Prezygotic Reproductive Isolation Research Articles

Prezygotic barriers effectively limit hybridization in a rapid evolutionary radiation.

Hybridization is increasingly recognized as an important evolutionary process across the tree of life. In many clades, phylogenomic approaches have permitted unparalleled insight into the extent and frequency of hybridization. However, we continue to lack a deep understanding of the factors that limit and shape patterns of hybridization, especially in evolutionary radiations. In this study, we characterized patterns of introgression across Costus (Costaceae), a young evolutionary radiation of tropical understory plants that maintain widespread interfertility despite exhibiting strong prezygotic reproductive isolation. We analyzed a phylogenomic dataset of 756 genes from 54 Costus species using multiple complementary approaches - D-statistics, gene-tree-based tests, and phylogenetic network analyses - to detect and characterize introgression events throughout the evolutionary history of the radiation. Our results identified a moderate number of introgression events, including a particularly ancient, well-supported event spanning one of the deepest divergences in the clade. Most introgression events occurred between taxa or ancestral lineages that shared the same pollination syndrome (bee-pollinated or hummingbird-pollinated). These findings suggest that prezygotic barriers, including pollinator specialization, have been key to the balance between introgression and reproductive isolation in Costus.

The genetic basis of traits associated with the evolution of serpentine endemism in monkeyflowers.

The floras on chemically and physically challenging soils, such as gypsum, shale, and serpentine, are characterized by narrowly endemic species. The evolution of edaphic endemics may be facilitated or constrained by genetic correlations among traits contributing to adaptation and reproductive isolation across soil boundaries. The yellow monkeyflowers in the Mimulus guttatus species complex are an ideal system in which to examine these evolutionary patterns. To determine the genetic basis of adaptive and prezygotic isolating traits, we performed genetic mapping experiments with F2 hybrids derived from a cross between a serpentine endemic, M. nudatus, and its close relative M. guttatus. Few large effect and many small effect QTL contribute to interspecific divergence in life history, floral, and leaf traits, and a history of directional selection contributed to trait divergence. Loci contributing to adaptive traits and prezygotic reproductive isolation overlap, and their allelic effects are largely in the direction of species divergence. These loci contain promising candidate genes regulating flowering time and plant organ size. Together, our results suggest that genetic correlations among traits can facilitate the evolution of adaptation and speciation and may be a common feature of the genetic architecture of divergence between edaphic endemics and their widespread relatives.

Israeli Oncocyclus irises: Phylogenetic relationships and evolutionary history

Abstract Oncocyclus is a large group of bearded irises with poorly known phylogeny and evolutionary history. In Israel, Iris sect. Oncocyclus comprises eight species belonging to three aggregates. We used a combination of approaches to resolve the phylogenetic relationships of these species and indicate the evolutionary forces responsible for their origin. We sequenced the whole chloroplast genomes of species and integrated a phylogenetic tree with results of genetic (AFLP) divergence, degree of reproductive isolation, and species distribution modeling. Our findings suggest that quantitative and even qualitative morphological characters, such as flower color, are unreliable diagnostic traits for Oncocyclus taxonomy; that some recognized species comprise more than one species; and that group evolution did not involve the origin of distinct flower aggregates. A lack of pre-zygotic reproductive isolation agrees with the very low variability of the Oncocyclus plastome, suggesting that Israeli Oncocyclus species are very young. Homoploid hybridization followed or preceded by long periods of geographic isolation, and local selection likely contributed to speciation in Oncocyclus. In the group evolutionary history, importance of homoploid hybridization and local selection differed among species, but limited gene flow played a crucial role for all species.

Limited song-mixing without genomic gene flow in a contact zone between two songbird species.

