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Abstract Females mate multiply despite numerous costs. It is well established that polyandry can result in sexual conflict, favoring male adaptations that prevent sperm competition often to the disadvantage of the female. Such adaptations are extreme in spiders with one-shot genitalia of which parts break off and act as mating plugs, rendering them dysfunctional. In the spider Argiope bruennichi, mating plugs effectively prevent further males from inseminating and males that inseminate and plug both genital openings of a female secure exclusive paternity. However, females frequently prevent monopolization by attacking and cannibalizing males during their first copulation, leaving their second spermatheca free for another male. Here, we test whether the high frequency of sexual cannibalism evolved as a female adaptation to resist monopolization and secure indirect benefits of polyandry. To standardize conditions, we double-mated females either with the same or two different males and prevented male consumption. Using a split-brood design, we raised offspring to maturity under poor and rich food conditions and measured their survival, duration of juvenile phase, and adult body mass. Under low food, daughters of polyandrous mothers matured later but slightly heavier than daughters of monandrous females. Since the adaptive value of this combination is unclear, these findings lend no conclusive support to our hypothesis. We discuss the stereotypic nature of the female attack in the context of antagonistic co-evolution considering previous studies that found modest direct benefits of cannibalism as well as a potential for non-additive benefits. Significance statement Sexual conflict is extreme in spiders where sexual cannibalism impairs male mating rates. Males of the spider Argiope bruennichi possess one-shot genitalia which they break off to plug female genital openings. They gain exclusive paternity with a female if two copulations are achieved and both genital openings plugged. Females, however, stereotypically attack every male at the onset of copulation, limiting most males to single copulation but retaining the option to secure potential benefits of polyandry. Previous studies revealed weak direct and non-additive indirect benefits of multiple mating. In this study, we tested for the presence of additive genetic benefits but again found only inconclusive evidence for adaptive differences in offspring quality between monandrous and polyandrous females. All results combined, we here speculate that the stereotypic female attack might be a ghost of a past antagonistic co-evolution.

Rapidly changing environments impose novel selection pressures on organisms, and sometimes adaptive phenotypic plasticity allows organisms to survive and reproduce in the face of environmental change. However, plastic responses can also be maladaptive. In this study, we investigate whether male reproductive investment responds plastically to varied experience with traffic noise. We exposed male crickets chronically to one of three noise treatments from the 2nd-3rd instar until their natural death: masking traffic noise (including noise that overlaps in frequency with the male crickets’ mating calls), non-masking traffic noise (an identical traffic noise track from which we digitally removed the frequencies that mask the crickets’ mating call), and silence. We dissected and weighed their testes and spermatophore molds. Controlling for body mass, we found that the spermatophore molds of crickets reared in masking and non-masking noise were 29% and 24% lighter, respectively, than those of crickets reared in silence. There were no differences in body mass-adjusted testes mass among treatments. If spermatophore mold mass is positively associated with male reproductive output, this reduction in size could have negative fitness consequences for animals exposed to traffic noise. We encourage future work to investigate impacts of noise on reproductive investment in other study systems that are likely sensitive to anthropogenic noise (e.g., birds, frogs, singing insects). Anthropogenic noise is a pervasive pollutant and chronic noise can negatively affect fitness. How does anthropogenic noise influence reproductive investment? Phenotypically plastic responses to noise may increase survival and reproduction in noisy environments. Traffic noise masks the sounds crickets make, potentially changing conspecifics’ perception of population density, mate availability, and the risk of sperm competition. We found that male crickets reared in silence developed significantly larger spermatophore molds (a reproductive structure that delivers the sperm containing packet to the female) than those reared in traffic noise. If the spermatophore mold influences male reproductive output, this reduction in size may have negative impacts on reproductive success of animals exposed to masking traffic noise.

Parents are expected to reduce offspring investment when confronted with reliable cues of compromised parentage, yet establishing which cues are reliable is an empirical challenge. Presenting a potential cuckolder to a breeding male is often used in experiments as an indirect cue of paternity loss. However, determining the reliability and hence the utility of this cue is an important but often-overlooked research step. Furthermore, cues of compromised parentage are typically manipulated only during the narrow time window(s) when copulations take place, and so we currently have a poor understanding of whether these cues also convey useful information at other critical timepoints in the reproductive cycle, such as during nest site selection. Here, we present a series of field and laboratory studies using a paternal care giving toadfish, the plainfin midshipman fish (Porichthys notatus) to address these questions. We tested whether the presence of a potential cuckolder near a potential nesting site reduces the odds that males will choose to nest at that site, or reduces the amount of care they provide for offspring. Overall, we found no clear effect of cuckolder presence on the likelihood that a male would occupy nor abandon a nesting site, nor on the amount of paternal care provided. The presentation of a single sneaker male may have been too weak a signal of cuckoldry to elicit a response from guarder males. Alternatively, a single sneaker male may not represent a severe enough threat to paternity to warrant a response. We highlight the importance of considering the diverse range of natural history and ecological factors that underlie paternity cue utility across different model organisms. Breeding decisions, such as which nesting sites to occupy or how much to invest into offspring, may be affected by external cues of parentage loss (e.g. cuckoldry) or risk thereof. Here, we tested whether the presence of a cuckolder male in the breeding environment affects male nest site selection and paternal care. Despite being a commonly used putative indirect cue of sperm competition and paternity loss, we found no clear evidence that the presence of a single sneaker male affected these breeding decisions. Our results underscore the importance of first establishing the utility of various cues, which involves considering the ecological context from which the cues arise, before using them to assess the mechanisms underlying animal decision-making.

