Rapid divergent evolution of internal female genitalia and the coevolution of male genital morphology revealed by micro-computed tomography.

Animal genitalia are thought to evolve rapidly and divergently in response to sexual selection. Studies of genital evolution have focused largely on male genitalia. The paucity of work on female genital morphology is probably due to problems faced in quantifying shape variation, due to their composition and accessibility. Here we use a combination of micro-computed tomography, landmark free shape quantification and phylogenetic analysis to quantify the rate of female genital shape evolution among 29 species of Antichiropus millipedes, and their coevolution with male genitalia. We found significant variation in female and male genital shape among species. Male genital shape showed a stronger phylogenetic signal than female genital shape, although the phylogenetic signal effect sizes did not differ significantly. Male genital shape was found to be evolving 1.2 times faster than female genital shape. Female and male genital shape exhibited strong correlated evolution, indicating that genital shape changes in one sex are associated with corresponding changes in the genital shape of the other sex. This study adds novel insight into our growing understanding of how female genitalia can evolve rapidly and divergently, and highlights the advantages of three-dimensional techniques and multivariate analyses in studies of female genital evolution.

Dung beetles increase plant growth: a meta-analysis.

The ecosystem services provided by dung beetles are well known and valued. Dung beetles bury dung for feeding and breeding, and it is generally thought that the process of burying dung increases nutrient uptake by plant roots, which promotes plant growth. Many studies have tested the effects of dung beetles on plant growth, but there has been no quantitative synthesis of these studies. Here we use a multi-level meta-analysis to estimate the average effect of dung beetles on plant growth and investigate factors that moderate this effect. We identified 28 publications that investigated dung beetle effects on plant growth. Of these, 24 contained the minimum quantitative data necessary to include in a meta-analysis. Overall, we found that dung beetles increased plant growth by 17%; the 95% CI for possible values for the true increase in plant growth that were most compatible with our data, given our statistical model, ranged from 1% to 35%. We found evidence that the dung beetle-plant growth relationship is influenced by the plant measurement type and the number of beetles accessing the dung. However, beetles did not increase plant growth in all quantitative trials, as individual effect sizes ranged from -72% to 806%, suggesting important context-dependence in the provision of ecosystem services.

Aggressive interactions influence cognitive performance in Western Australian magpies.

Extensive research has investigated the relationship between the social environment and cognition, suggesting that social complexity may drive cognitive evolution and development. However, evidence for this relationship remains equivocal. Group size is often used as a measure of social complexity, but this may not capture intraspecific variation in social interactions. Social network analysis can provide insight into the cognitively demanding challenges associated with group living at the individual level. Here, we use social networks to investigate whether the cognitive performance of wild Western Australian magpies (Gymnorhina tibicen dorsalis) is related to group size and individual social connectedness. We quantified social connectedness using four interaction types: proximity, affiliative, agonistic and vocal. Consistent with previous research on this species, individuals in larger groups performed better on an associative learning task. However, social network position was also related to cognitive performance. Individuals receiving aggressive interactions performed better, while those involved in aggressive interactions with more group members performed worse. Overall, this suggests that cognitive performance is related to specific types of social interaction. The findings from this study highlight the value of considering fine-grained metrics of sociality that capture the challenges associated with social life when testing the relationship between the social environment and cognition.

Intergenerational response to sperm competition risk in an invasive mammal.

Studies of socially mediated phenotypic plasticity have demonstrated adaptive male responses to the 'competitive' environment. Despite this, whether variation in the paternal social environment also influences offspring reproductive potential in an intergenerational context has not yet been examined. Here, we studied the descendants of wild-caught house mice, a destructive pest species worldwide, to address this knowledge gap. We analysed traits that define a 'competitive' phenotype in the sons of males (sires) that had been exposed to either a high-male density (competitive) or high-female density (non-competitive) environment. We report disparate reproductive strategies among the sires: high-male density led to a phenotype geared for competition, while high-female density led to a phenotype that would facilitate elevated mating frequency. Moreover, we found that the competitive responses of sires persisted in the subsequent generation, with the sons of males reared under competition having elevated sperm quality. As all sons were reared under common-garden conditions, variation in their reproductive phenotypes could only have arisen via nongenetic inheritance. We discuss our results in relation to the adaptive advantage of preparing sons for sperm competition and suggest that intergenerational plasticity is a previously unconsidered aspect in invasive mammal fertility control.

