Long-sperm precedence and other cryptic female choices in Drosophila melanogaster.

Females that mate multiply make postmating choices about which sperm fertilize their eggs (cryptic female choice); however, the male characteristics they use to make such choices remain unclear. In this study, we sought to understand female sperm use patterns by evaluating whether Drosophila melanogaster females adjust sperm use (second male paternity) in response to four main factors: male genotype, male courtship effort, male pheromone alteration, and male postmating reproductive morphology. Our experiment was replicated across four different D. melanogaster lines, in a full factorial design, including a pheromone manipulation in which second males were perfumed to resemble heterospecific (D. yakuba) males. We found that females prefer longer sperm-regardless of mating order-in almost all contexts; this observed pattern of 'long-sperm precedence' is consistent with female postcopulatory choice of high-fitness male traits. Nonetheless, we also found that this general preference can be plastically altered by females in response to effects including perfuming treatment; this differential female sperm use is between otherwise identical males, and therefore solely female-mediated. Furthermore, our finding that females exercise choice using diverse criteria suggests a possible mechanism for the maintenance of variation in sexually selected male traits.

Genomic Approaches Are Improving Taxonomic Representation in Genetic Studies of Speciation.

Until recently, our understanding of the genetics of speciation was limited to a narrow group of model species with a specific set of characteristics that made genetic analysis feasible. Rapidly advancing genomic technologies are eliminating many of the distinctions between laboratory and natural systems. In light of these genomic developments, we review the history of speciation genetics, advances that have been gleaned from model and non-model organisms, the current state of the field, and prospects for broadening the diversity of taxa included in future studies. Responses to a survey of speciation scientists across the world reveal the ongoing division between the types of questions that are addressed in model and non-model organisms. To bridge this gap, we suggest integrating genetic studies from model systems that can be reared in the laboratory or greenhouse with genomic studies in related non-models where extensive ecological knowledge exists.

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.

CAGEE: Computational Analysis of Gene Expression Evolution.

Despite the increasing abundance of whole transcriptome data, few methods are available to analyze global gene expression across phylogenies. Here, we present a new software package (Computational Analysis of Gene Expression Evolution [CAGEE]) for inferring patterns of increases and decreases in gene expression across a phylogenetic tree, as well as the rate at which these changes occur. In contrast to previous methods that treat each gene independently, CAGEE can calculate genome-wide rates of gene expression, along with ancestral states for each gene. The statistical approach developed here makes it possible to infer lineage-specific shifts in rates of evolution across the genome, in addition to possible differences in rates among multiple tissues sampled from the same species. We demonstrate the accuracy and robustness of our method on simulated data and apply it to a data set of ovule gene expression collected from multiple self-compatible and self-incompatible species in the genus Solanum to test hypotheses about the evolutionary forces acting during mating system shifts. These comparisons allow us to highlight the power of CAGEE, demonstrating its utility for use in any empirical system and for the analysis of most morphological traits. Our software is available at https://github.com/hahnlab/CAGEE/.

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.

A shift to shorter cuticular hydrocarbons accompanies sexual isolation among Drosophila americana group populations.

Because sensory signals often evolve rapidly, they could be instrumental in the emergence of reproductive isolation between species. However, pinpointing their specific contribution to isolating barriers, and the mechanisms underlying their divergence, remains challenging. Here, we demonstrate sexual isolation due to divergence in chemical signals between two populations of Drosophila americana (SC and NE) and one population of D. novamexicana, and dissect its underlying phenotypic and genetic mechanisms. Mating trials revealed strong sexual isolation between Drosophila novamexicana males and SC Drosophila americana females, as well as more moderate bi-directional isolation between D. americana populations. Mating behavior data indicate SC D. americana males have the highest courtship efficiency and, unlike males of the other populations, are accepted by females of all species. Quantification of cuticular hydrocarbon (CHC) profiles-chemosensory signals that are used for species recognition and mate finding in Drosophila-shows that the SC D. americana population differs from the other populations primarily on the basis of compound carbon chain-length. Moreover, manipulation of male CHC composition via heterospecific perfuming-specifically perfuming D. novamexicana males with SC D. americana males-abolishes their sexual isolation from these D. americana females. Of a set of candidates, a single gene-elongase CG17821-had patterns of gene expression consistent with a role in CHC differences between species. Sequence comparisons indicate D. novamexicana and our Nebraska (NE) D. americana population share a derived CG17821 truncation mutation that could also contribute to their shared "short" CHC phenotype. Together, these data suggest an evolutionary model for the origin and spread of this allele and its consequences for CHC divergence and sexual isolation in this group.