In many insects, males transfer nuptial gifts to females prior to, during, or after copulation. Nuptial gifts comprise any form of non-gametic material transfer, such as glandular secretions, prey items, and spermatophores. These gifts can increase male fitness by facilitating copulation or sperm transfer or by promoting the female’s use of the focal male’s sperm. However, when a female mates with multiple males, the relationships between the relative size, number, or quality of nuptial gifts that each male donates and the proportion of offspring that each male sires are unknown in many cases. Here, we assessed these relationships in the Hawaiian swordtail cricket Laupala cerasina. We experimentally manipulated the number of gifts a female received from two male mating partners in sequence, with the first male giving either more, fewer, or an equal number of gifts relative to the second male. We found that there was a significant positive relationship between nuptial gift number and paternity share. In addition, we found considerable first-male sperm precedence, regardless of gift contribution. Finally, offspring from second males tended to be produced later and to be part of larger families. These results show that both nuptial gift number and mating order play important roles in sperm use patterns and fertilization success in this species. In many insects, males transfer nuptial gifts (a token, often nutritional, gift) to females prior to, during, or after copulation. In the Hawaiian swordtail cricket Laupala cerasina, males transfer a series of nuptial gifts in the form of spermless spermatophores, to females during courtship. Moreover, males experience sperm competition as females of this species are known to mate multiply. By manipulating the number of gifts transferred, we tested the hypothesis that males who transfer more nuptial gifts achieve higher fitness by fathering more offspring. We found a significant positive relationship between gift number and the proportion of offspring a male sires. In addition, controlling for gift number, the first male to mate with a female achieves more paternity. These results show that both nuptial gift number and mating order affect sperm use patterns and fertilization success in this species.

Behavioural diversity is a basic component of biodiversity, with implications in ecological interactions at the intra- and interspecific levels. The reproductive behaviour of Odonata (dragonflies and damselflies) is unique among insects and conditioned by the anatomical separation between the male’s reproductive organs and the intromittent organ. Prior to mating, males must translocate sperm from the genital pore in the ninth abdominal segment to the seminal vesicle located ventrally in the second abdominal segment. This behaviour, exclusive to odonates, is known as intra-male sperm translocation (ST). Here, we review the literature on ST and use phylogenetic comparative analyses to investigate the evolution of ST within the Odonata. Information on ST was compiled for 176 species, with the commonest variant being ST once per mating, after tandem formation (66%). Other variants found were ST involving precopulatory genital touching (10%), ST by the male alone before tandem (16%) or after copulation (5%), and repetition of ST during the same copulation (3%). The precopulatory genital touching might have evolved to detect female receptivity. ST before tandem formation might be favoured when mating opportunities are scarce and copulations are brief. ST after mating might be favoured if males need to be ready to copulate fast. Finally, repeated ST could have evolved through postcopulatory sexual selection in males with limited sperm removal ability, as a means to improve their sperm competition. The most plausible scenario for the evolution of ST is that the ancestors of the Odonata produced a spermatophore and attached it to the body, leading towards the evolution of the secondary genitalia in males. Our study emphasises the role of behavioural diversity to understand behavioural evolution. Unique behaviours are exclusive of a few individuals, populations and/or species. The intra-male sperm translocation (ST) of dragonflies and damselflies is a unique behaviour in animals: before mating, males need to transfer sperm from the primary to the secondary genitalia, which are anatomically separated. Thus, the viability and quality of sperm (i.e. fertility) will depend on the timing of ST relative to copulation. Our literature review found a variety of ST variants, being ST in tandem and before copulation the ancestral strategy. We discuss putative evolutionary routes for all the variants found and emphasise the importance of retrieving detailed observations of such unique behaviours in the field, which could help to better understand behavioural evolution in this insect group. Behavioural diversity is rarely addressed by conservation strategies, despite unique behaviours being at a higher risk of extinction.

Bright colouration appearing in one sex only can be driven by components of sexual selection including female choice, male competition or mate recognition. Male Litoria wilcoxii undergo rapid colour change from brown to yellow during amplexus, however, the function, if any, is unknown. We tested possible behavioural functions by observing breeding aggregations and behavioural responses (colour change, movement, call and amplexus duration) to varying stimuli (including model male and female frogs). We also examined whether colour change was a by-product of hormone release by comparing spermatic urine of frogs injected with epinephrine (colour change hormone) and hCG (triggers spermiation). Finally, the predation cost of being bright yellow was examined by placing frog models (yellow and brown) in the field and measuring predator attack rate. The behavioural responses of males to model females, brown/brown models (female with amplexing brown male), and brown/yellow models (female with amplexing yellow male), were similar to reactions towards real females, with the important exception that males did not attempt amplexus with brown/yellow models. Epinephrine injections triggered colour change but not sperm release in male frogs, while hCG induced sperm release but not colour change. Attack rates were low in predation trials with no difference in attack rates between yellow and brown models observed. Our study presents a novel function for rapid dynamic colour change as an intrasexual signal during amplexus that could avert sperm competition and displacement by other males. Colour displays during breeding are believed to have evolved through mechanisms of sexual selection (female choice, male–male competition or sexual recognition). Stony creek frogs (Litoria wilcoxii) have been observed to rapidly change colour from brown (similar to female colouration) to bright yellow during amplexus, which is unusual as the colour change occurs after mate selection. Behavioural experiments were used to test hypotheses on the evolutionary function of colour change in this species. In L.wilcoxii, colour functions as an intrasexual signal during amplexus, we hypothesise that this could avert sperm competition and/or displacement by other males during amplexus. The function presented here is novel among amphibians, however as data on dynamic colour change in amphibians is lacking, this trait may be more common.

