Polyandrous females provide sons with more competitive sperm: Support for the sexy‐sperm hypothesis in the rattlebox moth (Utetheisa ornatrix)

Given the costs of multiple mating, why has female polyandry evolved? Utetheisa ornatrix moths are well suited for studying multiple mating in females because females are highly polyandrous over their life span, with each male mate transferring a substantial spermatophore with both genetic and nongenetic material. The accumulation of resources might explain the prevalence of polyandry in this species, but another, not mutually exclusive, possibility is that females mate multiply to increase the probability that their sons will inherit more‐competitive sperm. This latter “sexy‐sperm” hypothesis posits that female multiple mating and male sperm competitiveness coevolve via a Fisherian runaway process. We tested the sexy‐sperm hypothesis by using competitive double matings to compare the sperm competition success of sons of polyandrous versus monandrous females. In accordance with sexy‐sperm theory, we found that in 511 offspring across 17 families, the male whose polyandrous mother mated once with each of three different males sired significantly more of all total offspring (81%) than did the male whose monandrous mother was mated thrice to a single male. Interestingly, sons of polyandrous mothers had a significantly biased sex ratio of their brood toward sons, also in support of the hypothesis.     

Microsatellite evidence for sperm storage and multiple paternity in the marine gastropod Crepidula coquimbensis

In gregarious species with copulation and internal fertilization, male-male competition and female cryptic choice may affect reproductive success of both sexes. We carried out a molecular analysis to study paternity and sperm use by females in the protandrous marine brooding gastropod Crepidula coquimbensis. In the field, a single female inhabits an empty hosting shell with up to six males. This gregarious behavior may promote intra-brood multiple paternity if females can store sperm from several consecutive copulations with the surrounding males. To study female sperm usage, the males sharing shelters with five different adult females were collected and preserved for paternity analysis. Females were transported alive to the laboratory and isolated for six months. After that, an additional male was offered to each of the five study females. Once the females had laid capsules, a total of 528 embryos from the five females were assigned paternity based on five microsatellite loci. Paternity analysis showed that every male sharing the empty hosting shell of a female as well as the additional male were assigned fatherhood of embryos laid by this specific female. Females can thus use sperms from multiple males including sperms stored for at least six months. In addition, in four out of the five offspring arrays, a similar contribution of each male to the brood was observed, a pattern associated with the close relationship between the number of fathers observed and the effective paternity index calculated. These results contrast with those of paternity analyses carried out in another species of the same genus, C. fornicata which is characterized by a stacking behavior in which the closest male to the female achieves the highest reproductive success. Male reproductive success may be largely influenced by the aggregation pattern and male mating opportunities in the Crepidula complex, a hypothesis to be examined further by studying other species exhibiting different grouping behavior.     

Sperm Competition in a Viviparous Fish

We investigated multiple paternity and sperm precedence in the Amarillo fish, Girardinichthys multiradiatus (Goodeidae). We allowed females to mate with two different-sized males consecutively and assessed the paternity of the ensuing broods using allozyme electrophoresis. We presented one-half of the females the larger, and the other half the smaller, male first. Allozyme variation among individuals was low, yielding conservative estimates of multiple paternity. Half the broods were of mixed paternity, but one male always sired more than 70% of the embryos in each brood. The proportion of the brood sired was not related to mating sequence, but when we classified males by relative size, the larger male of each pair usually fathered greater proportions of offspring than the smaller male. This association disappeared when we used the actual size of the males in the analysis. Instead, for any pair of males, the difference in number of offspring sired was correlated to differences in the rate of courtship displays, rather than size differences, suggesting that courtship intensity is a better predictor of paternity than male size. Within a pair, the larger male usually displayed more than the smaller one, but there was no correlation between male size and display rate across all males. Parsimony suggests a correlation between courtship rate and sperm production, but we cannot rule out the possibility that females allocate paternity according to the relative merits of the males.     

