Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.     

Pre‐ and postcopulatory sexual selection act in concert to determine male reproductive success in Tribolium castaneum

To capture how sexual selection shapes male reproductive success across different stages of reproduction in Tribolium castaneum (Coleoptera, Tenebrionidae), we combined sequential sperm defence (P1) and sperm offence (P2) trials with additional trials where both males were added simultaneously to the female. We found a positive correlation between the relative paternity share in simultaneous male–male competition trials and the P2 trial. This suggests that males preferred by females as sires achieve superior fertilization success during sperm competition in the second male position. In simultaneous male–male competition trials, where pre‐, peri‐ and postcopulatory sexual selection were all allowed to act, the relative paternity share of preferred males was more than 20% higher than in P2 sperm competition trials where precopulatory female choice was disabled. Additional behavioural observations revealed that mating with more attractive males resulted significantly more frequently in offspring production than mating with less attractive males. Thus, by comparing male fertilization success in trials where precopulatory choice was turned off with more inclusive estimates of fertilization success where pre‐ and pericopulatory choice could occur, we show that female mate choice may effectively inhibit sperm competition. Female mate choice and sperm competition (P2) are positively correlated, which is consistent with directional sexual selection in this species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 67–75.     

Misregulation of spermatogenesis genes in Drosophila hybrids is lineage‐specific and driven by the combined effects of sterility and fast male regulatory divergence

Hybrid male sterility is a common outcome of crosses between different species. Gene expression studies have found that a number of spermatogenesis genes are differentially expressed in sterile hybrid males, compared with parental species. Late‐stage sperm development genes are particularly likely to be misexpressed, with fewer early‐stage genes affected. Thus, a link has been posited between misexpression and sterility. A more recent alternative explanation for hybrid gene misexpression has been that it is independent of sterility and driven by divergent evolution of male‐specific regulatory elements between species (faster male hypothesis). The faster male hypothesis predicts that misregulation of spermatogenesis genes should be independent of sterility and approximately the same in both hybrids, whereas sterility should only affect gene expression in sterile hybrids. To test the faster male hypothesis vs. the effect of sterility on gene misexpression, we analyse spermatogenesis gene expression in different species pairs of the Drosophila phylogeny, where hybrid male sterility occurs in only one direction of the interspecies cross (i.e. unidirectional sterility). We find significant differences among genes in misexpression with effects that are lineage‐specific and caused by sterility or fast male regulatory divergence.     

Assessing the potential for post‐ejaculatory female choice in a polyandrous beach‐spawning fish

In species with limited opportunities for pre‐ejaculatory sexual selection (behavioural components), post‐ejaculatory mechanisms may provide opportunities for mate choice after gametes have been released. Recent evidence from a range of taxa has revealed that cryptic female choice (i.e., female‐mediated differential fertilization bias), through chemical cues released with or from eggs, can differentially regulate the swimming characteristics of sperm from various males and ultimately determine male fertilization success under sperm competition. We assessed the potential role that such female‐modulated chemical cues play in influencing sperm swimming characteristics in beach‐spawning capelin (Mallotus villosus), an externally fertilizing fish that mates as couples (one male and one female) or threesomes (two males and one female) with presumably limited opportunities for pre‐ejaculatory sexual selection. We assayed sperm swimming characteristics under varying doses and donor origins of egg cues and also examined the possibility of assortative mating based on body size. We found mating groups were not associated by size, larger males did not produce better quality ejaculates, and egg cues (regardless of dosage or donor identity) did not influence sperm swimming characteristics. Our findings suggest that intersexual pre‐ejaculatory sexual selection and cryptic female choice mediated by female chemical cues are poorly developed in capelin, possibly due to unique natural selection constraints on reproduction.     

Sperm competition and the evolution of precopulatory weapons: Increasing male density promotes sperm competition and reduces selection on arm strength in a chorusing frog

Sperm competition theory assumes a trade‐off between precopulatory traits that increase mating success and postcopulatory traits that increase fertilization success. Predictions for how sperm competition might affect male expenditure on these traits depend on the number of competing males, the advantage gained from expenditure on weapons, and the level of sperm competition. However, empirical tests of sperm competition theory rarely examine precopulatory male expenditure. We investigated how variation in male density affects precopulatory sexual selection on male weaponry and the level of sperm competition in the chorusing frog Crinia georgiana, where males use their arms as weapons in male–male combat. We measured body size and arm girth of 439 males, and recorded their mating success in the field. We found density‐dependent selection acting on arm girth. Arm girth was positively associated with mating success, but only at low population densities. Increased male density was associated with higher risk and intensity of sperm competition arising from multimale amplexus, and a reversal in the direction of selection on arm girth. Opposing patterns of pre‐ and postcopulatory selection may account for the negative covariation between arm girth and testes across populations of this species.     