Populations of a single species may vary substantially in sexually selected traits. However, the majority of studies investigating such variation across populations have focused on traits important for pre-copulatory reproductive success, such as male ornamentation or song. Relatively fewer studies have investigated whether traits important in post-copulatory sexual selection vary across populations. In this study, we examine how sperm and ejaculate quantity and quality in the American horseshoe crab, Limulus polyphemus differ at six sites, from Maine to Florida. Horseshoe crabs from these sites differ in many respects, including average body size, age, and the risk and intensity of sperm competition as measured by the operational sex ratio. We find that sperm and ejaculate quantity and quality significantly differ across this range and that this variation persists after taking body size and age into account. Sperm traits also do not follow strictly linear latitudinal clines. Finally, we find that the site-specific operational sex ratio correlates with changes in sperm and ejaculate quantity. This latter result may indicate that the same processes that lead to variation in post-copulatory sexually selected traits across related species may also promote trait divergence across populations of the same species. This study describes variation in sperm and ejaculate traits from horseshoe crabs collected at six sites from Maine to Florida. While geographic variation has been described for many traits, relatively few studies have demonstrated that sperm and ejaculate traits follow similar patterns. The variation we found does not follow simple latitudinal patterns, suggesting that other forces may be driving sperm and ejaculate trait divergence across sites. We also have data that suggest sexual selection may play a role in driving sperm trait variation, but we caution that additional sites need to be sampled.

Density effects can have a strong influence over both the mating system of a species and the reproductive strategies of individuals. The way in which males respond to an increase in the density of other male competitors is generally explained by sperm competition theory. When the perceived risk of sperm competition is high, males increase reproductive effort to aspects of mate searching, copulation and mate-guarding that will ensure reproductive success. In species with little likelihood of female defence, male competition is largely restricted to a scramble for access to females and sperm competition. In such species, the ability to search quickly and locate females will be under strong selection. Millipedes (Arthropoda: Myriapoda: Diplopoda) are classic scramble competitors, although their utility in the study of scramble competition has been generally overlooked. Here we investigate the Australian Polydesmidan millipede Gigantowales chisholmi and describe their mating behaviour. We manipulated male density, exposing individual males to either high or low male density treatments, and compared aspects of mate searching and copulation. We found that males from high-density treatments searched at faster speeds and copulated for shorter durations, than those kept alone. We also found that larger males achieved higher mating success but copulated for shorter durations. Our data support the idea that for scramble competitors, males who are more likely to achieve mating success (in this case large males) will use mating tactics that emphasise increased efforts towards mate acquisition rather than investment in their current mates. In an extraordinary number of animal species, the males who are most successful are those who can efficiently locate females, mate with them and move on in search of additional mates. This is scramble competition and is relatively unstudied compared with other animal mating systems such as those where males aggressively fight for mates. Millipedes, also relatively unstudied, represent great systems for the study of scramble competition. Here we show that males of the Australian millipede, G. chisholmi, search for females at higher speeds and mate more quickly when they are kept at higher density. Larger males are more successful but mate for shorter durations. This suggests that for scramble competitors, those more likely to achieve mating success (in this case large males) use tactics that increase mate acquisition rather than investment in their current mates.

In the marine snail Littorina saxatilis females are highly polyandrous, resulting in simultaneous broods of offspring sired by ∼20 males. Such high polyandry, in combination with female sperm storage, is likely to promote postcopulatory sexual selection among males. In addition, females may gain direct reproductive benefits from multiple mating. Investigating sexual selection in males and female benefits in relation to the number of sires, we put single virgin females in aquaria with one, two, five or ten males for 80 days, during which each female mated a majority of available males. After removal of the males, females continued to produce offspring during >1 year. Offspring genotypes of 27 families showed multiple paternity with 76 % of males contributing to juveniles, but paternity deviated from random with one or a few males siring the majority of the offspring. Larger males tended to be overrepresented among the sires. Female reproductive output (newborn or juvenile stage) did, however, not increase with number of sires, and females seemed not limited by the number of available males. Because previous studies have shown that females are seemingly indiscriminative in their mate choice, and given that they mate many hundreds of times in their lifespan, this suggests there is comparatively limited scope for premating sexual selection in this species. Hence, we interpret these results mainly in the light of postcopulatory sexual selection. Sexual selection is a main component of evolution. In that context, mating behaviour is central, although it sometimes appears puzzling, even maladaptive. For example, matings are costly; still, in some species, females mate multiple times and with numerous males. Why this behaviour has evolved is intriguing. Extensive numbers of matings suggest less efficient precopulatory mate choice, opening for postcopulatory sexual selection through cryptic female choice and sperm competition. Using a highly promiscuous snail species with internal fertilisation and long-term sperm storage, we identified the fathers of 549 offspring. We found that while females mated most males available, the offspring were non-randomly distributed among mated males and large males were overrepresented among the sires. This suggests that sexual selection predominantly occurs after mating and that males benefit from size-related effects. Yet, we could not find any female benefits from mating multiple males.