High prevalence of multiple paternity in the invasive crayfish species,Procambarus clarkii

Reproductive strategy is a central feature of the ecology of invasive species as it determines the potential for population increase and range expansion. The red swamp crayfish, Procambarus clarkii, has invaded many countries and caused serious problems in freshwater ecosystems. However, little is known about the effects of environmental conditions on crayfish paternity and offspring traits in the wild. We studied these reproductive characteristics of P. clarkii in wild populations from two different habitats (ponds and ditches) in three locations with different environmental conditions in China. Genotyping of 1,436 offspring and 30 mothers of 30 broods was conducted by using four microsatellites. An analysis of genotyping results revealed that gravid females were the exclusive mother of the progeny they tended. Twenty-nine of 30 mothers had mated with multiple (2-4) males, each of which contributed differently to the number of offspring in a brood. The average number of fathers per brood and the number of offspring per brood were similar (P > 0.05) among six sampling sites, indicating that in P. clarkii multiple paternity and offspring number per brood are independent of environmental conditions studied. Indirect benefits from increasing the genetic diversity of broods, male and sperm competition, and cryptic female choice are a possible explanation for the high level multiple paternity and different contribution of fathers to offspring in this species.     

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.     

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

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

Strong reproductive skew among males in the multiply mated swordtail Xiphophorus multilineatus (Teleostei)

Male swordtails in the genus Xiphophorus display a conspicuous ventral elongation of the caudal fin, the sword, which arose through sexual selection due to female preference. Females mate regularly and are able to store sperm for at least 6 months. If multiple mating is frequent, this would raise the intriguing question about the role of female choice and male-male competition in shaping the mating system of these fishes. Size-dependent alternate mating strategies occur in Xiphophorus; one such strategy is courtship with a sigmoid display by large dominant males, while the other is gonopodial thrusting, in which small subordinate males sneak copulations. Using microsatellite markers, we observed a frequency of multiple paternity in wild-caught Xiphophorus multilineatus in 28% of families analyzed, but the actual frequency of multiple mating suggested by the correction factor PrDM was 33%. The number of fathers contributing genetically to the brood ranged from one to three. Compared to other species in the family Poeciliidae, both frequency and degree of multiple paternity were low. Paternity was found to be highly skewed, with one male on average contributing more than 70% to the offspring. Hence in this Xiphophorus mating system, typically one male dominates and sneaker males do not appear to be particularly effective. Postcopulatory mechanisms, however, such as sperm competition, are also indicated by our data, using sex-linked phenotypes among the offspring.     

Sperm mobility determines the outcome of sperm competition in the domestic fowl

The aim of this study was to establish whether the mobility of sperm of the domestic fowl, as measured by an in vitro assay, predicted the outcome of sperm competition. Thirteen pairs of New Hampshire roosters, comprising one male categorized as having high-mobility sperm and the other as having average-mobility sperm, were used. Each male provided 25 x 10(6) sperm, which were mixed and artificially inseminated into between four and seven New Hampshire hens, each of which produced 2-11 offspring. The experiment was conducted twice, such that the same pair of males inseminated the same females. Paternity was assigned by using microsatellite markers. There was a clear effect of sperm-mobility phenotype on the outcome of sperm competition: in all 13 pairs the high-mobility male fathered the majority of offspring (75.3% overall; p < 0.0001). The proportion of offspring fathered by the high-mobility male within pairs varied significantly between male pairs (p < 0.0005). This effect was associated with the difference in sperm-mobility scores between males within pairs; there was a significant positive relationship between the proportion of offspring fathered by the high-mobility male and the ratio of mobility scores between males (p < 0.05). In addition, compared with their success predicted from the non-competitive situation, in the competitive situation high-mobility males were disproportionately successful in fertilizing eggs compared with average-mobility males. This may occur because female sperm storage is limited in some way and a greater proportion of high-mobility sperm gain access to the female's sperm storage tubules. There was no evidence that female effects accounted for any of the variation in paternity.     

Reproductive costs of mating with a sibling male: sperm depletion and multiple mating in Cephalonomia hyalinipennis

Polygynous parasitoid males may be limited by the amount of sperm they can transmit to females, which in turn may become sperm limited. In this study, I tested the effect of male mating history on copula duration, female fecundity, and offspring sex ratio, and the likelihood that females will have multiple mates, in the gregarious parasitoid Cephalonomia hyalinipennis Ashmead (Hymenoptera: Bethylidae: Epyrinae), a likely candidate for sperm depletion due to its local mate competition system. Males were eager to mate with the seven females presented in rapid succession. Copula duration did not differ with male mating history, but latency before a first mating was significantly longer than before consecutive matings. Male mating history had no bearing on female fecundity (number of offspring), but significantly influenced offspring sex ratio. The last female to mate with a given male produced significantly more male offspring than the first one, and eventually became sperm depleted. In contrast, the offspring sex ratio of first‐mated females was female biased, denoting a high degree of sex allocation control. Once‐mated females, whether sperm‐depleted or not, accepted a second mating after a period of oviposition. Sperm‐depleted females resumed production of fertilized eggs after a second mating. Young, recently mated females also accepted a second mating, but extended in‐copula courtship was observed. Carrying out multiple matings in this species thus seems to reduce the cost of being constrained to produce only haploid males after accepting copulation with a sperm‐depleted male. I discuss the reproductive fitness costs that females experience when mating solely with their sibling males and the reproductive fitness gain of males that persist in mating, even when almost sperm‐depleted. Behavioural observations support the hypothesis that females monitor their sperm stock. It is concluded that C. hyalinipennis is a species with a partial local mating system.     

