Male courtship signals and female signal assessment in Photinus greeni fireflies

The evolutionary dynamic of courtship signaling systems is drivenby the interaction between male trait distributions and femalepreferences. This interaction is complex because females maychoose mates based on multiple components of male signals, andfemale preference functions may vary depending on mate availability,female reproductive state, and environmental conditions. InPhotinus fireflies (Coleoptera: Lampyridae), flying males emitbioluminescent flash signals to locate sedentary females, whichreply selectively to attractive male flash signals with theirown response flash. In this study, we first examined temporalvariation in the paired-pulse flash patterns produced by Photinusgreeni males in the field and found significant among-male variation(70% of total variation) in interpulse intervals (IPIs). Therewas no significant relationship between male IPI and spermatophoresize, suggesting that P. greeni male courtship signals do notprovide females with reliable indicators of male material resources.In laboratory playback experiments, we presented P. greeni femaleswith simulated flash signals to assess how IPI and pulse durationindependently affected the likelihood of female flash response.We also examined the effects of female body mass and time duringthe mating season on female preference functions, hypothesizingthat females would be less discriminating when they were heavier(more fecund) and when mate availability declined. We foundthat P. greeni females discriminated among signals within theirspecies' range based primarily on flash pattern IPI. Neitherthe time during the mating season nor female weight alteredfemale preference functions for IPI, although season did influencefemale response to pulse duration. These results reveal thatP. greeni females discriminate among conspecific males basedprimarily on male IPIs, the same signal character previouslyshown to be important for firefly species recognition. Fieldplayback experiments indicated that female responsiveness peakednear the average IPI given by males at different ambient temperatures,suggesting that fireflies exhibit temperature coupling similarto that seen in many acoustically signaling animals.     

Multiple paternity in reptiles: patterns and processes

The evolution of female promiscuity poses an intriguing problem as benefits of mating with multiple males often have to arise via indirect, genetic, effects. Studies on birds have documented that multiple paternity is common in natural populations but strong evidence for selection via female benefits is lacking. In an attempt to evaluate the evidence more broadly, we review studies of multiple paternity in natural populations of all major groups of nonavian reptiles. Multiple paternity has been documented in all species investigated so far and commonly exists in over 50% of clutches, with particularly high levels in snakes and lizards. Marine turtles and lizards with prolonged pair-bonding have relatively low levels of multiple paternity but levels are nevertheless higher than in many vertebrates with parental care. There is no evidence that high levels of polyandry are driven by direct benefits to females and the evidence that multiple paternity arises from indirect genetic benefits is weak. Instead, we argue that the most parsimonious explanation for patterns of multiple paternity is that it represents the combined effect of mate-encounter frequency and conflict over mating rates between males and females driven by large male benefits and relatively small female costs, with only weak selection via indirect benefits. A crucial step for researchers is to move from correlative approaches to experimental tests of assumptions and predictions of theory under natural settings, using a combination of molecular techniques and behavioural observations.     

High levels of polyandry,but limited evidence for multiple paternity,in wild populations of the western rock lobster (Panulirus cygnus)

Polyandry, where multiple mating by females results in the temporal and spatial overlap of ejaculates from two or more males, is taxonomically widespread and occurs in varying frequencies within and among species. In decapods (crabs, lobsters, crayfish, and prawns), rates of polyandry are likely to be variable, but the extent to which patterns of multiple paternity reflect multiple mating, and thus are shaped by postmating processes that bias fertilization toward one or a subset of mated males, is unclear. Here, we use microsatellite markers to examine the frequency of multiple mating (the presence of spermatophores from two or more males) and patterns of paternity in wild populations of western rock lobster (Panulirus cygnus). Our data confirm that >45% of females had attached spermatophores arising from at least two males (i.e., confirming polyandry), but we found very limited evidence for multiple paternity; among 24 clutches sampled in this study, only two arose from fertilizations by two or more males. Single inferred paternal genotypes accounted for all remaining progeny genotypes in each clutch, including several instances when the mother had been shown to mate with two or more males. These findings highlight the need for further work to understand whether polyandry is adaptive and to uncover the mechanisms underlying postmating paternity biases in this system.     