The study of sexual selection is being revolutionised by the realisation that most populations exhibit some degree of polyandry, i.e. females mating with multiple males. Polyandry can drastically change the operation of sexual selection on males as it reduces the reproductive success that males derive by mating with different females, by forcing their ejaculates to compete for fertilisation after copulation (sperm competition). Variation in polyandry within a population means that the impact of polyandry can differ drastically across males, depending on the polyandry of their own mating partners. Because the patterns through which males share mates within a population may have strong repercussions for variation in male reproductive success, measuring such patterns is critical to study the operation of sexual selection. Several methods have been proposed to measure the pattern of mate sharing at the population level. Here, we develop a new method (sperm competition intensity correlation, SCIC) and compare its performance against two established methods (Newman’s assortativity and nestedness), using both idealised model populations and random simulated populations, across a range of biologically relevant population parameters: (i) population size, (ii) sex ratio and (iii) the ‘mating density’ of the population. We conclude that SCIC may be the most promising approach, as it is both internally consistent and robust across the parameter range. We discuss some important caveats and provide advice regarding the choice of method for future studies of sexual selection. Electronic supplementary material The online version of this article (doi:10.1007/s00265-015-2041-7) contains supplementary material, which is available to authorized users.

For polyandrous species where females have sperm storage structures, males develop several strategies to avoid sperm competition and thus to maximize the number of eggs fertilized. On the other hand, females may receive several benefits from multiple paternity (indirect and directly), and a potential sexual conflict can arise. This research describes the mating systems of an exploited crab species (Metacarcinus edwardsii), integrating (1) the individual level by assessing the mating behavior in a scenario of potential polyandry, (2) the organ level by examining histological sections of seminal receptacles from localities with scenarios of contrasting sex ratios, and (3) the genetic level by measuring the number of parents involved in egg clutches. We found that females can mate with multiple males under experimental conditions. Further, in all localities, we found histological evidences that sperm receptacles stored ejaculates from more than one male. However, contrary to expectations, genetic analysis revealed high probability of single male paternity of all progeny in each egg clutch. In this mating system, males compete to be the single male that mates with a receptive female, investing energy in guarding behavior and foregoing opportunities to mate with other females, all in order to ensure their paternity. However, females benefit from multiple mating (or potential for it) by prolonged guarding behavior, protecting them from predation after molt (soft-shelled period). The mating system of M. edwardsii can be defined as polygamous (where both sexes can mate multiple times) with genetic monogamy.

In systems with male alternative reproductive tac- tics,discretebehavioralpatternsoftenleadtounequallevelsof sperm competition across tactics. In many of these systems, the disadvantaged tactic shows adaptations to sperm traits that increase the competitive ability of its sperm. However, many studieshavefoundthatnotall spermtraitsdifferacrosstactics, and in some systems no sperm trait adaptations are found. Why some systems and some sperm traits display tactic- specific differences and others do not is not always clear. Here, we investigate three sperm traits—ejaculate size, sperm concentration, and sperm velocity—in the American horse- shoe crab, Limulus polyphemus, a system where males have condition-dependent reproductive tactics and females influ- ence the amount of sperm competition males face during spawning. Contrary to our predictions that the disadvantaged tactic would show sperm trait adaptations, we found that horseshoe crabs of the tactic that faces the least amount of sperm competition had more concentrated sperm than the males of the other tactics.We propose a number of hypotheses to explain this result and suggest that future studies of alter- native reproductive tactics might benefit from considering the role of females in influencing tactic-specific sperm trait adaptations.

Animals of diverse taxa show different conditional mating strategies: they adjust their behaviour according to social and environmental situations, which may bring diverse (fitness) advantages for an individual. Especially in primates, mating is often associated with other social behaviours, such as grooming. Here, we study Barbary macaque post-copulatory grooming: we investigate for the first time whether males and females modify their grooming initiations after mating depending to the type of copulation (i.e. with or without ejaculation) and female lactation state. Our results show that males and females adjust grooming initiations conditional on copulation type, with males initiating grooming after copulations with ejaculation and females after non-ejaculatory mating. Moreover, lactating females tend to start grooming the males they just mated with more than vice versa, whereas there is no such a difference for grooming initiations after copulations with non-lactating females. These data indicate that Barbary macaques show post-mating grooming strategies that vary depending on the type of copulation that occurred and in dependence of female reproductive state. These grooming initiation patterns may reflect sex-specific mating interests and potentially serve to increase fitness: females, in particular lactating females, may benefit from initiating grooming to secure protection for themselves and their offspring and reduce harassment. Males may profit from grooming females after ejaculatory copulation by keeping them from mating with another male and thus potentially decreasing sperm competition.

