Female behaviour and the interaction of male and female genital traits mediate sperm transfer during mating

Natural selection and post‐copulatory sexual selection, including sexual conflict, contribute to genital diversification. Fundamental first steps in understanding how these processes shape the evolution of specific genital traits are to determine their function experimentally and to understand the interactions between female and male genitalia during copulation. Our experimental manipulations of male and female genitalia in red‐sided garter snakes (Thamnophis sirtalis parietalis) reveal that copulation duration and copulatory plug deposition, as well as total and oviductal/vaginal sperm counts, are influenced by the interaction between male and female genital traits and female behaviour during copulation. By mating females with anesthetized cloacae to males with spine‐ablated hemipenes using a fully factorial design, we identified significant female–male copulatory trait interactions and found that females prevent sperm from entering their oviducts by contracting their vaginal pouch. Furthermore, these muscular contractions limit copulatory plug size, whereas the basal spine of the male hemipene aids in sperm and plug transfer. Our results are consistent with a role of sexual conflict in mating interactions and highlight the evolutionary importance of female resistance to reproductive outcomes.     

Mating behavior,insemination and sperm transfer in the ground beetle Carabus insulicola

Mating behavior and the processes of insemination and sperm transfer in the ground beetle Carabus insulicola were analyzed. C. insulicola has elaborate genitalia, in which the strongly sclerotized male copulatory piece is inserted into the female vaginal appendix in copula. During mating, I observed pre-copulatory struggles of males and females, as well as delays in ejaculation, suggesting the presence of intersexual conflicts. Insemination was achieved with a spermatophore, which strongly adhered to the openings of the spermatheca, common oviduct, and vaginal appendix. The spermatophore dissolved after copulation, and sperm were transferred into the spermatheca within three hours after copulation. Sperm bundles were contained within the testes and spermatophores, but free spermatozoa were found in the spermatheca.     

Heterochrony and growth rate variation mediate the development of divergent genital morphologies in closely related Ohomopterus ground beetles

The rapid divergence of genital morphology is well studied in the context of sexual selection and speciation; however, little is known about the developmental mechanisms underlying divergence in genitalia. Ground beetles in the subgenus Ohomopterus genus Carabus have species‐specific genitalia that show coevolutionary divergence between the sexes. In this study, using X‐ray microcomputed tomography, we examined the morphogenesis of male and female genitalia in two closely related Ohomopterus species with divergent genital morphologies. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar in the two species. The male aedeagus and internal sac and female bursa copulatrix were partially formed at pupation and developed gradually thereafter. The species‐specific genital parts, male copulatory piece, and female vaginal appendix differed in the timing and rate of development. The relatively long copulatory piece of Carabus maiyasanus began to develop earlier, but subsequent rates of growth were similar in the two species. The timing of the formation of the vaginal appendix and initial growth rates were similar, but subsequent rapid growth led to a longer vaginal appendix in C. maiyasanus. Thus, substantial interspecific differences in the size of genital parts were mediated by different underlying developmental mechanisms between the sexes (i.e., a shift in the developmental schedule in males and a change in growth rate in females). These results revealed the spatio–temporal dynamics of species‐specific genital structure development, providing a novel platform for evo–devo studies of the diversification of genital morphologies.     

Significance of constraints on genital coevolution: Why do female Drosophila appear to cooperate with males by accepting harmful matings?

The mechanisms driving the coevolution of male and female genital morphologies are still debated. Female genitalia in Drosophila species bear membranous “pouches” or hardened “shields,” which the male genital armature contact during copulation. Although shield‐like structures likely serve to mitigate the effects of harmful mating, some authors have suggested that soft pouches, which do not prevent male genitalia from inflicting wounds, represent a congruent sensory organ. To elucidate the evolutionary forces responsible for the development of such organs, I examined the effects of artificial damage to various genital parts of female Drosophila erecta on reproductive success. Despite a high survival rate among females, damage to the ovipositor plate resulted in frequent failure of insemination and in the embedment of eggs into the substrate. Damage to the vaginal shield resulted in increased mortality and frequent failure of egg embedment, with an egg blocking the vagina under the damaged shield in some females. Wounding of the pouch had less of an effect on both mating and oviposition success, suggesting that the structure “lures” the male trauma‐causing organs to areas where the resultant wounds do not interfere with insemination or oviposition. These data show that the dual functions of female genitalia (mating and oviposition) mediate genital coevolution.     

