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.     

A time-sequence functional analysis of mating behaviour and genital coupling in Drosophila: role of cryptic female choice and male sex-drive in the evolution of male genitalia

Male genitalia in Drosophila exemplify strikingly rapid and divergent evolution, whereas female genitalia are relatively invariable. Whereas precopulatory and post-copulatory sexual selection has been invoked to explain this trend, the functional significance of genital structures during copulation remains obscure. We used time-sequence analysis to study the functional significance of external genitalic structures during the course of copulation, between D. melanogaster and D. simulans. This functional analysis has provided new information that reveals the importance of male-driven copulatory mechanics and strategies in the rapid diversification of genitalia. The posterior process, which is a recently evolved sexual character and present only in males of the melanogaster clade, plays a crucial role in mounting as well as in genital coupling. Whereas there is ample evidence for precopulatory and/or post-copulatory female choice, we show here that during copulation there is little or no physical female choice, consequently, males determine copulation duration. We also found subtle differences in copulatory mechanics between very closely related species. We propose that variation in male usage of novel genitalic structures and shifts in copulatory behaviour have played an important role in the diversification of genitalia in species of the Drosophila subgroup.     

Intraspecific evidence from guppies for correlated patterns of male and female genital trait diversification

The role of sexual selection in fuelling genital evolution is becoming increasingly apparent from comparative studies revealing interspecific divergence in male genitalia and evolutionary associations between male and female genital traits. Despite this, we know little about intraspecific variance in male genital morphology, or how male and female reproductive traits covary among divergent populations. Here we address both topics using natural populations of the guppy, Poecilia reticulata, a livebearing fish that exhibits divergent patterns of male sexual behaviour among populations. Initially, we performed a series of mating trials on a single population to examine the relationship between the morphology of the male's copulatory organ (the gonopodium) and the success of forced matings. Using a combination of linear measurements and geometric morphometrics, we found that variation in the length and shape of the gonopodium predicted the success of forced matings in terms of the rate of genital contacts and insemination success, respectively. We then looked for geographical divergence in these traits, since the relative frequency of forced matings tends to be greater in high-predation populations. We found consistent patterns of variation in male genital size and shape in relation to the level of predation, and corresponding patterns of (co)variation in female genital morphology. Together, these data enable us to draw tentative conclusions about the underlying selective pressures causing correlated patterns of divergence in male and female genital traits, which point to a role for sexually antagonistic selection.     

Coevolution between Male and Female Genitalia in the Drosophila melanogaster Species Subgroup

In contrast to male genitalia that typically exhibit patterns of rapid and divergent evolution among internally fertilizing animals, female genitalia have been less well studied and are generally thought to evolve slowly among closely-related species. As a result, few cases of male-female genital coevolution have been documented. In Drosophila, female copulatory structures have been claimed to be mostly invariant compared to male structures. Here, we re-examined male and female genitalia in the nine species of the D. melanogaster subgroup. We describe several new species-specific female genital structures that appear to coevolve with male genital structures, and provide evidence that the coevolving structures contact each other during copulation. Several female structures might be defensive shields against apparently harmful male structures, such as cercal teeth, phallic hooks and spines. Evidence for male-female morphological coevolution in Drosophila has previously been shown at the post-copulatory level (e.g., sperm length and sperm storage organ size), and our results provide support for male-female coevolution at the copulatory level.     

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.     

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.     

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.     

Origins of female genital diversity: Predation risk and lock‐and‐key explain rapid divergence during an adaptive radiation

The study of male genital diversity has long overshadowed evolutionary inquiry of female genitalia, despite its nontrivial diversity. Here, we identify four nonmutually exclusive mechanisms that could lead to genital divergence in females, and potentially generate patterns of correlated male–female genital evolution: (1) ecological variation alters the context of sexual selection (“ecology hypothesis”), (2) sexually antagonistic selection (“sexual‐conflict hypothesis”), (3) female preferences for male genitalia mediated by female genital traits (“female‐choice hypothesis”), and (4) selection against inter‐population mating (“lock‐and‐key hypothesis”). We performed an empirical investigation of all four hypotheses using the model system of Bahamas mosquitofish inhabiting blue holes that vary in predation risk. We found unequivocal support for the ecology hypothesis, with females exhibiting a smaller genital opening in blue holes containing piscivorous fish. This is consistent with stronger postmating female choice/conflict when predators are present, but greater premating female choice in their absence. Our results additionally supported the lock‐and‐key hypothesis, uncovering a pattern of reproductive character displacement for genital shape. We found no support for the sexual conflict or female choice hypotheses. Our results demonstrate a strong role for ecology in generating female genital diversity, and suggest that lock‐and‐key may provide a viable cause of female genital diversification.     