It is now generally recognized that it is necessary to examine how sexual selection operates across the lifespan of a male, in order to understand the total sexual selection in action. However, less attention has been paid to the fact that selection pressures can change in response to varying environmental conditions. Here, we examine male allocation to trait subject to pre- and post-copulatory sexual selection in Callosobruchus chinensis. We find evidence of a trade-off between dispersal ability and testes size and sperm transfer, across geographical strains. In addition, male mating success of strains with larger testes and better dispersal ability, respectively, changed in response to environmental conditions. Males with better dispersal ability had greater success in securing mates, compared to males with larger testes, under conditions where it was harder to find females. There was no difference in male ability to secure mates when it was easy for them to find females. However, we suggest that males with larger testes are likely to have an advantage in sperm competition under conditions where it is easier to find females, and when females are thus likely to mate multiply. Our results indicate that environmental fluctuations and trade-offs can work in conjunction to maintain alternative male behavioral reproductive strategies.

Polygynous males must balance their limited ejaculate resources between current copulations, with putative future ones. Working on olive flies (Bactrocera oleae), our objectives were to determine (a) how males budget sperm to consecutive copulations, (b) what costs consecutive copulations incur, and (c) how male mating history affects female fecundity, fertility, and longevity. We allowed males to copulate with virgin females on successive days and monitored the fertility and fecundity of these females, and the longevity of both sexes. We found that as males gained sexual experience, latency to mate declined significantly, while copula duration increased. The number of sperm stored by females declined according to the sexual history of her mate—as males gained experience, significantly fewer sperm cells were transferred. Mated males suffered a significant longevity cost compared to virgin ones, but this cost was not compounded by additional matings. Male sexual experience affected both female fecundity and fertility. Furthermore, mating with an experienced male incurred a longevity cost to females. We conclude that non-sperm components of the male ejaculate are responsible for reducing female fecundity, fertility, and longevity, and predict that females should prefer to mate with virgin males.

Examining how the response to sperm competition risk varies in a population is essential in order to understand variation in reproductive success and mating system. In polyandrous butterflies, male ...

Emasculation—males becoming effectively sterile by self-removing their genitals—has long been considered a peculiar evolutionary phenomenon with unknown function, taxonomically restricted to few spiders and flies. In spiders, emasculation results in half or full eunuchs when males sever one or both sperm transferring organs, palps. Three types of emasculation, pre-maturation, mating, and post-mating are known in spiders, all having evolved multiple times. Males practicing pre-maturation emasculation sever one of their palps while still immature, then engage in strict monogyny via genital plugging and spontaneous death. Emasculation during mating also results in genital plugs, but half eunuchs have another chance to mate. So far, the behavior of those males that become eunuchs post-mating by self-removing disfigured palps has not been investigated empirically. We test the mechanism and adaptive significance of post-mating emasculation in coin spiders (Herennia multipuncta) and use phylogenetic reconstruction to understand its evolutionary history. Our laboratory assays corroborate three hypotheses related to mate monopolization: (1) The plugging hypothesis—predicting genital plugs to prevent female remating; (2) The better-fighter hypothesis—predicting enhanced eunuch aggressiveness toward rivals; and (3) The gloves-off hypothesis—predicting increased eunuch endurance. The support for these hypotheses in spiders practicing emasculation during and after mating reinforces recent phylogenetic interpretations of these two emasculation types being evolutionarily linked in the family Nephilidae. We weigh the evidence in support of three different, but equally parsimonious scenarios of nephilid emasculation evolution. We conclude that emasculation is an adaptive, sexually selected trait that calls for further comparative and experimental research.

An obvious means to secure paternity is the production of a mating plug that blocks the female genital opening after mating. Although the mechanical efficacy and persistence of plugs on/in the female genital openings are key traits that determine the degree of paternity protection, these factors have hardly been explored. We therefore investigated the influence of the size of the amorphous plug material (experimentally terminated mating duration as a proxy) and age of the mating plug (time interval between copulations with two successive males) on the efficacy of the plug by analysing the mating success of subsequent males in the dwarf spider Oedothorax retusus (Linyphiidae: Erigoninae). Overall, subsequent males attempted to mate in 82 % of trials but only 32.5 % of these resulted in copulation, demonstrating that the plugs are effective safeguards against remating. Remating probability was significantly higher after previous short copulations (~small plug size) compared to long copulations (~large plug size). In the small plug group, fresh plugs (short remating intervals) were significantly less effective compared to older plugs. In the large plug group, remating probability was similarly low over all remating intervals. The observed copulations, however, do not necessarily result in sperm transfer, since sperm masses were found on the plugged female genital area. Our study on O. retusus shows that mating plugs are a powerful mechanical safeguard whose efficacy varies with plug size and age. We discuss these findings in the light of theoretical considerations on the evolution of effective mating plugs.

