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

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

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Benefits of polyandry: Molecular evidence from field‐caught dung beetles

When females mate with multiple males, they set the stage for postcopulatory sexual selection via sperm competition and/or cryptic female choice. Surprisingly little is known about the rates of multiple mating by females in the wild, despite the importance of this information in understanding the potential for postcopulatory sexual selection to drive the evolution of reproductive behaviour, morphology and physiology. Dung beetles in the genus Onthophagus have become a laboratory model for studying pre‐ and postcopulatory sexual selection, yet we still lack information about the reproductive behaviour of female dung beetles in natural populations. Here, we develop microsatellite markers for Onthophagus taurus and use them to genotype the offspring of wild‐caught females and to estimate natural rates of multiple mating and patterns of sperm utilization. We found that O. taurus females are highly polyandrous: 88% of females produced clutches sired by at least two males, and 5% produced clutches with as many as five sires. Several females (23%) produced clutches with significant paternity skew, indicating the potential for strong postcopulatory sexual selection in natural populations. There were also strong positive correlations between the number of offspring produced and both number of fathers and paternity skew, which suggests that females benefit from mating polyandrously by inciting postcopulatory mechanisms that bias paternity towards males that can sire more viable offspring. This study evaluates the fitness consequences of polyandry for an insect in the wild and provides strong evidence that female dung beetles benefit from multiple mating under natural conditions.     

Male insemination decisions and sperm quality influence paternity in the golden orb-weaving spider

In polyandrous species, paternity may be influenced by the timingand frequency of mating. Female spiders possess 2 genital openingsthat lead to separate sperm-storage structures. Thus, even whenmating with a previously mated female, a male may reduce directsperm competition by inseminating the opposite opening to herfirst mate. Such morphology may provide females with greatercontrol over paternity. We examined simultaneously whether malesavoided already inseminated female genital openings and whetherthis behavior varied with the time between successive matings.To explore these questions, we mated female golden orb weaverspiders, Nephila edulis, each to 2 males and manipulated thetiming of their second mating. We documented male inseminationpatterns and explored the influence of male mating decisionson paternity success using the irradiated male technique. Wefound that 60% of males avoided sperm competition by discriminatingagainst inseminated genital openings. Moreover, male matingbehavior had a dramatic impact on the paternity success of irradiatedmales. When males inseminated the same genital opening, thecompetitive ability of the irradiated male's sperm was dramaticallyreduced resulting in lower paternity success. In contrast, whenthe 2 males inseminated opposite genital openings both malessired equal proportions of offspring regardless of their radiationstatus. There was no evidence that the timing of the secondmating affected patterns of paternity. Our data suggest thatdifferences in sperm quality may influence paternity successof N. edulis males under a sperm-competitive scenario. In contrast,females appear to have limited postmating control over paternity.     

Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Seasonal monogamy and multiple paternity in a wild population of a territorial reptile (tuatara)

Investigating the mating system of a population provides insight into the evolution of reproductive patterns, and can inform conservation management of threatened or endangered species. Combining behavioural and genetic data is necessary to fully understand the mating system and factors affecting male reproductive success, yet behavioural data are often difficult to collect for threatened species. In the present study, we use behavioural data and paternity analyses to characterize the mating system of a high density population of a long-lived, ancient reptile (tuatara, Sphenodon punctatus ). We further investigate the phenotypic traits (including body size, body condition, tail length, and ectoparasite load) that affect male reproductive success. Our behavioural data reflect a seasonally monogamous system with low levels of polyandry and polygyny that are consistent with male mate guarding. Male reproduction is highly skewed (only 25–30% of males are successful), and body size is the primary predictor of male reproductive success. Based on the genetic data, multiple paternity was found in only 8% of clutches, and the results of the paternity analyses showed monandrous clutches from socially polyandrous females. Our behavioural and genetic results revealed complexities in female mating patterns that support the potential for cryptic female choice or sperm competition. This warrants further experimental investigation into the mechanisms underlying reptile fertilization and the disparities between social and genetic polyandry in wild populations.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 161–170.     

