FUNCTIONAL INCOMPATIBILITY BETWEEN THE FERTILIZATION SYSTEMS OF TWO ALLOPATRIC POPULATIONS OF CALLOSOBRUCHUS MACULATUS (COLEOPTERA: BRUCHIDAE)

Abstract Recent studies indicate that postcopulatory sexual selection may represent an important component of the speciation process by initiating reproductive isolation via the evolutionary divergence of fertilization systems. Using two geographically isolated populations of the polyandrous beetle Callosobruchus maculatus, we investigated divergence in fertilization systems by determining the extent of postcopulatory functional incompatibility. Through reciprocal, cross‐population matings we were able to separately estimate the effects of male and female population origin and their interaction on the extent of last‐male sperm precedence, female receptivity to further copulation and female oviposition. Our results indicate partial incompatibility between the fertilization systems of the two populations at all three functional levels. Males derived from the same population as females outcompete rival, allopatric males with respect to sperm preemption, sperm protection, and ability to stimulate female oviposition. This pattern is reciprocated in both populations indicating that postcopulatory, prezygotic events represent important mechanisms by which between‐population gene flow is reduced. We suggest the partial gametic isolation observed is a by‐product of the coevolution of male and female fertilization systems by a process of cryptic female choice. Our results are consistent with a mechanism akin to conventional mate choice models although they do not allow us to reject antagonistic sexual coevolution as the mechanism of cryptic female choice.     

Competitive PCR reveals the complexity of postcopulatory sexual selection in Teleogryllus commodus

The outcome of mate choice depends on complex interactions between males and females both before and after copulation. Although the competition between males for access to mates and premating choice by females are relatively well understood, the nature of interactions between cryptic female choice and male sperm competition within the female reproductive tract is less clear. Understanding the complexity of postcopulatory sexual selection requires an understanding of how anatomy, physiology and behaviour mediate sperm transfer and storage within multiply mated females. Here we use a newly developed molecular technique to directly quantify mixed sperm stores in multiple mating females of the black field cricket, Teleogryllus commodus. In this species, female postcopulatory choice is easily observed and manipulated as females delay the removal of the spermatophore in favour of preferred males. Using twice‐mated females, we find that the proportion of sperm in the spermatheca attributed to the second male to mate with a female (S₂) increases linearly with the time of spermatophore attachment. Moreover, we show that the insemination success of a male increases with its attractiveness and decreases with the size of the female. The effect of male attractiveness in this context suggests a previously unknown episode of mate choice in this species that reinforces the sexual selection imposed by premating choice and conflicts with the outcome of postmating male harassment. Our results provide some of the clearest evidence yet for how sperm transfer and displacement in multiply mated females can lead directly to cryptic female choice, and that three distinct periods of sexual selection operate in black field crickets.     

Female crickets assess relatedness during mate guarding and bias storage of sperm towards unrelated males

Recent evidence shows that females exert a post‐copulatory fertilization bias in favour of unrelated males to avoid the genetic incompatibilities derived from inbreeding. One of the mechanisms suggested for fertilization biases in insects is female control over transport of sperm to the sperm‐storage organs. We investigated post‐copulatory inbreeding‐avoidance mechanisms in females of the cricket Teleogryllus oceanicus. We assessed the relative contribution of related and unrelated males to the sperm stores of double‐mated females. To demonstrate unequivocally that biased sperm storage results from female control rather than cryptic male choice, we manipulated the relatedness of mated males and of males performing post‐copulatory mate guarding. Our results show that when guarded by a related male, females store less sperm from their actual mate, irrespective of the relatedness of the mating male. Our data support the notion that inhibition of sperm storage by female crickets can act as a form of cryptic female choice to avoid the severe negative effects of inbreeding.     