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.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Introgression shapes fruit color convergence in invasive Galápagos tomato.

Invasive species represent one of the foremost risks to global biodiversity. Here, we use population genomics to evaluate the history and consequences of an invasion of wild tomato-Solanum pimpinellifolium-onto the Galápagos Islands from continental South America. Using >300 archipelago and mainland collections, we infer this invasion was recent and largely the result of a single event from central Ecuador. Patterns of ancestry within the genomes of invasive plants also reveal post-colonization hybridization and introgression between S. pimpinellifolium and the closely related Galápagos endemic Solanum cheesmaniae. Of admixed invasive individuals, those that carry endemic alleles at one of two different carotenoid biosynthesis loci also have orange fruits-characteristic of the endemic species-instead of typical red S. pimpinellifolium fruits. We infer that introgression of two independent fruit color loci explains this observed trait convergence, suggesting that selection has favored repeated transitions of red to orange fruits on the Galápagos.

Reproductive Proteins Evolve Faster Than Non-reproductive Proteins Among Solanum Species.

Elevated rates of evolution in reproductive proteins are commonly observed in animal species, and are thought to be driven by the action of sexual selection and sexual conflict acting specifically on reproductive traits. Whether similar patterns are broadly observed in other biological groups is equivocal. Here, we examine patterns of protein divergence among wild tomato species (Solanum section Lycopersicon), to understand forces shaping the evolution of reproductive genes in this diverse, rapidly evolving plant clade. By comparing rates of molecular evolution among loci expressed in reproductive and non-reproductive tissues, our aims were to test if: (a) reproductive-specific loci evolve more rapidly, on average, than non-reproductive loci; (b) 'male'-specific loci evolve at different rates than 'female'-specific loci; (c) genes expressed exclusively in gametophytic (haploid) tissue evolve differently from genes expressed in sporophytic (diploid) tissue or in both tissue types; and (d) mating system variation (a potential proxy for the expected strength of sexual selection and/or sexual conflict) affects patterns of protein evolution. We observed elevated evolutionary rates in reproductive proteins. However, this pattern was most evident for female- rather than male-specific loci, both broadly and for individual loci inferred to be positively selected. These elevated rates might be facilitated by greater tissue-specificity of reproductive proteins, as faster rates were also associated with more narrow expression domains. In contrast, we found little evidence that evolutionary rates are consistently different in loci experiencing haploid selection (gametophytic-exclusive loci), or in lineages with quantitatively different mating systems. Overall while reproductive protein evolution is generally elevated in this diverse plant group, some specific patterns of evolution are more complex than those reported in other (largely animal) systems, and include a more prominent role for female-specific loci among adaptively evolving genes.

Inferring the Genetic Basis of Sex Determination from the Genome of a Dioecious Nightshade.

Dissecting the genetic mechanisms underlying dioecy (i.e., separate female and male individuals) is critical for understanding the evolution of this pervasive reproductive strategy. Nonetheless, the genetic basis of sex determination remains unclear in many cases, especially in systems where dioecy has arisen recently. Within the economically important plant genus Solanum (∼2,000 species), dioecy is thought to have evolved independently at least 4 times across roughly 20 species. Here, we generate the first genome sequence of a dioecious Solanum and use it to ascertain the genetic basis of sex determination in this species. We de novo assembled and annotated the genome of Solanum appendiculatum (assembly size: ∼750 Mb scaffold N50: 0.92 Mb; ∼35,000 genes), identified sex-specific sequences and their locations in the genome, and inferred that males in this species are the heterogametic sex. We also analyzed gene expression patterns in floral tissues of males and females, finding approximately 100 genes that are differentially expressed between the sexes. These analyses, together with observed patterns of gene-family evolution specific to S. appendiculatum, consistently implicate a suite of genes from the regulatory network controlling pectin degradation and modification in the expression of sex. Furthermore, the genome of a species with a relatively young sex-determination system provides the foundational resources for future studies on the independent evolution of dioecy in this clade.

