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.     

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.     

Male courtship attractiveness and paternity success in Photinus greeni fireflies

Although female mate choice and male sperm competition have separately attracted much attention, few studies have addressed how precopulatory and postcopulatory episodes of sexual selection might interact to drive the evolution of male traits. In Photinus fireflies, females preferentially respond to males based on their bioluminescent courtship signals, and females gain direct benefits through male nuptial gifts acquired during multiple matings over several nights. We experimentally manipulated matings of P. greeni fireflies to test the hypothesis that postcopulatory paternity success might be biased toward males that are more attractive during courtship interactions. We first measured male courtship attractiveness to individual females using field behavioral assays. Females were then assigned to two double-mating treatments: (1) least attractive second male-females were first mated with their most attractive male, followed by their least attractive male, or (2) most attractive second male-females mated with males in reverse order. Larval offspring produced by each female following these double matings were genotyped using random amplified polymorphic DNA (RAPD) markers, and male paternity was determined. Contrary to prediction, firefly males that were more attractive to females based on their bioluminescent courtship displays subsequently showed significantly lower paternity, reflecting possible male trade-offs or sexual conflict. Differences in male paternity were not related to male body condition, testes or accessory gland mass, or to variation in female spermathecal size. Additionally, this study suggests that changes in phenotypic selection gradients may occur during different reproductive stages. These results indicate that it is crucial for future studies on sexual selection in polyandrous species to integrate both precopulatory and postcopulatory episodes to fully understand the evolution of male traits.     

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.     

Polyandry reduces sperm length variation in social insects

Postcopulatory sexual selection, either in the form of sperm competition or cryptic female choice, is an important selective force that is thought to have generated the enormous variation in sperm morphology observed interspecifically. However, the evolutionary significance of intraspecific variation in sperm morphology, and the role that postcopulatory sexual selection plays in influencing this variation, remains poorly investigated in invertebrates. Here, we tested the hypothesis that postcopulatory sexual selection reduces variation in sperm morphology, both between and within males, in 27 species of eusocial ants and bees. These eusocial species offer an unusual opportunity to assess how selection acts on variance in sperm morphology, as haploid males produce clonal, haploid sperm that does not experience haploid-diploid conflict. We provide solid evidence that males of polyandrous ant and bee species indeed produce less-variable sperm, indicating that sperm competition selected for sperm of superior quality. Our results offer a mechanistic explanation for the evolution of high-quality sperm and provide comprehensive evidence that sperm morphology of social insects is influenced by sexual selection.     

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.     

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.     

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.     

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.     

DOES GENETIC RELATEDNESS OF MATES INFLUENCE COMPETITIVE FERTILIZATION SUCCESS IN GUPPIES?

A growing number of studies highlight the nontransitive properties of ejaculates when they are in competition to fertilize a female's eggs. Increasingly, these studies suggest that postcopulatory processes act as a filter against sperm from closely related males or those with similar genotypes, limiting the deleterious effects of inbreeding on offspring fitness. We investigated the potential for such postcopulatory mechanisms of inbreeding avoidance in the guppy (Poecilia reticulata), a promiscuous livebearing fish. We used artificial insemination as a method of delivering to a female the combined ejaculates from a first cousin (relatedness coefficient r = 0.125) and an unrelated male. This method of sperm delivery controls behavioral processes of pre- and postcopulatory female choice, which can bias paternity toward unrelated males. Our genetic analysis revealed no effect of parental relatedness on paternity outcomes. The observed mean paternity share for related males (0.47) and associated variance did not differ significantly from an expected binomial distribution that assumes no biased use of sperm with respect to relatedness (0.5). Although our data provide no evidence for postcopulatory mechanisms of inbreeding avoidance, the ability of female guppies to influence ejaculate transfer and retention offers an alternative and easily testable mechanism of inbreeding avoidance in this species.     

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.     

