Sperm competition is a pervasive selective force in evolution, shaping reproductive anatomy, physiology and behaviour. Here, we present comparative evidence that varying sperm competition levels account for variation in the male reproductive anatomy of rodents, the largest and most diverse mammalian order. We focus on the sperm-producing testes and the accessory reproductive glands, which produce the seminal fluid fraction of the ejaculate. We demonstrate a positive association between relative testis size and the prevalence of within-litter multiple paternity, consistent with previous analyses in which relative testis size has been found to correlate with sperm competition levels inferred from social organization and mating systems. We further demonstrate an association between sperm competition level and the relative size of at least two accessory reproductive glands: the seminal vesicles and anterior prostate. The size of the major product of these glands-the copulatory plug-is also found to vary with sperm competition level. Our findings thus suggest that selection for larger plugs under sperm competition may explain variation in accessory gland size, and highlight the need to consider both sperm and non-sperm components of the male ejaculate in the context of post-copulatory sexual selection.

An elevated rate of substitution characterizes the molecular evolution of reproductive proteins from a wide range of taxa. Although the selective pressures explaining this rapid evolution are yet to be resolved, recent evidence implicates sexual selection as a potentially important explanatory factor. To investigate this hypothesis, we sought evidence of a high rate of adaptive gene evolution linked to postcopulatory sexual selection in muroid rodents, a model vertebrate group displaying a broad range of mating systems. Specifically, we sequenced 7 genes from diverse rodents that are expressed in the testes, prostate, or seminal vesicles, products of which have the potential to act in sperm competition. We inferred positive Darwinian selection in these genes by estimation of the ratio of nonsynonymous (d(N), amino acid changing) to synonymous (d(S), amino acid retaining) substitution rates (omega = d(N)/d(S)). Next, we tested whether variation in this ratio among lineages could be attributed to interspecific variation in mating systems, as inferred from the variation in these rodents' relative testis sizes (RTS). Four of the 7 genes examined (Prm1, Sva, Acrv1, and Svs2, but not Svp2, Msmb, or Spink3) exhibit unambiguous evidence of positive selection. One of these, the seminal vesicle-derived protein Svs2, also shows some evidence for a concentration of positive selection in those lineages in which sperm competition is common. However, this was not a general trend among all the rodent genes we examined. Using the same methods, we then reanalyzed previously published data on 2 primate genes, SEMG1 and SEMG2. Although SEMG2 also shows evidence of positive selection concentrated in lineages subject to high levels of sperm competition, no such trend was found for SEMG1. Overall, despite a high rate of positive selection being a feature of many ejaculate proteins, these results indicate that the action of sexual selection potentially responsible for elevated evolutionary rates may be difficult to detect on a gene-by-gene basis. Although the extreme diversity of reproductive phenotypes exhibited in nature attests to the power of sexual selection, the extent to which this force predominates in driving the rapid molecular evolution of reproductive genes therefore remains to be determined.

Hermaphrodites combine the male and female sex functions into a single individual, either sequentially or simultaneously. This simple fact means that they exhibit both similarities and differences in the way in which they experience, and respond to, sexual conflict compared to separate-sexed organisms. Here, we focus on clarifying how sexual conflict concepts can be adapted to apply to all anisogamous sexual systems and review unique (or especially important) aspects of sexual conflict in hermaphroditic animals. These include conflicts over the timing of sex change in sequential hermaphrodites, and in simultaneous hermaphrodites, over both sex roles and the postmating manipulation of the sperm recipient by the sperm donor. Extending and applying sexual conflict thinking to hermaphrodites can identify general evolutionary principles and help explain some of the unique reproductive diversity found among animals exhibiting this widespread but to date understudied sexual system.

Sperm competition theory predicts that males should invest prudently in ejaculates according to levels of female promiscuity. Males may therefore be sensitive to cues in their social environment associated with sexual competition, and tailor investment in sperm production accordingly. We tested this idea experimentally for the first time, to our knowledge, in a mammal by comparing reproductive traits of male house mice (Mus musculus domesticus) that had experienced contrasting encounter regimes with potential sexual competitors. We found that daily sperm production and numbers of sperm in the caput epididymis were significantly higher in subjects that had experienced a high encounter rate of social cues from three other males compared to those that had experienced a low encounter rate of social cues from just one other male. Epididymal sperm counts were negatively correlated with the frequency of scentmarking behaviour across all males in our study, suggesting that investment in ejaculate production may be traded off against traits that function in gaining copulations, although there was no difference in overall levels of scent marking between treatment groups. We conclude that social experience-mediated phenotypic plasticity in mammalian spermatogenesis is likely to be adaptive under sperm competition, enabling males to balance the energetic costs and paternity-enhancing benefits of ejaculate production, and is a potentially widespread explanation for intraspecific variation in ejaculate expenditure.

During insemination, males of internally fertilizing species transfer a complex array of seminal fluid proteins to the female reproductive tract. These proteins can have profound effects on female reproductive physiology and behavior and are thought to mediate postcopulatory sexual selection and intersexual conflict. Such selection may cause seminal fluid to evolve rapidly, with potentially important consequences for speciation. Here we investigate the evolution of seminal fluid proteins in a major mammalian radiation, the muroid rodents, by quantifying diversity in seminal fluid proteome composition for the first time across a broad range of closely related species. Using comparative proteomics techniques to identify and cross-match proteins, we demonstrate that rodent seminal fluid is highly diverse at the level of both proteomes and individual proteins. The striking interspecific heterogeneity in seminal fluid composition revealed by our survey far exceeds that seen in a second proteome of comparable complexity, skeletal muscle, indicating that the complement of proteins expressed in seminal fluid may be subject to rapid diversification. We further show that orthologous seminal fluid proteins exhibit substantial interspecific variation in molecular mass. Because this variation cannot be attributed to differential glycosylation or radical differences in termination sites, it is strongly suggestive of rapid amino acid divergence. Sperm competition is implicated in generating such divergence for at least one major seminal fluid protein in our study, SVS II, which is responsible for copulatory plug formation via transglutaminasecatalyzed cross-linking after insemination. We show that the molecular mass of SVS II is positively correlated with relative testis size across species, which could be explained by selection for an increased number of cross-linking sites involved in the formation of the copulatory plug under sperm competition.