From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

When and how can we predict adaptive responses to climate change?

Predicting if, when, and how populations can adapt to climate change constitutes one of the greatest challenges in science today. Here, we build from contributions to the special issue on evolutionary adaptation to climate change, a survey of its authors, and recent literature to explore the limits and opportunities for predicting adaptive responses to climate change. We outline what might be predictable now, in the future, and perhaps never even with our best efforts. More accurate predictions are expected for traits characterized by a well-understood mapping between genotypes and phenotypes and traits experiencing strong, direct selection due to climate change. A meta-analysis revealed an overall moderate trait heritability and evolvability in studies performed under future climate conditions but indicated no significant change between current and future climate conditions, suggesting neither more nor less genetic variation for adapting to future climates. Predicting population persistence and evolutionary rescue remains uncertain, especially for the many species without sufficient ecological data. Still, when polled, authors contributing to this special issue were relatively optimistic about our ability to predict future evolutionary responses to climate change. Predictions will improve as we expand efforts to understand diverse organisms, their ecology, and their adaptive potential. Advancements in functional genomic resources, especially their extension to non-model species and the union of evolutionary experiments and "omics," should also enhance predictions. Although predicting evolutionary responses to climate change remains challenging, even small advances will reduce the substantial uncertainties surrounding future evolutionary responses to climate change.

Heat stress reveals a fertility debt owing to postcopulatory sexual selection.

Climates are changing rapidly, demanding equally rapid adaptation of natural populations. Whether sexual selection can aid such adaptation is under debate; while sexual selection should promote adaptation when individuals with high mating success are also best adapted to their local surroundings, the expression of sexually selected traits can incur costs. Here we asked what the demographic consequences of such costs may be once climates change to become harsher and the strength of natural selection increases. We first adopted a classic life history theory framework, incorporating a trade-off between reproduction and maintenance, and applied it to the male germline to generate formalized predictions for how an evolutionary history of strong postcopulatory sexual selection (sperm competition) may affect male fertility under acute adult heat stress. We then tested these predictions by assessing the thermal sensitivity of fertility (TSF) in replicated lineages of seed beetles maintained for 68 generations under three alternative mating regimes manipulating the opportunity for sexual and natural selection. In line with the theoretical predictions, we find that males evolving under strong sexual selection suffer from increased TSF. Interestingly, females from the regime under strong sexual selection, who experienced relaxed selection on their own reproductive effort, had high fertility in benign settings but suffered increased TSF, like their brothers. This implies that female fertility and TSF evolved through genetic correlation with reproductive traits sexually selected in males. Paternal but not maternal heat stress reduced offspring fertility with no evidence for adaptive transgenerational plasticity among heat-exposed offspring, indicating that the observed effects may compound over generations. Our results suggest that trade-offs between fertility and traits increasing success in postcopulatory sexual selection can be revealed in harsh environments. This can put polyandrous species under immediate risk during extreme heat waves expected under future climate change.

Intra-specific correlations between ejaculate traits and competitive fertilization success: a meta-analysis across species and fertilization modes.

Understanding of how selection can act on traits that improve competitiveness and subsequent paternity has advanced, including the idea that internal and external fertilization presents different environments that may select differentially on ejaculate traits. However, no studies have quantitatively synthesized the intra-specific relationships between these traits and paternity. Therefore, we conducted a meta-analysis across 52 papers to determine which ejaculate traits positively correlate with paternity share and how these correlations vary with fertilization mode. Overall, most ejaculate traits were positively associated with paternity, with the notable exception of sperm length. Sub-analyses on sperm number, sperm length, and sperm velocity revealed no statistical differences between fertilization modes in the relationship between traits and paternity when all effect sizes across species were combined. However, in a sub-analysis on fish species only, we found evidence that sperm velocity may be more important in external fertilizers. We also observed differences in the importance of phylogenetic relatedness and some species-specific differences. Our results suggest that while most ejaculate traits should be under positive directional selection in both internal and external fertilizers, sperm length may be subject to more nuanced selection pressures. Overall, we highlight important patterns of intra-specific relationships between ejaculate traits and competitive fertilization success.

