Measuring polyandry in wild populations: a case study using promiscuous crickets

Mating rate has important implications for patterns of sexual selection and sexual conflict and hence for issues such as speciation and the maintenance of genetic diversity. Knowledge of natural mating rates can provide insights into the factors driving female mating behaviour. We investigated the level of polyandry in a Spanish population of the field cricket Gryllus bimaculatus using microsatellite markers. Two approaches were employed: (i) genotyping the offspring of wild-caught gravid females to determine the number of males siring the brood and (ii) genotyping sperm stored in the spermathecae of females mated in the wild to estimate the number of mating partners. We compared existing methods for inferring the minimum and probable number of fathers and described a novel probabilistic technique estimating the number of mates by genotyping stored sperm. Using the most conservative allele-counting method, 71% of females produced offspring sired by at least two males (a minimum mean of 2.4 fathers per clutch), and all females had mated to at least two males with minimum mean estimates of 2.7-5.1 mates per female. Our study reveals high levels of polyandry in the wild and suggests that females mate with more males than sire their offspring.     

Fertility assurance through extrapair fertilizations and male paternity defense

Extrapair paternity has been observed in many formally monogamous species. Male pursuit of extrapair fertilizations is explained by the advantages of having offspring that receive essential paternal care from other males. Since females are capable of exercising a degree of control over the post-copulatory sperm competition, extrapair paternity cannot persist unless it confers fitness benefits on cuckolding females. Thus, extrapair paternity involves cooperation between mated females and extrapair males. On the other hand, paired males frequently exhibit strategies that minimize their loss of paternity and/or conserve paternal investment if paternity is lost. Hence, extrapair attributes of diverse species and populations reported in the literature are particular solutions of evolutionary games involving gender-specific cuckolding/anti-cuckolding strategies. Here we use methods of evolutionary game theory to study the role of male paternity guarding strategies in situations where females seek extrapair fertilizations for reasons of genetic compatibility and/or in pursuit of genetic diversity for their offspring. Our results indicate that in these circumstances pursuit of extrapair fertilizations is the only evolutionary stable female strategy. Males, on the other hand, have two, mutually exclusive, evolutionary stable strategies: full time pursuit of extrapair fertilizations and a compromise strategy wherein they protect in-pair paternity during their mate's fertile periods and pursue extrapair paternity the rest of the time. The relative merits of these two strategies are determined by the efficiency of male in-pair paternity defense, breeding synchrony, fitness advantages of extrapair over in-pair offspring, and the intensity of competition for extrapair fertilizations from floater males.     

Influence of female reproductive anatomy on the outcome of sperm competition in Drosophila melanogaster

Females as well as males can influence the outcome of sperm competition, and may do so through the anatomy of their reproductive tracts. Female Drosophila melanogaster store sperm in two morphologically distinct organs: a single seminal receptacle and, normally, two spermathecae. These organs have different temporal roles in sperm storage. To examine the association between sperm storage organ morphology and sperm competition, we used a mutant type of female with three spermathecae. Although the common measure of sperm competition, P(2), did not differ between females with two and three spermathecae, the pattern of sperm use over time indicated that female morphology did affect male reproductive success. The rate of offspring production by females with three spermathecae rose and fell more rapidly than by females with two spermathecae. If females remate or die before using up second male sperm, then second male reproductive success will be higher when they mate with females with three spermathecae. The results indicate that temporal patterns of sperm use as well as P(2) should be taken into account when measuring the outcome of sperm competition.     

Cryptic female choice in the yellow dung fly Scathophaga stercoraria (L.)

Both female choice and male-male competition may take place during reproduction in many species. Female choice tends to be less obvious than male-male competition and consequently has received less attention from researchers. The opportunity for cryptic female choice arises after multiple insemination. Through postcopulatory processes, a female could alter the pattern of paternity among her offspring so that it does not directly reflect the different contributions of sperm made by her mates. To be able to determine if a female alters the relative sperm contributions of her mates, the behaviors and influences of the males must therefore be first taken into account. The interest of each male is to father all the offspring, and the interest of each female is to maximize paternal quality. Female yellow dung flies have complex internal reproductive tracts that may give them considerable control over the fertilization success of stored sperm from different males. In laboratory trials to date, the last male to mate has usually been most successful. In the present study, cryptic choice occurred in Scathophaga stercoraria and the pattern of choice was consistent with previously reported results. The fertilization success of a female's second mate (P2) was substantially larger if a female was kept at constant temperature and if the second male was genetically similar to her at the phosphoglucomutase (Pgm) locus. Females from the field normally have three spermathecae, but some have four. Lines were bred to have either three or four spermathecae. Flies from the different lines were crossed to generate females with similar genetic backgrounds that had either three or four spermathecae. P2 was significantly lower for high-quality females, that is, those that laid larger-than-average-clutches, with four spermathecae than for low-quality females with four spermathecae; female quality had no influence on P2 for females with three spermathecae. The results suggest that only large females may benefit from increased spermathecae number by being able to act against male interests. Females may only have three spermathecae, even though genetic variation for more is present, because selection for more spermathecae is weak.     