Song is considered to play an important role in the maintenance of prezygotic reproductive isolation between closely related songbird species. Therefore, song-mixing in a contact zone between closely related species is often considered as evidence of hybridization. The Sichuan Leaf Warbler Phylloscopus forresti and the Gansu Leaf Warbler P. kansuensis, which diverged two million years ago, have formed a contact zone in the south of the Gansu Province of China, where mixed-songs have been observed. In this study, we investigated the potential causes and consequences of song-mixing by integrating bioacoustic, morphological, mitochondrial, and genomic data with field ecological observations. We found that the two species display no apparent morphological differences, while their songs differ dramatically. We demonstrated that approximately 11% of the males in the contact zone sang mixed-songs. Two males singing mixed-song were genotyped, and both were found to be P. kansuensis. Despite the presence of mixed-singers, population genomic analyses detected no signs of recent gene flow between the two species, although two possible cases of mitochondrial introgression were identified. We conclude that the rather limited song-mixing does not lead to, or result from, hybridization, and hence does not result in the breakdown of reproductive barriers between these cryptic species.

Males and females contribute differently to the evolution of habitat segregation driven by hybridization.

Costly heterospecific mating interactions, such as hybridization, select for prezygotic reproductive isolation. One of the potential traits responding to the selection arising from maladaptive hybridization is habitat preference, whose divergence results in interspecific habitat segregation. Theoretical studies have so far assumed that habitat preference is a sexually shared trait. However, male and female habitat preferences can experience different selection pressures. Here, by combining analytical and simulation approaches, we theoretically examine the evolution of sex-specific habitat preferences. Habitat segregation can have demographic consequences, potentially generating eco-evolutionary dynamics. We thus explicitly consider demography in the simulation model. We also vary the degrees of species discrimination to examine how mate choice influences the evolution of habitat preferences. Results show that both sexes can reduce hybridisation risk by settling in the habitats where abundant conspecific mates reside. However, when females can discriminate species, excess conspecific male aggregation intensifies male-male competition for mating opportunities, posing an obstacle to conspecific aggregation. Meanwhile, conspecific female aggregation attracts conspecific males, by offering the mating opportunity. Therefore, under effective species discrimination, females play a leading role in initiating habitat use divergence. Simulations typically result in either the coexistence with established habitat segregation or the extinction of one of the species. The former result is especially likely when the species differ to some extent in habitat preferences upon secondary contact. Our results disentangle the selection pressures acting on male and female habitat preferences, deepening our understanding of the evolutionary process of habitat segregation due to hybridization.

The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe

Appendicularian tunicates are some of the most abundant mesozooplankton organisms with key roles in marine trophic webs and global carbon flux. Like most appendicularians with cosmopolitan distributions, Oikopleura dioica Fol, 1872 is considered a single species worldwide based on morphological features that distinguish them from other appendicularians. Despite their abundance, however, there are still only ~ 70 described appendicularian species, compared to over 2800 ascidian tunicates. Here we perform a molecular phylogenetic, morphological, and reproductive assessment of O. dioica specimens collected from the Ryukyu Archipelago, mainland Japan, and Europe. The specimens are morphologically very similar, with only detailed examination of the oikoplastic epithelium and quantitative measurements revealing minor distinguishing characteristics. Phylogenetic analyses of the ribosomal gene loci and mitochondrial cytochrome oxidase I (COI) gene strongly indicate that they form three separate genetic clades despite their morphological similarities. Finally, in vitro crosses between the Ryukyu and mainland Japanese specimens show total prezygotic reproductive isolation. Our results reveal that the current taxonomic O. dioica classification likely hides multiple cryptic species, highlighting the genetic diversity and complexity of their population structures. Cryptic organisms are often hidden under a single species name because their morphological similarities make them difficult to distinguish and their correct identification is fundamental to understanding Earth’s biodiversity. O. dioica is an attractive model to understand how morphological conservation can be maintained despite pronounced genetic divergence.

The strength of reproductive isolating barriers in seed plants: Insights from studies quantifying premating and postmating reproductive barriers over the past 15 years.

