Sperm competition, alternative mating tactics and context-dependent fertilization success in the burying beetle, Nicrophorus vespilloides

Fertilization success in sperm competition is often determined by laboratory estimates of the proportion of offspring sired by the first (P1) or second (P2) male that mates. However, inferences from such data about how sexual selection acts on male traits in nature may be misleading if fertilization success depends on the biological context in which it is measured. We used the sterile male technique to examine the paternity of the same male in two mating contexts in the burying beetle, Nicrophorus vespilloides, a species where males have alternative mating strategies based on the presence or absence of resources. We found no congruence in the paternity achieved by a given male when mating under different social conditions. P2 estimates were extremely variable under both conditions. Body size was unrelated to success in sperm competition away from a carcass but, most probably through pre-copulatory male-male competition, influenced fertilization success on a carcass. The contribution of sperm competition is therefore dependent on the conditions under which it is measured. We discuss our findings in relation to sperm competition theory and highlight the need to consider biological context in order to link copulation and fertilization success for competing males.     

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.     

Testosterone, testes size, and mating success in birds: a comparative study

Reproductive behaviors of vertebrates are often underpinned by temporal patterns of hormone secretion. We investigated interspecific patterns of circulating testosterone in male birds to test the hypothesis that testosterone plays a crucial role in sexual selection as determined by degree of polygyny and extra-pair paternity. We predicted that the evolution of increased levels of polygyny and extra-pair paternity would have resulted in the evolution of increased levels of testosterone to allow males more efficiently to compete for mates. This hypothesis was tested in comparative analyses of 116 species of birds using Generalized Least Squares Models. We assessed the importance of latitudinal distribution, because this can confound the relationship between testosterone and mating success. There were weak positive phylogenetic correlations between measures of testosterone and estimates of mating success at the social level, but this association appeared to be confounded by latitudinal distribution, a significant correlate of testosterone titers. However, we found a significantly positive relationship between peak and residual peak testosterone (which is the peak testosterone level that is controlled for the baseline level) and extra-pair paternity independent of latitude. These results suggest that selection pressures arising from social and sexual mating differently affected testosterone levels with the former being mediated by factors associated with latitudinal distribution. An analysis of residual testes size revealed a positive association between peak and residual testosterone and testes size relative to body size. In a path analysis, we show that relative testis size primarily evolved in association with intense sperm competition and thus high sperm production, and these mechanisms had a secondary impact on blood testosterone levels at a phylogenetic scale. Our results suggest that sperm competition has played an important role in the evolution of reproductive mechanisms in birds.     

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.     

Multiple post‐mating barriers to hybridization in field crickets

Mechanisms that prevent different species from interbreeding are fundamental to the maintenance of biodiversity. Barriers to interspecific matings, such as failure to recognize a potential mate, are often relatively easy to identify. Those occurring after mating, such as differences in the how successful sperm are in competition for fertilisations, are cryptic and have the potential to create selection on females to mate multiply as a defence against maladaptive hybridization. Cryptic advantages to conspecific sperm may be very widespread and have been identified based on the observations of higher paternity of conspecifics in several species. However, a relationship between the fate of sperm from two species within the female and paternity has never been demonstrated. We use competitive microsatellite PCR to show that in two hybridising cricket species, Gryllus bimaculatus and G. campestris, sequential cryptic reproductive barriers are present. In competition with heterospecifics, more sperm from conspecific males is stored by females. Additionally, sperm from conspecific males has a higher fertilisation probability. This reveals that conspecific sperm precedence can occur through processes fundamentally under the control of females, providing avenues for females to evolve multiple mating as a defence against hybridization, with the counterintuitive outcome that promiscuity reinforces isolation and may promote speciation.     

The outcome of sperm competition is affected by behavioural and anatomical reproductive traits in a simultaneously hermaphroditic land snail

Sperm competition is important in species with reproductive strategies that involve multiple mating and prolonged sperm storage such as the simultaneously hermaphroditic land snail Cornu aspersum. Double mating trials in this species have revealed that mating order and courtship behaviour affect paternity success. We investigated the effect of behavioural and anatomical reproductive traits on paternity success from triple mating trials. Triple mating resulted in triple fertilization in 58% of the cases whereas zero paternity was observed in 16% of sperm donors. Third sperm donors achieved higher paternity followed by first and second sperm donors. Snails with a longer epiphallus, the spermatophore forming organ, sired more offspring regardless of their mating order. Genetic compatibility between sperm donor and recipient did not influence paternity success. The results of the present study identified mating order and epiphallus length, as traits affecting the outcome of sperm competition in this species.     