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.

Selection on the Fly: Short-Term Adaptation to an Altered Sexual Selection Regime in Drosophila pseudoobscura.

Experimental evolution studies are powerful approaches to examine the evolutionary history of lab populations. Such studies have shed light on how selection changes phenotypes and genotypes. Most of these studies have not examined the time course of adaptation under sexual selection manipulation, by resequencing the populations' genomes at multiple time points. Here, we analyze allele frequency trajectories in Drosophila pseudoobscura where we altered their sexual selection regime for 200 generations and sequenced pooled populations at 5 time points. The intensity of sexual selection was either relaxed in monogamous populations (M) or elevated in polyandrous lines (E). We present a comprehensive study of how selection alters population genetics parameters at the chromosome and gene level. We investigate differences in the effective population size-Ne-between the treatments, and perform a genome-wide scan to identify signatures of selection from the time-series data. We found genomic signatures of adaptation to both regimes in D. pseudoobscura. There are more significant variants in E lines as expected from stronger sexual selection. However, we found that the response on the X chromosome was substantial in both treatments, more pronounced in E and restricted to the more recently sex-linked chromosome arm XR in M. In the first generations of experimental evolution, we estimate Ne to be lower on the X in E lines, which might indicate a swift adaptive response at the onset of selection. Additionally, the third chromosome was affected by elevated polyandry whereby its distal end harbors a region showing a strong signal of adaptive evolution especially in E lines.

Fertilization mode differentially impacts the evolution of vertebrate sperm components.

Environmental change frequently drives morphological diversification, including at the cellular level. Transitions in the environment where fertilization occurs (i.e., fertilization mode) are hypothesized to be a driver of the extreme diversity in sperm morphology observed in animals. Yet how fertilization mode impacts the evolution of sperm components-head, midpiece, and flagellum-each with different functional roles that must act as an integrated unit remains unclear. Here, we test this hypothesis by examining the evolution of sperm component lengths across 1103 species of vertebrates varying in fertilization mode (external vs. internal fertilization). Sperm component length is explained in part by fertilization mode across vertebrates, but how fertilization mode influences sperm evolution varies among sperm components and vertebrate clades. We also identify evolutionary responses not influenced by fertilization mode: midpieces evolve rapidly in both external and internal fertilizers. Fertilization mode thus influences vertebrate sperm evolution through complex component- and clade-specific evolutionary responses.

Experimental sexual selection reveals rapid evolutionary divergence in sex-specific transcriptomes and their interactions following mating.

Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.

Female fruit flies cannot protect stored sperm from high temperature damage.

Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. Here, we test whether females from two fruit fly species can protect stored sperm from a high temperature stress. We find that sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is damaging stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to damage mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.

Experimental sexual selection affects the evolution of physiological and life-history traits.

Sexual selection and sexual conflict are expected to affect all aspects of the phenotype, not only traits that are directly involved in reproduction. Here, we show coordinated evolution of multiple physiological and life-history traits in response to long-term experimental manipulation of the mating system in populations of Drosophila pseudoobscura. Development time was extended under polyandry relative to monogamy in both sexes, potentially due to higher investment in traits linked to sexual selection and sexual conflict. Individuals (especially males) evolving under polyandry had higher metabolic rates and locomotor activity than those evolving under monogamy. Polyandry individuals also invested more in metabolites associated with increased endurance capacity and efficient energy metabolism and regulation, namely lipids and glycogen. Finally, polyandry males were less desiccation- and starvation resistant than monogamy males, suggesting trade-offs between resistance and sexually selected traits. Our results provide experimental evidence that mating systems can impose selection that influences the evolution of non-sexual phenotypes such as development, activity, metabolism and nutrient homeostasis.

SpermTree, a species-level database of sperm morphology spanning the animal tree of life.