CRYPTIC FEMALE CHOICE: CRITERIA FOR ESTABLISHING FEMALE SPERM CHOICE

In this paper, I consider the criteria necessary to demonstrate the postcopulatory ability of females to favor the sperm of one conspecific male over another, that is, sperm choice. In practice it is difficult to distinguish between sperm competition and sperm choice, and sperm choice can be demonstrated only if the effects of sperm competition can be controlled. Few studies have used experimental protocols that do this, so evidence for sperm choice is limited. Moreover, in those studies in which sperm choice occurs, it does so to avoid incompatible genetic combinations and is therefore unlikely to result in directional sexual selection.     

The evolution of sperm morphometry in pheasants

Post-copulatory sexual selection is thought to be a potent evolutionary force driving the diversification of sperm shape and function across species. In birds, insemination and fertilization are separated in time and sperm storage increases the duration of sperm-female interaction and hence the opportunity for sperm competition and cryptic female choice. We performed a comparative study of 24 pheasant species (Phasianidae, Galliformes) to establish the relative importance of sperm competition and the duration of sperm storage for the evolution of sperm morphometry (i.e. size of different sperm traits). We found that sperm size traits were negatively associated with the duration of sperm storage but were independent of the risk of sperm competition estimated from relative testis mass. Our study emphasizes the importance of female reproductive biology for the evolution of sperm morphometry particularly in sperm-storing taxa.     

Directional postcopulatory sexual selection is associated with female sperm storage in Trinidadian guppies

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded‐off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.     

FEMALE CONTROL OF SPERM TRANSFER AND INTRASPECIFIC VARIATION IN SPERM PRECEDENCE: ANTECEDENTS TO THE EVOLUTION OF A COURTSHIP FOOD GIFT

Manipulation of ejaculates is believed to be an important avenue of female choice throughout the animal kingdom, but evidence of its importance to sexual selection remains scarce. In crickets, such manipulation is manifest in the premature removal of the externally attached spermatophore, which may afford females an important means of postcopulatory mate choice. We tested the hypothesis that premature spermatophore removal contributes significantly to intraspecific variation in sperm precedence by (1) experimentally manipulating spermatophore attachment durations of competing male Gryllodes sigillatus and (2) employing protein electrophoresis to determine the paternity of doubly mated females. The relative spermatophore attachment durations of competing males had a significant influence on male paternity, but the pattern of sperm precedence deviated significantly from the predictions of an ideal lottery. Instead, paternity data and morphological evidence accorded best with a model of partial sperm displacement derived here. Our model is similar to a displacement model of Parker et al. in that sperm of the second male mixes instantaneously with that of the first throughout the displacement process, but the novel feature of our model is that the number of sperm displaced is only a fraction of the number of sperm transferred by the second male. Regardless of the underlying mechanism, female G. sigillatus can clearly alter the paternity of their offspring through their spermatophore-removal behavior, and employ such cryptic choice in favoring larger males and those providing larger courtship food gifts. We discuss how female control of sperm transfer and intraspecific variation in sperm precedence may be important precursors to the evolution of gift giving in insects.     

Male genital morphology and its influence on female mating preferences and paternity success in guppies

In internally fertilizing species male genitalia often show a higher degree of elaboration than required for simply transferring sperm to females. Among the hypotheses proposed to explain such diversity, sexual selection has received the most empirical support, with studies revealing that genital morphology can be targeted by both pre-and postcopulatory sexual selection. Until now, most studies have focused on these two episodes of selection independently. Here, we take an alternative approach by considering both components simultaneously in the livebearing fish, Poecilia reticulata. We allowed females to mate successively (and cooperatively) with two males and determined whether male genital length influenced the female's propensity to mate with a male (precopulatory selection, via female choice) and whether male genital size and shape predicted the relative paternity share of subsequent broods (postcopulatory selection, via sperm competition/cryptic female choice). We found no evidence that either episode of sexual selection targets male genital size or shape. These findings, in conjunction with our recent work exposing a role of genital morphology in mediating unsolicited (forced) matings in guppies, further supports our prior speculation that sexual conflict may be an important broker of genital evolution in this species.     

Multiple paternity and postcopulatory sexual selection in a hermaphrodite: what influences sperm precedence in the garden snail Helix aspersa?