Speciation is driven by the evolution of reproductive isolating barriers that reduce, and ultimately prevent, substantial gene flow between lineages. Despite its central role in evolutionary biology, the process can be difficult to study because it proceeds differently among groups and may occur over long timescales. Due to this complexity, we typically rely on generalizations of empirical data to describe and understand the process. Previous reviews of reproductive isolation (RI) in flowering plants have suggested that prezygotic or extrinsic barriers generally have a stronger effect on reducing gene flow compared to postzygotic or intrinsic barriers. Past conclusions have rested on relatively few empirical estimates of RI; however, RI data have become increasingly abundant over the past 15 years. We analyzed data from recent studies quantifying multiple pre- and postmating barriers in plants and compared the strengths of isolating barriers across 89 taxa pairs using standardized RI metrics. Individual prezygotic barriers were on average stronger than individual postzygotic barriers, and the total strength of prezygotic RI was approximately twice that of postzygotic RI. These findings corroborate that ecological divergence and extrinsic factors, as opposed to solely the accumulation of genetic incompatibilities, are important to speciation and the maintenance of species boundaries in plants. Despite an emphasis in the literature on asymmetric postmating and postzygotic RI, we found that prezygotic barriers acted equally asymmetrically. Overall, substantial variability in the strengths of 12 isolating barriers highlights the great diversity of mechanisms that contribute to plant diversification.

Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria

BackgroundSpeciation genes contribute disproportionately to species divergence, but few examples exist, especially in vertebrates. Here we test whether Zan, which encodes the sperm acrosomal protein zonadhesin that mediates species-specific adhesion to the egg’s zona pellucida, is a speciation gene in placental mammals.ResultsGenomic ontogeny reveals that Zan arose by repurposing of a stem vertebrate gene that was lost in multiple lineages but retained in Eutheria on acquiring a function in egg recognition. A 112-species Zan sequence phylogeny, representing 17 of 19 placental Orders, resolves all species into monophyletic groups corresponding to recognized Orders and Suborders, with <5% unsupported nodes. Three other rapidly evolving germ cell genes (Adam2, Zp2, and Prm1), a paralogous somatic cell gene (TectA), and a mitochondrial gene commonly used for phylogenetic analyses (Cytb) all yield trees with poorer resolution than the Zan tree and inferior topologies relative to a widely accepted mammalian supertree. Zan divergence by intense positive selection produces dramatic species differences in the protein’s properties, with ordinal divergence rates generally reflecting species richness of placental Orders consistent with expectations for a speciation gene that acts across a wide range of taxa. Furthermore, Zan’s combined phylogenetic utility and divergence exceeds those of all other genes known to have evolved in Eutheria by positive selection, including the only other mammalian speciation gene, Prdm9.ConclusionsSpecies-specific egg recognition conferred by Zan’s functional divergence served as a mode of prezygotic reproductive isolation that promoted the extraordinary adaptive radiation and success of Eutheria.

Better time of flower opening, a good time to improve the efficiency of hybrid seed production

The proper time of flower opening, which ensures successful pollination by exposure of stamens and gynoecia via petal movement, is locally adapted to diverse environmental changes among different plants (Cheng et al., 2021). Consequently, flower opening time may differ among divergent populations as a result of long-term adaptation, thereby promoting prezygotic reproductive isolation by preventing mating and hybridization.

Molecular signatures of parallel adaptive divergence causing reproductive isolation and speciation across two genera