Wild yellow dung fly females may not select sperm based on dung pat microclimate but could nevertheless benefit from polyandry

Molecular techniques have substantially improved our knowledge of postcopulatory sexual selection. Nevertheless, studies examining sperm utilization in natural populations of nonsocial insects are rare, support for sperm selection (biased use of stored sperm, e.g. to match offspring genotypes to prevailing environmental conditions) is elusive, and its relevance within natural populations unknown. We performed an oviposition site choice experiment in the field where female yellow dung flies Scathophaga stercoraria could deposit eggs into three different microenvironments on a dung pat (the east–west ridge, north- or south-exposed side), and genotyped the offspring and sperm remaining in storage after oviposition. Females exhibited plasticity in the number of eggs deposited according to pat age. Additionally, temperature strongly influenced egg placement: the warmer the temperature, the higher the proportion of eggs laid into the north-exposed side of dung. The number of ejaculates in storage differed amongst spermathecae, and females stored sperm from more males than fathered their offspring (2.11 sires vs. 2.84 males within sperm stores). Mean last male paternity was 83.4%, roughly matching previous laboratory estimates. Importantly, we found no evidence that females selectively lay eggs of different genotypes, by biasing paternity towards certain males, depending on offspring’s microclimate. Thus, while we show female choice over number of eggs and where these are deposited, there was no evidence for sperm selection. We further revealed positive effects of multiple mating on total number of offspring and proportion of offspring emerging from the dung. We argue that the integration of field studies and laboratory experiments is essential to promote our understanding of polyandry and cryptic female choice.     

The effect of cryptic female choice on sex allocation in simultaneous hermaphrodites

Sex allocation theory for simultaneous hermaphrodites has focused primarily on the effects of sperm competition, but the role of mate choice has so far been neglected. We present a model to study the coevolution of cryptic female choice and sex allocation in simultaneous hermaphrodites. We show that the mechanism of cryptic female choice has a strong effect on the evolutionary outcome: if individuals remove a fixed proportion of less-preferred sperm, the optimal sex allocation is more female biased (i.e. more biased towards egg production) than without cryptic female choice; conversely, if a fixed amount of sperm is removed, sex allocation is less female-biased than without cryptic female choice, and can easily become male biased (i.e. biased towards sperm production). Under male-biased sex allocation, hermaphroditism can become unstable and the population can split into pure males and hermaphrodites with a female-biased allocation. We discuss the idea that the evolution of sex allocation may depend on the outcome of sexual conflict over the fate of received sperm: the sperm donor may attempt to manipulate or by-pass cryptic female choice and the sperm recipient is expected to resist such manipulation. We conclude that cryptic female choice can have a strong influence on sex allocation in simultaneous hermaphrodites and strongly encourage empirical work on this question.     

Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae)

Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism.     

An integrative view of sexual selection in Tribolium flour beetles

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.     

Sperm competitive ability and genetic relatedness in Drosophila melanogaster: similarity breeds contempt

Abstract.— Offspring of close relatives often suffer severe fitness consequences. Previous studies have demonstrated that females, when given a choice, will choose to avoid mating with closely related males. But where opportunities for mate choice are limited or kin recognition is absent, precopulatory mechanisms may not work. In this case, either sex could reduce the risks of inbreeding through mechanisms that occur during or after copulation. During mating, males or females could commit fewer gametes when mating with a close relative. After mating, females could offset the effects of mating with a closely related male through cryptic choice. Few prior studies of sperm competition have examined the effect of genetic similarity, however, and what studies do exist have yielded equivocal results. In an effort to resolve this issue, we measured the outcome of sperm competition when female Drosophila melanogaster were mated to males of four different degrees of genetic relatedness and then to a standardized competitor. We provide the strongest evidence to date that sperm competitive ability is negatively correlated with relatedness, even after controlling for inbreeding depression.     