Mating Behavior, Sexual Selection, and Copulatory Courtship in a Promiscuous Beetle

The function of male movements during copulation is unclear. These movements may be a result of the necessary mechanics of insemination, or they may also have further function, for instance, stimulating or courting a female during mating, perhaps influencing female mate choice. We present data from three experiments exploring the mating behavior and copulatory movements of the highly promiscuous beetle Psilothrix viridicoeruleus. Male mating success in the struggle over mating was not related to male or female size (measured by weight) but successful males were more vigorous in terms of copulatory movements. These males took longer to mount females but copulated longer and remained mounted longer. We discuss these results in terms of the mating system of Psilothrix and also in terms of observations of the timing of insemination during copulation. We suggest that copulatory movements in this species are best understood as copulatory courtship.     

Evaluating the post‐copulatory sexual selection hypothesis for genital evolution reveals evidence for pleiotropic harm exerted by the male genital spines of Drosophila ananassae

The contemporary explanation for the rapid evolutionary diversification of animal genitalia is that such traits evolve by post‐copulatory sexual selection. Here, we test the hypothesis that the male genital spines of Drosophila ananassae play an adaptive role in post‐copulatory sexual selection. Whereas previous work on two Drosophila species shows that these spines function in precopulatory sexual selection to initiate genital coupling and promote male competitive copulation success, further research is needed to evaluate the potential for Drosophila genital spines to have a post‐copulatory function. Using a precision micron‐scale laser surgery technique, we test the effect of spine length reduction on copulation duration, male competitive fertilization success, female fecundity and female remating behaviour. We find no evidence that male genital spines in this species have a post‐copulatory adaptive function. Instead, females mated to males with surgically reduced/blunted genital spines exhibited comparatively greater short‐term fecundity relative to those mated by control males, indicating that the natural (i.e. unaltered) form of the trait may be harmful to females. In the absence of an effect of genital spine reduction on measured components of post‐copulatory fitness, the harm seems to be a pleiotropic side effect rather than adaptive. Results are discussed in the context of sexual conflict mediating the evolution of male genital spines in this species and likely other Drosophila.     

Mating behavior and the function of the male genital spine in the ground beetle Carabus clathratus

The morphologies of male genitalia often appear harmful or aggressive, as if they may inflict physical damage upon females during copulation. Such male genitalia are often thought to function in intra- and intersexual interactions during mating. In the carabid genus Carabus, division Spinulati, males possess a spine (spinula) on the intromittent organ, of which function is unknown. To reveal the function of the spinula, we studied the mating behavior and genital coupling of a Spinulati species, Carabus (Limnocarabus) clathratus. The males positioned the spinula along the inner wall of the vaginal opening throughout copulation. This placement created a small dent and subsequently a melanized patch (wound) on the vaginal wall, but the spinula rarely penetrated the vaginal wall. The spinula did not reach the innermost part of the vagina where the spermatophore is deposited. These results suggest that the spinula is not used for inflicting damage on female genitalia or manipulating spermatophores of rival males. During spermatophore formation, the male partially withdrew the aedeagus, and only the aedeagal tip and endophallus remained within the female. By placing the spinula against the vaginal wall, the male could hold the endophallus within the vaginal chamber in the unstable copulatory posture. Thus, our observations suggest that the spinula primarily functions as an "anchor" to maintain the coupling of the male and female genitalia and thereby ensure insemination.     

Genital damage in the orb-web spider Argiope bruennichi (Araneae: Araneidae) increases paternity success

The morphology of male genitalia often suggests functions besidessperm transfer that may have evolved under natural or sexualselection. In several species of sexually cannibalistic spiders,males damage their paired genitalia during mating, limitingthem to one copulation per pedipalp. Using a triple-mating experiment,we tested if genital damage in the orb-web spider Argiope bruennichiincreases male fitness either through facilitating his escapefrom an aggressive female or by obstructing the female's inseminationducts against future copulation attempts from other males. Wefound no survival advantage for males damaging their pedipalps;however, copulations into a previously used insemination ductwere significantly shorter when the previous male had left partsof his genitalia inside the insemination duct. Because copulationduration determines paternity in this species, our result suggeststhat male genital damage in A. bruennichi is sexually selected.By breaking off parts of their intromittent organs inside avirgin female, males can reduce sperm competition and therebyincrease their paternity success.     

