Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.

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’.

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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.

word count: 270 23 24 Total word count: 5085 25 2 ABSTRACT 26 Despite holding a central role for fertilisation success, reproductive traits often show elevated 27 rates of evolution and diversification. The rapid evolution of seminal fluid proteins (Sfps) within 28populations is predicted to cause mis-signalling between the male ejaculate and female 29 reproductive tract between populations resulting in postmating prezygotic (PMPZ) isolation. 30Crosses between populations of Drosophila montana show PMPZ isolation in the form of 31 reduced fertilisation success in both noncompetitive and competitive contexts. Here we test 32 whether male ejaculate proteins deriving from either the accessory glands or the ejaculatory 33 bulb differ between populations using liquid chromatography tandem mass spectrometry. We 34 find more than 150 differentially abundant proteins between populations which may contribute 35 to PMPZ isolation. These proteins include a number of proteases and peptidases, and several 36 orthologs of D. melanogaster Sfps, all known to mediate fertilisation success and which mimic 37 PMPZ isolation phenotypes. Males of one population typically produced greater quantities of 38 Sfps and the strongest PMPZ isolation occurs in this direction. The accessory glands and 39 ejaculatory bulb have different functions and the ejaculatory bulb contributes more to 40 population differences than the accessory glands. Proteins with a secretory signal, but not 41 Sfps, evolve faster than non-secretory proteins although the conservative criteria used to 42 define Sfps may have impaired the ability to identify rapidly evolving proteins. We take 43 advantage of quantitative proteomics data from three Drosophila species to determine shared 44 and unique functional enrichments of Sfps that could be subject to selection between taxa and 45 subsequently mediate PMPZ isolation. Our study provides the first high throughput quantitative 46 proteomic evidence showing divergence of reproductive proteins implicated in the emergence 47 IMPACT SUMMARY (word count: 300) 51Identifying traits that prevent successful interbreeding is key to understanding early stages of 52 the formation of new species, or speciation. Reproductive isolation arising prior to and during 53 fertilisation frequently involves differences in how the sexes interact. In internally fertilising taxa, 54 such interactions are mediated between the female reproductive tract where fertilisation occurs 55 and the receipt of the ejaculate necessary for fertilisation. Because ejaculate proteins are at 56 least partially responsible for these interactions, differences in male ejaculate protein 57 composition could negatively impact fertilisation success, generating reproductive isolation. 58While the biological classes of ejaculate proteins are shared across all animal taxa, proteins 59 that are secreted by males tend to show rapid evolution in gene expression and genetic 60 sequence. Thus, reproductive proteins are suggested as prime targets facilitating reproductive 61 iso...