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.     

Multiple Mating Increases Fecundity, Fertility and Relative Clutch Mass in the Female Leopard Gecko (Eublepharis macularius)

Females across many taxa may mate with several males or mate more than once with the same male within one reproductive event. Although many researchers have discussed the effects of multiple mating on reproductive success of females, few studies have attempted to disentangle whether the reproductive success of females differs with respect to whether females mate with multiple males or mate more than once with one male. In this study, we hypothesized that female leopard geckos (Eublepharis macularius) increase aspects of their reproductive success, such as fecundity, fertility and relative clutch mass, by mating more than once within one reproductive event, either by mating repeatedly with the same male or multiply mating with different males. We controlled for the potentially confounding variables of mating frequency and mate number by allowing females to mate once with one male, twice with the same male, or twice with two different males. We found that females that mated with more than one male laid more clutches, exhibited increased egg fertility and invested more in clutches relative to females that mated only once with one male, whereas females that mated twice to the same male were intermediate for these variables. Thus, reproductive success is higher among female leopard geckos that mated with more than one male compared to female leopard geckos that mated only once.     

Genetic parentage assessment in the crayfish Orconectes placidus,a high-fecundity invertebrate with extended maternal brood care

Microsatellite data have recently been introduced in the context of genetic maternity and paternity assignments in high-fecundity fish species with single-parent-tended broods. Here we extend such analyses to an aquatic invertebrate, the crayfish Orconectes placidus, in which gravid females carry large numbers of offspring. Genetic parentage analyses of more than 900 progeny from 15 wild crayfish broods revealed that gravid females were invariably the exclusive dams of the offspring they tended (i.e. there was no allomaternal care), and that most of the females had mated with multiple (usually two) males who contributed sometimes highly skewed numbers of offspring to a brood. Within any multiply sired brood, the unhatched eggs (or the hatched juveniles) from different fathers were randomly distributed across the mother's brood space. All of these genetic findings are discussed in the light of observations on the mating behaviours and reproductive biology of crayfish.     

Single paternity of clutches and sperm storage in the promiscuous green turtle (Chelonia mydas)

Paternity of 22 green turtle ( Chelonia mydas ) clutches from 13 females of the southern Great Barrier Reef breeding population was determined through microsatellite analyses at five loci, including the analysis of successive clutches for nine of the females. A large number of alleles per locus (10–40) provided probabilities of detecting multiple paternity that were quite high, particularly at all loci combined (99.9%). Although green turtles are promiscuous breeders and there was an expectation of finding extensive multiple paternity, only two clutches were multiply sired and, in these, very few eggs had been fertilized by a secondary male. The rarity of multiple paternity may reflect either a low proportion of multiple matings by females in this population, or sperm competition, possibly resulting from a first-male sperm preference. Additionally, the analysis of > 900 offspring provided data on mutations, which included 20 mutation events that were observed in 27 offspring and involved both maternal and paternal lineages. Most mutations ( n = 16) occurred at a single highly variable locus and their presence emphasizes the need to use multiple loci in paternity studies.     

Single and multiple mating reduces longevity of female dumpling squid (Euprymna tasmanica)

For many species, mating is a necessary yet costly activity. The costs involved can have an important influence on the evolution of life histories and senescence. Females of many species mate multiply, and this behaviour can inflict a longevity cost. Most studies investigating the effects of multiple mating on female survival have been conducted on insects, and the effects in other taxa are largely unknown. We investigate the effects of both a single mating and a second mating on longevity in female dumpling squid (Euprymna tasmanica), a species in which both sexes mate multiply. Through comparing the longevity of virgin, once‐mated and twice‐mated females, we found that a single mating reduced female life span by 15 days on average. A second mating resulted in an additional 8 day (on average) longevity cost, despite no difference in total clutch mass, number of clutches, single egg mass or number of eggs per clutch between once‐mated and twice‐mated females. This demonstrates a cost to multiple mating which may be independent of the cost of egg production. Furthermore, total clutch mass and female life span were positively correlated, whereas female life span decreased with increasing average water temperature. The presence of an additive effect of reproduction on longevity suggests that multiple mating in cephalopods may have benefits that outweigh these costs, or that there is a conflict in optimal mating frequency between males and females.     