Parallel evolution of reproductive isolation (PERI) provides strong evidence for natural selection playing a fundamental role in the origin of species. However, PERI has been rarely demonstrated for well established species drawn from different genera. In particular, parallel molecular signatures for the same genes in response to similar habitat divergence in such different lineages is lacking. Here, based on whole-genome sequencing data, we first explore the speciation process in two sister species of Carpinus (Betulaceae) in response to divergence for temperature and soil-iron concentration in habitats they occupy in northern and southwestern China, respectively. We then determine whether parallel molecular mutations occur during speciation in this pair of species and also in another sister-species pair of the related genus, Ostryopsis, which occupy similarly divergent habitats in China. We show that gene flow occurred during the origin of both pairs of sister species since approximately 9.8 or approximately 2 million years ago, implying strong natural selection during divergence. Also, in both species pairs we detected concurrent positive selection in a gene (LHY) for flowering time and in two paralogous genes (FRO4 and FRO7) of a gene family known to be important for iron tolerance. These changes were in addition to changes in other major genes related to these two traits. The different alleles of these particular candidate genes possessed by the sister species of Carpinus were functionally tested and indicated likely to alter flowering time and iron tolerance as previously demonstrated in the pair of Ostryopsis sister species. Allelic changes in these genes may have effectively resulted in high levels of prezygotic reproductive isolation to evolve between sister species of each pair. Our results show that PERI can occur in different genera at different timescales and involve similar signatures of molecular evolution at genes or paralogues of the same gene family, causing reproductive isolation as a consequence of adaptation to similarly divergent habitats.

Laboratory-Based Mate Choices of Two Eccritotarsus Species (Hemiptera: Miridae) Used as Biological Control Agents of Water Hyacinth, Pontederia (Eichhornia) crassipes (Pontederiaceae), in South Africa

Abstract Many biological control agents have been released to manage the water hyacinth, Pontederia (Eichhornia) crassipes (C. Martius) Solms-Laubach (Pontederiaceae). They include two Eccritotarsus sources from Brazil and Peru. Distinguishing features between the two were found, necessitating their redescription. The Brazil source remained Eccritotarsus catarinensis (Carvalho), whereas the Peru source is Eccritotarsus eichhorniae Henry. Our objectives in this laboratory study were to assess the mate choice in the two species and, thus, determine which species performs better than the other. Mate choice in the form of no-choice, bichoice, and multichoice tests were conducted within and between species in a 3:1, 2:1, and 1:1 sex ratios. The E. catarinensis pair had short copula latency but long copula duration in multichoice experiments, whereas the E. eichhorniae pair had short copula latency but long copula duration in no-choice experiments. In no-choice and bichoice experiments, E. eichhorniae♀ and E. eichhorniae♂ outcompeted E. catarinensis♀ and E. catarinensis♂. In multichoice experiments, E. catarinensis♂ outcompeted E. eichhorniae♂, whereas E. eichhorniae♀ outcompeted E. catarinensis♀. Despite being shown to be more fecund, E. eichhorniae is likely not to outcompete E. catarinensis where the two species co-occur and that E. catarinensis♂ and E. eichhorniae♀ will outcompete their counterparts. Previous research indicated that when E. catarinensis♂ and E. eichhorniae♀ are crossed, they produce few offspring, underscoring the need to confirm if the two species will coexist, compete, or displace each other in their natural habitats. Our findings further support that prezygotic reproductive isolation mechanisms led to the speciation of these species.

First successful hybridization experiment between native European weatherfish (Misgurnus fossilis) and non-native Oriental weatherfish (M. anguillicaudatus) reveals no evidence for postzygotic barriers

The European weatherfish Misgurnus fossilis (Linnaeus, 1758) is a threatened freshwater species in large parts of Europe and might come under pressure from currently establishing exotic weatherfish species. Additional threats might arise if those species hybridize which has been questioned in previous research. Regarding the hybridization of M. fossilis × M. anguillicaudatus (Cantor, 1842), we demonstrate that despite the considerable genetic distance between parental species, the estimated long divergence time and different ploidy levels do not represent a postzygotic barrier for hybridization of the European and Oriental weatherfish. The paternal species can be easily differentiated based on external pigment patterns with hybrids showing intermediate patterns. No difference in standard metabolic rate, indicating a lack of hybrid vigour, renders predictions of potential threats to the European weatherfish from hybridization with the Oriental weatherfish difficult. Therefore, the genetic and physiological basis of invasiveness via hybridization remains elusive in Misgurnus species and requires further research. The existence of prezygotic reproductive isolation mechanisms and the fertility of F1 hybrids remains to be tested to predict the potential threats of globally invasive Oriental weatherfish species.