Male responses to sperm competition risk associated with increased macronutrient intake and reduced lifespan.

Increased expenditure on the ejaculate is a taxonomically widespread male response to sperm competition. Increased ejaculate expenditure is assumed to come at a cost to future reproduction, otherwise males should always invest maximally. However, the life-history costs of strategic ejaculation are not well documented. Macronutrient intake is known to affect the trade-off between reproduction and lifespan. Intakes of protein and carbohydrate that maximize reproduction often differ from those that maximize lifespan. Here, we asked whether strategic expenditure on the ejaculate by male crickets, Teleogryllus oceanicus, is mediated by macronutrient intake, and whether it comes at a cost of reduced lifespan. Males were exposed to rival song throughout their lifespan or were held in a silent non-competitive environment. Males exposed to song had a higher intake of both protein and carbohydrate, they reached adulthood sooner, produced ejaculates of higher quality, and died sooner than males living in a silent environment. Our findings provide a rare example of both the mechanisms and life-history costs associated with strategic ejaculation.

Bateman gradients reflect variation in sexual selection in a species with dynamic sex roles.

Bateman gradients, the slope of the regression of reproductive success on mating success, are among the most commonly reported measures of sexual selection. They are particularly insightful in species with reversed sex roles, where females are expected to be under sexual selection. We measured Bateman gradients in replicate experimental populations of the spermatophore gift-giving bushcricket Kawanaphila nartee (Orthoptera: Tettigoniidae). In this species, the operational sex ratio (OSR) and thus the sex competing for mates varies depending on the availability of pollen food resources: under pollen-limited regimens females are more competitive, whereas under pollen-rich regimens males are more competitive. We maintained populations in enclosures with either limited or supplemented pollen and calculated Bateman gradients for males and females under both conditions. Bateman gradients were significantly positive in males, and the slope was steeper in pollen-supplemented populations where the OSR was more male-biased. Bateman gradients for females were shallow and nonsignificant regardless of pollen availability. Our results show that the strength of sexual selection on males can depend on environmental context. The lack of significant gradients among females may reflect experimental limitations on our ability to estimate Bateman gradients in female K nartee.

Ontogeny can provide insight into the roles of natural and sexual selection in cricket cuticular hydrocarbon evolution.

The often complex cocktails of hydrocarbon compounds found on the cuticles of insects can serve both naturally and sexually selected functions, contributing to an individual's ability to withstand water loss and attract mating partners. However, whether natural and sexual selection act synergistically or antagonistically on a species' cuticular hydrocarbon (CHC) profile remains unclear. Here, we examined the ontogeny of the CHC profile in a species of cricket, Teleogryllus oceanicus, while manipulating humidity during development. We predicted that juvenile crickets should produce only those compounds that contribute to desiccation resistance, while those compounds contributing specifically to male attractiveness should be produced only at sexual maturity. Further, if attractive CHCs come at a cost to desiccation resistance as predicted by some models of sexual selection, then males reared under low humidity should be constrained to invest less in attractive CHCs. Crickets reared under low humidity produced more long-chain methyl-branched alkanes, alkenes and alkadienes than did crickets reared under high humidity. The abundance of n-alkanes was unaffected by humidity treatment. Sexual dimorphism in the CHC profile was not apparent until adult emergence and became exaggerated 10 days after emergence, when crickets were sexually mature. Males produced more of the same compounds that were increased in both sexes under low humidity, but the humidity treatment did not interact with sex in determining CHC abundance. The data suggest that CHC profiles which protect crickets from desiccation might have synergistic effects on male attractiveness, as there was no evidence to suggest males trade-off a CHC profile produced in response to low humidity for one associated with sexual signalling.

Sexual Selection Shapes Seminal Vesicle Secretion Gene Expression in House Mice.

Reproductive proteins typically have high rates of molecular evolution, and are assumed to be under positive selection from sperm competition and cryptic female choice. However, ascribing evolutionary divergence in the genome to these processes of sexual selection from patterns of association alone is problematic. Here, we use an experimental manipulation of postmating sexual selection acting on populations of house mice and explore its consequences for the expression of seminal vesicle secreted (SVS) proteins. Following 25 generations of selection, males from populations subjected to postmating sexual selection had evolved increased expression of at least two SVS genes that exhibit the signature of positive selection at the molecular level, SVS1 and SVS2. These proteins contribute to mating plug formation and sperm survival in the female reproductive tract. Our data thereby support the view that sexual selection is responsible for the evolution of these seminal fluid proteins.