The assessment of insemination success in yellow dung flies using competitive PCR

In spite of considerable interest in postcopulatory sexual selection, separating the effects of sperm competition from cryptic female choice remains difficult because mechanisms underlying postcopulatory processes are poorly understood. One methodological challenge is to quantify insemination success for individual males within the sperm stores of multiply mated females to discover how insemination translates into eventual paternity. Any proposed method must be applicable in organisms without extensive DNA sequence information (which include the majority of model species for sexual selection). Here, we describe the development and application of microsatellite competitive-multiplex-PCR for quantifying relative contributions to a small number of sperm in storage. We studied how DNA template characteristics affect PCR amplification of known concentrations of mixed DNA and generated regressions for correcting observations of allelic signal strength based on such characteristics. We used these methods to examine patterns of sperm storage in twice-mated female yellow dung flies, Scathophaga stercoraria. We confirm previous findings supporting sperm displacement and demonstrate that average paternity for the last mate accords with the mean proportion of sperm stored. We further find consistent skew in storage across spermathecae, with more last male sperm stored in the singlet spermatheca on one side of the body than in the doublet on the opposite side. We also show that the time between copulations may be important for effectively sorting sperm. Finally, we demonstrate that male size may influence the opportunity for sperm choice, suggesting future work to disentangle the roles of male competition and cryptic female choice.     

Multiple mating,sperm transfer and oviposition pattern in the giant sperm species,Drosophila bifurca

Many diverse traits are involved in gamete systems, and several models have analysed sperm length variation in terms of the intensity of sperm competition. This study investigates mating, sperm transfer and oviposition patterns in Drosophila bifurca, which possesses the longest sperm in the animal kingdom (about 6 cm). The prediction is that sperm gigantism should prevent male–male interaction. In this study, we examine how sperm transfer varies as males mate with a series of females, and how female receptivity changes with time after mating. As predicted, we found an extremely limited overlap of ejaculates owing to (1) reduced sperm transfer to females that had already mated, and (2) female remating depended both on the amount of sperm transferred and the modes of egg laying. The amount of sperm transferred to the female is discussed in relation to the peculiar morphology of the male reproductive tract and to sexual dimorphism and ecological hypotheses.     

Gametic interactions promote inbreeding avoidance in house mice

Reproduction among related individuals is generally maladaptive. Inbreeding imposes significant costs on individual reproductive success, and can decrease population fitness. Theory predicts that polyandrous females can avoid inbreeding by exploiting paternity‐biasing mechanisms that enable differential sperm ‘use’. Evidence of sperm selection is difficult to demonstrate because patterns of non‐random paternity can be generated by a variety of different mechanisms. Here, using in vitro fertilisation in mice, we provide evidence of sperm selection at the gametic level. We mixed the sperm of sibling and non‐sibling males, and observed a fertilisation bias towards the sperm of non‐sibling males. The number of motile sperm and sperm swimming performance did not differ between competitors among the replicate assays. Therefore, our result can only be ascribed to egg‐driven sperm selection against related sperm. We conclude that the expression or secretion of gametic proteins could provide the molecular basis for this mechanism of cryptic female choice.     

Genetic divergence of the seminal signal-receptor system in houseflies: the footprints of sexually antagonistic coevolution?

To understand fully the significance of cryptic female choice, we need to focus on each of those postmating processes in females which create variance in fitness among males. Earlier studies have focused almost exclusively on the proportion of a female's eggs fertilized by different males (sperm precedence). Yet, variance in male postmating reproductive success may also arise from differences in ability to stimulate female oviposition and to delay female remating. Here, we present a series of reciprocal mating experiments among genetically differentiated wild-type strains of the housefly Musca domestica. We compared the effects of male and female genotype on oviposition and remating by females. The genotype of each sex affected both female oviposition and remating rates, demonstrating that the signal-receptor system involved has indeed diverged among these strains. Further, there was a significant interaction between the effects of male and female genotype on oviposition rate. We discuss ways in which the pattern of such interactions provides insights into the coevolutionary mechanism involved. Females in our experiments generally exhibited the weakest, rather than the strongest, response to males with which they are coevolved. These results support the hypothesis that coevolution of male seminal signals and female receptors is sexually antagonistic.     