No evidence for external genital morphology affecting cryptic female choice and reproductive isolation in Drosophila

Genitalia are one of the most rapidly diverging morphological features in animals. The evolution of genital morphology is proposed to be driven by sexual selection via cryptic female choice, whereby a female selectively uptakes and uses a particular male's sperm on the basis of male genital morphology. The resulting shifts in genital morphology within a species can lead to divergence in genitalia between species, and consequently to reproductive isolation and speciation. Although this conceptual framework is supported by correlative data, there is little direct empirical evidence. Here, we used a microdissection laser to alter the morphology of the external male genitalia in Drosophila, a widely used genetic model for both genital shape and cryptic female choice. We evaluate the effect of precision alterations to lobe morphology on both interspecific and intraspecific mating, and demonstrate experimentally that the male genital lobes do not affect copulation duration or cryptic female choice, contrary to long‐standing assumptions regarding the role of the lobes in this model system. Rather, we demonstrate that the lobes are essential for copulation to occur. Moreover, slight alterations to the lobes significantly reduced copulatory success only in competitive environments, identifying precopulatory sexual selection as a potential contributing force behind genital diversification.     

Sexual conflict over mating in red-sided garter snakes (Thamnophis sirtalis) as indicated by experimental manipulation of genitalia

Sexual conflict over mating can result in sex-specific morphologies and behaviours that allow each sex to exert control over the outcome of reproduction. Genital traits, in particular, are often directly involved in conflict interactions. Via genital manipulation, we experimentally investigated whether genital traits in red-sided garter snakes influence copulation duration and formation of a copulatory plug. The hemipenes of male red-sided garter snakes have a large basal spine that inserts into the female cloaca during mating. We ablated the spine and found that males were still capable of copulation but copulation duration was much shorter and copulatory plugs were smaller than those produced by intact males. We also anaesthetized the female cloacal region and found that anaesthetized females copulated longer than control females, suggesting that female cloacal and vaginal contractions play a role in controlling copulation duration. Both results, combined with known aspects of the breeding biology of red-sided garter snakes, strongly support the idea that sexual conflict is involved in mating interactions in this species. Our results demonstrate the complex interactions among male and female traits generated by coevolutionary processes in a wild population. Such complexity highlights the importance of simultaneous examination of male and female traits.     

Female walking during copulation reduces the likelihood of sperm transfer from males in the sweet potato weevil,Cylas formicarius

Behaviour during copulation can alter the fate of sperm of the mating males. This behaviour may exert selective pressure, resulting in the evolution of diverse reproductive behaviour, morphology, and physiology. This study examined the role of female copulatory behaviour on sperm fate in the sweet potato weevil, Cylas formicarius (Fabricius) (Coleoptera: Brentidae). In this species, males mount the female during copulation. The female frequently walks during copulation, carrying the male on her back. Here, we describe and quantify the copulatory behaviour of mating pairs and examine the sperm fate. Insemination success, as determined by the presence of sperm in the spermatheca, was lower when females walked for longer periods during copulation. This result emphasizes the value of studying variation in female copulatory behaviour in order to understand the factors that influence sperm fate. We discuss the implications of these results on sexual selection and utility in programs applying sterile insect techniques.     

Copulatory Plug Displacement Evidences Sperm Competition in Lemur catta

Male displacement of copulatory (sperm) plugs from female vaginas provides further evidence for sperm competition in ring-tailed lemurs (Lemur catta), a gregarious prosimian species with a multimale, multifemale mating system. During two mating seasons, I studied two groups of free-ranging ring-tailed lemurs on St. Catherines Island, GA, USA. I observed 22 mating pairs in which males achieved penile intromission. Copulatory plug displacement by males occurred in 9 cases. Plugs were displaced during copulation by male penes upon withdrawl following deep vaginal thrusting. In every case of copulatory plug displacement, the male displacing a plug mated to ejaculation with the estrous female. In a mating system in which females typically mate with more than one male during estrous, often in succession, copulatory plug displacement may function to disrupt or preclude other males' successful insemination of estrous females. The effects of sperm plug displacement on paternity in Lemur catta are unknown, as no study had heretofore documented copulatory plug displacement in this species. The first-male mating advantage suggested for Lemur catta should be re-evaluated where mating order is known, and copulatory plug displacement during mating, or lack thereof, is identified. Because there is a tendency for first-mating males to mate-guard for longer periods of time in Lemur catta, the latency period between the first mate's ejaculation and that of subsequent mates may be an important determinant of male fertilization success.     