SPERM MIXING IN THE COCKROACH DIPLOPTERA PUNCTATA

Females of the viviparous cockroach Diploptera punctata store sperm from their first mating, and do not remate until after giving birth to their first batch of young. The irradiated male technique was used to determine the outcome of sperm competition in the second batch of eggs of females mated sequentially to normal and irradiated males. It is estimated that the second male to mate with a female fertilizes approximately two thirds of the eggs in a female's second batch of eggs. Direct evidence for sperm mixing was obtained. Undeveloped eggs (fertilized by irradiated sperm) and developing embryos (fertilized by normal sperm) were found interspersed throughout oothecae that were extruded from females, demonstrating that normal and irradiated sperm were released from the spermathecae at oviposition and that they competed for fertilizations.     

Reproductive Benefits from Female Promiscuous Mating in a Small Mammal

Promiscuous systems where both males and females mate several times with different individuals are widespread among mammalian species. As a consequence, females obtain sperm from more than one male and paternity is decided by sperm competition. In theory, females might gain ‘genetic benefits’ for their offspring from this mechanism. In a mating experiment we now demonstrate in the promiscuous rodent Galea musteloides that females which were paired with four males, and became pregnant, weaned more surviving offspring than females which were paired with a single male. Litter sizes did not differ between the two groups. The data support the hypothesis that promiscuous females copulate with several males to induce sperm competition and/or to enforce cryptic female choice and thereby increase the viability of their offspring.     

Male age and its association with reproductive traits in captive and wild house sparrows

Evolutionary theory predicts that females seek extra‐pair fertilizations from high‐quality males. In socially monogamous bird species, it is often old males that are most successful in extra‐pair fertilizations. Adaptive models of female extra‐pair mate choice suggest that old males may produce offspring of higher genetic quality than young males because they have proven their survivability. However, old males are also more likely to show signs of reproductive senescence, such as reduced sperm quality. To better understand why old males account for a disproportionally large number of extra‐pair offspring and what the consequences of mating with old males are, we compared several sperm traits of both captive and wild house sparrows, Passer domesticus. Sperm morphological traits and cloacal protuberance volume (a proxy for sperm load) of old and young males did not differ substantially. However, old males delivered almost three times more sperm to the female's egg than young males. We discuss the possibility of a post‐copulatory advantage for old over young males and the consequences for females mated with old males.     

A cost of cryptic female choice in the yellow dung fly

Female dung flies Scathophaga stercoraria (L.) store sperm from several males in three or four spermathecae. Selection on the number of spermathecae was successful and the morphological intermediate stages in the evolution from three to four spermathecae are illustrated. The genetic quality of a male from a female's perspective depends on an interaction between their genotypes and the microhabitat in which the offspring will grow. Females influence the paternity pattern of their offspring, and do this differently in different microhabitats. Females with four spermathecae are better able to influence paternity than are those with three spermathecae. However, there must be a cost to building and maintaining an extra spermatheca. We estimate, using the animal model on pedigree data, that this cost is approximately five eggs per clutch, i.e. around 8% of the mean clutch size. This is a substantial cost and such costs should not be ignored in discussions of the benefits to females of assessing the genetic qualities of their mating partners. We suggest that the number of spermathecae in the study population is stable because the relative benefits in quality of offspring through cryptic female choice is balanced by the costs in total numbers of offspring.     