Sperm storage mediated by cryptic female choice for nuptial gifts

Polyandrous females are expected to discriminate among males through postcopulatory cryptic mate choice. Yet, there is surprisingly little unequivocal evidence for female-mediated cryptic sperm choice. In species in which nuptial gifts facilitate mating, females may gain indirect benefits through preferential storage of sperm from gift-giving males if the gift signals male quality. We tested this hypothesis in the spider Pisaura mirabilis by quantifying the number of sperm stored in response to copulation with males with or without a nuptial gift, while experimentally controlling copulation duration. We further assessed the effect of gift presence and copulation duration on egg-hatching success in matings with uninterrupted copulations with gift-giving males. We show that females mated to gift-giving males stored more sperm and experienced 17% higher egg-hatching success, compared with those mated to no-gift males, despite matched copulation durations. Uninterrupted copulations resulted in both increased sperm storage and egg-hatching success. Our study confirms the prediction that the nuptial gift as a male signal is under positive sexual selection by females through cryptic sperm storage. In addition, the gift facilitates longer copulations and increased sperm transfer providing two different types of advantage to gift-giving in males.     

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.     

Polyandry and postcopulatory sexual selection in a wild population

When females mate multiply, postcopulatory sexual selection can occur via sperm competition and cryptic female choice. Although postcopulatory selection has the potential to be a major force in driving evolution, few studies have estimated its strength in natural populations. Likewise, although polyandry is widespread across taxa and is the focus of a growing body of research, estimates of natural female mating rates are still limited in number. Microsatellites can be used to estimate the number of mates represented in females' sperm stores and the number of sires contributing to their offspring, enabling comparisons both of polyandry and of two components of postcopulatory selection: the proportion of males that mate but fail to sire offspring, and the degree of paternity skew among the males that do sire offspring. Here, we estimate the number of mates and sires among wild females in the Hawaiian swordtail cricket Laupala cerasina. We compare these estimates to the actual mating rates and paternity shares we observed in a semi‐natural population. Our results show that postcopulatory sexual selection operates strongly in this species: wild females mated with an average minimum of 3.6 males but used the sperm from only 58% of them. Furthermore, among the males that did sire offspring, paternity was significantly skewed. These patterns were similar to those observed in the field enclosure, where females mated with an average of 5.7 males and used the sperm from 62% of their mates, with paternity significantly skewed among the sires.     

Sexual conflict and cryptic female choice in the black field cricket, Teleogryllus commodus

The prevalence and evolutionary consequences of cryptic female choice (CFC) remain highly controversial, not least because the processes underlying its expression are often concealed within the female reproductive tract. However, even when female discrimination is relatively easy to observe, as in numerous insect species with externally attached spermatophores, it is often difficult to demonstrate directional CFC for certain male phenotypes over others. Using a biological assay to separate male crickets into attractive or unattractive categories, we demonstrate that females strongly discriminate against unattractive males by removing their spermatophores before insemination can be completed. This results in significantly more sperm being transferred by attractive males than unattractive males. Males respond to CFC by mate guarding females after copulation, which increases the spermatophore retention of both attractive and unattractive males. Interestingly, unattractive males who suffered earlier interruption of sperm transfer benefited more from mate guarding, and they guarded females more vigilantly than attractive males. Our results suggest that postcopulatory mate guarding has evolved via sexual conflict over insemination times rather than through genetic benefits of biasing paternity toward vigorous males, as has been previously suggested.     

Cryptic female preference for colorful males in guppies

Cryptic female choice (CFC) refers to female-mediated processes occurring during or after copulation that result in biased sperm use in favor of preferred or compatible males. Despite recent empirical support for this hypothesis, evidence that CFC contributes towards the evolution of male body ornaments, in the same way that precopulatory female choice does, is currently lacking. Here, we tested the possibility that CFC selects for increased male attractiveness in the guppy Poecilia reticulata, a freshwater fish exhibiting internal fertilization. Specifically, we examined whether females are able to manipulate the number of sperm transferred or retained at copulation in favor of relatively attractive males. In support of this prediction, we found that following solicited copulations the number of sperm inseminated is influenced exclusively by the female's perception of relative male coloration, independent of any direct manipulation of males themselves. Because females prefer brightly colored males during precopulatory mate choice, our finding that colorful males are also favored as a consequence of enhanced insemination success indicates that cryptic female choice can reinforce precopulatory preferences for extravagant male ornaments.     

