The neurodevelopmental genes alan shepard and Neuroglian contribute to female mate preference in African Drosophila melanogaster.

Mate choice is a key trait that determines fitness for most sexually reproducing organisms, with females often being the choosy sex. Female preference often results in strong selection on male traits that can drive rapid divergence of traits and preferences between lineages, leading to reproductive isolation. Despite this fundamental property of female mate choice, very few loci have been identified that contribute to mate choice and reproductive isolation. We used a combination of population genetics, quantitative complementation tests, and behavioral assays to demonstrate that alan shepard and Neuroglian contribute to female mate choice, and could contribute to partial reproductive isolation between populations of Drosophila melanogaster. Our study is among the first to identify genes that contribute to female mate preference in this historically important system, where female preference is an active premating barrier to reproduction. The identification of loci that are primarily known for their roles in neurodevelopment provides intriguing questions of how female mate preference evolves in populations via changes in sensory system and higher learning brain centers.

Synthesis and Scope of the Role of Postmating Prezygotic Isolation in Speciation.

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation-a barrier that acts after gamete release but before zygote formation-is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation.

Patterns of Population Structure and Introgression Among Recently Differentiated Drosophila melanogaster Populations.

Despite a century of genetic analysis, the evolutionary processes that have generated the patterns of exceptional genetic and phenotypic variation in the model organism Drosophila melanogaster remains poorly understood. In particular, how genetic variation is partitioned within its putative ancestral range in Southern Africa remains unresolved. Here, we study patterns of population genetic structure, admixture, and the spatial structuring of candidate incompatibility alleles across a global sample, including 223 new accessions, predominantly from remote regions in Southern Africa. We identify nine major ancestries, six that primarily occur in Africa and one that has not been previously described. We find evidence for both contemporary and historical admixture between ancestries, with admixture rates varying both within and between continents. For example, while previous work has highlighted an admixture zone between broadly defined African and European ancestries in the Caribbean and southeastern USA, we identify West African ancestry as the most likely African contributor. Moreover, loci showing the strongest signal of introgression between West Africa and the Caribbean/southeastern USA include several genes relating to neurological development and male courtship behavior, in line with previous work showing shared mating behaviors between these regions. Finally, while we hypothesized that potential incompatibility loci may contribute to population genetic structure across the range of D. melanogaster; these loci are, on average, not highly differentiated between ancestries. This work contributes to our understanding of the evolutionary history of a key model system, and provides insight into the partitioning of diversity across its range.

Hybrid incompatibility between Drosophila virilis and D. lummei is stronger in the presence of transposable elements.

Mismatches between parental genomes in selfish elements are frequently hypothesized to underlie hybrid dysfunction and drive speciation. However, because the genetic basis of most hybrid incompatibilities is unknown, testing the contribution of selfish elements to reproductive isolation is difficult. Here, we evaluated the role of transposable elements (TEs) in hybrid incompatibilities between Drosophila virilis and D. lummei by experimentally comparing hybrid incompatibility in a cross where active TEs are present in D. virilis (TE+) and absent in D. lummei, to a cross where these TEs are absent from both D. virilis (TE-) and D. lummei genotypes. Using genomic data, we confirmed copy number differences in TEs between the D. virilis (TE+) strain and both the D. virilis (TE-) strain and D. lummei. We observed F1 postzygotic reproductive isolation exclusively in the interspecific cross involving TE+ D. virilis but not in crosses involving TE- D. virilis. This mirrors intraspecies dysgenesis where atrophied testes only occur when TE+ D. virilis is the paternal parent. A series of backcross experiments, that accounted for alternative models of hybrid incompatibility, showed that both F1 hybrid incompatibility and intrastrain dysgenesis are consistent with the action of TEs rather than genic interactions. Thus, our data suggest that this TE mechanism manifests as two different incompatibility phenotypes. A further Y-autosome interaction contributes to additional, sex-specific, inviability in one direction of this cross-combination. These experiments demonstrate that TEs that cause intraspecies dysgenesis can increase reproductive isolation between closely related lineages, thereby adding to the processes that consolidate speciation.

Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility.