Sperm competition has been studied in many gonochoric animals but little is known about its occurrence in simultaneous hermaphrodites, especially in land snails. The reproductive behaviour of the land snail Helix aspersa involves several features, like multiple mating, long-term sperm storage and dart-shooting behaviour, which may promote sperm competition. Cryptic female choice may also occur through a spermatheca subdivided into tubules, which potentially allows compartmentalized sperm storage of successive mates. In order to determine the outcome of postcopulatory sexual selection in this species, we designed a cross-breeding experiment where a recipient ('female') mated with two sperm donors ('males'). Mates came from either the same population as the recipient or from a distinct one. To test for the influence a recipient can have on the paternity of its offspring, we excluded the effects of dart shooting by using only virgin snails as sperm donors because they do not shoot any dart before their first copulation. We measured the effects of size of mates as well as time to first and second mating on second mate sperm precedence (P2; established using microsatellite markers). Multiple paternity was detected in 62.5% of clutches and overall there was first-mate sperm precedence with a mean P2 of 0.24. Generalized linear modelling revealed that the best predictors of paternity were the time between matings and the time before first mating. Overall, both first and second mates that copulated quickly got greater parentage, which may suggest that postcopulatory events influence patterns of sperm precedence in the garden snail.     

Reproductive success by large, closely related males facilitated by sperm storage in an aggregate breeding amphibian

The outcome of sexual selection on males may depend on female mate choice and male–male competition as well as postcopulatory processes such as cryptic female choice and sperm competition. We studied the outcome of sexual selection in the spotted salamander ( Ambystoma maculatum ), specifically examining the role of body size and relatedness on male reproductive success. Using controlled mating experiments in the field, we gave females access to three males of different sizes. We used seven microsatellite loci to determine paternity in the resulting larvae, estimate relatedness ( r ) between females and their mates, and calculate md ² (a measure of within-individual genomic divergence), heterozygosity, and standardized heterozygosity in the larvae. Both body size and relatedness to the female were significant predictors of male reproductive success. The relatedness of the males available to a female did not influence the amount of stored sperm she used to sire her larvae. Nonetheless, computer simulations showed that the average md ², heterozygosity, and standardized heterozygosity of the offspring were lower than expected by random mating. These differences are due to the use of stored sperm to fertilize some eggs; md ², heterozygosity, and standardized heterozygosity of larvae sired by stored sperm were significantly lower than those of larvae sired by the experimental males. These results suggest that relatedness may further influence a male's long-term reproductive success by determining whether his sperm is stored for later breeding seasons. Sexual selection in this salamander likely involves a complex interaction among many factors and may act over many seasons.     

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Cryptic female choice in the yellow dung fly Scathophaga stercoraria (L.)

Both female choice and male-male competition may take place during reproduction in many species. Female choice tends to be less obvious than male-male competition and consequently has received less attention from researchers. The opportunity for cryptic female choice arises after multiple insemination. Through postcopulatory processes, a female could alter the pattern of paternity among her offspring so that it does not directly reflect the different contributions of sperm made by her mates. To be able to determine if a female alters the relative sperm contributions of her mates, the behaviors and influences of the males must therefore be first taken into account. The interest of each male is to father all the offspring, and the interest of each female is to maximize paternal quality. Female yellow dung flies have complex internal reproductive tracts that may give them considerable control over the fertilization success of stored sperm from different males. In laboratory trials to date, the last male to mate has usually been most successful. In the present study, cryptic choice occurred in Scathophaga stercoraria and the pattern of choice was consistent with previously reported results. The fertilization success of a female's second mate (P2) was substantially larger if a female was kept at constant temperature and if the second male was genetically similar to her at the phosphoglucomutase (Pgm) locus. Females from the field normally have three spermathecae, but some have four. Lines were bred to have either three or four spermathecae. Flies from the different lines were crossed to generate females with similar genetic backgrounds that had either three or four spermathecae. P2 was significantly lower for high-quality females, that is, those that laid larger-than-average-clutches, with four spermathecae than for low-quality females with four spermathecae; female quality had no influence on P2 for females with three spermathecae. The results suggest that only large females may benefit from increased spermathecae number by being able to act against male interests. Females may only have three spermathecae, even though genetic variation for more is present, because selection for more spermathecae is weak.