Sneaker “jack” males outcompete dominant “hooknose” males under sperm competition in Chinook salmon (Oncorhynchus tshawytscha)

In a variety of taxa, males deploy alternative reproductive tactics to secure fertilizations. In many species, small “sneaker” males attempt to steal fertilizations while avoiding encounters with larger, more aggressive, dominant males. Sneaker males usually face a number of disadvantages, including reduced access to females and the higher likelihood that upon ejaculation, their sperm face competition from other males. Nevertheless, sneaker males represent an evolutionarily stable strategy under a wide range of conditions. Game theory suggests that sneaker males compensate for these disadvantages by investing disproportionately in spermatogenesis, by producing more sperm per unit body mass (the “fair raffle”) and/or by producing higher quality sperm (the “loaded raffle”). Here, we test these models by competing sperm from sneaker “jack” males against sperm from dominant “hooknose” males in Chinook salmon. Using two complementary approaches, we reject the fair raffle in favor of the loaded raffle and estimate that jack males were ~1.35 times as likely as hooknose males to fertilize eggs under controlled competitive conditions. Interestingly, the direction and magnitude of this skew in paternity shifted according to individual female egg donors, suggesting cryptic female choice could moderate the outcomes of sperm competition in this externally fertilizing species.     

Seasonal variation in genital and body size, sperm displacement ability, female mating rate, and male harassment in two calopterygid damselflies (Odonata: Calopterygidae)

Sperm competition is a pervasive force. One adaptation is the male ability to displace the rivals' sperm that females have stored from previous copulations. In the damselfly, Calopteryx haemorrhoidalis asturica , males with wider aedeagi displace more spermathecal sperm. The present study documents that the same mechanism operates in another damselfly, Hetaerina americana . However, this genital width in both species decreases along the season, but late-emerging females have more sperm displaced than early-emerging females. Because territorial males mated more and were larger in body and genital size than nonterritorial males, late-season females mated with considerably larger males with respect to female size and this produced higher sperm displacement. Assuming female benefits from storing sperm but that such benefit does not prevail if males displace sperm, it is predicted that, along the season, females will mate less and male harassment (in terms of male mating attempts and oviposition duration) will increase. These predictions were corroborated. In H. americana , it was also tested whether spermathecal sperm became less viable along the season. The results obtained did not corroborate this. This is the first evidence indicating that season affects sperm displacement ability and female mating frequency due to changes in male body and genital size.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 815–829.     

Effects of Sexual Selection Upon Sperm Morphology and Sexual Skin Morphology in Primates

We review possible effects of sexual selection upon sperm morphology, and sexual skin morphology, in primates. Comparative morphometric studies, involving 31 species representing 21 primate genera, revealed a positive relationship between volume of the sperm midpiece, occurrences of multiple partner matings by females, and large relative testes sizes, which indicate sperm competition. The midpiece houses the mitochondria required to power sperm motility. Hence, sperm competition may have influenced the evolution of increased mitochondrial loading in species where females mate with multiple partners during the fertile period. Females of some Old World monkey species and female chimpanzees exhibit large estrogen-dependent sexual skin swellings during the follicular phase of the menstrual cycle. Studies of mandrills support the conclusion that swellings act primarily as sexually attractive, graded signals and that swelling size may indicate current reproductive quality. Measurements of the genitalia in chimpanzees indicate a secondary function for female swellings. The swelling increases the operating depth of the female's vagina by 50% during the fertile phase of her cycle. Males have evolved long, filiform penes capable of placing sperm close to the os cervix during competitive multipartner matings. This may exemplify how morphologic specializations in females can influence the coevolution of advantageous genitalic specializations in males: the phenomenon that Eberhard (1985) dubbed cryptic female choice.