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.     

Does long-term storage of spermatozoa occur in the adder (Vipera berus)?

In the adder ( Vipera berus) multiple inseminations during the spring mating season have been demonstrated, with fertilization taking place several weeks after mating activities have ceased. Subsequent sperm competition in the female reproductive tract can result in within-season mixed paternity. Prolonged sperm storage between mating seasons has been suggested as another mechanism of sperm competition in the adder. Competition between new and old spermatozoa would obstruct efforts to reveal reproductive strategies that correlate observed behaviour to paternity in natural populations. In the present study, controlled breeding experiments with adders were performed and DNA fingerprinting was used to determine paternity among all offspring of a total of 15 litters. The offspring of 11 large females, that had most probably also mated in a previous season, were sired exclusively by enclosure males. This result and earlier empirical data support the hypothesis that long-term sperm storage, over one or several reproductive cycles, does not occur in the adder.     

The frequency of multiple paternity predicts variation in testes size among island populations of house mice

Polyandry generates selection on males through sperm competition, which has broad implications for the evolution of ejaculates and male reproductive anatomy. Comparative analyses across species and competitive mating trials within species have suggested that sperm competition can influence the evolution of testes size, sperm production and sperm form and function. Surprisingly, the intraspecific approach of comparing among population variation for investigating the selective potential of sperm competition has rarely been explored. We sampled seven island populations of house mice and determined the frequency of multiple paternity within each population. Applying the frequency of multiple paternity as an index of the risk of sperm competition, we looked for selective responses in male reproductive traits. We found that the risk of sperm competition predicted testes size across the seven island populations of house mice. However, variation in sperm traits was not explained by sperm competition risk. We discuss these findings in relation to sperm competition theory, and other intrinsic and extrinsic factors that might influence ejaculate quality.     

Phenotypic engineering of sperm-production rate confirms evolutionary predictions of sperm competition theory

Sperm production is a key male reproductive trait and an important parameter in sperm competition theory. Under sperm competition, paternity success is predicted to depend strongly on male allocation to sperm production. Furthermore, because sperm production is inherently costly, individuals should economize in sperm expenditure, and conditional adjustment of the copulation frequency according to their sperm availability may be expected. However, experimental studies showing effects of sperm production on mating behaviour and paternity success have so far been scarce, mainly because sperm production is difficult to manipulate directly in animals. Here, we used phenotypic engineering to manipulate sperm-production rate, by employing dose-dependent RNA interference (RNAi) of a spermatogenesis-specific gene, macbol1, in the free-living flatworm Macrostomum lignano. We demonstrate (i) that our novel dose-dependent RNAi approach allows us to induce high variability in sperm-production rate; (ii) that a reduced sperm-production rate is associated with a decreased copulation frequency, suggesting conditional adjustment of mating behaviour; and (iii) that both sperm production and copulation frequency are important determinants of paternity success in a competitive situation, as predicted by sperm competition theory. Our study clearly documents the potential of phenotypic engineering via dose-dependent RNAi to test quantitative predictions of evolutionary theory.     

Multiple paternity in the common frog (Rana temporaria): genetic evidence from tadpole kin groups

Very few studies have investigated the occurrence of multiple paternity and sperm competition in amphibians. We studied genetic relatedness within kin groups of tadpoles of an aquatically breeding anuran Rana temporaria using allozymes. We collected samples from 52 naturally fertilized spawn clumps produced by single females at three breeding sites in two populations. We estimated relatedness (r) within kin groups, and compared the observed genotype distributions of the tadpoles (on average 23 individuals in each group) with the expected distributions based on single mating. Average relatedness over five polymorphic loci was 0.44 and 0.43 in the two populations, the latter being significantly smaller than that expected by single mating (0.5). The number of patrilines, calculated from relatedness estimates, was 1.3 in one population and 1.4 in the other. Genotype distributions deviated significantly from the expected in half of the kin groups and at all breeding sites. The results show that egg clutches of R. temporaria commonly contain multiply sired offspring. We suggest that communal breeding may affect paternity patterns in R. temporaria as well as in anurans in general.     

Sperm competition experiments between lines of crickets producing different sperm lengths.