In some species, sperm is stored within the female reproductive tract for months to years, and yet remains viable to fertilize eggs and produce offspring. Female red-sided garter snakes store sperm for over 7 months of winter dormancy. In previous work, we demonstrated that these stored sperm account for an average of 25 % paternity of a litter when the female mates with a male at spring emergence. Here, we tested whether last-male sperm precedence was prevalent when a female mates with two males during the spring. On average, paternity was shared equally among the first (P1 proportion of paternity of the first male to mate) and second males (P2) to mate in the spring, and stored sperm (Pss), but the variance in paternity was high. Thus, last male sperm precedence may diminish when a female has more than two mates. Male size did not affect paternity, but, as the interval between matings increased, P1 increased at the expense of Pss. Interestingly, as the second spring male’s copulation duration increased, P1 also increased at the expense of P2. This result suggests that female influence over sperm and/or copulatory plug transfer during matings may also affect which male fathers her offspring in response to coercive matings as we assisted females to mate for their second mating. Finally, all females were spring “virgins”; consequently, sperm stored from autumn matings (and/or previous spring matings) remain competitive even when faced with two rivals in sperm competition and is likely the driver of the evolution of sperm longevity.

Sexual selection may drive speciation, but most research focuses on pre-copulatory sexual selection, overlooking post-copulatory processes. Post-copulatory sexual selection in allopatric populations could drive divergence in post-copulatory pre-zygotic (PCPZ) phenotypes, limiting gene flow upon secondary contact. Here, we performed in vitro experiments examining one potential PCPZ barrier between two closely related passerine species, house sparrows (Passer domesticus) and Spanish sparrows (Passer hispaniolensis). In birds, crossing in the vagina may be particularly challenging for sperm, so we tested the effect of female reproductive tract fluids on sperm swimming speed and motility. If a PCPZ barrier exists at this stage of the fertilization process, heterospecific female fluids are predicted to reduce sperm swimming speed or motility relative to conspecific female fluid. We found that house sparrow female fluids affected the two species’ sperm asymmetrically, depending on the control sperm velocity and male species. Overall, however, sperm performed equally in conspecific and heterospecific female fluids, and the species had similar sperm morphology and sperm swimming performance. Low divergence in PCPZ phenotypes between species, perhaps because post-copulatory sexual selection is stabilizing or only moderately strong in these taxa, may be insufficient to cause an overall PCPZ barrier. Reinforcement may be unlikely to drive PCPZ barriers for this species pair, because relatively effective pre-copulatory barriers exist between the species, and because hybrids can be quite successful. Testing the role of PCPZ barriers in birds with more divergent PCPZ phenotypes will improve our understanding of speciation in passerines.

Some theoretical models of sperm competition make the assumption that in fish species with external fertilisation, sperm length relates positively to swimming speed at the expense of sperm longevity. Few studies have tested this assumption. We used the three-spined stickleback, Gasterosteus aculeatus L., to study functional sperm morphology. In this study, the relationship between males’ mean sperm length and fertilisation rate was investigated in vitro in a non-competitive situation. Fertilisation at different time points after sperm release was taken into account, and sperm morphology was quantified from scanning electron microscopy images. The time series of artificial fertilisations demonstrated that males which produced sperm with a longer tail fertilised faster, but their sperm had a shorter lifespan (or activity period). It was further suggested that males that produced sperm with a larger midpiece had greater fertilisation chances later on in the fertilisation process. Thus, in sticklebacks, there exists functional variation in sperm morphology, and sperm tail length is traded off against sperm longevity (or activity).

Males are limited in sperm and seminal fluid, which can lead to prudent sperm allocation across consecutive matings. While sperm depletion is usually considered a population characteristic, we investigated an individual size-related variation in sperm depletion in the promiscuous sea slug Chelidonura sandrana. We found that sperm counts substantially declined across four successive copulations, indicating sperm depletion. Sperm depletion occurred at a disproportionally fast rate in larger sperm donors. Based on our finding of body size-dependent sperm depletion, we further investigated whether this translates into prudent sperm allocation across ejaculates. We found no evidence that slugs adjusted the number of delivered sperm in response to partner body weight, a tight proxy of fecundity. Instead, sperm counts increased with sperm donor body weight. Sperm donation independent of partner body size and—as found in previous work—partner novelty leaves scope for currently unidentified traits like the sperm competition environment to affect male sperm allocation. Alternatively, we propose that, in C. sandrana, a low optimal male mating rate close to the female optimum as indicated by our data may ultimately render indiscriminate sperm allocation the most beneficial male mating strategy.

Multiple mating is a common reproductive strategy among mammals, and rodents living in communal, mixed sex social groups are predisposed to a polygamous existence. The sandy inland mouse is a naturally polyandrous species that occurs across most of Australia’s arid region. Females typically have greater reproductive restrictions compared with males and are therefore expected to acquire substantial fitness benefits from copulating with more than one male. Here, I show that the reproductive output of female sandy inland mice did not differ between females mated monandrously (single male) or polyandrously (two males). Paternity data obtained from the polyandrous litters revealed that in most cases, there was a first male-to-mate advantage. I discuss this result in relation to the chastity enforcement hypothesis for the evolution of the copulatory plug. Finally, I compared ejaculate traits of competing males and found that the paternity loss of males that mated first was attributable to their own sperm density and sperm quality, and not to that of their rivals. The sperm data also revealed that second males gained greater paternity representation when sperm velocities and motilities were higher in first-mated males. This investigation indicates that mating position is a critical determinant of male fitness in mammalian sperm competition.