Regional differences in the abiotic environment contribute to genomic divergence within a wild tomato species.

The wild currant tomato Solanum pimpinellifolium inhabits a wide range of abiotic habitats across its native range of Ecuador and Peru. Although it has served as a key genetic resource for the improvement of domestic cultivars, little is known about the genetic basis of traits underlying local adaptation in this species, nor what abiotic variables are most important for driving differentiation. Here we use redundancy analysis (RDA) and other multivariate statistical methods (structural equation modelling [SEM] and generalized dissimilarity modelling [GDM]) to quantify the relationship of genomic variation (6,830 single nucleotide polymorphisms [SNPs]) with climate and geography, among 140 wild accessions. RDA, SEM and GDM each identified environment as explaining more genomic variation than geography, suggesting that local adaptation to heterogeneous abiotic habitats may be an important source of genetic diversity in this species. Environmental factors describing temporal variation in precipitation and evaporative demand explained the most SNP variation among accessions, indicating that these forces may represent key selective agents. Lastly, by studying how SNP-environment associations vary throughout the genome (44,064 SNPs), we mapped the location and investigated the functions of loci putatively contributing to climatic adaptations. Together, our findings indicate an important role for selection imposed by the abiotic environment in driving genomic differentiation between populations.

Assessing biological factors affecting postspeciation introgression.

An increasing number of phylogenomic studies have documented a clear "footprint" of postspeciation introgression among closely related species. Nonetheless, systematic genome-wide studies of factors that determine the likelihood of introgression remain rare. Here, we propose an a priori hypothesis-testing framework that uses introgression statistics-including a new metric of estimated introgression, D p-to evaluate general patterns of introgression prevalence and direction across multiple closely related species. We demonstrate this approach using whole genome sequences from 32 lineages in 11 wild tomato species to assess the effect of three factors on introgression-genetic relatedness, geographical proximity, and mating system differences-based on multiple trios within the "ABBA-BABA" test. Our analyses suggest each factor affects the prevalence of introgression, although our power to detect these is limited by the number of comparisons currently available. We find that of 14 species pairs with geographically "proximate" versus "distant" population comparisons, 13 showed evidence of introgression; in 10 of these cases, this was more prevalent between geographically closer populations. We also find modest evidence that introgression declines with increasing genetic divergence between lineages, is more prevalent between lineages that share the same mating system, and-when it does occur between mating systems-tends to involve gene flow from more inbreeding to more outbreeding lineages. Although our analysis indicates that recent postspeciation introgression is frequent in this group-detected in 15 of 17 tested trios-estimated levels of genetic exchange are modest (0.2-2.5% of the genome), so the relative importance of hybridization in shaping the evolutionary trajectories of these species could be limited. Regardless, similar clade-wide analyses of genomic introgression would be valuable for disentangling the major ecological, reproductive, and historical determinants of postspeciation gene flow, and for assessing the relative contribution of introgression as a source of genetic variation.

Intraspecific Genetic Variation Underlying Postmating Reproductive Barriers between Species in the Wild Tomato Clade (Solanum sect. Lycopersicon).

A goal of speciation genetics is to understand how the genetic components underlying interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has been observed among populations within species in the wild tomato clade. In some cases, molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the mechanistic connection between these intra- and inter-specific pollen rejection behaviors is poorly understood in most instances. We generated an F2 population between SI and SC genotypes of a single species, Solanum pennellii, to examine the genetic basis of intraspecific variation in UI against other species, and to determine whether loci underlying SI are genetically associated with this variation. We found that F2 individuals vary in the rate at which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-determining S-locus. Moreover, individuals that expressed S-RNase-the S-locus protein involved in SI pollen rejection-in their styles had much more rapid UI responses compared with those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice loci-in this case at loci that prevent self-fertilization-can contribute to variation in the expression of interspecific isolation, including postmating prezygotic barriers. Understanding the nature of such intraspecific variation can provide insight into the accumulation of these barriers between diverging lineages.

Constitutive and Plastic Gene Expression Variation Associated with Desiccation Resistance Differences in the Drosophila americana Species Group.