Crosses between Drosophila simulans females and Drosophila melanogaster males produce viable F1 sons and poorly viable F1 daughters. Unlike most hybrid incompatibilities, this hybrid incompatibility violates Haldane's rule, the observation that incompatibilities preferentially affect the heterogametic sex. Furthermore, it has a different genetic basis than hybrid lethality in the reciprocal cross, with the causal allele in Drosophila melanogaster being a large species-specific block of complex satellite DNA on its X chromosome known as the 359-bp satellite, rather than a protein-coding locus. The causal allele(s) in Drosophila simulans are unknown but likely involve maternally expressed genes or factors since the F1 females die during early embryogenesis. The maternal haploid (mh) gene is an intriguing candidate because it is expressed maternally and its protein product localizes to the 359-bp repeat. We found that this gene has diverged extensively between Drosophila melanogaster and Drosophila simulans. This observation led to the hypothesis that Drosophila melanogaster mh may have coevolved with the 359-bp repeat and that hybrid incompatibility thus results from the absence of a coevolved mh allele in Drosophila simulans. We tested for the functional divergence of mh by creating matched transformants of Drosophila melanogaster and Drosophila simulans orthologs in both Drosophila melanogaster and Drosophila simulans strains. Surprisingly, we find that Drosophila simulans mh fully complements the female sterile phenotype of Drosophila melanogaster mh mutations. Contrary to our hypothesis, we find no evidence that adding a Drosophila melanogaster mh gene to Drosophila simulans increases hybrid viability.

Divergent selection on behavioural and chemical traits between reproductively isolated populations of Drosophila melanogaster.

Speciation is driven by traits that can act to prevent mating between nascent lineages, including male courtship and female preference for male traits. Mating barriers involving these traits evolve quickly because there is strong selection on males and females to maximize reproductive success, and the tight co-evolution of mating interactions can lead to rapid diversification of sexual behaviour. Populations of Drosophila melanogaster show strong asymmetrical reproductive isolation that is correlated with geographic origin. Using strains that capture natural variation in mating traits, we ask two key questions: which specific male traits are females selecting, and are these traits under divergent sexual selection? These questions have proven extremely challenging to answer, because even in closely related lineages males often differ in multiple traits related to mating behaviour. We address these questions by estimating selection gradients for male courtship and cuticular hydrocarbons for two different female genotypes. We identify specific behaviours and particular cuticular hydrocarbons that are under divergent sexual selection and could potentially contribute to premating reproductive isolation. Additionally, we report that a subset of these traits are plastic; males adjust these traits based on the identity of the female genotype they interact with. These results suggest that even when male courtship is not fixed between lineages, ongoing selection can act on traits that are important for reproductive isolation.

Testing potential mechanisms of conspecific sperm precedence in Drosophila pseudoobscura.

Drosophila pseudoobscura females that co-occur with sister species D. persimilis show elevated fertilization by conspecific sperm when they mate with both a heterospecific and a conspecific male. This phenomenon, known as conspecific sperm precedence (CSP), has evolved as a mechanism to avoid maladaptive hybridization with D. persimilis. In this study, we assessed pericopulatory (during mating) and postcopulatory (after mating) traits in crosses with sympatric or allopatric D. pseudoobscura females and conspecific or heterospecific males to evaluate potential mechanisms of CSP in this system. We observed shorter copulation duration in crosses with sympatric females, but found no difference in quantity of sperm transferred or female reproductive tract toxicity between sympatry and allopatry. Our data show some support for the hypothesis that parasperm, a short, sterile sperm morph, can protect fertile eusperm from the D. pseudoobscura female reproductive tract, though it is unclear how this might affect patterns of sperm use in sympatry vs. allopatry. Overall, these results suggest that copulation duration could potentially contribute to the elevated CSP observed in sympatry.

Regulation of male fertility and accessory gland gene expression by the Drosophila HR39 nuclear receptor.

Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. Here we show that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, our studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility.

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

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

Moving Speciation Genetics Forward: Modern Techniques Build on Foundational Studies in Drosophila.

