequila did not show a significantly greater estimate of theta. We used the Beaumont approach to infer population declines, and found that both species showed clear evidence of a decline in effective population size, although this was stronger and probably occurred over a longer period of time in Z. tequila than in A. splendens. The decline in Z. tequila probably occurred before captive populations were established. We discuss implications for the conservation of critically endangered populations.

Morphological and genetic divergence of intralacustrine stickleback morphs in Iceland: a case for selective differentiation?

The evolutionary processes involved in population divergence and local adaptation are poorly understood. Theory predicts that divergence of adjacent populations is possible but depends on several factors including gene flow, divergent selection, population size and the number of genes involved in divergence and their distribution on the genome. We analyse variation in neutral markers, markers linked to putative quantitative trait loci and morphological traits in a recent (<10000 years) zone of primary divergence between stickleback morphs in Lake Thingvallavatn, Iceland. Environmental factors, especially predation, are clearly implicated in reducing gene flow between morphs. There is continuous morphological and genetic variation between habitats with a zone centre similar to secondary contact zones. Individual microsatellite loci are implicated as being linked to adaptive variation by direct tests as well as by differences in cline shape. Patterns of linkage disequilibria indicate that the morphs have diverged at several loci. This divergence shows parallels and differences with the well-studied limnetic-benthic stickleback morphs, both in phenotypic divergence and at the genomic level.

Sex and differentiation: population genetic divergence and sexual dimorphism in Mexican goodeid fish.

Genetic differentiation arises due to the interaction between natural and sexual selection, migration and genetic drift. A potential role of sexual selection in speciation has received much interest, although comparative studies are inconsistent in finding supporting evidence. A poorly tested prediction is that species subject to a higher intensity of sexual selection should show greater genetic differentiation amongst populations because females from these populations should be more choosy in mate choice. The Goodeinae is a group of endemic Mexican fishes in which female choice has driven some species to be morphologically sexually dimorphic, whereas others are relatively monomorphic. Here, we measured population divergence, using microsatellite loci, within four goodeid species which show contrasting levels of sexual dimorphism. We found higher levels of differentiation between populations of the more dimorphic species, implying less gene flow between populations. We also found evidence of higher levels of genetic differences between the sexes within populations of the dimorphic species, consistent with greater dispersal in males. Adjusted for geographic distance, the mean F(ST) for the dimorphic species is 0.25 compared with 0.16 for the less dimorphic species. We conclude that population differentiation is accelerated in more sexually dimorphic species, and that comparative phylogeography may provide a more powerful approach to detecting processes, such as an influence of sexual selection on differentiation, than broad-scale comparative studies.