Female extrapair mate choice in a cooperative breeder: trading sex for help and increasing offspring heterozygosity

Sexual conflict between males and females over mating is common. Females that copulate with extrapair mates outside the pair-bond may gain (i) direct benefits such as resources or increased paternal care, (ii) indirect genetic benefits for their offspring, or (iii) insurance against infertility in their own social mate. Few studies have been able to demonstrate the different contexts in which females receive varying types of benefits from extrapair mates. Here, I examined sexual conflict, female extrapair mate choice, and patterns of extrapair paternity in the cooperatively breeding superb starling Lamprotornis superbus using microsatellite markers. Although extrapair paternity was lower than many other avian cooperative breeders (14% of offspring and 25% of nests), females exhibited two distinct mating patterns: half of the extrapair fertilizations were with males from inside the group, whereas half were with males from outside the group. Females with few potential helpers copulated with extrapair mates from within their group and thereby gained direct benefits in the form of additional helpers at the nest, whereas females paired to mates that were relatively less heterozygous than themselves copulated with extrapair mates from outside the group and thereby gained indirect genetic benefits in the form of increased offspring heterozygosity. Females did not appear to gain fertility insurance from copulating with extrapair mates. This is the first study to show that individuals from the same population mate with extrapair males and gain both direct and indirect benefits, but that they do so in different contexts.     

MHC‐dependent survival in a wild population: evidence for hidden genetic benefits gained through extra‐pair fertilizations

Females should prefer to be fertilized by males that increase the genetic quality of their offspring. In vertebrates, genes of the major histocompatibility complex (MHC) play a key role in the acquired immune response and have been shown to affect mating preferences. They are therefore important candidates for the link between mate choice and indirect genetic benefits. Higher MHC diversity may be advantageous because this allows a wider range of pathogens to be detected and combated. Furthermore, individuals harbouring rare MHC alleles might better resist pathogen variants that have evolved to evade common MHC alleles. In the Seychelles warbler, females paired with low MHC‐diversity males elevate the MHC diversity of their offspring to levels comparable to the population mean by gaining extra‐pair fertilizations. Here, we investigate whether increased MHC diversity results in higher life expectancy and whether there are any additional benefits of extra‐pair fertilizations. Our 10‐year study found a positive association between MHC diversity and juvenile survival, but no additional survival advantage of extra‐pair fertilizations. In addition, offspring with a specific allele (Ase‐ua4) had a fivefold longer life expectancy than offspring without this allele. Consequently, the interacting effects of sexual selection and pathogen‐mediated viability selection appear to be important for maintaining MHC variation in the Seychelles warbler. Our study supports the prediction that MHC‐dependent extra‐pair fertilizations result in genetic benefits for offspring in natural populations. However, such genetic benefits might be hidden and not necessarily apparent in the widely used fitness comparison of extra‐ and within‐pair offspring.     

Copulation patterns in the golden orb-web spider Nephila madagascariensis

A consequence of multiple mating by females can be that the sperm of two or more males directly compete for the fertilisation of ova inside the female reproductive tract. Selection through sperm-competition favours males that protect their sperm against that of rivals and strategically allocate their sperm, e.g., according to the mating status of the female and the morphology of the spermatheca. In the majority of spiders, we encounter the otherwise unusual situation that females possess two independent insemination ducts, both ending in their own sperm storage organ, the spermatheca. Males have paired mating organs, but generally can only fill one spermatheca at a time. We investigated whether males of the African golden orb-web spider Nephila madagascariensis can prevent rival males from mating into the same spermatheca and whether the mating status of the female and/or the spermatheca causes differences in male mating behaviour. There was no significant difference in the duration of copulations into unused spermathecae of virgin and mated females. We found that copulations into previously inseminated spermathecae were generally possible, but shorter than copulations into the unused side of mated females or with virgins. Thus, male N. madagascariensis may have an advantage when they mate with virgins, but cannot prevent future males from mating. However, in rare instances, parts of the male genitals can completely obstruct a female genital opening.     

Shorter sperm confer higher competitive fertilization success

Spermatozoa exhibit taxonomically widespread patterns of divergent morphological evolution. However, the adaptive significance of variation in sperm morphology remains unclear. In this study we examine the role of natural variation in sperm length on fertilization success in the dung beetle Onthophagus taurus. We conducted sperm competition trials between males that differed in the length of their sperm and determined the paternity of resulting offspring using amplified fragment length polymorphism (AFLP) markers. We also quantified variation in the size and shape of the female's sperm storage organ to determine whether female morphology influenced the competitiveness of different sperm morphologies. We found that fertilization success was biased toward males with relatively shorter sperm, but that selection on sperm length was dependent on female tract morphology; selection was directional for reduced sperm length across most of the spermathecal size range, but stabilizing in females with the smallest spermathecae. Our data provide empirical support for the theory that sperm competition should favor the evolution of numerous tiny sperm. Moreover, because sperm length is both heritable and genetically correlated with condition, our results are consistent with a process by which females can accrue genetic benefits for their offspring from the incitement of sperm competition and/or cryptic female choice, as proposed by the "sexy sperm" and "good sperm" models for the evolution of polyandry.     