Multiyear multiple paternity and mate fidelity in the American alligator, Alligator mississippiensis

We examined multiple paternity during eight breeding events within a 10-year period (1995–2005) for a total of 114 wild American alligator nests in Rockefeller Wildlife Refuge in south-west Louisiana. Our goals included examining (i) within population variation in multiple paternity among years, (ii) variation in multiple paternity in individual females and (iii) the potential for mate fidelity. To accomplish this, in the current study, eggs were sampled from 92 nests over 6 years and analysed along with 22 nests from a previous 2-year study. Genotypes at five microsatellite loci were generated for 1802 alligator hatchlings. Multiple paternity was found in 51% of clutches and paternal contributions to these clutches were highly skewed. Rates of multiple paternity varied widely among years and were consistently higher in the current study than previously reported for the same population. Larger females have larger clutches, but are not more likely to have multiply sired nests. However, small females are unlikely to have clutches with more than two sires. For 10 females, nests from multiple years were examined. Seven (70%) of these females exhibited long-term mate fidelity, with one female mating with the same male in 1997, 2002 and 2005. Five females exhibiting partial mate fidelity (71%) had at least one multiple paternity nest and thus mated with the same male, but not exclusively. These patterns of mate fidelity suggest a potential role for mate choice in alligators.     

Repeated mating with the same male increases female longevity and fecundity in a polyandrous leaf beetle Galerucella birmanica (Coleoptera: Chrysomelidae)

Female multiple mating (polyandry) is widespread across Insecta, even if mating can be costly to females. To explain the evolution and maintenance of polyandry, several hypotheses, mainly focusing on the material (direct) and/or the genetic (indirect) benefits, have been proposed and empirically tested in many species. Considering only the direct benefits, repeatedly‐mated females are expected to exhibit the same fitness as multiply‐mated females under the same mating frequency. In the present study, we compare the fitness of females received monandrous repeated mating (MM) and polyandrous multiple mating (PM) in a polyandrous leaf beetle Galerucella birmanica and assess female mate preference with regard to polyandry or monandry. Our data indicate that the longevity and the egg‐laying duration of MM females are significantly longer than that of PM females. MM females produce significantly more hatched eggs than PM females over their lifetime under the same mating frequency, which results from the high hatching rate of eggs produced by MM females. PM females mated with novel virgin males in the second mating suffer decreased longevity and lifetime fecundity compared with PM females mated with novel mated males in the second mating. Once‐mated females are more likely to re‐mate with familiar males than novel males. By contrast to expectations, the results of the present study suggest that repeated mating provides females with more direct benefits than multiple mating in G. birmanica, and females prefer to re‐mate with familiar males. The possible causes of this finding are discussed.     

Genetic monandry in 6 viviparous species of true sea snakes

Using a suite of 10 highly variable microsatellite loci, we conducted genetic paternity analyses for 76 embryos in the broods of 12 pregnant females representing 6 viviparous species of true sea snakes (Hydrophis clade) in the family Elapidae. To our surprise, we uncovered no evidence for multiple paternity within any of the clutches despite the fact that the genetic markers showed high intraspecific heterozygosities and as many as 20 conspecific alleles per locus. This outcome stands in sharp contrast to the rather high (but also variable) frequency of multiple paternity previously reported in many other reptilian species. However, because our study appears to be the first assessment of genetic parentage for any sea snake species (and indeed for any member of the elapid clade), we can only speculate as to whether this apparent monandry by females is a broader phylogenetic characteristic of elapid snakes or whether it might relate somehow to the sea snakes' peculiar lifestyle that uniquely combines viviparity with a marine existence.     