Sperm numbers can be important determinants of fertilization success in sperm competition. However, the importance of variation in sperm size is less well understood. Sperm size varies significantly both between and within species and comparative studies have suggested that some of this variance can be explained by sperm competition. In this study we examine whether variation in sperm length has consequences for fertilization precedence using controlled sperm competition experiments in the field cricket Gryllus bimaculatus. This species is an ideal model for such investigations because the mechanism of sperm competition generates complete mixing of different males' spermatozoa in the female (thereby allowing individual sperm to express their own competitive abilities). We successfully bred lines of crickets, the males of which produced short, medium and long sperm types with narrow and non-overlapping distributions. Males of different lines were then sequentially mated with control females in order to create two-male sperm competitions. The paternity outcomes of these competitions were measured after matings using an irradiated male technique (with a full reciprocal design that controls for natural fertility and any irradiation effects on gamete competitiveness) over a 12 day oviposition period. However, having successfully bred diverging sperm length lines and competing males that differed in sperm length, we found no evidence that a male's sperm size explained any of the variation in their relative fertilization success. Males from lines producing longer sperm showed no fertilization advantage over males producing shorter sperm across 97 double matings. There was also no advantage for males producing a sperm length close to the population mean over those competitors whose sperm length had been selectively diverged across 63 matings.     

Sperm length influences fertilization success during sperm competition in the snail Viviparus ater

Sperm form and size is tremendously variable within and across species. However, a general explanation for this variation is lacking. It has been suggested that sperm size may influence sperm competition, and there is evidence for this in some taxa but not others. In addition to normal fertilizing sperm, a number of molluscs and insects produce nonfertile sperm that are also extremely morphologically variable, and distinct from fertilizing forms. There is evidence that nonfertile sperm play an indirect role in sperm competition by decreasing female remating propensity in Lepidopterans, but in most taxa the function of parasperm is unknown. We investigated the role of nonfertile (oligopyrene) sperm during sperm competition in the fresh water snail Viviparus ater. Previous studies found that the proportion of oligopyrene sperm increased with the risk of sperm competition, and hence it seems likely that these sperm influence fertilization success during competitive matings. In mating experiments in which females were sequentially housed with males, we examined a range of male characteristics which potentially influence fertilization success. We found that the size of oligopyrene sperm was the best predictor of fertilization success, with males having the longer sperm siring the highest proportion of offspring. Furthermore, we found a positive shell size and sperm concentration effect on paternity, and females with multiply sired families produced more offspring than females mating with only one male. This result suggests polyandry is beneficial for female snails.     

HOW WELL DO MATING FREQUENCY AND DURATION PREDICT PATERNITY SUCCESS IN THE POLYGYNANDROUS WATER STRIDER AQUARIUS REMIGIS?

Abstract.— The relationship between mating success and paternity success is a key component of sexual selection but has seldom been estimated for species in which both sexes mate with many partners (polygynandry). We used a modification of Parker's sterile male technique to measure this relationship for the water strider Aquarius remigis in 47 laboratory populations simulating natural conditions of polygynandry. We also tested the hypothesis that prolonged copulation, a characteristic of this species, enhances paternity success. Mating behavior and paternity success were assayed for four days while males and females freely interacted. Paternity success was also assayed for an additional 7 days when females were isolated from males. Mating success significantly predicted paternity success and accounted for ≤ 36% of the variance. Copulation duration was negatively related to both mating success and paternity success and did not explain any of the residual variance in paternity success. Thus, we found no evidence that prolonged copulation functions as a paternity assurance strategy in this species. Comparisons of sterile and fertile males suggested that paternity success is directly influenced by the quantity of sperm transferred. Our results support previous studies that have used mating success to estimate sexual selection, but also highlight the potential importance of sperm competition and other postinsemination processes.     

Value of male remating and functional sterility in redback spiders

In the Australian redback spider, Latrodectus hasselti, males typically use their paired copulatory organs (palps) to copulate twice with a single female then sacrifice themselves to their cannibalistic mates in a strategy that increases their paternity in that one mating, but leads to death. This type of terminal investment in one mating is predicted only if the expected value of future matings is low for males relative to the value of repeated mating with the same female. In this laboratory study, we quantified the reproductive value of mating more than once with the same female (repeated mating) and mating with more than one female (multiple mating) for male redback spiders. We tested two natural selection hypotheses for repeated mating, sperm limitation and reproductive insurance, but found no support for either hypothesis. We show that, in the absence of sperm competition or cannibalism, male lifetime reproductive output is the same whether a male copulates once, twice, or several times with a given female. Repeated mating does not increase the probability of successful fertilization, nor does it increase the number of offspring produced in successful matings. Although male repeated mating is not favoured because of increased fertility of mates, other studies suggest it may be important in sperm competition. Here we show that the relative reproductive value of the first two copulations is very high for redback males because they are functionally sterile after each palp has been used once; nonvirgin males are unable to father offspring. Functional sterility and repeated mating by male redbacks may be favoured by the same factors that lead to male sacrifice behaviour: ecological constraints on multiple mating combined with competitive benefits of maximal investment in the first mating. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Polyandry and postcopulatory sexual selection in a wild population