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.     

The coevolution of male and female genitalia in a mammal: A quantitative genetic insight

Male genitalia are among the most phenotypically diverse morphological traits, and sexual selection is widely accepted as being responsible for their evolutionary divergence. Studies of house mice suggest that the shape of the baculum (penis bone) affects male reproductive fitness and experimentally imposed postmating sexual selection has been shown to drive divergence in baculum shape across generations. Much less is known of the morphology of female genitalia and its coevolution with male genitalia. In light of this, we used a paternal half-sibling design to explore patterns of additive genetic variation and covariation underlying baculum shape and female vaginal tract size in house mice (Mus musculus domesticus). We applied a landmark-based morphometrics approach to measure baculum size and shape in males and the length of the vaginal tract and width of the cervix in females. Our results reveal significant additive genetic variation in house mouse baculum morphology and cervix width, as well as evidence for genetic covariation between male and female genital measures. Our data thereby provide novel insight into the potential for the coevolutionary divergence of male and female genital traits in a mammal.     

Selection of male traits during and after copulation in the seedbug Lygaeus simulans (Heteroptera, Lygaeidae)

In order to evaluate selection of male morphological traits during copulation, a laboratory experiment was performed with the promiscuous seedbug Lygaeus simulans. Three male traits suspected as putative targets of selection were measured: weight, fluctuating asymmetry of a measure on the forewings, and length of a conspicuous genital structure, the processus gonopori. As fitness measures we considered total fecundity (number of fertilized eggs), insemination and fertilization success (established if a female laid fertilized eggs after copulation), and the interval between copulation and oviposition. Eighty-four males were allowed a single copulation with one virgin female each. Out of 67 copulations, 27 (40.2%) resulted in fertilized eggs and the oviposition latency ranged from 6 to 26 days. Regressions of male traits on the fitness measures showed significant phenotypical selection of two male traits: (1) males of average weight are more likely to achieve fertilization and (2) the oviposition latency was shorter for males with lower asymmetry. The copulation-oviposition interval may be especially important for male fertilization success because Lygaeus males perform copulatory mate guarding and the last male copulating with a female fertilizes most of the eggs. No selection of the genitalic trait was detected.     

Rapid diversification of male genitalia and mating strategies in Ohomopterus ground beetles

We analysed evolutionary diversification and covariation in male genitalia and four mating traits related to sexual selection, i.e. testis size, spermatophore size, copulation duration and post-copulatory guarding duration, in Ohomopterus ground beetles using phylogenetically independent contrasts. Male genital size and mating duration have evolved more rapidly than body size and the other traits studied. Male genital size was negatively correlated with copulation duration, suggesting that elongated male genitalia may enable decreased time investment in a single copulation because it is more effective at facilitating spermatophore deposition. Male genital size was positively correlated with spermatophore size, suggesting coevolution between offensive and defensive male mating tactics because the elongated male genitalia may be advantageous in displacement of rivals' plug-like spermatophores, and decreased mating duration may intensify sperm competition. Thus, the remarkable diversity of male genitalia in Ohomopterus may have been facilitated by the interplay between inter- and intrasexual selection processes.     

QTL for the species-specific male and female genital morphologies in Ohomopterus ground beetles

Animals with internal fertilization often exhibit marked diversification in genital morphology among closely related species. However, our knowledge of the genetic architecture underlying genital evolution is still limited. We constructed genetic linkage maps and analysed quantitative trait loci (QTL) for F₂ hybrids of two closely related species of the carabid beetles Carabus (Ohomopterus) iwawakianus and C. (O.) maiyasanus, which show matching male and female genital shapes within species, but marked differences in genital morphologies between species. The linkage maps comprised both amplified fragment length polymorphism and microsatellite markers. Composite interval mapping to detect QTL for three traits of male copulatory piece (length, width, weight) and two traits for female vaginal appendix (length, width) resulted in the detection of one to five significant QTL for each trait. The QTL explained large proportions of phenotypic variance. Thus, the interspecific difference in the genital morphologies appeared to be determined by relatively small numbers of genes with large genetic effects. QTL of different traits for the same or different sexes co‐occurred on five of eight linkage groups with significant QTL; in particular, three QTL for different male and female genital traits occurred almost at the same position. Each of the male genital traits showed uniform signs of additive genetic effects, suggesting that directional selection has led to species‐specific morphologies. However, the signs of additive genetic effects in each female genital trait were not uniform, suggesting that coevolution between sexes is not necessarily concerted. This result requires further assessment because the sample size of F₂ females was small.     