Patterns of reproductive isolation in a haplodiploid mite, Amphitetranychus viennensis: prezygotic isolation, hybrid inviability and hybrid sterility

BackgroundEvolution of reproductive isolation is an important process, generating biodiversity and driving speciation. To better understand this process, it is necessary to investigate factors underlying reproductive isolation through various approaches but also in various taxa. Previous studies, mainly focusing on diploid animals, supported the prevalent view that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection by showing a positive relationship between the degree of reproductive isolation and genetic distance. Haplodiploid animals are expected to generate additional insight into speciation, but few studies investigated the prevalent view in haplodiploid animals. In this study, we investigate whether the relationship also holds in a haplodiploid spider mite, Amphitetranychus viennensis (Zacher).ResultsWe sampled seven populations of the mite in the Palaearctic region, measured their genetic distance (mtDNA) and carried out cross experiments with all combinations. We analyzed how lack of fertilization rate (as measure of prezygotic isolation) as well as hybrid inviability and hybrid sterility (as measures of postzygotic isolation) varies with genetic distance. We found that the degree of reproductive isolation varies among cross combinations, and that all three measures of reproductive isolation have a positive relationship with genetic distance. Based on the mtDNA marker, lack of fertilization rate, hybrid female inviability and hybrid female sterility were estimated to be nearly complete (99.0–99.9% barrier) at genetic distances of 0.475–0.657, 0.150–0.209 and 0.145–0.210, respectively. Besides, we found asymmetries in reproductive isolation.ConclusionsThe prevalent view on the evolution of reproductive barriers is supported in the haplodiploid spider mite we studied here. According to the estimated minimum genetic distance for total reproductive isolation in parent population crosses in this study and previous work, a genetic distance of 0.15–0.21 in mtDNA (COI) appears required for speciation in spider mites. Variations and asymmetries in the degree of reproductive isolation highlight the importance of reinforcement of prezygotic reproductive isolation through incompatibility and the importance of cytonuclear interactions for reproductive isolation in haplodiploid spider mites.

Concordance of the spectral properties of dorsal wing scales with the phylogeographic structure of European male Polyommatus icarus butterflies

The males of more than 80% of the Lycaenidae species belonging to the tribe Polyommatini exhibit structural coloration on their dorsal wing surfaces. These colors have a role in reinforcement in prezygotic reproductive isolation. The species-specific colors are produced by the cellular self-assembly of chitin/air nanocomposites. The spectral position of the reflectance maximum of such photonic nanoarchitectures depends on the nanoscale geometric dimensions of the elements building up the nanostructure. Previous work showed that the coloration of male Polyommatus icarus butterflies in the Western and Eastern Palearctic exhibits a characteristic spectral difference (20 nm). We investigated the coloration and the de novo developed DNA microsatellites of 80 P. icarus specimens from Europe from four sampling locations, spanning a distance of 1621 km. Remarkably good concordance was found between the spectral properties of the blue sexual signaling color (coincident within 5 nm) and the population genetic structure as revealed by 10 microsatellites for the P. icarus species.

Calling Phenology and Call Structure of Sympatric Treefrogs in Eastern Texas

Anurans often call in acoustically complex choruses, which can consist of multiple closely related species. Closely related sympatric species often differ in phenology and the acoustic properties of male advertisement calls, which, in some species, are phenotypically plastic. The tetraploid Eastern Gray Treefrog (Hyla versicolor) originates from multiple, separate hybridization events of the diploid Cope's Gray Treefrog (Hyla chrysoscelis) with now-extinct diploid anurans. The two species are visually indistinguishable, exhibit highly similar ecology and behavior, and co-occur frequently across their range. Males of these species can be distinguished from each other by the structure of their advertisement calls. Daily audio recordings and weather data were collected for five consecutive years from eight breeding sites of sympatric H. versicolor and H. chrysoscelis. We examined spatial and temporal patterns in calling phenology and responses of both species to environmental cues. Additionally, we measured three advertisement call properties to evaluate differences in call structure based on community composition. The phenologies of the two gray treefrog species were nearly identical, and the presence of the other species did not significantly affect call pulse rate or dominant frequency in either species. Chorus composition affected call duration in both species, but in H. chrysoscelis, this effect was temperature dependent. Our results indicate that while calling phenology does not contribute to reproductive isolation in sympatric gray treefrogs, modulation of phenotypically plastic call properties could affect male mating success and prezygotic reproductive isolation.

