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.     

DISENTANGLING PRECOPULATORY AND POSTCOPULATORY SEXUAL SELECTION IN POLYANDROUS SPECIES

Sexual selection operates on a sequence of events, from mating to offspring production. Which stages in this sequence undergo stronger selection, especially the relative importance of pre‐ versus postcopulatory processes, are intensely debated issues. Unequal siring success among mates of polyandrous females is classically taken as evidence for a large contribution of postcopulatory processes to the variance in male reproductive success (var(RS_m)). However, paternity skews also depend on the timing and number of copulations, a source of variation that should be considered precopulatory rather than postcopulatory. We develop a method for decomposing var(RS_m) accounting for copulatory activity and apply it to experimental mating groups of the snail Physa acuta. In our experiment, 40% of var(RS_m) emerges at the precopulatory stage, only half of which depends on variation in mating success (number of partners). Ignoring copulation characteristics can therefore lead to severe underestimation of precopulatory sexual selection. Moreover, although only 36% of var(RS_m) arises at the postcopulatory stage, this is when sexual selection on body weight mostly occurs. Finally, trade‐offs were detected between different components of precopulatory success, whereas pre‐ and postcopulatory success appear independent. Our study opens the way to a detailed quantitative understanding of sexual selection in polyandrous species.     

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.     

High opportunity for postcopulatory sexual selection under field conditions

In polygamous systems, male fitness is determined not only by mating success but also by fertilization success. Despite the growing interest over the past several decades in postcopulatory sexual selection, its relative importance compared to precopulatory sexual selection remains a subject of debate. Here, we use extensive behavioral observations of a seminatural population of Hawaiian swordtail crickets, Laupala cerasina, and molecular paternity assignment to measure the opportunities for pre‐ and postcopulatory selection. Because postcopulatory selection has the potential to operate at multiple stages, we also separately attribute its effects to factors specific to mating events versus factors specific to males. We find that variance in postcopulatory success is over four times as great as variance in precopulatory success, with most of it unexplained by male mating order or the number of nuptial gifts given. Surprisingly, we also find that male singing effort is under postcopulatory selection, suggesting that males who sing more frequently also have more competitive ejaculates. Our results are consistent with the hypothesis that high polyandry levels promote greater relative postcopulatory selection. They also highlight the need for detailed behavioral observations under conditions as natural as possible when measuring mating and reproductive success.     

No evidence that polyandry benefits females in Drosophila melanogaster

Understanding the evolution of polyandry (mating with multiple males) is a major issue in the study of animal breeding systems. We examined the adaptive significance of polyandry in Drosophila melanogaster, a species with well-documented costs of mating in which males generally cannot force copulations. We found no direct fitness advantages of polyandry. Females that mated with multiple males had no greater mean fitness and no different variance in fitness than females that mated repeatedly with the same male. Subcomponents of reproductive success, including fecundity, egg hatch rate, larval viability, and larval development time, also did not differ between polyandrous and monogamous females. Polyandry had no affect on progeny sex ratios, suggesting that polyandry does not function against costly sex-ratio distorters. We also found no evidence that polyandry functions to favor the paternity of males successful in precopulatory sexual selection. Experimentally controlled opportunities for precopulatory sexual selection had no effect on postcopulatory sperm precedence. Although these results were generally negative, they are supported with substantial statistical power and they help narrow the list of evolutionary explanations for polyandry in an important model species.     

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.     

The contrasting role of male relatedness in different mechanisms of sexual selection in red junglefowl

In structured populations, competition for reproductive opportunities should be relaxed among related males. The few tests of this prediction often neglect the fact that sexual selection acts through multiple mechanisms, both before and after mating. We performed experiments to study the role of within‐group male relatedness across pre‐ and postcopulatory mechanisms of sexual selection in social groups of red junglefowl, Gallus gallus, in which two related males and one unrelated male competed over females unrelated to all the males. We confirm theoretical expectations that, after controlling for male social status, competition over mating was reduced among related males. However, this effect was contrasted by other sexual selection mechanisms. First, females biased male mating in favor of the unrelated male, and might also favor his inseminations after mating. Second, males invested more—rather than fewer—sperm in postcopulatory competition with relatives. A number of factors may contribute to explain this counterintuitive pattern of sperm allocation, including trade‐offs between male investment in pre‐ versus postcopulatory competition, differences in the relative relatedness of pre‐ versus postcopulatory competitors, and female bias in sperm utilization in response to male relatedness. Collectively, these results reveal that within‐group male relatedness may have contrasting effects in different mechanisms of sexual selection.     