Sexual selection and the evolution of genital shape and complexity in water striders

Animal genitalia show two striking but incompletely understood evolutionary trends: a great evolutionary divergence in the shape of genitalic structures, and characteristic structural complexity. Both features are thought to result from sexual selection, but explicit comparative tests are hampered by the fact that it is difficult to quantify both morphological complexity and divergence in shape. We undertake a comparative study of multiple nongenitalic and male genital traits in a clade of 15 water strider species to quantify complexity and shape divergence. We show that genital structures are more complex and their shape more divergent among species than nongenital traits. Further, intromittent genital traits are more complex and have evolved more divergently than nonintromittent genital traits. More importantly, shape and complexity of nonintromittent genital traits show correlated evolution with indices of premating sexual selection and intromittent genital traits with postmating sexual selection, suggesting that the evolution of different components of genital morphology are shaped independently by distinct forms of sexual selection. Our quantitative results provide direct comparative support for the hypothesis that sexual selection is associated with morphological complexity in genitalic traits and highlight the importance of quantifying morphological shape and complexity, rather than size in studies of genital evolution.     

One size fits all? Determinants of sperm transfer in a highly dimorphic orb‐web spider

The evolutionary significance of widespread hypo‐allometric scaling of genital traits in combination with rapid interspecific genital trait divergence has been of key interest to evolutionary biologists for many years and remains poorly understood. Here, we provide a detailed assessment of quantitative genital trait variation in males and females of the sexually highly dimorphic and cannibalistic orb‐weaving spider Argiope aurantia. We then test how this trait variation relates to sperm transfer success. In particular, we test specific predictions of the one‐size‐fits‐all and lock‐and‐key hypotheses for the evolution of genital characters. We use video‐taped staged matings in a controlled environment with subsequent morphological microdissections and sperm count analyses. We find little support for the prediction of the one‐size‐fits‐all hypothesis for the evolution of hypo‐allometric scaling of genital traits, namely that intermediate trait dimensions confer highest sperm transfer success. Likewise, our findings do not support the prediction of the lock‐and‐key hypothesis that a tight fit of male and female genital traits mediates highest sperm transfer success. We do, however, detect directional effects of a number of male and female genital characters on sperm transfer, suggesting that genital trait dimensions are commonly under selection in nature. Importantly, even though females are much larger than males, spermatheca size limits the number of sperm transferred, contradicting a previous hypothesis about the evolutionary consequences of genital size dimorphism in extremely size‐dimorphic taxa. We also find strong positive effects of male body size and copulation duration on the probability of sperm transfer and the number of sperm transferred, with implications for the evolution of extreme sexual size dimorphism and sexual cannibalism in orb weavers.     

BOTH MALE AND FEMALE NOVEL TRAITS PROMOTE THE CORRELATED EVOLUTION OF GENITALIA BETWEEN THE SEXES IN AN ARTHROPOD

The correlated evolution of genitalia between sexes has been demonstrated in many taxa. However, it remains unclear whether female rather than male genitalia can play a key role in the correlated evolution of male and female genitalia. We conducted an extensive cross‐population analysis of the divergence patterns of genital structures, weights of whole genital organs, and the bodies of both sexes, and male genital length in a group of xystodesmid millipedes showing diverse genital morphologies. We demonstrate that the correlated evolution of male and female genitalia toward exaggerated states has occurred in the millipedes, which have evolved novel traits in both males (forceps‐like gonopods) and females (retractable bellows). Enlargement and elongation of forceps‐like gonopods may be advantageous in sperm competition, whereas enlargement and elongation of the bellows may facilitate acceptance/rejection of insemination for ensuring the female's fitness. These male and female genital parts have affected the correlated evolution in the opposite sex, resulting in diversification and exaggeration of genital morphology. Our study suggests that evolutionary novel traits in not only males but also in females could play an important role in the correlated evolution of genitalia between the sexes.     