Quantifying variation in female internal genitalia: no evidence for plasticity in response to sexual conflict risk in a seed beetle.

Sexually antagonistic coevolution can drive the evolution of male traits that harm females, and female resistance to those traits. While males have been found to vary their harmfulness to females in response to social cues, plasticity in female resistance traits remains to be examined. Here, we ask whether female seed beetles Callosobruchus maculatus are capable of adjusting their resistance to male harm in response to the social environment. Among seed beetles, male genital spines harm females during copulation and females might resist male harm via thickening of the reproductive tract walls. We develop a novel micro computed tomography imaging technique to quantify female reproductive tract thickness in three-dimensional space, and compared the reproductive tracts of females from populations that had evolved under high and low levels of sexual conflict, and for females reared under a social environment that predicted either high or low levels of sexual conflict. We find little evidence to suggest that females can adjust the thickness of their reproductive tracts in response to the social environment. Neither did evolutionary history affect reproductive tract thickness. Nevertheless, our novel methodology was capable of quantifying fine-scale differences in the internal reproductive tracts of individual females, and will allow future investigations into the internal organs of insects and other animals.

Evolutionary, proteomic, and experimental investigations suggest the extracellular matrix of cumulus cells mediates fertilization outcomes†.

Studies of fertilization biology often focus on sperm and egg interactions. However, before gametes interact, mammalian sperm must pass through the cumulus layer; in mice, this consists of several thousand cells tightly glued together with hyaluronic acid and other proteins. To better understand the role of cumulus cells and their extracellular matrix, we perform proteomic experiments on cumulus oophorus complexes (COCs) in house mice (Mus musculus), producing over 24,000 mass spectra to identify 711 proteins. Seven proteins known to stabilize hyaluronic acid and the extracellular matrix were especially abundant (using spectral counts as an indirect proxy for abundance). Through comparative evolutionary analyses, we show that three of these evolve rapidly, a classic signature of genes that influence fertilization rate. Some of the selected sites overlap regions of the protein known to impact function. In a follow-up experiment, we compared COCs from females raised in two different social environments. Female mice raised in the presence of multiple males produced COCs that were smaller and more resistant to dissociation by hyaluronidase compared to females raised in the presence of a single male, consistent with a previous study that demonstrated such females produced COCs that were more resistant to fertilization. Although cumulus cells are often thought of as enhancers of fertilization, our evolutionary, proteomic, and experimental investigations implicate their extracellular matrix as a potential mediator of fertilization outcomes.

Males adjust their manipulation of female remating in response to sperm competition risk.

To reduce the potential for sperm competition, male insects are thought to inhibit the post-mating reproductive behaviour of females through receptivity-inhibiting compounds transferred in the ejaculate. Selection is expected to favour phenotypic plasticity in male post-copulatory expenditure, with males investing strategically in response to their perceived risk of sperm competition. However, the impact that socially cued strategic allocation might have on female post-mating behaviour has rarely been assessed. Here, we varied male perception of sperm competition risk, both prior to and during mating, to determine if a male's competitive environment impacts the extent to which he manipulates female remating behaviour. We found that female Australian field crickets (Teleogryllus oceanicus) mated to males that were reared under sperm competition risk emerged from a shelter in search of male song sooner than did females mated to males reared without risk, but only when mating occurred in a risk-free environment. We also found that females reared in a silent environment where potential mates were scarce emerged from the shelter sooner than females exposed to male calls during development. Collectively, our findings suggest complex interacting effects of male and female sociosexual environments on female post-mating sexual receptivity.

No evidence for divergence in male harmfulness or female resistance in response to changes in the opportunity for dispersal.