Polyandry enhances offspring viability with survival costs to mothers only when mating exclusively with virgin males in Drosophila melanogaster

A prominent hypothesis for polyandry says that male–male competitive drivers induce males to coerce already‐mated females to copulate, suggesting that females are more likely to be harassed in the presence of multiple males. This early sociobiological idea of male competitive drive seemed to explain why sperm‐storing females mate multiply. Here, we describe an experiment eliminating all opportunities for male–male behavioral competition, while varying females’ opportunities to mate or not with the same male many times, or with many other males only one time each. We limited each female subject's exposure to no more than one male per day over her entire lifespan starting at the age at which copulations usually commence. We tested a priori predictions about relative lifespan and daily components of RS of female Drosophila melanogaster in experimental social situations producing lifelong virgins, once‐mated females, lifelong monogamous, and lifelong polyandrous females, using a matched‐treatments design. Results included that (1) a single copulation enhanced female survival compared to survival of lifelong virgins, (2) multiple copulations enhanced the number of offspring for both monogamous and polyandrous females, (3) compared to females in lifelong monogamy, polyandrous females paired daily with a novel, age‐matched experienced male produced offspring of enhanced viability, and (4) female survival was unchallenged when monogamous and polyandrous females could re‐mate with age‐ and experienced‐matched males. (5) Polyandrous females daily paired with novel virgin males had significantly reduced lifespans compared to polyandrous females with novel, age‐matched, and experienced males. (6) Polyandrous mating enhanced offspring viability and thereby weakened support for the random mating hypothesis for female multiple mating. Analyzes of nonequivalence of variances revealed opportunities for within‐sex selection among females. Results support the idea that females able to avoid constraints on their behavior from simultaneous exposure to multiple males can affect both RS and survival of females and offspring.     

Why do females mate multiply? A review of the genetic benefits

The aim of this review is to consider the potential benefits that females may gain from mating more than once in a single reproductive cycle. The relationship between non-genetic and genetic benefits is briefly explored. We suggest that multiple mating for purely non-genetic benefits is unlikely as it invariably leads to the possibility of genetic benefits as well. We begin by briefly reviewing the main models for genetic benefits to mate choice, and the supporting evidence that choice can increase offspring performance and the sexual attractiveness of sons. We then explain how multiple mating can elevate offspring fitness by increasing the number of potential sires that compete, when this occurs in conjunction with mechanisms of paternity biasing that function in copula or post-copulation. We begin by identifying cases where females use pre-copulatory cues to identify mates prior to remating. In the simplest case, females remate because they identify a superior mate and 'trade up' genetically. The main evidence for this process comes from extra-pair copulation in birds. Second, we note other cases where pre-copulatory cues may be less reliable and females mate with several males to promote post-copulatory mechanisms that bias paternity. Although a distinction is drawn between sperm competition and cryptic female choice, we point out that the genetic benefits to polyandry in terms of producing more viable or sexually attractive offspring do not depend on the exact mechanism that leads to biased paternity. Post-copulatory mechanisms of paternity biasing may: (1) reduce genetic incompatibility between male and female genetic contributions to offspring; (2) increase offspring viability if there is a positive correlation between traits favoured post-copulation and those that improve performance under natural selection; (3) increase the ability of sons to gain paternity when they mate with polyandrous females. A third possibility is that genetic diversity among offspring is directly favoured. This can be due to bet-hedging (due to mate assessment errors or temporal fluctuations in the environment), beneficial interactions between less related siblings or the opportunity to preferentially fertilise eggs with sperm of a specific genotype drawn from a range of stored sperm depending on prevailing environmental conditions. We use case studies from the social insects to provide some concrete examples of the role of genetic diversity among progeny in elevating fitness. We conclude that post-copulatory mechanisms provide a more reliable way of selecting a genetically compatible mate than pre-copulatory mate choice. Some of the best evidence for cryptic female choice by sperm selection is due to selection of more compatible sperm. Two future areas of research seem likely to be profitable. First, more experimental evidence is needed demonstrating that multiple mating increases offspring fitness via genetic gains. Second, the role of multiple mating in promoting assortative fertilization and increasing reproductive isolation between populations may help us to understand sympatric speciation.     