Reproductive success in Zygogramma bicolorata: A role of post-insemination association of male and female

Reproductive success is attained by various mechanisms in insects. Prolonged post insemination association is one such mechanism to increase the reproductive success. The present study was conducted to assess the role of post insemination association of mating partners on reproductive performance in Chrysomelidae beetle, Zygogramma bicolorata Pallister. The matings were disrupted at different time intervals and fecundity and percent egg viability of the females were recorded. In addition, the mounting attempts, mating attempts, time to commencement of mating and latent period were also recorded. It was hypothesized that: (1) the mounting and mating attempts would not exist, (2) copulation duration, would not affect the reproductive performance, and (3) the beetle would not exhibit the mate guarding behaviour. Interestingly, results revealed that 6.00 ± 1.3 and 6.59 ± 0.93 mounting and mating attempts are needed to establish successful mating. The results revealed that males improved their percent egg viability with a mating duration ranging from nearly 30–50 min. While fecundity increased with a mating duration of above 30 min and up to a duration of 60 min. This result concluded that males of this beetle display post copulatory mate guarding behaviour after 60 min in which male rides on female’s back with his aedeagus inserted in the female genital tract.     

Complex copulatory behavior and the proximate effect of genital and body size differences on mechanical reproductive isolation in the millipede genus Parafontaria

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.     

The combined effects of pre‐ and post‐copulatory processes are masking sexual conflict over mating rate in Gerris buenoi

In polygynandrous animals, post‐copulatory processes likely interfere with precopulatory sexual selection. In water striders, sexual conflict over mating rate and post‐copulatory processes are well documented, but their combined effect on reproductive success has seldom been investigated. We combine genetic parentage analyses and behavioural observations conducted in a competitive reproductive environment to investigate how pre‐ and post‐copulatory processes influence reproductive success in Gerris buenoi Kirkaldy. Precopulatory struggles had antagonistic effects on male and female reproductive success: efficiently gaining copulations was beneficial for males, whereas efficiently avoiding copulations was profitable for females. Also, high mating rates and an intermediate optimal resistance level of females supported the hypothesis of convenience polyandry. Contrary to formal predictions, high mating rates (i.e. the number of copulations) did not increase reproductive success in males or decrease reproductive success in females. Instead, the reproductive success of both sexes was higher when offspring were produced with several partners and when there were few unnecessary matings. Thus, male and female G. buenoi displayed different interests in reproduction, but post‐copulatory processes were masking the effects of copulatory mating success on reproductive success. Given the high mating rates observed, sperm competition could easily counter the effect of mating rates, perhaps in interaction with cryptic female choice and/or fecundity selection. Our study presents a complex but realistic overview of sexual selection forces at work in a model organism for the study of sexual conflict, confirming that insights are gained from investigating all episodes in the reproduction cycle of polygynandrous animals.     

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.     

Broken Copulatory Organs are Low-Cost Adaptations to Sperm Competition in Redback Spiders

In some spiders, a discrete portion of the male's copulatory organ (the apical sclerite) breaks off during copulation and remains in the female's reproductive tract. Apical sclerites may prevent insemination by rivals (sperm competition), stimulate females to favourably bias paternity (cryptic choice) or breakage may reflect sexual conflict over copulation duration with little direct effect on paternity. It has been assumed that any benefits of organ breakage are balanced by a large cost (male sterility) in species where males could otherwise mate multiply, but this has never been experimentally tested. We examined these ideas in the Australian redback spider (Latrodectus hasselti Thorell 1870, Araneae: Theridiidae), a species where males are functionally sterile after one normal mating. We experimentally removed sclerites and found males were able to mate, had similar copulation durations and transferred similar numbers of sperm as males with intact sclerites. Benefits of organ breakage were examined by forcing intact, rival males to inseminate the same or opposite reproductive tracts (female have paired, independent tracts in this taxon) and assessing paternity as a function of sclerite location. As predicted, apical sclerites were typically deposited at the entrance to the female's sperm storage organ, where they could physically block insemination by rivals. First male precedence was common when males inseminated the same tract and deposited sclerites at the entrance to the spermatheca, but not when sclerites were found elsewhere in the tract, or when rivals inseminated opposite tracts (where physically blocking rivals was impossible). Our data show that, in redbacks, copulatory organ breakage is not a side‐effect of sexual conflict, is unlikely to be a cue for cryptic female choice, but allows males to avoid sperm competition. Moreover, copulatory organ damage can have minimal reproductive cost for males, so assumptions of sterility after organ breakage are unjustified without supporting data.     