When females mate multiply, postcopulatory sexual selection can occur via sperm competition and cryptic female choice. Although postcopulatory selection has the potential to be a major force in driving evolution, few studies have estimated its strength in natural populations. Likewise, although polyandry is widespread across taxa and is the focus of a growing body of research, estimates of natural female mating rates are still limited in number. Microsatellites can be used to estimate the number of mates represented in females' sperm stores and the number of sires contributing to their offspring, enabling comparisons both of polyandry and of two components of postcopulatory selection: the proportion of males that mate but fail to sire offspring, and the degree of paternity skew among the males that do sire offspring. Here, we estimate the number of mates and sires among wild females in the Hawaiian swordtail cricket Laupala cerasina. We compare these estimates to the actual mating rates and paternity shares we observed in a semi‐natural population. Our results show that postcopulatory sexual selection operates strongly in this species: wild females mated with an average minimum of 3.6 males but used the sperm from only 58% of them. Furthermore, among the males that did sire offspring, paternity was significantly skewed. These patterns were similar to those observed in the field enclosure, where females mated with an average of 5.7 males and used the sperm from 62% of their mates, with paternity significantly skewed among the sires.     

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.     

Multiple paternity and postcopulatory sexual selection in a hermaphrodite: what influences sperm precedence in the garden snail Helix aspersa?

Sperm competition has been studied in many gonochoric animals but little is known about its occurrence in simultaneous hermaphrodites, especially in land snails. The reproductive behaviour of the land snail Helix aspersa involves several features, like multiple mating, long-term sperm storage and dart-shooting behaviour, which may promote sperm competition. Cryptic female choice may also occur through a spermatheca subdivided into tubules, which potentially allows compartmentalized sperm storage of successive mates. In order to determine the outcome of postcopulatory sexual selection in this species, we designed a cross-breeding experiment where a recipient ('female') mated with two sperm donors ('males'). Mates came from either the same population as the recipient or from a distinct one. To test for the influence a recipient can have on the paternity of its offspring, we excluded the effects of dart shooting by using only virgin snails as sperm donors because they do not shoot any dart before their first copulation. We measured the effects of size of mates as well as time to first and second mating on second mate sperm precedence (P2; established using microsatellite markers). Multiple paternity was detected in 62.5% of clutches and overall there was first-mate sperm precedence with a mean P2 of 0.24. Generalized linear modelling revealed that the best predictors of paternity were the time between matings and the time before first mating. Overall, both first and second mates that copulated quickly got greater parentage, which may suggest that postcopulatory events influence patterns of sperm precedence in the garden snail.     

INFERENCE OF SPERM COMPETITION FROM BROODS OF FIELD-CAUGHT DROSOPHILA

In field studies of multiple mating and sperm competition there typically is no experimental control over the number of times that a female mates, the interval between matings, or the genetic identity of multiple fathers contributing to a brood. Irrespective of this complexity, high-resolution molecular markers can be used to assign paternity with considerable confidence. This study employed two highly heterozygous microsatellite loci to assess multiple paternity and sperm displacement in a sample of broods taken from a natural population of Drosophila melanogaster. The large number of alleles present at each of the loci makes it difficult to derive explicit maximum-likelihood estimates for multiple paternity and sperm displacement from brood samples. Monte Carlo simulations were used to estimate maximum-likelihood parameters for the distribution of female remating frequency and the proportion of offspring sired by the second or subsequent mating males. Estimates were made based on genotypes scored at two distinct marker loci because they were found to give statistically homogeneous results. Fitting a Poisson distribution of number of matings, the mean number of males mated by a female was 1.82. The sperm displacement parameter estimated from doubly mated females were 0.79 and 0.86 for the two loci (0.83 for the joint estimate). The overall probability that a multiply mated female will be misclassified as singly mated was only 0.006, which indicates that microsatellites can provide excellent resolution for identifying multiple mating. In addition, microsatellites can be used to generate relatively precise estimates of sperm precedence in brood-structured samples from a natural population.