Male courtship attractiveness and paternity success in Photinus greeni fireflies

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

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

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

High frequency of polyandry in a lek mating system

The adaptive significance of polyandry by female birds in theabsence of direct benefits remains unclear. We determined thefrequencies of polyandrous mating and multiple paternity inthe ruff, a lekking shorebird with a genetic dimorphism inmale mating behavior. More than half of female ruffs mate with, and have clutches fertilized by, more than one male. Individualfemales mate with males of both behavioral morphs more oftenthan expected. Polyandrous mating was more likely followingcopulation interference, but interference was uncommon. Themultiple paternity rate of ruffs is the highest known for avian lekking species and for shorebirds. The general hypothesis thatpair-bond constraints are the major selective factor favoringmultiple mating in birds does not predict our findings. Activegenetic diversification, which has been widely dismissed asa functional explanation for polyandrous mating in birds, mayapply with respect to the behavioral polymorphism in ruffs becauseof a Mendelian genetic basis for male behavioral morph determinationand aspects of male—male cooperation and female choice.However, rates of multiple paternity in other species of lekkingbirds are higher than generally realized, and the potentialbenefits of diversification in general deserve further consideration.     

The effect of an inversion system and the time interval between matings on postcopulatory sexual selection in the seaweed fly, Coelopa frigida

The seaweed fly, Coelopa frigida, exhibits LMSP. A large chromosomal inversion system affects many traits including egg-to-adult viability via heterosis. Consequently, there is also considerable potential for cryptic female mate choice to operate on the basis of sperm karyotype. Here, we investigated the effect of time interval and chromosomal inversion karyotype on postcopulatory sexual selection. Homokaryotypic females were mated with a male of the same and a male of the opposite homokaryotype. The order of the matings was varied so cryptic female mate choice could operate either in concert or antagonistically with LMSP. LMSP was found when there was a 24 h time interval between matings, irrespective of the order in which the males were mated. However, when the males were mated in quick succession the order of mating was important. When LMSP and cryptic female mate choice work in concert a high level of LMSP was found. However, when the male of opposite homokaryotype mated first, then first male sperm precedence was observed. This suggests that polyandrous females might be able to bias paternity but only when matings occur in quick succession. Consequently, population density is likely to affect the operation of postcopulatory sexual selection.     

Sexual selection and assortative mating: an experimental test

Mate choice and mate competition can both influence the evolution of sexual isolation between populations. Assortative mating may arise if traits and preferences diverge in step, and, alternatively, mate competition may counteract mating preferences and decrease assortative mating. Here, we examine potential assortative mating between populations of Drosophila pseudoobscura that have experimentally evolved under either increased (‘polyandry’) or decreased (‘monogamy’) sexual selection intensity for 100 generations. These populations have evolved differences in numerous traits, including a male signal and female preference traits. We use a two males: one female design, allowing both mate choice and competition to influence mating outcomes, to test for assortative mating between our populations. Mating latency shows subtle effects of male and female interactions, with females from the monogamous populations appearing reluctant to mate with males from the polyandrous populations. However, males from the polyandrous populations have a significantly higher probability of mating regardless of the female's population. Our results suggest that if populations differ in the intensity of sexual selection, effects on mate competition may overcome mate choice.     