Insights into the mating habits of the tiger salamander (Ambystoma tigrinum tigrinum) as revealed by genetic parentage analyses

Among urodeles, ambystomatid salamanders are particularly amenable to genetic parentage analyses because they are explosive aggregate breeders that typically have large progeny arrays. Such analyses can lead to direct inferences about otherwise cryptic aspects of salamander natural history, including the rate of multiple mating, individual reproductive success, and the spatial distribution of clutches. In 2002, we collected eastern tiger salamander (Ambystoma tigrinum tigrinum) egg masses (> 1000 embryos) from a approximately 80 m linear transect in Indiana, USA. Embryos were genotyped at four variable microsatellite loci and the resulting progeny array data were used to reconstruct multilocus genotypes of the parental dams and sires for each egg mass. UPGMA analysis of genetic distances among embryos resolved four instances of egg mass admixture, where two or more females had oviposited at exactly the same site resulting in the mixing of independent cohorts. In total, 41 discrete egg masses were available for parentage analyses. Twenty-three egg masses (56%) consisted exclusively of full-siblings (i.e. were singly sired) and 18 (44%) were multiply sired (mean 2.6 males/clutch). Parentage could be genetically assigned to one of 17 distinct parent pairs involving at least 15 females and 14 different males. Reproductive skew was evident among males who sired multiply sired clutches. Additional evidence of the effects of sexual selection on male reproductive success was apparent via significant positive correlations between male mating and reproductive success. Females frequently partitioned their clutches into multiple discrete egg masses that were separated from one another by as many as 43 m. Collectively, these data provide the first direct evidence for polygynandry in a wild population of tiger salamanders.     

Multiple paternity in Ixodes ricinus (Acari: Ixodidae), assessed by microsatellite markers

This investigation examines multiple paternity in Ixodes ricinus (Acari: Ixodidae). Previous studies have shown that multiple mating occurs in this tick, but this is the first evaluation of multiple paternity. Three family groups were examined by a panel of polymorphic microsatellite loci; all ticks were bred from wild-collected engorged females with a copulating male attached. For most larvae, the attached males could be excluded as possible sire, and in the 3 tested families, at least 2 of 3 females mated successfully with more than 1 male. This finding suggests that multiple paternity is a common reproductive strategy in I. ricinus, which may have consequences for the ticks' dispersal success by increasing the genetic diversity in broods from single females colonizing new sites.     

Microsatellite analysis of female mating behaviour in lek-breeding sage grouse

We used microsatellite DNA markers to genotype chicks in 10 broods of lek-breeding sage grouse, Centrocercus urophasianus, whose mothers' behaviour was studied by radio-tracking and observing leks. Previous behavioural studies suggested that almost all matings are performed by territorial males on leks and that multiple mating is rare. Two broods (20%) were sired by more than one male. Genetic analyses of the broods of eight females that visited an intensively studied lek were consistent with behavioural observations. Four females observed mating produced singly sired broods and males other than the individual observed copulating were excluded as sires for most or all of their chicks. Territorial males at the study lek were excluded as sires of broods of four other females that visited the lek but were not observed mating there. Radio-tracking suggested that two of these females mated at other leks. Our results confirm the reliability of mating observations at leks, but do not rule out a possible unseen component of the mating system.     

Multiple paternity in an aggregate breeding amphibian: the effect of reproductive skew on estimates of male reproductive success

Aggregate, or explosive, breeding is widespread among vertebrates and likely increases the probability of multiple paternity. We assessed paternity in seven field-collected clutches of the explosively breeding spotted salamander (Ambystoma maculatum) using 10 microsatellite loci to determine the frequency of multiple paternity and the number of males contributing to a female's clutch. Using the Minimum Method of allele counts, multiple paternity was evident in 70% of these egg masses. Simple allele counts underestimate the number of contributing males because this method cannot distinguish multiple fathers with common or similar alleles. Therefore, we used computer simulations to estimate from the offspring genotypes the most likely number of contributing fathers given the distributions of allele frequencies in this population. We determined that two to eight males may contribute to A. maculatum clutches; therefore, multiple paternity is a common strategy in this aggregate breeding species. In aggregate mating systems competition for mates can be intense, thus differential reproductive success (reproductive skew) among males contributing to a female's clutch could be a probable outcome. We use our data to evaluate the potential effect of reproductive skew on estimates of the number of contributing males. We simulated varying scenarios of differential male reproductive success, ranging from equal contribution to high reproductive skew among contributing sires in multiply sired clutches. Our data suggest that even intermediate levels of reproductive skew decrease confidence substantially in estimates of the number of contributing sires when parental genotypes are unknown.     