Female preference for male courtship flashes in Photinus ignitus fireflies

In Photinus fireflies, males produce spontaneous bioluminescentcourtship flashes. Females preferentially respond to particularmale flashes with flashes of their own. This study exploredvariation in female flash responsiveness as a function of maleflash duration, female condition, lantern size, and lanterndistance, as well as the relationship between male characteristicsand spermatophore mass in Photinus ignitus fireflies. We determinedfemale preference by scoring female flash response to simulatedmale flashes and determined variation in overall female flashresponsiveness for laboratory-mated, laboratory-fed, and controlP. ignitus females. Flash duration, lantern size, and body masswere recorded for field-collected males. Males were then matedto determine spermatophore mass. Females exhibited greater preferencefor artificial flashes representing the upper range of conspecificmale flash duration and lantern size as well as flashes producedat a closer distance. Both laboratory-mated and laboratory-fedP. ignitus females showed lower overall responsiveness acrossall flash durations relative to control females that did notmate or feed in the laboratory. Male flash duration predicteda significant proportion of the variation in spermatophore massfor early-season males. These results suggest that female Photinusignitus may prefer long flashes in order to obtain the directbenefit of larger spermatophores and may adjust their overallflash responsiveness as the relative importance of this benefitvarieswith changing female condition.     

Paternity analysis of wild‐caught females shows that sperm package size and placement influence fertilization success in the bushcricket Pholidoptera griseoaptera

In species where females store sperm, males may try to influence paternity by the strategic placement of sperm within the female's sperm storage organ. Sperm may be mixed or layered in storage organs, and this can influence sperm use beyond a ‘fair raffle’. In some insects, sperm from different matings is packaged into discrete packets (spermatodoses), which retain their integrity in the female's sperm storage organ (spermatheca), but little is known about how these may influence patterns of sperm use under natural mating conditions in wild populations. We examined the effect of the size and position of spermatodoses within the spermatheca and number of competing ejaculates on sperm use in female dark bushcrickets (Pholidoptera griseoaptera) that had mated under unmanipulated field conditions. Females were collected near the end of the mating season, and seven hypervariable microsatellite loci were used to assign paternity of eggs laid in the laboratory. Females contained a median of three spermatodoses (range 1–6), and only six of the 36 females contained more than one spermatodose of the same genotype. Both the size and relative placement of the spermatodoses within the spermatheca had a significant effect on paternity, with a bias against smaller spermatodoses and those further from the single entrance/exit of the spermatheca. A higher number of competing males reduced the chances of siring offspring for each male. Hence, both spermatodose size and relative placement in the spermatheca influence paternity success.     

Male reproductive allocation in fireflies (Photinus spp.)

Abstract. Resource allocation between somatic and reproductive structures has important fitness consequences for individuals, and optimal trade-offs are expected to depend not only on mating system differences among species but also on levels of resource stress within species. We tested the prediction that polyandry (associated with increased sperm competition) will increase male reproductive allocation in bioluminescent fireflies in Photinus spp. by comparing the relative mass of testes, seminal vesicles, and reproductive accessory glands among a monandrous and several polyandrous species. In addition, we examined a single population of a polyandrous species, Photinus greeni , to see how reproductive allocation might shift between years in response to different levels of larval resource stress. As predicted, males of P. collustrans , a monandrous species, showed a fivefold lower allocation to sperm production and a 100-fold lower allocation to reproductive accessory glands compared with males from polyandrous species. We also found evidence within P. greeni of a trade-off between allocation either to reproduction or to somatic tissue; following larval resource stress, males eclosed at significantly shorter body lengths, yet showed a 35% increase in their reproductive allocation. These results demonstrate that mating systems strongly influence male allocation to reproductive accessory glands as well as to sperm production. Furthermore, these results suggest that under larval resource stress males of Photinus spp. increase their allocation to reproduction at the expense of somatic tissue, thus maximizing their ability to produce nuptial gifts required for reproductive success.     

Mechanisms underlying the sperm quality advantage in Drosophila melanogaster

Contrary to early predictions of sperm competition theory, postcopulatory sexual selection favoring increased investment per sperm (e.g., sperm size, sperm quality) has been demonstrated in numerous organisms. We empirically demonstrate for Drosophila melanogaster that both sperm quality and sperm quantity independently contribute to competitive male fertilization success. In addition to these independent effects, there was a significant interaction between sperm quality and quantity that suggests an internal positive reinforcement on selection for sperm quality, with selection predicted to intensify as investment per sperm increases and the number of sperm competing declines. The mechanism underlying the sperm quality advantage is elucidated through examination of the relationship between female sperm-storage organ morphology and the differential organization of different length sperm within the organ. Our results exemplify that primary sex cells can bear secondary sexual straits.     