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.     

An integrative mating system assessment of a nonmodel,economically important Pacific rockfish (Sebastes melanops) reveals nonterritorial polygamy and conservation implications for a large species flock

Characterizing the mating systems of long‐lived, economically important Pacific rockfishes comprising the viviparous Sebastes species flock is crucial for their conservation. However, direct assignment of mating success to sires is precluded by open, offshore populations and high female fecundity. We addressed this challenge by integrating paternity‐assigned mating success of females with the adult sex ratio (ASR) of the population, male evolutionary responses to receptive females, and reproductive life history traits—in the framework of sexual selection theory—to assess the mating system of Sebastes melanops. Microsatellite parentage analysis of 17 pregnant females, 1,256 of their progeny, and 106 adults from the population yielded one to four sires per brood, a mean of two sires, and a female mate frequency distribution with a truncated normal (random) pattern. The 11 multiple paternity broods all contained higher median allele richness than the six single paternity broods (Wilcoxon test: W = 0, p < .001), despite similar levels of average heterozygosity. By sampling sperm and alleles from different males, polyandrous females gain opportunities to enhance their sperm supply and to lower the cost of mating with genetically incompatible males through reproductive compensation. A mean of two mates per mated female with a variance of one, an ASR = 1.2 females per male, and the expected population mean of 2.4 mates for mated males (and the estimated 35 unavailable sires), fits polygamous male mate frequency distributions that distinguish polygynandry and polyandrogyny mating systems, that is, variations of polygamy, but not polyandry. Inference for polygamy is consistent with weak premating sexual selection on males, expected in mid‐water, schooling S. melanops, owing to polyandrous mating, moderately aggregated receptive females, an even ASR, and no territories and nests used for reproduction. Each of these characteristics facilitates more mating males and erodes conspicuous sexual dimorphism. Evaluation of male evolutionary responses of demersal congeners that express reproductively territorial behavior revealed they have more potential mechanisms for producing premating sexual selection, greater variation in reproductive success, and a reduced breeding effective population size of adults and annual effective size of a cohort, compared to S. melanops modeled with two mates per adult. Such divergence in behavior and mating system by territorial species may differentially lower their per capita birth rates, subsequent population growth, and slow their recovery from exploitation.     

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.     

The Effects of Perceived Mating Opportunities on Patterns of Reproductive Investment by Male Guppies

Males pay considerable reproductive costs in acquiring mates (precopulatory sexual selection) and in producing ejaculates that are effective at fertilising eggs in the presence of competing ejaculates (postcopulatory sexual selection). Given these costs, males must balance their reproductive investment in a given mating to optimise their future reproductive potential. Males are therefore expected to invest in reproduction prudently according to the likelihood of obtaining future matings. In this study we tested this prediction by determining whether male reproductive investment varies with expected future mating opportunities, which were experimentally manipulated by visually exposing male guppies (Poecilia reticulata) to high or low numbers of females in the absence of competing males. Our experiment did not reveal consistent effects of perceived future mating opportunity on either precopulatory (male mate choice and mating behaviour) or postcopulatory (sperm quality and quantity) investment. However, we did find that male size and female availability interacted to influence mating behaviour; large males visually deprived of females during the treatment phase became more choosy and showed greater interest in their preferred female than those given continuous visual access to females. Overall, our results suggest males tailor pre- rather than postcopulatory traits according to local female availability, but critically, these effects depend on male size.     