Identification of a novel sperm class and its role in fertilization in Drosophila

In many species, males have evolved to produce a sterile sperm (parasperm) in conjunction with fertilizing sperm (eusperm). Here, we document evidence of males depositing two morphologically distinct types of parasperm (1 and 2) into the female reproductive tract in Drosophila pseudoobscura. These parasperm differ in length, shape, amount produced, amount in long‐term storage and may have separate roles in ensuring male fertilization success. Although both parasperm types protect eusperm from female spermicides, only parasperm 2, which has a corkscrew shape, is associated with sperm competition. Increased production of parasperm 2 is also negatively correlated with the eusperm and parasperm 1 production. Thus, selection may be acting on parasperm production in the presence of sperm competition. Our findings show how both sperm competition and cryptic female choice may be acting in conjunction to influence the evolution of ejaculate composition. Our identification and characterization of two distinct parasperm morphs will enhance the ability for further evaluation of parasperm's role in fertilization.     

The contrasting role of male relatedness in different mechanisms of sexual selection in red junglefowl

In structured populations, competition for reproductive opportunities should be relaxed among related males. The few tests of this prediction often neglect the fact that sexual selection acts through multiple mechanisms, both before and after mating. We performed experiments to study the role of within‐group male relatedness across pre‐ and postcopulatory mechanisms of sexual selection in social groups of red junglefowl, Gallus gallus, in which two related males and one unrelated male competed over females unrelated to all the males. We confirm theoretical expectations that, after controlling for male social status, competition over mating was reduced among related males. However, this effect was contrasted by other sexual selection mechanisms. First, females biased male mating in favor of the unrelated male, and might also favor his inseminations after mating. Second, males invested more—rather than fewer—sperm in postcopulatory competition with relatives. A number of factors may contribute to explain this counterintuitive pattern of sperm allocation, including trade‐offs between male investment in pre‐ versus postcopulatory competition, differences in the relative relatedness of pre‐ versus postcopulatory competitors, and female bias in sperm utilization in response to male relatedness. Collectively, these results reveal that within‐group male relatedness may have contrasting effects in different mechanisms of sexual selection.     

Sex‐biased transcriptome divergence along a latitudinal gradient

Sex‐dependent gene expression is likely an important genomic mechanism that allows sex‐specific adaptation to environmental changes. Among Drosophila species, sex‐biased genes display remarkably consistent evolutionary patterns; male‐biased genes evolve faster than unbiased genes in both coding sequence and expression level, suggesting sex differences in selection through time. However, comparatively little is known of the evolutionary process shaping sex‐biased expression within species. Latitudinal clines offer an opportunity to examine how changes in key ecological parameters also influence sex‐specific selection and the evolution of sex‐biased gene expression. We assayed male and female gene expression in Drosophila serrata along a latitudinal gradient in eastern Australia spanning most of its endemic distribution. Analysis of 11 631 genes across eight populations revealed strong sex differences in the frequency, mode and strength of divergence. Divergence was far stronger in males than females and while latitudinal clines were evident in both sexes, male divergence was often population specific, suggesting responses to localized selection pressures that do not covary predictably with latitude. While divergence was enriched for male‐biased genes, there was no overrepresentation of X‐linked genes in males. By contrast, X‐linked divergence was elevated in females, especially for female‐biased genes. Many genes that diverged in D. serrata have homologs also showing latitudinal divergence in Drosophila simulans and Drosophila melanogaster on other continents, likely indicating parallel adaptation in these distantly related species. Our results suggest that sex differences in selection play an important role in shaping the evolution of gene expression over macro‐ and micro‐ecological spatial scales.     

Sperm competitive advantage of a rare mitochondrial haplogroup linked to differential expression of mitochondrial oxidative phosphorylation genes

Maternal inheritance of mitochondria creates a sex‐specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2‐carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA‐Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22‐fold upregulation of atp8 in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated.     

Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles

Sexual selection is thought to favor the evolution of secondary sexual traits in males that contribute to mating success. In species where females mate with more than one male, sexual selection also continues after copulation in the form of sperm competition and cryptic female choice. Theory suggests that sperm competition should favor traits such as testes size and sperm production that increase a male's competitive fertilization success. Studies of experimental evolution offer a powerful approach for assessing evolutionary responses to variation in sexual selection pressures. Here we removed sexual selection by enforcing monogamy on replicate lines of a naturally polygamous horned beetle, Onthophagus taurus, and monitoring male investment in their testes for 21 generations. Testes size decreased in monogamous lines relative to lines in which sexual selection was allowed to continue. Differences in testes size were dependent on selection history and not breeding regime. Males from polygamous lines also had a competitive fertilization advantage when in sperm competition with males from monogamous lines. Females from polygamous lines produced sons in better condition, and those from monogamous lines increased their sons condition by mating polygamously. Rather than being costly for females, multiple mating appears to provide females with direct and/or indirect benefits. Neither body size nor horn size diverged between our monogamous and polygamous lines. Our data show that sperm competition does drive the evolution of testes size in onthophagine beetles, and provide general support for sperm competition theory.     

Reconstructing paternal genotypes to infer patterns of sperm storage and sexual selection in the hawksbill turtle

Postcopulatory sperm storage can serve a range of functions, including ensuring fertility, allowing delayed fertilization and facilitating sexual selection. Sperm storage is likely to be particularly important in wide‐ranging animals with low population densities, but its prevalence and importance in such taxa, and its role in promoting sexual selection, are poorly known. Here, we use a powerful microsatellite array and paternal genotype reconstruction to assess the prevalence of sperm storage and test sexual selection hypotheses of genetic biases to paternity in one such species, the critically endangered hawksbill turtle, Eretmochelys imbricata. In the majority of females (90.7%, N = 43), all offspring were sired by a single male. In the few cases of multiple paternity (9.3%), two males fertilized each female. Importantly, the identity and proportional fertilization success of males were consistent across all sequential nests laid by individual females over the breeding season (up to five nests over 75 days). No males were identified as having fertilized more than one female, suggesting that a large number of males are available to females. No evidence for biases to paternity based on heterozygosity or relatedness was found. These results indicate that female hawksbill turtles are predominantly monogamous within a season, store sperm for the duration of the nesting season and do not re‐mate between nests. Furthermore, females do not appear to be using sperm storage to facilitate sexual selection. Consequently, the primary value of storing sperm in marine turtles may be to uncouple mating and fertilization in time and avoid costly re‐mating.     

Female remating in butterflies: interaction between female genotype and nonfertile sperm

Female mating rate is fundamental to evolutionary biology as it determines the pattern of sexual selection and sexual conflict. Despite its importance, the genetic basis for female remating rate is largely unknown and has only been demonstrated in one species. In paternally investing species there is often a conflict between the sexes over female mating rate, as females remate to obtain male nutrient donations and males try to prevent female remating to ensure high fertilization success. Butterflies produce two types of sperm: fertilizing, eupyrene sperm, and large numbers of nonfertile, apyrene sperm. The function of apyrene sperm in the polyandrous, paternally investing green‐veined white butterfly, Pieris napi, is to fill the female’s sperm storage organ thereby reducing her receptivity. However, there is large variation in number of apyrene sperm stored. Here, I examine the genetic basis to this variation, and if variation in number of apyrene sperm stored is related to females’ remating rate. The number of apyrene sperm stored at the time of remating has a genetic component and is correlated with female remating tendency, whereas no such relationship is found for fertilizing sperm. The duration of the nonreceptivity period in P. napi also has a genetic component and is inversely related to the degree of polyandry. Sexual conflict over female remating rate appears to be present in this species, with males using their apyrene sperm to exploit a female system designed to monitor sperm in storage. Ejaculates with a high proportion of nonfertile sperm may have evolved to induce females to store more of these sperm, thereby reducing remating. As a counter‐adaptation, females have evolved a better detection system to regain control over their remating rate. Sexually antagonistic co‐evolution of apyrene sperm number and female sperm storage may be responsible for ejaculates with predominantly nonfertile sperm in this butterfly.     