Polyandry reduces sperm length variation in social insects

Postcopulatory sexual selection, either in the form of sperm competition or cryptic female choice, is an important selective force that is thought to have generated the enormous variation in sperm morphology observed interspecifically. However, the evolutionary significance of intraspecific variation in sperm morphology, and the role that postcopulatory sexual selection plays in influencing this variation, remains poorly investigated in invertebrates. Here, we tested the hypothesis that postcopulatory sexual selection reduces variation in sperm morphology, both between and within males, in 27 species of eusocial ants and bees. These eusocial species offer an unusual opportunity to assess how selection acts on variance in sperm morphology, as haploid males produce clonal, haploid sperm that does not experience haploid-diploid conflict. We provide solid evidence that males of polyandrous ant and bee species indeed produce less-variable sperm, indicating that sperm competition selected for sperm of superior quality. Our results offer a mechanistic explanation for the evolution of high-quality sperm and provide comprehensive evidence that sperm morphology of social insects is influenced by sexual selection.     

No evidence for postcopulatory inbreeding avoidance in Drosophila melanogaster

Selection to avoid inbreeding is predicted to vary across species due to differences in population structure and reproductive biology. Over the past decade, there have been numerous investigations of postcopulatory inbreeding avoidance, a phenomenon that first requires discrimination of mate (or sperm) relatedness and then requires mechanisms of male ejaculate tailoring and/or cryptic female choice to avoid kin. The number of studies that have found a negative association between male-female genetic relatedness and competitive fertilization success is roughly equal to the number of studies that have not found such a relationship. In the former case, the underlying mechanisms are largely unknown. The present study was undertaken to verify and expand upon a previous report of postcopulatory inbreeding avoidance in D. melanogaster, as well as to resolve underlying mechanisms of inbreeding avoidance using transgenic flies that express a sperm head-specific fluorescent tag. However, siblings did not have a lower fertilization success as compared to unrelated males in either the first (P(1) ) or second (P(2) ) mate role in sperm competition with a standard unrelated competitor male in our study population of D. melanogaster. Analyses of mating latency, copulation duration, egg production rate, and remating interval further revealed no evidence for inbreeding avoidance.     

Geographic variation in sperm traits reflects predation risk and natural rates of multiple paternity in the guppy

Guppies (Poecilia reticulata) are models for understanding the interplay between natural and sexual selection. In particular, predation has been implicated as a major force affecting female sexual preferences, male mating tactics and the level of sperm competition. When predation is high, females typically reduce their preferences for showy males and engage more in antipredator behaviours, whereas males exploit these changes by switching from sexual displays to forced matings. These patterns are thought to account for the relatively high levels of multiple paternity in high‐predation populations compared to low‐predation populations. Here, we assess the possible evolutionary consequences of these patterns by asking whether variation in sperm traits reflect differences in predation intensity among four pairs of Trinidadian populations: four that experience relatively low levels of predation from a gape‐limited predator and four that experience relatively high levels of predation from a variety of piscivores. We found that males in high‐predation populations had faster swimming sperm with longer midpieces compared to males in low‐predation populations. However, we found no differences among males in high‐ and low‐predation populations with respect to sperm number, sperm head length, flagellum length and total sperm length.     

Male sperm storage compromises sperm motility in guppies

Sperm senescence can have important evolutionary implications due to its deleterious effects on sperm quality and offspring performance. Consequently, it has been argued that polyandry (female multiple mating) may facilitate the selection of younger, and therefore competitively superior, sperm when ejaculates from multiple males compete for fertilization. Surprisingly, however, unequivocal evidence that sperm ageing influences traits that underlie sperm competitiveness is lacking. Here, we used a paired experimental design that compares sperm quality between ‘old’ and ‘young’ ejaculates from individual male guppies (Poecilia reticulata). We show that older sperm exhibit significant reductions in sperm velocity compared with younger sperm from the same males. We found no evidence that the brightness of the male''s orange (carotenoid) spots, which are thought to signal resistance to oxidative stress (and thus age-related declines in sperm fitness), signals a male''s ability to withstand the deleterious effects of sperm ageing. Instead, polyandry may be a more effective strategy for females to minimize the likelihood of being fertilized by aged sperm.     

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.     

Cryptic female preference for colorful males in guppies

Cryptic female choice (CFC) refers to female-mediated processes occurring during or after copulation that result in biased sperm use in favor of preferred or compatible males. Despite recent empirical support for this hypothesis, evidence that CFC contributes towards the evolution of male body ornaments, in the same way that precopulatory female choice does, is currently lacking. Here, we tested the possibility that CFC selects for increased male attractiveness in the guppy Poecilia reticulata, a freshwater fish exhibiting internal fertilization. Specifically, we examined whether females are able to manipulate the number of sperm transferred or retained at copulation in favor of relatively attractive males. In support of this prediction, we found that following solicited copulations the number of sperm inseminated is influenced exclusively by the female's perception of relative male coloration, independent of any direct manipulation of males themselves. Because females prefer brightly colored males during precopulatory mate choice, our finding that colorful males are also favored as a consequence of enhanced insemination success indicates that cryptic female choice can reinforce precopulatory preferences for extravagant male ornaments.     