Paternity analyses in wild‐caught and laboratory‐reared Caribbean cricket females reveal the influence of mating environment on post‐copulatory sexual selection

Polyandry is ubiquitous in insects and provides the conditions necessary for male‐ and female‐driven forms of post‐copulatory sexual selection to arise. Populations of Amphiacusta sanctaecrucis exhibit significant divergence in portions of the male genitalia that are inserted directly into the female reproductive tract, suggesting that males may exercise some post‐copulatory control over fertilization success. We examine the potential for male–male and male–female post‐copulatory interactions to influence paternity in wild‐caught females of A. sanctaecrucis and contrast our findings with those obtained from females reared in a high‐density laboratory environment. We find that female A. sanctaecrucis exercise control by mating multiple times (females mount males), but that male–male post‐copulatory interactions may influence paternity success. Moreover, post‐copulatory interactions that affect reproductive success of males are not independent of mating environment: clutches of wild‐caught females exhibit higher sire diversity and lower paternity skew than clutches of laboratory‐reared females. There was no strong evidence for last male precedence in either case. Most attempts at disentangling the contributions of male–male and male–female interactions towards post‐copulatory sexual selection have been undertaken in a laboratory setting and may not capture the full context in which they take place – such as the relationship between premating and post‐mating interactions. Our results reinforce the importance of designing studies that can capture the multifaceted nature of sexual selection for elucidating the role of post‐copulatory sexual selection in driving the evolution of male and female reproductive traits, especially when different components (e.g. precopulatory and post‐copulatory interactions) do not exert independent effects on reproductive outcomes.     

Determinants of reproductive success across sequential episodes of sexual selection in a firefly

Because females often mate with multiple males, it is critical to expand our view of sexual selection to encompass pre-, peri- and post-copulatory episodes to understand how selection drives trait evolution. In Photinus fireflies, females preferentially respond to males based on their bioluminescent courtship signals, but previous work has shown that male paternity success is negatively correlated with flash attractiveness. Here, we experimentally manipulated both the attractiveness of the courtship signal visible to female Photinus greeni fireflies before mating and male nuptial gift size to determine how these traits might each influence mate acceptance and paternity share. We also measured pericopulatory behaviours to examine their influence on male reproductive success. Firefly males with larger spermatophores experienced dual benefits in terms of both higher mate acceptance and increased paternity share. We found no effect of courtship signal attractiveness or pericopulatory behaviour on male reproductive success. Taken together with previous results, this suggests a possible trade-off for males between producing an attractive courtship signal and investing in nuptial gifts. By integrating multiple episodes of sexual selection, this study extends our understanding of sexual selection in Photinus fireflies and provides insight into the evolution of male traits in other polyandrous species.     

Postcopulatory inbreeding avoidance by female crickets only revealed by molecular markers

Multiple mating is thought to provide an opportunity for females to avoid the costs of genetic incompatibility by postcopulatory selection of compatible sperm haplotypes. Few studies have tested the genetic incompatibility hypothesis directly. Here we experimentally manipulated the compatibility of females with their mates using the gryllid cricket Teleogryllus oceanicus. We recorded the hatching success of eggs laid by females mated with two nonsibling males, two siblings, or one nonsibling male and one sibling. In contrast with two previous studies on crickets that have adopted this approach, the hatching success of eggs did not differ between females mated with two full siblings and females mated with two unrelated males, indicating that embryo viability was not a cost of inbreeding in this species. We assigned paternity to offspring produced by females mated to both a sibling and a nonsibling male using microsatellite markers. As in previous studies of this species, we were unable to detect any difference in the proportion of offspring sired by the 1st and the 2nd male to mate with a female when females were unrelated to their mates. However, in our experimental matings the proportion of offspring sired by the nonsibling male depended on his sequence position. Paternity was biased toward the nonsibling male when he mated first. Our data show that molecular analyses of paternity are essential to detect subtle mechanisms of postcopulatory sexual selection.     