Tactic‐specific benefits of polyandry in Chinook salmon Oncorhynchus tshawytscha

This study examined whether polyandrous female Chinook salmon Oncorhynchus tshawytscha obtain benefits compared with monandrous females through an increase in hatching success. Both of the alternative reproductive tactics present in male O. tshawytscha (large hooknoses and small, precocious jacks) were used, such that eggs were either fertilized by a single male (from each tactic) or multiple males (using two males from the same or different tactics). The results show that fertilized eggs from the polyandrous treatments had a significantly higher hatching success than those from the monandrous treatments. It is also shown that sperm speed was positively related with offspring hatching success. Finally, there were tactic‐specific effects on the benefits females received. The inclusion of jacks in any cross resulted in offspring with higher hatching success, with the cross that involved a male from each tactic providing offspring with the highest hatching success than any other cross. This study has important implications for the evolution of multiple mating and why it is so prevalent across taxa, while also providing knowledge on the evolution of mating systems, specifically those with alternative reproductive tactics.     

Potential fitness benefits from mate selection in the Atlantic cod (Gadus morhua)

Little evidence of benefits from female mate choice has been found when males provide no parental care or resources. Yet, good genes models of sexual selection suggest that elaborated male sexual characters are reliable signals of mate quality and that the offspring of males with elaborate sexual ornaments will perform better than those of males with less elaborate ornaments. We used cod (Gadus morhua L.), an externally fertilizing species where males provide nothing but sperm, to examine the potential of optimal mate selection with respect to offspring survival. By applying in vitro fertilizations, we crossed eight females with nine males in all possible combinations and reared each of the 72 sib groups. We found that offspring survival was dependent on which female was mated with which male and that optimal mate selection has the potential to increase mean offspring survival from 31.9 to 55.6% (a 74% increase). However, the size of the male sexual ornaments and sperm quality (i.e. sperm velocity and sperm density) could not predict offspring survival. Thus, even if there may be large fitness benefits of mate selection, we might not yet have identified the male characteristics generating high offspring survival.     

Genetic dissimilarity predicts paternity in the smooth newt (Lissotriton vulgaris)

Under sperm competition, paternity is apportioned by polyandrous females according to the order of matings and the genetic quality of the inseminating males. In order to distinguish between these two effects, we sequentially paired 12 female smooth newts (Lissotriton vulgaris) with each of two males and, where possible, repeated the same procedure in reverse order of the identical males after assumed sperm depletion. For a total of 578 offspring, amplified fragment length polymorphisms genetic markers revealed multiple paternities in all matings, without significant first- or second-male sperm precedence. The paternity share of individual males was transitive across the two trials with male order switch, and successful males had a significantly higher genetic dissimilarity to the female than expected by chance. We argue that patterns of paternity in natural newt populations are determined through a combination of good genes and relatedness.     

Hold on: Females modulate sperm depletion from storage sites in the fly Drosophila melanogaster

Among many species of insects, females gain fitness benefits by producing numerous offspring. Yet actions related to producing numerous offspring such as mating with multiple males, producing oocytes and placing offspring in sub-optimal environments incur costs. Females can decrease the magnitude of these costs by retaining gametes when suitable oviposition sites are absent. We used the pomace fly, Drosophila melanogaster, to explore how the availability of fresh feeding/oviposition medium influenced female fitness via changes in offspring survivorship and the modulation of gamete release. Availability of fresh medium affected the absolute number and temporal production of offspring. This outcome was attributable to both decreased larval survival under crowded conditions and to female modulation of gamete release. Direct examination of the number of sperm retained among the different female storage organs revealed that females ‘hold on’ to sperm, retaining more sperm in storage, disproportionately within the spermathecae, when exposed infrequently to fresh medium. Despite this retention, females with lower rates of storage depletion exhibited decreased sperm use efficiency shortly after mating. This study provides direct evidence that females influence the rate of sperm depletion from specific storage sites in a way that can affect both female and male fitness. The possible adaptive significance of selective gamete utilization by female Drosophila includes lowering costs associated with frequent remating and larval overcrowding when oviposition sites are limiting, as well as potentially influencing paternity when females store sperm from multiple males.     