The outcome of sexual conflict can depend on the social environment, as males respond to changes in the inclusive fitness payoffs of harmfulness and harm females less when they compete with familiar relatives. Theoretical models also predict that if limited male dispersal predictably enhances local relatedness while maintaining global competition, kin selection can produce evolutionary divergences in male harmfulness among populations. Experimental tests of these predictions, however, are rare. We assessed rates of dispersal in female and male seed beetles Callosobruchus maculatus, a model species for studies of sexual conflict, in an experimental setting. Females dispersed significantly more often than males, but dispersing males travelled just as far as dispersing females. Next, we used experimental evolution to test whether limiting dispersal allowed the action of kin selection to affect divergence in male harmfulness and female resistance. Populations of C. maculatus were evolved for 20 and 25 generations under one of three dispersal regimens: completely free dispersal, limited dispersal and no dispersal. There was no divergence among treatments in female reproductive tract scarring, ejaculate size, mating behaviour, fitness of experimental females mated to stock males or fitness of stock females mated to experimental males. We suggest that this is likely due to insufficient strength of kin selection rather than a lack of genetic variation or time for selection. Limited dispersal alone is therefore not sufficient for kin selection to reduce male harmfulness in this species, consistent with general predictions that limited dispersal will only allow kin selection if local relatedness is independent of the intensity of competition among kin.

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.

Sexual ornaments but not weapons trade off against testes size in primates.

Males must partition their limited reproductive investments between traits that promote access to females (sexual ornaments and weapons) and traits that enhance fertilization success, such as testes and ejaculates. Recent studies show that if the most weaponized males can monopolize access to females through contest competition, thereby reducing the risk of sperm competition, they tend to invest less in sperm production. However, how males invest in sexual ornaments relative to sperm production remains less clear. If male ornaments serve as badges of status, with high-ranking males attaining near-exclusive access to females, similar to monopolizing females through combat, their expression should also covary negatively with investment in post-mating traits. In a comparative study across primates, which exhibit considerable diversification in sexual ornamentation, male weaponry and testes size, we found relative testes size to decrease with sexual ornaments but increase with canine size. These contrasting evolutionary trajectories might be driven by differential selection, functional constraints or temporal patterns of metabolic investment between the different types of sexual traits. Importantly, however, our results indicate that the theory of relative investments between weapons and testes in the context of monopolizing females can extend to male ornaments.

Male responses to sperm competition when rivals vary in number and familiarity.

Males of many species adjust their reproductive investment to the number of rivals present simultaneously. However, few studies have investigated whether males sum previous encounters with rivals, and the total level of competition has never been explicitly separated from social familiarity. Social familiarity can be an important component of kin recognition and has been suggested as a cue that males use to avoid harming females when competing with relatives. Previous work has succeeded in independently manipulating social familiarity and relatedness among rivals, but experimental manipulations of familiarity are confounded with manipulations of the total number of rivals that males encounter. Using the seed beetle Callosobruchus maculatus, we manipulated three factors: familiarity among rival males, the maximum number of rivals encountered simultaneously and the total number of rivals encountered over a 48 h period. Males produced smaller ejaculates when exposed to more rivals in total, regardless of the maximum number of rivals they encountered simultaneously. Males did not respond to familiarity. Our results demonstrate that males of this species can sum the number of rivals encountered over separate days, and therefore the confounding of familiarity with the total level of competition in previous studies should not be ignored.

Natural and sexual selection on cuticular hydrocarbons: a quantitative genetic analysis.

While the reproductive benefits of sexual displays have been widely studied, we have relatively limited evidence of the fitness costs associated with most display traits. Insect cuticular hydrocarbon (CHC) profiles are sexually selected traits that also protect against desiccation. These two functions are thought to oppose each other, with investment in particular compounds believed to increase attractiveness at the expense of compounds that protect against water loss. We investigated this potential trade-off in a quantitative genetic framework using the Australian field cricket, Teleogryllus oceanicus. Several compounds were significantly genetically correlated with either attractiveness or desiccation resistance. Of these compounds, one was negatively genetically correlated with attractiveness but positively genetically correlated with desiccation resistance. Furthermore, scoring each individual's overall CHC profile for its level of attractiveness and desiccation resistance indicated a negative genetic correlation between these multivariate phenotypes. Together, our results provide evidence for a genetic trade-off between sexually and naturally selected functions of the CHC profile. We suggest that the production of an attractive CHC profile may be costly for males, but highlight the need for further work to support this finding experimentally. Genetic covariation between the CHC profile and attractiveness suggests that females can gain attractive sons through female choice.

The evolution of female genitalia.