The question of how new species evolve has been examined at every level, from macroevolutionary patterns of diversification to molecular population genetic analyses of specific genomic regions between species pairs. Drosophila has been at the center of many of these research efforts. Though our understanding of the speciation process has grown considerably over the past few decades, very few genes have been identified that contribute to barriers to reproduction. The development of advanced molecular genetic and genomic methods provides promising avenues for the rapid discovery of more genes that contribute to speciation, particularly those involving prezygotic isolation. The continued expansion of tools and resources, especially for species other than Drosophila melanogaster, will be most effective when coupled with comparative approaches that reveal the genetic basis of reproductive isolation across a range of divergence times. Future research programs in Drosophila have high potential to answer long-standing questions in speciation. These include identifying the selective forces that contribute to divergence between populations and the genetic basis of traits that cause reproductive isolation. The latter can be expanded upon to understand how the genetic basis of reproductive isolation changes over time and whether certain pathways and genes are more commonly involved.

Factors contributing to the accumulation of reproductive isolation: A mixed model approach.

The analysis of large datasets describing reproductive isolation between species has been extremely influential in the study of speciation. However, the statistical methods currently used for these data limit the ability to make direct inferences about the factors predicting the evolution of reproductive isolation. As a result, our understanding of iconic patterns and rules of speciation rely on indirect analyses that have clear statistical limitations. Phylogenetic mixed models are commonly used in ecology and evolution, but have not been applied to studies of reproductive isolation. Here I describe a flexible framework using phylogenetic mixed models to analyze data collected at different evolutionary scales, to test both categorical and continuous predictor variables, and to test the effect of multiple predictors on rates and patterns of reproductive isolation simultaneously. I demonstrate the utility of this framework by re-analyzing four classic datasets, from both animals and plants, and evaluating several hypotheses that could not be tested in the original studies: In the Drosophila and Bufonidae datasets, I found support for more rapid accumulation of reproductive isolation in sympatric species pairs compared to allopatric species pairs. Using Silene and Nolana, I found no evidence supporting the hypothesis that floral differentiation elevates postzygotic reproductive isolation. The faster accumulation of postzygotic isolation in sympatry is likely the result of species coexistence determined by the level of postzygotic isolation between species. In addition, floral trait divergence does not appear to translate into pleiotropic effects on postzygotic reproductive isolation. Overall, these methods can allow researchers to test new hypotheses using a single statistical method, while remedying the statistical limitations of several previous methods.

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura.

Reinforcement occurs when hybridization between closely related lineages produces low-fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male-female interactions than interactions with heterospecifics.

Male-female genotype interactions maintain variation in traits important for sexual interactions and reproductive isolation.

Prezygotic reproductive isolation can evolve quickly when sexual selection drives divergence in traits important for sexual interactions between populations. It has been hypothesized that standing variation for male/female traits and preferences facilitates this rapid evolution and that variation in these traits is maintained by male-female genotype interactions in which specific female genotypes prefer specific male traits. This hypothesis can also explain patterns of speciation when ecological divergence is lacking, but this remains untested because it requires information about sexual interactions in ancestral lineages. Using a set of ancestral genotypes that previously had been identified as evolving reproductive isolation, we specifically asked whether there is segregating variation in female preference and whether segregating variation in sexual interactions is a product of male-female genotype interactions. Our results provide evidence for segregating variation in female preference and further that male-female genotype interactions are important for maintaining variation that selection can act on and that can lead to reproductive isolation.

Assortative mating and self-fertilization differ in their contributions to reinforcement, cascade speciation, and diversification.

Cascade speciation and reinforcement can evolve rapidly when traits are pleiotropic and act as both signal/cue in nonrandom mating. Here, we examine the contribution of two key traits-assortative mating and self-fertilization-to reinforcement and (by extension) cascade speciation. First, using a population genetic model of reinforcement we find that both assortative mating and self-fertilization can make independent contributions to increased reproductive isolation, consistent with reinforcement. Self-fertilization primarily evolves due to its 2-fold transmission advantage when inbreeding depression (d) is lower (d < 0.45) but evolves as a function of the cost of hybridization under higher inbreeding depression (0.45 < d < 0.48). When both traits can evolve simultaneously, increased self-fertilization often prohibits the evolution of assortative mating. We infer that, under specific conditions, mating system transitions are more likely to lead to increased reproductive isolation and initiate cascade speciation, than assortative mating. Based on the results of our simulations, we hypothesized that transitions to self-fertilization could contribute to clade-wide diversification if reinforcement or cascade speciation is common. We tested this hypothesis with comparative data from two different groups. Consistent with our hypothesis, there was a trend towards uniparental reproduction being associated with increased diversification rate in the Nematode phylum. For the plant genus Mimulus, however, self-fertilization was associated with reduced diversification. Reinforcement driving speciation via transitions to self-fertilization might be short lived or unsustainable across macroevolutionary scales in some systems (some plants), but not others (such as nematodes), potentially due to differences in susceptibility to inbreeding depression and/or the ability to transition between reproductive modes.