Genetic breeding system and investment patterns within nests of Dawson's burrowing bee (Amegilla dawsoni) (Hymenoptera: Anthophorini)

Dawson's burrowing bee is a large solitary ground-nesting bee endemic to the arid zone of Western Australia. In this study, we use microsatellite markers to analyse the genotypes of offspring from individual nests to determine the number of effective mates for each female. From these data we have determined that females almost certainly mate only once which is consistent with male reproductive tactics that include protandry and intense male-male competition for access to virgin females. We also use the molecular data to show that the nesting female is the mother of all the offspring of her nest and that brood parasitism is unlikely in this species. The data indicate that females make daughters at the beginning of the season followed by large sons in the middle, and then small sons at the end. Females often place one brood cell directly above another. The distribution of sex and morph in these doublets follows a pattern with most containing a female on the bottom and a minor male on the top, followed by almost equal numbers of female on top of female and minor male on top of major male. This pattern is likely favoured by emergence patterns, with males emerging before females and minor males emerging before major males. We suggest that although minor males have low reproductive success, their production may nonetheless be beneficial in that minor males open up emergence tunnels for their larger and reproductively more valuable siblings. In addition, minor males may be a best of a bad job product arising from changes in the costs to nesting females of gathering brood provisions over the course of the flight season.     

Fitness effects of X chromosome drive in the stalk-eyed fly, Cyrtodiopsis dalmanni

Sex-ratio (SR) males produce predominantly female progeny because most Y chromosome sperm are rendered nonfunctional. The resulting transmission advantage of XSR chromosomes should eventually cause population extinction unless segregation distortion is masked by suppressors or balanced by selection. By screening male stalk-eyed flies, Cyrtodiopsis dalmanni, for brood sex ratio we found unique SR alleles at three X-linked microsatellite loci and used them to determine if SR persists as a balanced polymorphism. We found that XSR/XST females produced more offspring than other genotypes and that SR males had lower sperm precedence and exhibited lower fertility when mating eight females in 24 h. Adult survival was independent of SR genotype but positively correlated with eye span. We infer that the SR polymorphism is likely maintained by a combination of weak overdominance for female fecundity and frequency dependent selection acting on male fertility. Our discovery of two SR haplotypes in the same population in a 10-year period further suggests that this SR polymorphism may be evolving rapidly.     

Why make daughters larger? Maternal sex-allocation and sex-dependent selection for body size in a mass-provisioning wasp, Trypoxylon politum

Mass-provisioning wasps package maternal investment into broodcells, sealed structures that contain all the provisions necessaryfor an offspring's growth and development. Optimal sex-allocationtheory predicts that if maternal provisions determine the sizeof each offspring, and the amount of provisions available toeach offspring varies, females should allocate well-stockedbrood cells to the sex that benefits most from being large.I tested this hypothesis using observations of organ-pipe wasps,Trypoxylon politum, and dissections of their nests. A Mississippipopulation of T. politum was intensively studied from 1993 to1995. This population fit the assumptions of optimal sex-allocationmodels by Green and Brockmann and Grafen. Female weight at emergencewas 1.29 times that of males, and wing length was 1.15 timesthat of males. This discrepancy in size occurred because thevolume of parental provisions strongly influenced adult bodysize, and better-stocked brood cells were preferentially allocatedto daughters. Brood-cell volume correlated with both wing lengthand weight at emergence in both sexes, and the chance that agiven brood cell contained a female offspring increased withincreasing brood-cell volume. Fitness was positively relatedto body size for females, but I found no evidence of an advantageto large males. Although there was evidence of stabilizing selectionfor male wing length in one year, there was no evidence of anincreasing relationship between body size and fitness (directionalselection) for males in either 1993 or 1994. Female fecunditywas positively related to body size in both years, indicatingthat larger females have increased reproductive success. Therate at which females provisioned brood cells was also correlatedwith body size. Observed patterns of investment in brood cellsare quantitatively consistent with the predictions of optimalsex-allocation theory, but certain aspects of female provisioningbehavior suggest females are not following a single "optimal"strategy. Patterns of provisioning were variable among differentfemales at the study site during the same year. Large femalestended to produce larger offspring. Although Brockmann and Grafen'smodel predicts a single, population wide "switchpoint" fromthe production of male to female offspring, there was no evidencefor such a switchpoint     