How the length of genital parts affects copulation performance in a carabid beetle: implications for correlated genital evolution between the sexes

To identify factors leading to the correlated evolution of exaggerated male and female genitalia, we studied the effects of the variable dimensions of corresponding functional genital parts (male copulatory piece and female vaginal appendix) on copulatory performance in the polygamous carabid beetle Carabus (Ohomopterus) maiyasanus. We used mating pairs of individuals from two populations to increase the variances in genital dimensions and determined the copulation performance (insemination and spermatophore replacement, and copulation time) in single‐ and double‐mating situations. In single mating, insemination success was not affected by genital dimensions, although the copulation time was significantly shorter when the male aedeagus was longer. In the double‐mating experiment, insemination and replacement of spermatophores by the second male succeeded more frequently when the copulatory piece was shorter and the vaginal appendix was longer, and when the difference between the length of the copulatory piece and the vaginal appendix was smaller. Thus, a matching of the corresponding genital parts between the sexes increases the male's reproductive success in sperm competition, but elongation of the copulatory piece cannot be explained simply by the improvement in male reproductive success. We discuss possible factors for the elongation of genital parts in terms of sexual conflict and reproductive interference through interspecific copulation.     

Correlated divergence of female and male genitalia in replicated lineages with ongoing ecological speciation

Divergence of genital traits among lineages has the potential to serve as a reproductive isolating barrier when copulation, insemination, and fertilization are inhibited by incompatibilities between female and male genitalia. Despite widespread evidence for genital trait diversity among closely related lineages and coevolution of female and male genitalia within lineages, few studies have investigated genital evolution during the early stages of speciation. We quantified genital variation in replicated population pairs of Poecilia mexicana with ongoing ecological speciation between sulfidic (H₂S containing) and nearby nonsulfidic habitats. These analyses revealed rapid and correlated divergence of female and male genitalia across evolutionarily independent population pairs exposed to divergent selection regimes. Both sexes exhibited convergent evolution of genital traits among populations inhabiting similar habitat types. Our results demonstrate that genital evolution can occur during the early stages of speciation‐with‐gene‐flow, potentially as a result of variation in the intensity of sexual conflict among populations. Our results suggest genitalia may contribute to early stages of divergence and challenge the generality of previously suggested mechanisms of genital evolution in poeciliids.     

Distinctive developmental variability of genital parts in the sexually dimorphic beetle, Prosopocoilus inclinatus (Coleoptera: Lucanidae)

Recent comparative studies have revealed that the rapid diversity of genitalia is closely related to sexual selection and that genital development interacts with the development of different body parts. Hypotheses about developmental stability due to selection to genital parts were tested by estimating allometric relations in a sexually dimorphic stag beetle Prosopocoilus inclinatus . All genital parts of males scaled to body size with a slope of less than 1 and all but the median lobe (male intromittent organ) showed smaller variability than other body parts. This supported the 'one-size-fits-all' hypothesis, which suggests broad copulation opportunity by males of any size with females within a population. Nevertheless, we found large variation among different genital parts in coefficients of variation and in values of the switch point where the allometric relations varied significantly. These results strongly support the view that developmental trajectories of genital traits are not necessarily integrated. Among the genitalic traits, male intromittent organ and female genitalia exhibited large variability, suggesting a high responsiveness to the selective regimes and physical interaction during copulation. This may account for rapid diversification of genital morphology, even in closely-related populations in beetle species.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 573–581.     

Multivariate stabilizing sexual selection and the evolution of male and female genital morphology in the red flour beetle*

Male genitals are highly divergent in animals with internal fertilization. Most studies attempting to explain this diversity have focused on testing the major hypotheses of genital evolution (the lock-and-key, pleiotropy, and sexual selection hypotheses), and quantifying the form of selection targeting male genitals has played an important role in this endeavor. However, we currently know far less about selection targeting female genitals or how male and female genitals interact during mating. Here, we use formal selection analysis to show that genital size and shape is subject to strong multivariate stabilizing sexual selection in both sexes of the red flour beetle, Tribolium castaneum. Moreover, we show significant sexual selection on the covariance between the sexes for specific aspects of genital shape suggesting that male and female genitalia also interact to determine the successful transfer of a spermatophore during mating. Our work therefore highlights the important role that both male and female genital morphologies play in determining mating success and that these effects can occur independently, as well as through their interaction. Moreover, it cautions against the overly simplistic view that the sexual selection targeting genital morphology will always be directional in form and restricted primarily to males.