Sperm are the most morphologically variable cell type known, despite performing the same functional role of fertilizing eggs across all sexually reproducing species. Sperm morphology commonly varies among individuals, populations, closely related species, and across animal phyla. Sperm morphology has long been used as a tool for placing species in a phylogenetic context and a range of selective forces are hypothesized to influence sperm evolution and diversification. However, we currently lack robust examinations of macroevolutionary (i.e. across phyla) patterns of sperm evolution, due largely to the challenges of comparing sperm morphological data across the animal tree of life. Here we describe the SpermTree database, which currently represents 5,675 morphological descriptions of sperm morphology from 4,705 unique species from 27 animal phyla. This dataset includes measurements of sperm head, midpiece, flagellum and total length, the latter of which spans four orders of magnitude. All entries in the dataset are matched to currently accepted scientific names in taxonomic databases, facilitating the use of these data in analyses examining sperm evolution in animals.

Experimental evolution of local adaptation under unidimensional and multidimensional selection.

Local adaptation is a fundamental evolutionary process generating biological diversity and potentially enabling ecological speciation. Divergent selection underlies the evolution of local adaptation in spatially structured populations by driving their adaptation toward local optima. Environments rarely differ along just one environmental axis; therefore, divergent selection may often be multidimensional. How the dimensionality of divergent selection affects local adaptation is unclear: evolutionary theory predicts that increasing dimensionality will increase local adaptation when associated with stronger overall selection but may have less predictable effects if selection strengths are equal. Experiments are required that allow the effect of the dimensionality of selection on local adaptation to be tested independently of the total strength of selection. We experimentally evolved 32 pairs of monogonont rotifer populations under either unidimensional divergent selection (a single pair of stressors) or multidimensional divergent selection (three pairs of stressors), keeping the total strength of selection equal between treatments. At regular intervals, we assayed fitness in home and away environments to assess local adaptation. We observed an initial increase and subsequent decline of local adaptation in populations exposed to multidimensional selection, compared with a slower but eventually stronger increase in local adaptation in populations exposed to unidimensional selection. Our results contrast with existing predictions, such as the "weak multifarious" and "stronger selection" hypotheses. Instead, we hypothesize that adaptation to multidimensional divergent selection may favor generalist genotypes and only produce transient local adaptation.

The genetic basis and adult reproductive consequences of developmental thermal plasticity.

Increasing temperature and thermal variability generate profound selection on populations. Given the fast rate of environmental change, understanding the role of plasticity and genetic adaptation in response to increasing temperatures is critical. This may be especially true for thermal effects on reproductive traits in which thermal fertility limits at high temperatures may be lower than for survival traits. Consequences of changing environments during development on adult phenotypes may be particularly problematic for core traits such as reproduction that begin early in development. Here we examine the consequences of developmental thermal plasticity on subsequent adult reproductive traits and its genetic basis. We used a panel of Drosophila melanogaster (the Drosophila Genetic Reference Panel; DGRP) in which male fertility performance was previously defined as either showing relatively little (status = 'high'-performing lines) or substantial ('low'-performing lines) decline when exposed to increasing developmental temperatures. We used a thermal reaction norm approach to quantify variation in the consequences of developmental thermal plasticity on multiple adult reproductive traits, including sex-specific responses, and to identify candidate genes underlying such variation. Developmental thermal stress impacted the means and thermal reaction norms of all reproductive traits except offspring sex ratio. Mating success declined as temperature increased with no difference between high and low lines, whereas increasing temperature resulted in declines for both male and female fertility and productivity but depended on line status. Fertility and offspring number were positively correlated within and between the sexes across lines, but males were more affected than females. We identified 933 SNPs with significant evolved genetic differentiation between high and low lines. In all, 54 of these lie within genomic windows of overall high differentiation, have significant effects of genotype on the male thermal reaction norm for productivity and are associated with 16 genes enriched for phenotypes affecting reproduction, stress responses and autophagy in Drosophila and other organisms. Our results illustrate considerable plasticity in male thermal limits on several reproductive traits following development at high temperature, and we identify differentiated loci with relevant phenotypic effects that may contribute to this population variation. While our work is on a single population, phenotypic results align with an increasing number of studies demonstrating the potential for stronger selection of thermal stress on reproductive traits, particularly in males. Such large fitness costs may have both short- and long-term consequences for the evolution of populations in response to a warming world.