雌性动物多次交配行为的机制及进化

雌性动物的后代数量不可能超过她的卵子数。在理论上, 一个生殖季节内, 一次或几次交配就足够使雌性所有卵子受精, 最大化其生殖潜能。但与理论预测相反, 许多物种的雌性经常与同一个或多个雄性发生多次交配。交配通常要付出较高的代价, 所以很难理解为什么雌性动物要反复进行多次交配。本文综述了解释此行为的一些适应性和非适应性假说。从获得直接收益和间接收益二个角度介绍了适应性假说。直接收益主要包括求偶喂食和“彩礼”、受精保证、亲代抚育、生殖刺激和护卫交配权等5 个方面。还着重介绍了多次交配对雌性后代的间接遗传受益, 即获得优质基因、提高后代遗传多样性和遗传互补性3 个假说。非适应性假说包括了遗传相关假说和顺从雄性行为假说。     

Sex ratio conflicts, mating frequency, and queen fitness in the ant Formica truncorum

We examined the effect of facultative sex allocation by workerson queen fitness in a Furnish population of the ant Formicatruncorum. Workers rear female-biased broods in colonies headedby a singly mated queen and male-biased broods in colonies headedby a multiply mated queen. As a result, multiply mated queenshave a 37% fitness advantage over singly mated queens. Neitherreproductive output nor worker population of colonies variedwith queen mating frequency. We suggest that singly mated queenspersist in the population because fitness benefits to multiplymated queens via sex allocation are balanced by costs of additionalmatings. Alternatively, singly mated queens may persist simplybecause some queens lack opportunities to mate multiply or becausemale control sometimes prevents additional matings by queens.     

Female loggerhead sea turtles (Caretta caretta L.) rarely remate during nesting season

The goal of this study was to assess the consequences of single versus multiple paternity by identifying paternity of clutches per female to identify whether there were detectable costs or benefits. Multiple mating can occur when the benefits of mating outweigh the costs, but if costs and benefits are equal, no pattern is expected. Previous research on loggerhead sea turtle (Caretta caretta) populations found male‐biased breeding sex ratios and multiple mating by many females nesting in southwestern Florida. A sample of nesting loggerhead females who laid more than one nest over the course of the season and a subset of their hatchlings were examined from 36 clutches in 2016 on Sanibel Island, Florida. Males that fathered hatchlings in the first clutch sampled were identified in subsequent clutches. Interestingly, 75% of the females analyzed had mated singly. No male was represented in more than one female's clutches. The results suggest that females likely mate at the beginning of the season and use stored sperm for multiple clutches. Evidence for mating between laying events was limited. There was no consistent pattern across the subsequent multiple paternity clutches, suggesting benefits to loggerhead females likely equal their costs and subsequent mating is likely determined by female preference.     

The effect of male mating history on paternal investment, fecundity and female remating in the seed beetle Callosobruchus maculatus

1. In many organisms, males provide nutrients to females via ejaculates that can influence female fecundity, longevity and mating behaviour. The effect of male mating history on male ejaculate size, female fecundity, female longevity and female remating behaviour in the seed beetle Callosobruchus maculatus was determined.
2. The quantity of ejaculate passed to females declined dramatically with successive matings. Despite the decline, a male's ability to fertilize a female fully did not appear to decline substantially until his fourth mating.
3. When females multiply mated with males of a particular mated status, the pattern of egg production was cyclic, with egg production increasing after mating. Females multiply mated to virgins had higher fecundity than females mated to non-virgins, and females mated to twice-mated males had disproportionately increased egg production late in their life.
4. Females that mated to multiple virgins, and consequently laid more eggs, experienced greater mortality than females mated only once or mated to non-virgins, suggesting that egg production is costly, and rather than ameliorating these costs, male ejaculates may increase them by allowing or stimulating females to lay more eggs.
5. Females mating with non-virgin males remated more readily than did females mated to virgins. Females given food supplements were less likely to remate than females that were nutritionally stressed, suggesting that females remate in part to obtain additional nutrients.     