Experimental evolution: Assortative mating and sexual selection, independent of local adaptation, lead to reproductive isolation in the nematode Caenorhabditis remanei.

Using experimental evolution, we investigated the contributions of ecological divergence, sexual selection, and genetic drift to the evolution of reproductive isolation in Caenorhabditis remanei. The nematodes were reared on two different environments for 100 generations. They were assayed for fitness on both environments after 30, 64, and 100 generations, and hybrid fitness were analyzed after 64 and 100 generations. Mating propensity within and between populations was also analyzed. The design allowed us to determine whether local adaptation was synchronous with pre- and postzygotic reproductive isolation. Prezygotic isolation evolved quickly but was unconnected with adaptation to the divergent environments. Instead, prezygotic isolation was driven by mate preferences favoring individuals from the same replicate population. A bottleneck treatment, meant to enhance the opportunity for genetic drift, had no effect on prezygotic isolation. Postzygotic isolation occurred in crosses where at least one population had a large fitness advantage in its "home" environment. Taken together, our results suggest that prezygotic isolation did not depend on drift or adaptation to divergent environments, but instead resulted from differences in sexual interactions within individual replicates. Furthermore, our results suggest that postzygotic isolation can occur between populations even when only one population has greater fitness in its home environment.

Intraspecific sperm competition genes enforce post-mating species barriers in Drosophila.

Sexual selection and sexual conflict are considered important drivers of speciation, based on both theoretical models and empirical correlations between sexually selected traits and diversification. However, whether reproductive isolation between species evolves directly as a consequence of intrapopulation sexual dynamics remains empirically unresolved, in part because knowledge of the genetic mechanisms (if any) connecting these processes is limited. Here, we provide evidence of a direct mechanistic link between intraspecies sexual selection and reproductive isolation. We examined genes with known roles in intraspecific sperm competition (ISC) in D. melanogaster and assayed their impact on conspecific sperm precedence (CSP). We found that two such genes (Acp36DE and CG9997) contribute to both offensive sperm competition and CSP; null/knockdown lines both had lower competitive ability against D. melanogaster conspecifics and were no longer able to displace heterospecific D. simulans sperm in competitive matings. In comparison, Sex Peptide (Acp70A)-another locus essential for ISC-does not contribute to CSP. These data indicate that two loci important for sperm competitive interactions have an additional role in similar interactions that enforce post-mating reproductive isolation between species, and show that sexual selection and sexual isolation can act on the same molecular targets in a gene-specific manner.

Interactions between a pollinating seed predator and its host plant: the role of environmental context within a population.

Plant-insect interactions often are important for plant reproduction, but the outcome of these interactions may vary with environmental context. Pollinating seed predators have positive and negative effects on host plant reproduction, and the interaction outcome is predicted to vary with density or abundance of the partners. We studied the interaction between Silene stellata, an herbaceous perennial, and Hadena ectypa, its specialized pollinating seed predator. Silene stellata is only facultatively dependent upon H. ectypa for pollination because other nocturnal moth co-pollinators are equally effective at pollen transfer. We hypothesized that for plants without conspecific neighbors, H. ectypa would have higher visitation rates compared to co-pollinators, and the plants would experience lower levels of H. ectypa pollen deposition. We predicted similar oviposition throughout the study site but greater H. ectypa predation in the area without conspecific neighbors compared to plants embedded in a naturally high density area. We found that H. ectypa had consistently higher visitation than moth co-pollinators in all host plant contexts. However, H. ectypa pollinator importance declined in areas with low conspecific density because of reduced pollen deposition, resulting in lower seed set. Conversely, oviposition was similar across the study site independent of host plant density. Greater likelihood of very high fruit predation combined with lower pollination by H. ectypa resulted in reduced S. stellata female reproductive success in areas with low conspecific density. Our results demonstrate local context dependency of the outcomes of pollinating seed predator interactions with conspecific host plant density within a population.