A reversal in sensory processing accompanies ongoing ecological divergence and speciation in Rhagoletis pomonella.

Changes in behaviour often drive rapid adaptive evolution and speciation. However, the mechanistic basis for behavioural shifts is largely unknown. The tephritid fruit fly Rhagoletis pomonella is an example of ecological specialization and speciation in action via a recent host plant shift from hawthorn to apple. These flies primarily use specific odours to locate fruit, and because they mate only on or near host fruit, changes in odour preference for apples versus hawthorns translate directly to prezygotic reproductive isolation, initiating speciation. Using a variety of techniques, we found a reversal between apple and hawthorn flies in the sensory processing of key odours associated with host fruit preference at the first olfactory synapse, linking changes in the antennal lobe of the brain with ongoing ecological divergence. Indeed, changes to specific neural pathways of any sensory modality may be a broad mechanism for changes in animal behaviour, catalysing the genesis of new biodiversity.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Postzygotic reproductive isolation among three Saccharomyces yeast species.

We have previously isolated heterothallic haploid strains from original homothallic diploids of two yeast species in the family Saccharomycetaceae. In this study, heterothallic haploid strains were isolated from an original homothallic diploid of Saccharomyces kudriavzevii type strain, followed by investigation of sexual interactions among these yeast strains, in addition to S. cerevisiae laboratory strains. It has been shown that prezygotic reproductive isolation was observed between Kazachstania naganishii and S. cerevisiae with α-factor mating pheromones representing crossaction with each other beyond the genus boundary. Using heterothallic strains, postzygotic reproductive isolation system was shown to reside in the genus Saccharomyces by mass mating and cell-cell contact experiments. In mass mating experiments, crossaction of α-factor and a-factor mating pheromones and sexual agglutination effectively occurred beyond species boundaries among S. kudriavzevii, S. paradoxus, and S. cerevisiae. When the fates of cell-cell pairs from these Saccharomyces yeast species were systematically chased one by one, interspecific F1 hybrids were effectively produced, while sporulations were partially prohibited, with spore germination perfectly blocked in the hybrids. These results indicated that postzygotic reproductive isolation definitively resides among these Saccharomyces yeast species and that disorder of chromosome organization had to some extent occurred in interspecific F1 hybrids.

Minimal prezygotic isolation between ecologically divergent sibling species.

Divergence in mating signals typically accompanies speciation. We examine two ecologically divergent sibling species of crickets to assess the degree and timing of the evolution of prezygotic reproductive isolation. Gryllus saxatilis occurs in rocky habitats throughout western North America with long-winged individuals capable of long-distance dispersal; Gryllus navajo is endemic to red-rock sandstone areas of south-eastern Utah and north-eastern Arizona and has short-winged individuals only capable of limited dispersal. Previous genetic work suggested some degree of introgression and/or incomplete lineage sorting is likely. Here we: (1) use restriction site associated DNA sequencing (RAD-seq) genetic data to describe the degree of genetic divergence among species and populations; (2) examine the strength of prezygotic isolation by (i) quantifying differences among male mating songs, and (ii) testing whether females prefer G. saxatilis or G. navajo calling songs. Our results show that genetically distinct "pure" species populations and genetically intermediate populations exist. Male mating songs are statistically distinguishable, but the absolute differences are small. In playback experiments, females from pure populations had no preference based on song; however, females from a genetically intermediate population preferred G. navajo song. Together these results suggest that prezygotic isolation is minimal, and mediated by female behaviour in admixed populations.

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.