Fluctuating heat stress during development exposes reproductive costs and putative benefits.

Temperature and thermal variability are increasing worldwide, with well-known survival consequences. However, effects on other potentially more thermally sensitive reproductive traits are less understood, especially when considering thermal variation. Studying the consequences of male reproduction in the context of climate warming and ability to adapt is becoming increasingly relevant. Our goals were to test how exposure to different average temperatures that either fluctuated or remained constant impacts different male reproductive performance traits and to assess adaptive potential to future heat stress. We took advantage of a set of Drosophila melanogaster isogenic lines of different genotypes, exposing them to four different thermal conditions. These conditions represented a benign and a stressful mean temperature, applied either constantly or fluctuating around the mean and experienced during development when heat stress avoidance is hindered because of restricted mobility. We measured subsequent male reproductive performance for mating success, fertility, number of offspring produced and offspring sex ratio, and calculated the influence of thermal stress on estimated heritability and evolvability of these reproductive traits. Both costs and benefits to different thermal conditions on reproductive performance were found, with some responses varying between genotypes. Mating success improved under fluctuating benign temperature conditions and declined as temperature stress increased regardless of genotype. Fertility and productivity were severely reduced at fluctuating mean high temperature for all genotypes, but some genotypes were unaffected at constant high mean temperature. These more thermally robust genotypes showed a slight increase in productivity under the fluctuating benign condition compared to constant high temperature, despite both thermal conditions sharing the same temperature for 6 hr daily. Increasing thermal stress resulted in higher heritability and evolvability. Overall, the effects of temperature on reproductive performance depended on the trait and genotype; performance of some traits slightly increased when high temperatures were experienced for short periods but decreased substantially even when experiencing a benign temperature for a portion of each day. While thermal stress increased genetic variation that could provide adaptive potential against climate warming, this is unlikely to compensate for the overall severe negative effect on reproductive performance as mean temperature and variance increase.

Experimental evolution supports signatures of sexual selection in genomic divergence.

Comparative genomics has contributed to the growing evidence that sexual selection is an important component of evolutionary divergence and speciation. Divergence by sexual selection is implicated in faster rates of divergence of the X chromosome and of genes thought to underlie sexually selected traits, including genes that are sex biased in expression. However, accurately inferring the relative importance of complex and interacting forms of natural selection, demography, and neutral processes that occurred in the evolutionary past is challenging. Experimental evolution provides an opportunity to apply controlled treatments for multiple generations and examine the consequent genomic divergence. Here, we altered sexual selection intensity, elevating sexual selection in polyandrous lines and eliminating it in monogamous lines, and examined patterns of allele frequency divergence in the genome of Drosophila pseudoobscura after more than 160 generations of experimental evolution. Divergence is not uniform across the genome but concentrated in "islands," many of which contain candidate genes implicated in mating behaviors and other sexually selected phenotypes. These are more often seen on the X chromosome, which also shows greater divergence in F ST than neutral expectations. There are characteristic signatures of selection seen in these regions, with lower diversity on the X chromosome than the autosomes, and differences in diversity on the autosomes between selection regimes. Reduced Tajima's D within some of the divergent regions may imply that selective sweeps have occurred, despite considerable recombination. These changes are associated with both differential gene expression between the lines and sex-biased gene expression within the lines. Our results are very similar to those thought to implicate sexual selection in divergence between species and natural populations, and hence provide experimental support for the likely role of sexual selection in driving such types of genetic divergence, but also illustrate how variable outcomes can be for different genomic regions.