Sperm performance in conspecific and heterospecific female fluid

Divergent sexual selection within allopatric populations may result in divergent sexual phenotypes, which can act as reproductive barriers between populations upon secondary contact. This hypothesis has been most tested on traits involved in precopulatory sexual selection, with less work focusing on traits that act after copulation and before fertilization (i.e., postcopulatory prezygotic traits), particularly in internally fertilizing vertebrates. However, postcopulatory sexual selection within species can also drive trait divergence, resulting in reduced performance of heterospecific sperm within the female reproductive tract. Such incompatibilities, arising as a by‐product of divergent postcopulatory sexual selection in allopatry, can represent reproductive barriers, analogous to species‐assortative mating preferences. Here, we tested for postcopulatory prezygotic reproductive barriers between three pairs of taxa with diverged sperm phenotypes and moderate‐to‐high opportunity for postcopulatory sexual selection (barn swallows Hirundo rustica versus sand martins Riparia riparia, two subspecies of bluethroats, Luscinia svecica svecica versus L. s. namnetum, and great tits Parus major versus blue tits Cyanistes caeruleus). We tested sperm swimming performance in fluid from the outer reproductive tract of females, because the greatest reduction in sperm number in birds occurs as sperm swim across the vagina. Contrary to our expectations, sperm swam equally well in fluid from conspecific and heterospecific females, suggesting that postcopulatory prezygotic barriers do not act between these taxon pairs, at this stage between copulation and fertilization. We therefore suggest that divergence in sperm phenotypes in allopatry is insufficient to cause widespread postcopulatory prezygotic barriers in the form of impaired sperm swimming performance in passerine birds.     

Male parental care, female reproductive success, and extrapair paternity

Birds differ considerably in the degree of male parental care,and it has been suggested that interspecific variation in extrapairpaternity is determined by the relative importance of benefitsto females from male parental care and good genes from extrapairsires. I estimated the relationship between extrapair paternityand the importance of male parental care for female reproductivesuccess mainly based on male removal studies, using a comparativeapproach. The reduction in female reproductive success causedby the absence of a male mate was positively correlated withthe male contribution to feeding offspring. The frequency ofextrapair paternity was negatively related to the reductionin female reproductive success caused by the absence of a mate.This was also the case when potentially confounding variablessuch as developmental mode of offspring and sexual dichromatismwere considered. A high frequency of extrapair paternity occursparticularly in bird species in which males play a minor rolein offspring provisioning and in which attractive males providerelatively little parental care. Bird species with frequentextrapair paternity thus appear to be those in which direct fitness benefits from male care are small, females can readilycompensate for the absence of male care, and indirect fitnessbenefits from extrapair sires are important.     

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.     

Quantitative genetic evidence that males trade attractiveness for ejaculate quality in guppies

Polyandry, where females mate with multiple males, means that a male''s reproductive success will depend both on his ability to acquire mates and the ability of his sperm to compete effectively for fertilizations. But, how do males partition their reproductive investment between these two episodes of selection? Theory predicts that increases in ejaculate investment will come at a cost to investment in other reproductive traits. Although evidence revealing such trade-offs is accumulating, we know little about their genetic basis. Here, I report patterns of genetic (co)variation for a range of traits subject to pre- and post-copulatory sexual selection in the guppy Poecilia reticulata, a promiscuous livebearing fish in which males alternate between courtship and sneak matings to obtain copulations. The analyses of genetic variation and covariation for these behaviours revealed a strong genetic predisposition for one tactic over the other. Both mating tactics were also strongly genetically integrated with the level of sexual ornamentation and ejaculate quality. Males that predominantly performed sneak matings were less ornamented but had faster swimming sperm than those that predominantly used courtship. These patterns of genetic variation and covariation reveal potential evolutionary constraints on the direction of selection of pre- and post-copulatory traits, and support sperm competition theory by revealing a trade-off between sexual attractiveness and investment in ejaculates.     

RAPID DIVERSIFICATION OF SPERM PRECEDENCE TRAITS AND PROCESSES AMONG THREE SIBLING DROSOPHILA SPECIES

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice‐mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within‐population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.