Reproductive delays between mating and birth may provide a previously unconsidered avenue for post-copulatory sexual selection in mammals. In particular, delayed fertilization could provide an enhanced opportunity for sperm competition by extending the time for ejaculates to interact in the female reproductive tract. We tested the prediction that species with delayed fertilization exhibit greater degrees of sperm competition than those without delays by examining testis volume (a proxy for sperm competition) in 38 species of bats. Examination of fluid-preserved museum specimens of bat species with and without delays revealed that species with delays (in particular those with delayed fertilization) had significantly larger testes than species without them. Although it predicts the presence of delayed fertilization, hibernation did not predict relative testis size. We conclude that, once they evolve, reproductive delays may facilitate sperm competition.

Postcopulatory sexual selection (PCSS) in internally fertilizing vertebrates is a topic of great interest, yet relatively little is known about the characteristics of sperm and ejaculates that confer an advantage in PCSS. In this study, we investigated several measures of sperm morphology that potentially contribute to fertilization success under PCSS. We tested whether sperm morphology related to success in PCSS (via extra-pair paternity) in house wrens (Troglodytes aedon). We found no evidence that sperm morphology differed between extra-pair sires and the within-pair males they cuckolded, nor that sperm morphology correlated with the proportion of within-pair offspring sired, the number of extra-pair offspring sired, or the total annual reproductive success. Male behavioral strategies may affect the probability that their sperm compete with other males’ sperm and that their sperm succeed under competition. Effects of these behavioral strategies, as well as differences between males in sperm number, could mask the effects of sperm morphology on the outcome of PCSS. Despite moderate levels of extra-pair paternity, selection on sperm may be relatively weak in house wrens. Further work is needed to understand general patterns in how sperm morphology relates to fertilization success within species.

Spermatozoa are among the most variable animal cell types, and much research is currently directed towards explaining inter- and intraspecific variation in sperm form and function. Recent comparative studies in passerine birds have found associations between the level of sperm competition and both sperm length and sperm velocity. In species with sperm competition, postcopulatory sexual selection may shape the morphology of sperm as adaptations to the female environment. The speed of evolutionary change in sperm morphology at the species level is largely unknown. In this study, we analysed variation in sperm morphology among morphologically distinct and geographically isolated bluethroat subspecies in Europe. Consistent with previous studies, our analyses of mtDNA and nuclear introns suggest recent divergence and lack of lineage sorting among the subspecies. We found significant divergence in total sperm length and in the length of some sperm components (i.e. head and midpiece). There was a significantly positive relationship between pairwise divergences in sperm morphology and mitochondrial DNA, suggesting a role for genetic drift in sperm divergence. The magnitude of sperm length divergence was considerably higher than that in other geographically structured passerines, and even higher than that observed between several pairs of sister species. We hypothesize that the rapid sperm evolution in bluethroats is driven by sperm competition, and that strong postcopulatory sexual selection on sperm traits can lead to rapid speciation through reproductive incompatibilities.

Investment into reproduction is influenced by multiple factors and varies substantially between males and females. Theory predicts that males should adjust their ejaculate size or quality in response to variation in female experience or phenotypic quality. In addition, sperm investment by males may also be influenced by their own status and experience. Although such adjustments of male ejaculate size can impact reproductive success (via fertilization success), fitness returns from male sperm investment may be influenced (either limited or facilitated) by the level of maternal investment. To test this hypothesis, we conducted an experiment that simultaneously evaluated the effect of paternal and maternal experience (which incorporates mating status, age, body size, and other related variables) on paternal sperm investment and maternal reproductive allocation in the lizard Anolis sagrei. During staged mating trials, experienced males were more likely to copulate with females, but these individuals were less likely to transfer sperm during mating than were naive individuals. Maternal experience had no impact on these mating behaviors. In contrast to expectations, experience and phenotypic quality (of both sexes) had no impact on male ejaculate size or quality (proportion of live sperm) or on maternal reproductive investment (in terms of egg size and yolk steroids). These findings were intriguing given the mating system and past evidence for differential maternal investment in relation to sire quality in A. sagrei. The results found in this study highlight the complexity of reproductive investment patterns, and we urge caution when applying general conclusions across populations or taxa.

To father offspring, a male must succeed at two processes of sexual selection: (1) mate with a female and (2) fertilize her eggs. We investigated the relationships between pre- and post-copulatory male traits and female mating responses in wild-captured and laboratory-reared spring field crickets, Gryllus veletis. The phenotype-linked fertility hypothesis suggests that females may receive a direct benefit, enhanced fertilization efficiency, by mating with males that signal attractively. We measured fine-scale components of male acoustic mate attraction signals as well as how much time males spent signalling, measured female preference for males in mating trials and then quantified sperm number and viability. We found no relationship between male signalling traits and male fertility or female preference, providing no evidence for the phenotype-linked fertility hypothesis. We also found no difference in sperm metrics between wild-captured and laboratory-reared males. While female crickets may receive benefits by choosing males based on acoustic signal characteristics, whether the benefits are a result of genetic quality, seminal fluid contents or some other male trait remains unknown.

Mating systems evolve with sexual size dimorphism (SSD) in many animals. Mating systems with males larger than females occur when males compete for female access or guard territories, while mating systems with group mating tend to occur in species where females are the same size or larger than males. In addition to variation in SSD with mating system, sperm competition varies among mating systems in predictable patterns. We examined the evolution of mating systems with SSD and testes mass in 111 North American Cyprinidae fishes using phylogenetic comparative methods. Our results demonstrate that the evolution of mating systems in Cyprinidae fishes is from ancestral taxa that are group spawners with females the same size or larger than males to pair spawning systems where males tend to be larger than females. We used an additive model to predict male and female body size from testes mass and mating system. Only mating system varied predictably with SSD. Our results for analyses of hyperallometry (Rensch's rule) were that individual species of Cyprinidae can have hyperallometry for SSD, but the pattern is not present across all taxa.