Fertilization mode drives sperm length evolution across the animal tree of life.

Evolutionary biologists have endeavoured to explain the extraordinary diversity of sperm morphology across animals for more than a century. One hypothesis to explain sperm diversity is that sperm length is shaped by the environment where fertilization takes place (that is, fertilization mode). Evolutionary transitions in fertilization modes may transform how selection acts on sperm length, probably by affecting postcopulatory mechanisms of sperm competition and the scope for cryptic female choice. Here, we address this hypothesis by generating a macro-evolutionary view of how fertilization mode (including external fertilizers, internal fertilizers and spermcasters) influences sperm length diversification among 3,233 species from 21 animal phyla. We show that sperm are shorter in species whose sperm are diluted in aquatic environments (that is, external fertilizers and spermcasters) and longer in species where sperm are directly transferred to females (that is, internal fertilizers). We also show that sperm length evolves faster and with a greater number of adaptive shifts in species where sperm operate within females (for example, spermcasters and internal fertilizers). Our results demonstrate that fertilization mode is a key driver in the evolution of sperm length across animals, and we argue that a complex combination of postcopulatory forces has shaped sperm length diversification throughout animal evolution.

Plastic responses of survival and fertility following heat stress in pupal and adult Drosophila virilis.

The impact of rising global temperatures on survival and reproduction is putting many species at risk of extinction. In particular, it has recently been shown that thermal effects on reproduction, especially limits to male fertility, can underpin species distributions in insects. However, the physiological factors influencing fertility at high temperatures are poorly understood. Key factors that affect somatic thermal tolerance such as hardening, the ability to phenotypically increase thermal tolerance after a mild heat shock, and the differential impact of temperature on different life stages are largely unexplored for thermal fertility tolerance. Here, we examine the impact of high temperatures on male fertility in the cosmopolitan fruit fly Drosophila virilis. We first determined whether temperature stress at either the pupal or adult life history stage impacts fertility. We then tested the capacity for heat-hardening to mitigate heat-induced sterility. We found that thermal stress reduces fertility in different ways in pupae and adults. Pupal heat stress delays sexual maturity, whereas males heated as adults can reproduce initially following heat stress, but become sterile within seven days. We also found evidence that while heat-hardening in D. virilis can improve high temperature survival, there is no significant protective impact of this same hardening treatment on fertility. These results suggest that males may be unable to prevent the costs of high temperature stress on fertility through heat-hardening, which limits a species' ability to quickly and effectively reduce fertility loss in the face of short-term high temperature events.

Within-population sperm competition intensity does not predict asymmetry in conpopulation sperm precedence.

Postcopulatory sexual selection can generate evolutionary arms races between the sexes resulting in the rapid coevolution of reproductive phenotypes. As traits affecting fertilization success diverge between populations, postmating prezygotic (PMPZ) barriers to gene flow may evolve. Conspecific sperm precedence is a form of PMPZ isolation thought to evolve early during speciation yet has mostly been studied between species. Here, we show conpopulation sperm precedence (CpSP) between Drosophila montana populations. Using Pool-seq genomic data we estimate divergence times and ask whether PMPZ isolation evolved in the face of gene flow. We find models incorporating gene flow fit the data best indicating populations experienced considerable gene flow during divergence. We find CpSP is asymmetric and mirrors asymmetry in non-competitive PMPZ isolation, suggesting these phenomena have a shared mechanism. However, we show asymmetry is unrelated to the strength of postcopulatory sexual selection acting within populations. We tested whether overlapping foreign and coevolved ejaculates within the female reproductive tract altered fertilization success but found no effect. Our results show that neither time since divergence nor sperm competitiveness predicts the strength of PMPZ isolation. We suggest that instead cryptic female choice or mutation-order divergence may drive divergence of postcopulatory phenotypes resulting in PMPZ isolation. This article is part of the theme issue 'Fifty years of sperm competition'.

Seminal fluid protein divergence among populations exhibiting postmating prezygotic reproductive isolation.

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