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

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

Wild yellow dung fly females may not select sperm based on dung pat microclimate but could nevertheless benefit from polyandry

Molecular techniques have substantially improved our knowledge of postcopulatory sexual selection. Nevertheless, studies examining sperm utilization in natural populations of nonsocial insects are rare, support for sperm selection (biased use of stored sperm, e.g. to match offspring genotypes to prevailing environmental conditions) is elusive, and its relevance within natural populations unknown. We performed an oviposition site choice experiment in the field where female yellow dung flies Scathophaga stercoraria could deposit eggs into three different microenvironments on a dung pat (the east–west ridge, north- or south-exposed side), and genotyped the offspring and sperm remaining in storage after oviposition. Females exhibited plasticity in the number of eggs deposited according to pat age. Additionally, temperature strongly influenced egg placement: the warmer the temperature, the higher the proportion of eggs laid into the north-exposed side of dung. The number of ejaculates in storage differed amongst spermathecae, and females stored sperm from more males than fathered their offspring (2.11 sires vs. 2.84 males within sperm stores). Mean last male paternity was 83.4%, roughly matching previous laboratory estimates. Importantly, we found no evidence that females selectively lay eggs of different genotypes, by biasing paternity towards certain males, depending on offspring’s microclimate. Thus, while we show female choice over number of eggs and where these are deposited, there was no evidence for sperm selection. We further revealed positive effects of multiple mating on total number of offspring and proportion of offspring emerging from the dung. We argue that the integration of field studies and laboratory experiments is essential to promote our understanding of polyandry and cryptic female choice.     

Fertility assurance through extrapair fertilization,and male parental effort

Extrapair paternity (EPP) has been observed in many formally monogamous species. Male pursuit of extrapair fertilizations (EPF) is explained by the advantages of having offspring that receive essential paternal care from other males. Because females are capable of exercising a degree of control over the post-copulatory sperm competition, EPP’s persistence indicates that females benefit from EPF. Thus, EPP involves cooperation between mated females and extrapair males. On the other hand, mated males exhibit a spectrum of anti-cuckolding strategies. Hence, extrapair attributes of diverse species and populations reported in the literature are particular solutions of evolutionary games involving gender-specific cuckolding/anti-cuckolding strategies. Here we use game theoretical methods to study the effect of male paternal effort conserving strategies in situations where females seek EPF for reasons of genetic compatibility and/or in pursuit of genetic diversity for their offspring. Our results indicate that in these circumstances pursuit of EPF is the only evolutionary stable female strategy. Males, on the other hand, have two, mutually exclusive, evolutionary stable strategies: males that restrict parental care regardless of their mate’s fidelity, and males that never restrict parental care. That is, when females seek EPF for reasons of fertility assurance and/or genetic diversity, the conditional male strategy—therein the male’s parental efforts are based on his certainty of paternity—loses in competition with the unconditional strategies.     

Genetic similarity, extrapair paternity, and offspring quality in Savannah sparrows (Passerculus sandwichensis)

The occurrence of extrapair paternity (EPP) in birds is oftenattributed to the action of good-genes sexual selection wherebyfemales "trade up" on male genetic quality by allocating fertilizationsto males with better genes than those possessed by their socialmate. To date, most studies of EPP in birds focus on absolutemeasures of male quality as a criterion for female choice, althoughmultiple mating by females in other taxa is more commonly ascribedto benefits associated with the individual optimization of offspringgenotypes. Here, we examine whether the genetic similarity ofsocial mates predicts female mating patterns in a populationof Savannah sparrows (Passerculus sandwichensis) where as manyas 70% of adults produce extrapair young (EPY). We considerthe influence of genetic similarity across all stages of a female'sdecision-making process, from pair formation through the decisionto produce EPY, to the allocation of fertilizations to specificextrapair sires. In a 4-year study of 175 males, 206 females,and 506 offspring, females were more likely to produce EPY whenpaired to genetically similar males, but they did not appearto be influenced by the size, age, mass, individual heterozygosity,and genetic diversity of their social mates. In paired comparisons,females were almost twice as likely to decrease their geneticsimilarity to males when producing EPY as they were to increaseit. Nonetheless, females did not select especially dissimilarmales when mating outside the pair-bond nor did they pair disassortativelywith respect to genetic similarity. Relative measures of malequality may influence mating patterns in birds, but only atsome points in a female's decision-making process.     