Selection should favor greater parental effort when the caring individual is more likely to be related to the offspring in its care. If certainty of paternity varies across broods, plasticity in the extent of paternal care may be advantageous, provided that cues of a male’s paternity are available. Consequently, to understand relationships between parentage and parental care, it is necessary to identify the proximate factors governing the decision of males to provide care. Here, we examined parental decision rules in burying beetles, Nicrophorus vespilloides, carrion breeders exhibiting elaborate biparental care. In this species, aggressive interactions that ensue upon the discovery of a carcass by more than one male usually result in a single dominant male that monopolizes access to the carcass and the resident female, relegating subordinates to sneak matings. In the current study, we removed the dominant male after the larvae had hatched and found that about half of the subordinates opted to care for the brood. The decision to provide care was affected by carcass size and the presence of the resident female during the post-hatching period. In a second experiment, we manipulated a subordinate’s access to the resident female and/or the carcass during the pre-hatching period. Surprisingly, we found that mating opportunities had no effect on the subordinate’s decision to provide care. Instead, it was the opportunity to access the carcass that triggered male parental behavior. Our results highlight the need for careful experimental manipulations to identify the cue(s) used by males to assess their certainty of paternity. Theory predicts that low confidence of paternity should favor a reduction in male parental care. However, empirical studies provide only mixed support for this tenet. A deeper understanding of the relationship between paternity assurance and parental care requires an understanding of the proximate mechanisms governing a male’s decision to care. Burying beetles reproduce on dead vertebrates and are known for their elaborate biparental care. We discovered, unexpectedly, that a male’s decision to care did not depend on his opportunities to mate with the resident female, but rather, on his opportunities to access the carcass. Males given access to the breeding resource cared for young even though they had been denied access to the resident female. Thus, deciphering the cues triggering parental behavior may reveal a “rule of thumb” that explains why paternal care can become unlinked from a male’s paternity.

Males advertise their intrinsic parental and/or genetic qualities by the size of secondary sexual ornaments. Moreover, they compete with one another for the best territory and males who arrive first at the breeding ground usually have an advantage in this competition. Females may consider multiple male qualities simultaneously and prefer the one most important for their fitness in the current context. They can further improve their fitness by selecting the best care-giver as their social mate and engaging in an extra-pair copulation with a genetically superior male. In such cases, sperm competition arises in the female reproductive tract and its outcome may be affected by the sperm morphology of both the social and extra-pair male. Here, we tested these ideas in the collared flycatcher (Ficedula albicollis), a species with context-dependent choice of social partners and frequent extra-pair paternity. We recorded male arrival to breeding sites, manipulated their forehead patches, and measured sperm size. In contrast to a previous study in a Swedish population, males with enlarged patches were non-significantly less successful late in the season while no such difference was found early in the season. Besides this tendential seasonal interaction, arrival date did not affect mating and paternity success or male fitness, and the same was true for sperm size. These results suggest different benefits of male ornamentation and female mate choice between populations and call for more replicated research within and between species. The fitness of a male of a migratory species might be affected by several pathways. First, early arrival should confer benefits in the form of best territory choice. Second, in a dichromatic and sexually promiscuous species, secondary sexual ornaments are considered by females both in the choice of social and extra-pair mates. Third, by modifying sperm traits, males may outmatch their rivals in sperm competition. These ideas have usually been tested in isolation. In this experimental study, we tested the joint effect of all of these factors on the genetic fitness of males. We found little evidence for the dependence of male reproductive success on either sperm morphology or plumage ornamentation which is in contrast to other populations of the species. Our study calls for replicated research both in well-established fields like mate choice and emerging ones like sperm competition.

The risk of disease transmission can affect female mating rate, and thus sexual conflict. Furthermore, the interests of a sexually transmitted organism may align or diverge with those of either sex, potentially making the disease agent a third participant in the sexual arms race. In Drosophila melanogaster, where sexual conflict over female mating rate is well established, we investigated how a common, non-lethal virus (sigma virus) might affect this conflict. We gave uninfected females the opportunity to copulate twice in no-choice trials: either with two uninfected males, or with one male infected with sigma virus followed by an uninfected male. We assessed whether females respond behaviorally to male infection, determined whether male infection affects either female or male reproductive success, and measured offspring infection rates. Male infection status did not influence time to copulation, or time to re-mating. However, male infection did affect male reproductive success: first males sired a significantly greater proportion of offspring, as well as more total offspring, when they were infected with sigma virus. Thus viral infection may provide males an advantage in sperm competition, or, possibly, females may preferentially use infected sperm. We found no clear costs of infection in terms of offspring survival. Viral reproductive success (the number of infected offspring) was strongly correlated with male reproductive success. Further studies are needed to demonstrate whether virus-induced changes in reproductive success affect male and female lifetime fitness, and whether virus-induced changes are under male, female, or viral control.