Polyandrous females produce sons that are successful at post‐copulatory competition

Some of the genetic benefit hypotheses put forward to explain multiple male mating (polyandry) predict that sons of polyandrous females will have an increased competitive ability under precopulatory or post‐copulatory competition via paternally inherited traits, such as attractiveness or fertilization efficiency. Here, we tested these predictions by comparing the competitive ability of sons of experimentally monandrous and polyandrous female bank voles (Myodes glareolus), while controlling for potential material and maternal effects. In female choice experiments, we found no clear preference for sons of either monandrous or polyandrous mothers. Moreover, neither male type was dominant over the other, indicating no advantage in precopulatory male contest competition. However, in competitive matings, sons of polyandrous mothers significantly increased their mating efforts (mating duration, intromission number). In line with this, paternity success was biased towards sons of polyandrous mothers. Because there was no evidence for maternal effects, our results suggest that female bank voles gain genetic benefits from polyandry.     

Multiple paternity is a shared reproductive strategy in the live‐bearing surfperches (Embiotocidae) that may be associated with female fitness

According to Bateman's principle, female fecundity is limited relative to males, setting the expectation that males should be promiscuous, while females should be choosy and select fewer mates. However, several surfperches (Embiotocidae) exhibit multiple paternity within broods indicating that females mate with multiple males throughout the mating season. Previous studies found no correlation between mating success and reproductive success (i.e., a Bateman gradient). However, by including samples from a broader range of reproductive size classes, we found evidence of a Bateman gradient in two surfperch species from distinct embiotocid clades. Using microsatellite analyses, we found that 100% of the spotfin surfperch families sampled exhibit multiple paternity (Hyperprosopon anale, the basal taxon from the only clade that has not previously been investigated) indicating that this tactic is a shared reproductive strategy among surfperches. Further, we detected evidence for a Bateman gradient in H. anale; however, this result was not significant after correction for biases. Similarly, we found evidence for multiple paternity in 83% of the shiner surfperch families (Cymatogaster aggregata) sampled. When we combine these data with a previous study on the same species, representing a larger range of reproductive size classes and associated brood sizes, we detect a Bateman gradient in shiner surfperch for the first time that remains significant after several conservative tests for bias correction. These results indicate that sexual selection is likely complex in this system, with the potential for conflicting optima between sexes, and imply a positive shift in fertility (i.e., increasing number) and reproductive tactic with respect to the mating system and number of sires throughout the reproductive life history of females. We argue that the complex reproductive natural history of surfperches is characterized by several traits that may be associated with cryptic female choice, including protracted oogenesis, uterine sac complexity, and sperm storage.     

The more the better – polyandry and genetic similarity are positively linked to reproductive success in a natural population of terrestrial salamanders (Salamandra salamandra)

Although classically thought to be rare, female polyandry is widespread and may entail significant fitness benefits. If females store sperm over extended periods of time, the consequences of polyandry will depend on the pattern of sperm storage, and some of the potential benefits of polyandry can only be realized if sperm from different males is mixed. Our study aimed to determine patterns and consequences of polyandry in an amphibian species, the fire salamander, under fully natural conditions. Fire salamanders are ideal study objects, because mating, fertilization and larval deposition are temporally decoupled, females store sperm for several months, and larvae are deposited in the order of fertilization. Based on 18 microsatellite loci, we conducted paternity analysis of 24 female‐offspring arrays with, in total, over 600 larvae fertilized under complete natural conditions. More than one‐third of females were polyandrous and up to four males were found as sires. Our data clearly show that sperm from multiple males is mixed in the female's spermatheca. Nevertheless, paternity is biased, and the most successful male sires on average 70% of the larvae, suggesting a ‘topping off’ mechanism with first‐male precedence. Female reproductive success increased with the number of sires, most probably because multiple mating ensured high fertilization success. In contrast, offspring number was unaffected by female condition and genetic characteristics, but surprisingly, it increased with the degree of genetic relatedness between females and their sires. Sires of polyandrous females tended to be genetically similar to each other, indicating a role for active female choice.     