Late‐breeding Great Cormorants Phalacrocorax carbo sinensis produce fewer young of the more vulnerable sex

We examined the brood sex ratio and offspring body mass in relation to the timing of breeding and brood size in the Great Cormorant Phalacrocorax carbo sinensis. The brood sex ratio was not related to brood size but it was significantly related to the hatching date, with a decreasing proportion of males in the brood in the course of the season. Male chicks had significantly lower body mass if they hatched later in the season, whereas there was no such relationship for female offspring. Assuming that environmental conditions deteriorate with progress of the breeding season, and male offspring may be more vulnerable to poor environmental conditions, the observed decline in the proportion of male offspring late in the season may be adaptive.     

Patterns of male reproductive success in Crepidula fornicata provide new insight for sex allocation and optimal sex change

The size-advantage model and sex-allocation theory are frequently invoked to explain the evolution and maintenance of sequential hermaphroditism in many taxa. A test of current theory requires quantitative estimates of reproductive success and knowledge of the relationship between reproduction and size for each gender. Reproductive success can be difficult to measure. In species where polyandry occurs, it can be quantified only by determining paternity of offspring. We employed microsatellite loci to establish paternity for 12 families of Crepidula fornicata, where a family is defined as a single female, her brood, and the males stacked on top of her. Genetic data were analyzed and paternity was assigned to a single potential father for more than 83% of the offspring tested. Estimates of reproductive success revealed that one male within the family fathered the majority of offspring and that he was usually the largest male and the one closest to the brooding female. The dominant male's success also tended to decrease as the number of mature males within the family increased. Our results suggest that sperm competition could be a driving force in determining male reproductive success and the timing of sex change in C. fornicata.     

17alpha-ethinylestradiol reduces the competitive reproductive fitness of the male guppy (Poecilia reticulata)

Whether endocrine disruption in an individual male is actually translated into reduced reproductive success in a natural competitive environment is extremely difficult to predict. Here, we have used paternity analysis to provide new information on the ability of an endocrine disruptor to deleteriously affect male guppy reproductive fitness by including the effect of intermale competition. Groups of male guppies were exposed to 10.5, 44.4, or 112 ng/L of the synthetic estrogen 17alpha-ethinylestradiol (EE2) from birth to adulthood. Subsequently, an exposed male competed against an unexposed male for the opportunity to fertilize a receptive female. The successful males siring the majority of the offspring in each brood were then identified using microsatellites in genetic paternity analysis. Only the highest dose of EE2 produced harmful effects with a significantly female-biased sex ratio, significant reductions in male sperm count, testis weight, body coloration and courtship behavior, and a significant increase in body size. These feminizing effects were translated into a highly significant reduction in fertility, where only 1 of the 17 exposed males sired offspring in competition with unexposed males. The evidence suggests that EE2-treated males have reduced reproductive fitness compared with untreated males, possibly the result of EE2 effects on multiple fitness traits. To our knowledge, this is the first study providing evidence of endocrine disruption at the population level that has included the ecologically highly relevant effect of sexual competition on male reproductive fitness.     

Reproductive strategies of the larval parasitoid Microplitis croceipes

Mate choice may have important consequences for offspring sex ratio and fitness of haplodiploid insects. Mate preference of females of the solitary larval parasitoid Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) for virgin and mated males, and vice versa, and the reproductive consequences (i.e., the sex ratio expressed as the proportion of male offspring) were examined in choice and non‐choice experiments. In addition, the effect of repeated rapid and daily copulation of an individual male on the sex ratio of offspring of the female mates was assessed. Males preferred virgins over mated females, whereas females copulated with a male irrespective of his mating status. In both the rapid and daily copulation assay, females copulating with a male that had copulated five times or more produced a higher sex ratio than females that had copulated with a virgin male. Females that copulated with virgin males once or twice produced a significantly and considerably lower sex ratio than females that first copulated with a sperm‐depleted male followed by a virgin male. This indicates that copulating with a sperm‐depleted male has costs and limits acquisition by the female of sperm from virgin males.