Female genitalia have been largely neglected in studies of genital evolution, perhaps due to the long-standing belief that they are relatively invariable and therefore taxonomically and evolutionarily uninformative in comparison with male genitalia. Contemporary studies of genital evolution have begun to dispute this view, and to demonstrate that female genitalia can be highly diverse and covary with the genitalia of males. Here, we examine evidence for three mechanisms of genital evolution in females: species isolating 'lock-and-key' evolution, cryptic female choice and sexual conflict. Lock-and-key genital evolution has been thought to be relatively unimportant; however, we present cases that show how species isolation may well play a role in the evolution of female genitalia. Much support for female genital evolution via sexual conflict comes from studies of both invertebrate and vertebrate species; however, the effects of sexual conflict can be difficult to distinguish from models of cryptic female choice that focus on putative benefits of choice for females. We offer potential solutions to alleviate this issue. Finally, we offer directions for future studies in order to expand and refine our knowledge surrounding female genital evolution.

Sex differences in nutrient intake can reduce the potential for sexual conflict over fitness maximization by female and male crickets.

As females and males have different roles in reproduction, they are expected to require different nutrients for the expression of reproductive traits. However, due to their shared genome, both sexes may be constrained in the regulation of nutrient intake that maximizes sex-specific fitness. Here, we used the Geometric Framework for nutrition to examine the effect of macronutrient and micronutrient intakes on lifespan, fecundity and cuticular hydrocarbons (CHCs) that signal mate quality to prospective mates in female field crickets, Teleogryllus oceanicus. In addition, we contrasted nutritional effects on life-history traits between males and females to determine how sex differences influence nutrient regulation. We found that carbohydrate intake maximized female lifespan and protein intake influenced CHC expression, while early life fecundity (cumulative fecundity at day 21) and lifetime fecundity were dependent on both macronutrient and micronutrient intakes. Fecundity required different nutrient blends to those required to optimize sperm viability in males, generating the potential for sexual conflict over macronutrient intake. The regulation of protein (P) and carbohydrate (C) intakes by virgin and mated females initially matched that of males, but females adjusted their intake to a higher P:C ratio, 1P:2C, that maximized fecundity as they aged. This suggests that a sex-specific, age-dependent change in intake target for sexually mature females, regardless of their mating status, adjusts protein consumption in preparation for oviposition. Sex differences in the regulation of nutrient intake to optimize critical reproductive traits in female and male T. oceanicus provide an example of how sexual conflict over nutrition can shape differences in foraging between the sexes.

Experimental evidence for the role of sexual selection in the evolution of cuticular hydrocarbons in the dung beetle, Onthophagus taurus.

A role for sexual selection in the evolution of insect cuticular hydrocarbons (CHCs) is suggested by observations of selection acting on male CHCs during female mate choice. However, evidence that CHCs evolve in response to sexual selection is generally lacking, and there is a need to extend our understanding beyond well-studied taxa. Experimental evolution offers a powerful approach to investigate the effect of sexual selection on the evolution of insect CHCs. We conducted such an experiment using the dung beetle, Onthophagus taurus. After six, 12 and 21 generations of experimental evolution, we measured the CHCs of beetles from three populations subject to sexual selection and three populations within which sexual selection had been removed via enforced monogamy. We found that the male CHC profile responded to the experimental removal of sexual selection. Conversely, the CHC profile of females responded to the presence of sexual selection but not to its removal. These results show that sexual selection can be an important mechanism affecting the evolution of insect CHCs and that male and female CHCs can evolve independently.

Experimental evolution reveals divergence in female genital teeth morphology in response to sexual conflict intensity in a moth.

The rapid evolutionary divergence of male genital structures under sexual selection is well documented. However, variation in female genital traits and the potential for sexual conflict to drive the coevolution between male and female traits has only recently received attention. In many lepidopterans, females possess genital teeth (collectively, signa). Comparative studies suggest these teeth, involved in the deflation of spermatophores, may have coevolved with male spermatophore thickness via sexually antagonistic coevolution in a contest over the rate of deflation of spermatophores within the reproductive tract. We tested the hypothesis that sexual conflict should generate coevolution between genital teeth and spermatophore morphology by examining these traits under experimental manipulation of sexual conflict intensity. Using micro-CT scanning, we examined spermatophore and teeth morphology in populations of the Indian moth, Plodia interpunctella, which had been evolving for 110 generations under different adult sex-ratio biases. We found divergence in female signa morphology in response to sexual conflict: females from female-biased populations (reduced sexual conflict) developed wider signa. However, we found no evidence of coevolution between signa traits and spermatophore thickness as reported from comparative studies.