Paternity costs from polyandry compensated by increased fecundity in the hide beetle

Polyandry-induced sperm competition is assumed to impose costson males through reduced per capita paternity success. In contrast,studies focusing on the consequences of polyandry for femalesreport increased oviposition rates and fertility. For thesespecies, there is potential for the increased female fecundityassociated with polyandry to offset the costs to males of sharedpaternity. We tested this hypothesis by comparing the proportionand number of offspring sired by males mated with monandrousand polyandrous females in the hide beetle, Dermestes maculates,both for males mating with different females and for males rematingwith the same female. In 4 mating treatments, monandrous femalesmated either once or twice with the same male and polyandrousfemales mated either twice with 2 different males or thricewith 2 males (where 1 male mated twice). Polyandrous and twice-matingmonandrous females displayed greater fecundity and fertilitythan singly mating monandrous females. Moreover, males rematedto the same female had greater paternity regardless of whetherthat female mated with another male. In both polyandrous treatments,male mating order did not affect paternity success. Finally,although the proportion of eggs sired decreased if a male matedwith a polyandrous female, multiply mating females or femalesthat remated with a previous mate laid significantly more eggsand thus the actual number of eggs sired was comparable. Thus,males do not necessarily accrue a net fitness loss when matingwith polyandrous females. This may explain the absence of anyobvious defensive paternity-protection traits in hide beetlesand other species.     

Postcopulatory female choice increases the fertilization success of novel males in the field cricket, Gryllus vocalis

Although recent studies have demonstrated that female crickets prefer novel males to previous mates, the relative contribution of pre- and postcopulatory behaviors to this advantage remain unknown, as do the reproductive consequences to males. I paired females either with previous or novel mates, and recorded the latency to mating and the time after mating at which the female removed the male's spermatophore, terminating sperm transfer. Females that mated with familiar males removed their spermatophores sooner than females that mated with novel males. Females paired with novel males also mated more quickly than females paired with familiar males, but this difference was not statistically significant. A molecular-based paternity analysis was used to determine whether the postcopulatory preference of females for novel males influences a male's fertilization success. Females were assigned to either mate three times with the same male and then once with a novel male, or four times with four different males. The paternity of the last male was higher when the female previously had mated repeatedly with the same male than when she had mated previously with different males. These results suggest that female spermatophore removal behavior influences male paternity such that novel males receive a fertility benefit.     

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.     

Number of mating males and mating interval affect last-male sperm precedence in Tenebrio molitor L.

Polyandrous females often mate with more than two males, and yet most studies of postcopulatory sexual selection involve only two males. In insects, second-male sperm precedence is usually taken as evidence of overall last-male sperm precedence despite some studies to the contrary. Furthermore, the processes or mechanisms causing the patterns are often unknown and yet are important when estimating how postcopulatory sexual selection might act on males. Whether the patterns and processes change in normal sperm competitive situations and the effects of other factors besides mating order need to be examined to better assess the evolutionary potential of postcopulatory sexual selection. In this study, I assessed the effects of mating interval and number of mating males on sperm precedence patterns and their causal mechanisms in the mealworm beetle, Tenebrio molitor. Last-male sperm precedence was the same when two or three males mated, but also depended on mating intervals and hence mechanisms of paternity bias. However, when females mated with many males, one of the mechanisms no longer created last-male sperm precedence. This example illustrates the importance of knowing both the patterns and mechanisms of paternity bias and whether they change depending on female mating frequency to make reasonable inferences about the potential for postcopulatory sexual selection on males. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.      

Sperm competition and the evolution of precopulatory weapons: Increasing male density promotes sperm competition and reduces selection on arm strength in a chorusing frog

Sperm competition theory assumes a trade‐off between precopulatory traits that increase mating success and postcopulatory traits that increase fertilization success. Predictions for how sperm competition might affect male expenditure on these traits depend on the number of competing males, the advantage gained from expenditure on weapons, and the level of sperm competition. However, empirical tests of sperm competition theory rarely examine precopulatory male expenditure. We investigated how variation in male density affects precopulatory sexual selection on male weaponry and the level of sperm competition in the chorusing frog Crinia georgiana, where males use their arms as weapons in male–male combat. We measured body size and arm girth of 439 males, and recorded their mating success in the field. We found density‐dependent selection acting on arm girth. Arm girth was positively associated with mating success, but only at low population densities. Increased male density was associated with higher risk and intensity of sperm competition arising from multimale amplexus, and a reversal in the direction of selection on arm girth. Opposing patterns of pre‐ and postcopulatory selection may account for the negative covariation between arm girth and testes across populations of this species.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.