GENITAL LOCK-AND-KEY AS A SELECTIVE AGENT AGAINST HYBRIDIZATION

We demonstrate experimentally that differences in genital characters impose a direct cost of interspecific copulation on two closely related carabid species, Carabus (Ohomopterus) maiyasanus and C. (O.) iwawakianus, that share a narrow hybrid zone. Males of both species attempted copulation indiscriminately between conspecific and heterospecific females. Females experiencing heterospecific mating often suffered mortality due to rupture of their vaginal membranes. Those without fatal injury laid eggs which developed into F₁ adults, but the fertilization rate was much lower than for intraspecific pairs. Males of C. maiyasanus, but not C. iwawakianus, often had broken genital parts (copulatory pieces) following interspecific copulations, which may prevent normal copulation in subsequent matings. Because of female mortality and low fertilization rate, the estimated fitness cost of interspecific mating was very large in terms of the reduction in the number of offspring (hatching larvae) for both sexes and both species. Thus, genital lock-and-key appears to exert significant selection against hybridization in the hybrid zone of these carabid beetles.     

Differential sperm expenditure reveals a possible role for post‐copulatory sexual selection in a lekking moth

Male reproductive success in the lesser wax moth Achroia grisella is strongly determined by pre‐copulatory mate choice, during which females choose among males aggregated in small leks based on the attractiveness of ultrasonic songs. Nothing is known about the potential of post‐copulatory mechanisms to affect male reproductive success. However, there is evidence that females at least occasionally remate with a second male and that males are unable to produce ejaculates quickly after a previous copulation. Here we investigated the effects of mating history on ejaculate size and demonstrate that the number of transferred sperm significantly decreased from first (i.e., virgin) to second (i.e., nonvirgin) copulation within individual males. For males of identical age, the number of sperm transferred was higher in virgin than in nonvirgin copulations, too, demonstrating that mating history, is responsible for the decrease in sperm numbers transferred and not the concomitant age difference. Furthermore, the number of transferred sperm was significantly repeatable within males. The demonstrated variation in ejaculate size both between subsequent copulations as well as among individuals suggests that there is allocation of a possibly limited amount of sperm. Because female fecundity is not limited by sperm availability in this system, post‐copulatory mechanisms, in particular sperm competition, may play a previously underappreciated role in the lesser wax moth mating system.     

Copulatory mechanism in a sexually cannibalistic spider with genital mutilation (Araneae: Araneidae: Argiope bruennichi)

Genitalia are among the fastest evolving morphological traits as evidenced by their common function as diagnostic traits in species identification. Even though the main function of genitalia is the successful transfer of spermatozoa, the presence of diverse structures that are obviously not necessary for this suggests that genitalia are a target of sexual selection. The male genitalia of many spider species are extremely complex and equipped with numerous sclerites, plates and spines whose functions are largely unknown. Selection on male genitalia may be particularly strong in sexually cannibalistic spiders, where mating success of males is restricted to a single female. We investigated the copulatory mechanism of the sexually cannibalistic orb weaving spider Argiope bruennichi by shock freezing mating pairs and revealed a complicated interaction between the appendices and sclerites that make up the male gonopods (paired pedipalps). The plate that covers the female genital opening (scape) is secured between two appendices of the male genital bulb, while three sclerites that bear the sperm duct are unfolded and extended into the female copulatory opening. During copulation, females attack and cannibalise the male and males mutilate their genitalia in about 80% of cases. Our study demonstrates that (i) genital coupling is largely accomplished on the external part of the female genitalia, (ii) that the mechanism requires an interaction between several non-sperm-transferring structures and (iii) that there are two predetermined breaking points in the male genitalia. Further comparative work on the genus Argiope will test if the copulatory mechanism with genital mutilation indeed is an adaptation to sexual cannibalism or if cannibalism is a female counter adaptation to male monopolisation through genital plugging.