Stress response mechanisms are ubiquitous and important for adaptation to heterogenous environments and could be based on constitutive or plastic responses to environmental stressors. Here we quantify constitutive and plastic gene expression differences under ambient and desiccation stress treatments, in males and females of three species of Drosophila known to differ in desiccation resistance. Drosophila novamexicana survives desiccation trials significantly longer than the two subspecies of Drosophila americana, consistent with its natural species range in the desert southwest USA. We found that desiccation stress reduces global expression differences between species-likely because many general stress response mechanisms are shared among species-but that all species showed plastic expression changes at hundreds of loci during desiccation. Nonetheless, D. novamexicana had the fewest genes with significant plastic expression changes, despite having the highest desiccation resistance. Of the genes that were significantly differentially expressed between species-either within each treatment (>200 loci), constitutively regardless of treatment (36 loci), or with different species-specific plasticity (26 loci)-GO analysis did not find significant enrichment of any major gene pathways or broader functions associated with desiccation stress. Taken together, these data indicate that if gene expression changes contribute to differential desiccation resistance between species, these differences are likely shaped by a relatively small set of influential genes rather than broad genome-wide differentiation in stress response mechanisms. Finally, among the set of genes with the greatest between-species plasticity, we identified an interesting set of immune-response genes with consistent but opposing reaction norms between sexes, whose potential functional role in sex-specific mechanisms of desiccation resistance remains to be determined.

Desiccation resistance and pigmentation variation reflects bioclimatic differences in the Drosophila americana species complex.

BACKGROUND: Disentangling the selective factors shaping adaptive trait variation is an important but challenging task. Many studies-especially in Drosophila-have documented trait variation along latitudinal or altitudinal clines, but frequently lack resolution about specific environmental gradients that could be causal selective agents, and often do not investigate covariation between traits simultaneously. Here we examined variation in multiple macroecological factors across geographic space and their associations with variation in three physiological traits (desiccation resistance, UV resistance, and pigmentation) at both population and species scales, to address the role of abiotic environment in shaping trait variation. RESULTS: Using environmental data from collection locations of three North American Drosophila species-D. americana americana, D. americana texana and D. novamexicana-we identified two primary axes of macroecological variation; these differentiated species habitats and were strongly loaded for precipitation and moisture variables. In nine focal populations (three per species) assayed for each trait, we detected significant species-level variation for both desiccation resistance and pigmentation, but not for UV resistance. Species-level trait variation was consistent with differential natural selection imposed by variation in habitat water availability, although patterns of variation differed between desiccation resistance and pigmentation, and we found little evidence for pleiotropy between traits. CONCLUSIONS: Our multi-faceted approach enabled us to identify potential agents of natural selection and examine how they might influence the evolution of multiple traits at different evolutionary scales. Our findings highlight that environmental factors influence functional trait variation in ways that can be complex, and point to the importance of studies that examine these relationships at both population- and species-levels.

A simple genetic architecture and low constraint allow rapid floral evolution in a diverse and recently radiating plant genus.

Genetic correlations among different components of phenotypes, especially those resulting from pleiotropy, can constrain or facilitate trait evolution. These factors could especially influence the evolution of traits that are functionally integrated, such as those comprising the flower. Indeed, pleiotropy is proposed as a main driver of repeated convergent trait transitions, including the evolution of phenotypically similar pollinator syndromes. We assessed the role of pleiotropy in the differentiation of floral and other reproductive traits between two species - Jaltomata sinuosa and J. umbellata (Solanaceae) - that have divergent suites of floral traits consistent with bee and hummingbird pollination, respectively. To do so, we generated a hybrid population and examined the genetic architecture (trait segregation and quantitative trait locus (QTL) distribution) underlying 25 floral and fertility traits. We found that most floral traits had a relatively simple genetic basis (few, predominantly additive, QTLs of moderate to large effect), as well as little evidence of antagonistic pleiotropy (few trait correlations and QTL colocalization, particularly between traits of different classes). However, we did detect a potential case of adaptive pleiotropy among floral size and nectar traits. These mechanisms may have facilitated the rapid floral trait evolution observed within Jaltomata, and may be a common component of rapid phenotypic change more broadly.

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.