Correlated responses to selection on female egg size in male reproductive traits in a butterfly

Genetic correlations between male and female traits can act as evolutionary constraints and, if involving reproductive traits, potentially influence sexual selection. Artificial selection on egg size in the tropical butterfly Bicyclus anynana has yielded highly divergent lines. Here we report evidence for correlated evolution in male traits. Males from the large-egg selected lines produced significantly heavier spermatophores independent of body size and tended to have more fertile sperm stored in their reproductive tracts than those from the small-egg selected lines. This may be due to an underlying genetic correlation in reproductive effort between the sexes. However, non-fertile sperm number and testis size remained unaffected by selection on egg size. Phenotypic correlations within an unselected population revealed that spermatophore mass and fertile sperm number, but not testis size and non-fertile sperm number, were positively related to male body size, and that larger spermatophores contained more fertile, but not non-fertile sperm. In addition, males provided larger females with bigger spermatophores and more fertile sperm, indicating males may be exercising mate choice during copulation.     

Sibling rivalry: Males with more brothers develop larger testes

When females mate with multiple partners in a reproductive cycle, the relative number of competing sperm from rival males is often the most critical factor in determining paternity. Gamete production is directly related to testis size in most species, and is associated with both mating behavior and perceived risk of competition. Deer mice, Peromyscus maniculatus, are naturally promiscuous and males invest significantly more in sperm production than males of P. polionotus, their monogamous sister‐species. Here, we show that the larger testes in P. maniculatus are retained after decades of enforced monogamy in captivity. While these results suggest that differences in sperm production between species with divergent evolutionary histories can be maintained in captivity, we also show that the early rearing environment of males can strongly influence their testis size as adults. Using a second‐generation hybrid population to increase variation within the population, we show that males reared in litters with more brothers develop larger testes as adults. Importantly, this difference in testis size is also associated with increased fertility. Together, our findings suggest that sperm production may be both broadly shaped by natural selection over evolutionary timescales and also finely tuned during early development.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

Spermatophore and Sperm Allocation in Males of the Monandrous Butterfly Pararge aegeria: the Female’s Perspective

In insects, spermatophore production represents a non‐trivial cost to a male. Non‐virgin males have been shown to produce small spermatophores at subsequent matings. Particularly in monandrous species, it may be an issue to receive a sufficiently large spermatophore at the first and typically only mating. Females of the monandrous Speckled wood butterfly Pararge aegeria (L.) produce fewer offspring after mating with a non‐virgin male. After mating, females spend all their active time selecting oviposition sites and typically ignore other males. Here, we show that females did not discriminate between a virgin male and a recently mated male in our laboratory experiments. We demonstrate that the number of eupyrene sperm bundles relative to spermatophore mass differed with subsequent male matings. Males transferred a significantly smaller spermatophore after the first copulation, but the spermatophore mass did not decrease further with subsequent matings. However, the number of eupyrene sperm bundles decreased linearly. Therefore, there was proportionally more eupyrene sperm in the male’s second spermatophore compared with the first and the later spermatophores. Such a pattern has been shown in polyandrous species. Hence, it suggests that differences in sperm allocation strategy between polyandrous and monandrous butterflies may be quantitative rather than qualitative. There was also a tendency for females that had mated with a recently mated male to have higher propensity to remate than did females that had mated with a virgin male. We discuss the results relative to the mating system in P. aegeria, including female remating opportunities in the field and male mate‐locating behaviour.     

Stabilizing selection on sperm number revealed by artificial selection and experimental evolution

Sperm competition is taxonomically widespread in animals and is usually associated with large sperm production, being the number of sperm in the competing pool the prime predictor of fertilization success. Despite the strong postcopulatory selection acting directionally on sperm production, its genetic variance is often very high. This can be explained by trade‐offs between sperm production and traits associated with mate acquisition or survival, that may contribute to generate an overall stabilizing selection. To investigate this hypothesis, we first artificially selected male guppies (Poecilia reticulata) for high and low sperm production for three generations, while simultaneously removing sexual selection. Then, we interrupted artificial selection and restored sexual selection. Sperm production responded to divergent selection in one generation, and when we restored sexual selection, both high and low lines converged back to the mean sperm production of the original population within two generations, indicating that sperm number is subject to strong stabilizing total sexual selection (i.e., selection acting simultaneously on all traits associated with reproductive success). We discuss the possible mechanisms responsible for the maintenance of high genetic variability in sperm production despite strong selection acting on it.