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.     

Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis

Eggs were sampled from 22 wild American alligator nests from the Rockefeller Wildlife Refuge in south-west Louisiana, along with the females guarding the nests. Three nests were sampled in 1995 and 19 were sampled in 1997. Females and offspring from all clutches were genotyped using five polymorphic microsatellite loci and the three nests from 1995 were also genotyped using one allozyme locus. Genotypes of the hatchlings were consistent with the guarding females being the mothers of their respective clutches. Multiple paternity was found in seven of the 22 clutches with one being fathered by three males, and the remaining six clutches having genotypes consistent with two males per clutch. Paternal contributions of multiply sired clutches were skewed. Some males sired hatchlings of more than one of the 22 clutches either as one of two sires of a multiple paternity clutch, as the sole sire of two different clutches, or as the sole sire of one clutch and one of two sires of a multiply sired clutch. There was no significant difference between females that had multiple paternity clutches and those that had singly sired clutches with respect to female total length (P = 0.844) and clutch size (P = 0.861). Also, there was no significant correlation between genetic relatedness of nesting females and pairwise nest distances (r2 = 0.003, F1,208 = 0.623, P = 0.431), indicating that females in this sample that nested close to one another were no more related than any two nesting females chosen at random. Eleven mutations were detected among hatchlings at the five loci over the 22 clutches. Most of these mutations (eight of 11) occurred at Ami(mu)-17, the only compound microsatellite locus of the five used in this study, corresponding to a mutation rate of 1.7 x 10-3. Finally, most of the mutations (82%) were homoplasious, i.e., mutating to an allelic state already present in this Louisiana population.     

Paternal alleles enhance female reproductive success in tropical pythons

The conventional view that female reproductive success is unlikely to benefit from multiple mating has come under strong challenge in recent years. In the present study, we demonstrate that the time wild-caught reproductive female water pythons (Liasis fuscus) spent in the laboratory prior to oviposition affected both hatching success and the number of male microsatellite alleles detected in the broods. A negative correlation between time in captivity and number of male alleles observed in the broods suggests that reduced hatching success was most likely not caused by environmental factors such as non-optimal temperatures, but rather by restricted mating opportunities. Maternal nutritional status and mean hatchling mass did not affect brood viability. However, our results revealed a positive correlation between number of male microsatellite alleles observed in the broods and hatching success, suggesting that increased paternal genetic variability enhanced female reproductive success. As microsatellite loci are unlikely to be direct targets of selection, we suggest that variability at these loci may cosegregate with other polymorphic genes directly linked to fitness.     

Paternal genotype reconstruction reveals multiple paternity and sex ratios in a breeding population of leatherback turtles (<Emphasis Type="Italic">Dermochelys</Emphasis><Emphasis Type="Italic">coriacea</Emphasis>)

When animals are difficult to observe while breeding, insights into the mating system may be gained by using molecular techniques. Patterns of extra-pair copulation, multiple paternity and parental genotype analysis may elucidate population characteristics that help improve knowledge of life history while informing management decisions. During the course of a long-term study of leatherback turtles, we assessed the level of multiple paternity in successive clutches for 12 known females nesting at Sandy Point National Wildlife Refuge (St. Croix, U.S. Virgin Islands). We used seven polymorphic microsatellite markers to genotype the females and 1,019 hatchlings representing 38 nests (3–4 clutches from each female). Using deductive genotype reconstruction and GERUD1.0, we identified the 12 mothers and 17 different fathers that were responsible for 38 nests. We found that seven females (58.3%) showed no evidence of multiple paternity in their clutches, while five females (41.7%) had mated with two males each. There was evidence of two fathers (polyandry) in successive clutches for these five females. Multiple fathers didn’t contribute to clutches equally. For clutches laid by an individual female, the primary father was responsible for 53.7 to 85.9% of the hatchlings. We demonstrate the feasibility of using male genotype reconstruction to characterize the male component of this breeding population and to assess operational sex ratios for breeding sea turtles.