Multiple sperm storage organs facilitate female control of paternity

It has been proposed that multiple sperm storage organs (spermathecae) could allow polyandrous females to control paternity. There is little conclusive evidence for this since insemination of individual spermathecae is generally not experimentally manipulable. Here, we examined sperm use patterns in the Australian redback spider (Latrodectus hasselti), which has paired, independent spermathecae. We assessed paternity when two rivals were forced to inseminate a single storage organ or opposite storage organs. When males inseminated a single spermatheca, mean paternity of the female's first mate was 79.8% (median 89.4%), and 38% of first mates achieved 100% paternity. In contrast, when males inseminated opposite organs, the mean paternity of the first mate was 49.3% (median 49.9%), only 10% of males achieved complete precedence, and paternity was normally distributed, suggesting sperm mixing. Males responded to this difference by avoiding previously inseminated female reproductive tracts. Complete sperm precedence can only be achieved if females permit males to copulate with both reproductive tracts. Females often cannibalize smaller males during their first copulation, thus limiting their paternity to 50%. These data show that multiple sperm storage organs can increase female control of paternity.     

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.     

Female-mediated differential sperm storage in a fly with complex spermathecae, Scatophaga stercoraria

Multiple spermathecae potentially allow selective sperm use, provided that sperm from rival males are stored differentially, that is, in different proportions across storage compartments. In the yellow dung fly, Scatophaga stercoraria, females have three spermathecae arranged as a doublet and singlet. To test whether females store the sperm of rival males actively and differentially, we mated fixed male pairs to three females. After copulation, females were (1) dissected immediately before they could start laying a clutch of eggs, (2) left awake for 30 min but prevented from oviposition, or (3) anaesthetized with carbon dioxide for 30 min to interfere with the muscular control presumably required for sperm transport from the site of insemination to the spermathecae. For each female, we estimated the proportion of the second male's sperm stored in her spermathecae (S(2)value), using sperm length as a male marker. After copulation, the S(2)values in the singlet and doublet spermathecae differed significantly, indicating differential sperm storage during copulation. Postcopulatory treatment affected differential sperm storage significantly. Females dissected immediately had lower S(2)values in the doublet than in the singlet spermatheca, while females left awake showed the reverse pattern for the same two males. This reversal did not occur when females were treated with carbon dioxide. The results indicate differential storage of sperm from different males during copulation and that female muscular activity can affect storage and separation of competing ejaculates beyond copulation. Copyright 2000 The Association for the Study of Animal Behaviour.     

Female and Male Moths Display Different Reproductive Behavior when Facing New versus Previous Mates

Multiple mating allows females to obtain material (more sperm and nutrient) and/or genetic benefits. The genetic benefit models require sperm from different males to fertilize eggs competitively or the offspring be fathered by multiple males. To maximize genetic benefits from multiple mating, females have evolved strategies to prefer novel versus previous mates in their subsequent matings. However, the reproductive behavior during mate encounter, mate choice and egg laying in relation to discrimination and preference between sexes has been largely neglected. In the present study, we used novel and previous mate treatments and studied male and female behavior and reproductive output in Spodoptera litura. The results of this study do not support the sperm and nutrient replenishment hypotheses because neither the number of mates nor the number of copulations achieved by females significantly increased female fecundity, fertility and longevity. However, females showed different oviposition patterns when facing new versus previous mates by slowing down oviposition, which allows the last male has opportunities to fertilize her eggs and the female to promote offspring diversity. Moreover, females that have novel males present called earlier and more than females that have their previous mates present, whereas no significant differences were found on male courtship between treatments. These results suggest that S. litura females can distinguish novel from previous mates and prefer the former, whereas males generally remate regardless of whether the female is a previous mate or not. In S. litura, eggs are laid in large clusters and offspring competition, inbreeding and disease transfer risks are thus increased. Therefore, offspring diversity should be valuable for S. litura, and genetic benefits should be the main force behind the evolution of female behavioral strategies found in the present study.