Evolutionary Implications of Mechanistic Models of TE-Mediated Hybrid Incompatibility.

New models of TE repression in plants (specifically Arabidopsis) have suggested specific mechanisms by which TE misregulation in hybrids might result in the expression of hybrid inviability. If true, these models suggest as yet undescribed consequences for (1) mechanistic connections between hybrid problems expressed at different postzygotic stages (e.g., inviability versus sterility), (2) the predicted strength, stage, and direction of isolation between diverging lineages that differ in TE activity, and (3) the association between species attributes that influence TE dynamics (e.g., mode of reproduction, geographical structure) and the rate at which they could accumulate incompatibilities. In this paper, we explore these implications and outline future empirical directions for generating data necessary to evaluate them.

Molecular evolution under increasing transposable element burden in Drosophila: a speed limit on the evolutionary arms race.

BACKGROUND: Genome architecture is profoundly influenced by transposable elements (TEs), and natural selection against their harmful effects is a critical factor limiting their spread. Genome defense by the piRNA silencing pathway also plays a crucial role in limiting TE proliferation. How these two forces jointly determine TE abundance is not well understood. To shed light on the nature of factors that predict TE success, we test three distinct hypotheses in the Drosophila genus. First, we determine whether TE abundance and relaxed genome-wide purifying selection on protein sequences are positively correlated. This serves to test the hypothesis that variation in TE abundance in the Drosophila genus can be explained by the strength of natural selection, relative to drift, acting in parallel against mildly deleterious non-synonymous mutations. Second, we test whether increasing TE abundance is correlated with an increased rate of amino-acid evolution in genes encoding the piRNA machinery, as might be predicted by an evolutionary arms race model. Third, we test whether increasing TE abundance is correlated with greater codon bias in genes of the piRNA machinery. This is predicted if increasing TE abundance selects for increased efficiency in the machinery of genome defense. RESULTS: Surprisingly, we find neither of the first two hypotheses to be true. Specifically, we found that genome-wide levels of purifying selection, measured by the ratio of non-synonymous to synonymous substitution rates (ω), were greater in species with greater TE abundance. In addition, species with greater TE abundance have greater levels of purifying selection in the piRNA machinery. In contrast, it appears that increasing TE abundance has primarily driven adaptation in the piRNA machinery by increasing codon bias. CONCLUSIONS: These results indicate that within the Drosophila genus, a historically reduced strength of selection relative to drift is unlikely to explain patterns of increased TE success across species. Other factors, such as ecological exposure, are likely to contribute to variation in TE abundances within species. Furthermore, constraints on the piRNA machinery may temper the evolutionary arms race that would drive increasing rates of evolution at the amino acid level. In the face of these constraints, selection may act primarily by improving the translational efficiency of the machinery of genome defense through efficient codon usage.

Molecular evolution in bacterial endosymbionts of fungi.

The prediction that progressive coupling of host and symbiont metabolic and reproductive interests leads to reduced mixing of symbiont lineages has been verified extensively in maternally transmitted bacterial endosymbionts of insects. To test whether this prediction is also applicable to associations of bacteria with fungi, we explored patterns of molecular evolution in two lineages of mutualistic endosymbionts of fungi: the Burkholderia endosymbionts of Rhizopus microsporus (Mucormycotina) and Candidatus Glomeribacter gigasporarum endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We compared these two lineages with the closely related Candidatus Tremblaya princeps endosymbionts of mealybugs (Hemiptera, Coccoidea, Pseudococcidae) and to free-living Burkholderia species. To make inferences about the life histories of the endosymbionts, we relied on the empirically validated predictions of the nearly neutral theory of molecular evolution that a reduction of the effective population size increases the rate of fixation of slightly deleterious mutations. Our analyses showed that the slightly deleterious mutation accumulation patterns in the Burkholderia endosymbionts of Rhizopus were nearly indistinguishable from those in their free-living relatives. In contrast, Ca. Glomeribacter showed unique patterns of molecular evolution that differentiated them from both the Burkholderia endosymbionts of Rhizopus and from the Ca. Tremblaya endosymbionts of insects. These findings imply that reduced mixing of symbiont lineages is not a universal feature of symbioses between fungi and endocellular bacteria.