Pre‐ and postcopulatory sexual selection act in concert to determine male reproductive success in Tribolium castaneum

To capture how sexual selection shapes male reproductive success across different stages of reproduction in Tribolium castaneum (Coleoptera, Tenebrionidae), we combined sequential sperm defence (P1) and sperm offence (P2) trials with additional trials where both males were added simultaneously to the female. We found a positive correlation between the relative paternity share in simultaneous male–male competition trials and the P2 trial. This suggests that males preferred by females as sires achieve superior fertilization success during sperm competition in the second male position. In simultaneous male–male competition trials, where pre‐, peri‐ and postcopulatory sexual selection were all allowed to act, the relative paternity share of preferred males was more than 20% higher than in P2 sperm competition trials where precopulatory female choice was disabled. Additional behavioural observations revealed that mating with more attractive males resulted significantly more frequently in offspring production than mating with less attractive males. Thus, by comparing male fertilization success in trials where precopulatory choice was turned off with more inclusive estimates of fertilization success where pre‐ and pericopulatory choice could occur, we show that female mate choice may effectively inhibit sperm competition. Female mate choice and sperm competition (P2) are positively correlated, which is consistent with directional sexual selection in this species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 67–75.     

Promiscuous females avoid inbreeding by controlling sperm storage

Recent studies in a variety of species have shown that polyandrous females are somehow able to bias paternity against their relatives postcopulation, although how they do so remains unknown. Field crickets readily mate with their siblings, but when also mated to an unrelated male, they produce disproportionately fewer inbred offspring. We use a new competitive microsatellite polymerase chain reaction technique to determine the contribution of males to stored sperm and subsequent paternity of offspring. Paternity is almost completely predicted by how much sperm from a particular male is stored, and unrelated males contribute more sperm to storage and have a corresponding higher paternity success.     

A theoretical study on the evolution of male parental care and female multiple mating: effects of female mate choice and male care bias

Male parental care and female multiple mating are seen in many species in spite of the cost they entail. Moreover, they even coexist in some species though polyandry, by reducing paternity confidence of caregiving males, seems to hinder the evolution of paternal care. Previous studies have investigated the coevolutionary process of paternal care and polyandry under various simplifying assumptions, including random mating and random provision of male care. We extend these models to examine possible effects of female mate choice and male care bias, assuming that (a) monandrous females mate preferentially with caregiving males while polyandrous females compromise their preference in order to mate with multiple males and (b) caregiving males tend to direct their care to offspring of monandrous females. Our models suggest that both the female preference and the male bias always favor caregiving males while they may not always facilitate the evolution of monandry.     

Effect of male body size on sperm precedence in the polyandrous butterfly Pieris napi L. (Lepidoptera: Pieridae)

Male Lepidoptera produce an ejaculate during copulation thatcontains both sperm and accessory gland nutrients and may functionas paternal investment and/or male mating effort Several studieshave examined how ejaculates function as paternal investment,but few have determined the influence of sperm competition onmale investment This study examines the effect of male bodysize on sperm precedence in the polyandrous butterfly Pierisnapi L. We used male body mass as an indicator of the size ofejaculate transferred and found that relative male size hada significant effect on paternity. The offspring of twice-matedfemales showed a low incidence of mixed paternity. Larger malesobtained the majority of fertilizations, and the degree of second-malesperm precedence was influenced by relative body size of matingmales. In general, second mates obtained fewer fertilizationsthe larger the size of the first mate. The interval betweenthe first and second mating was influenced by the size of thefirst male mate Females first mated to small males remated soonerthan females first mated to larger males Our results suggestthat large males may have a selective advantage over small maleswhen both a male's fertilization success and a female's refractoryperiod are influenced by the size of ejaculate transferred.Furthermore, the effect of male body size on the proportionof offspring sired lends support to the hypothesis that spermcompetition has played a major role in the evolution of ejaculatesize.