Unique multiple paternity in the endangered big‐headed turtle (Platysternon megacephalum) in an ex situ population in South China

Understanding the mating system and reproductive strategies of an endangered species is critical to the success of captive breeding. The big‐headed turtle (Platysternon megacephalum) is one of the most threatened turtle species in the world. Captive breeding and reintroduction are necessary to re‐establish wild populations of P. megacephalum in some of its historical ranges in China, where the original populations have been extirpated. However, the captive breeding of P. megacephalum is very difficult and this may be due to its mysterious reproductive strategies and special behavior (e.g., aggressive temperament and territoriality). In this study, we achieved successful captive breeding of P. megacephalum by creating a habitat that mimics natural conditions and then investigated its mating system using microsatellite makers. A total of 16 clutches containing 79 eggs of P. megacephalum were collected, and 52 were hatched successfully over two breeding seasons. Of the 15 effective clutches, 6 clutches (40%) exhibited multiple paternity. There was no significant correlation between clutch size and multiple paternity, and no significant difference in hatching success between multiple‐sired and single‐sired clutches. However, there was significant correlation between male body size and the number of offspring, with higher‐ranked males contributing to more clutches. Our results provide the first evidence of multiple paternity and male hierarchy in P. megacephalum. These findings suggest that multiple paternity and male hierarchy should be considered in captive breeding programs for P. megacephalum, and creating a habitat that mimics natural conditions is an effctive way to achieve successful captive breeding and investigate the mating systems of this species.     

Predominance of single paternity in the black spiny-tailed iguana: conservation genetic concerns for female-biased hunting

Because of female-biased illegal harvesting, knowledge about the genetic mating system of the black spiny-tailed iguana Ctenosaura pectinata is of primary interest for the conservation of this threatened species. Based on the high levels of multiple paternity found in clutches of many other reptiles, particularly in lizards, it is hypothesised that multiple paternity may also be common in black iguanas. This was investigated by using microsatellite DNA to estimate the number of males siring nine litters (9 mothers, 121 offspring genotyped at ten polymorphic loci) of black iguanas. Contrary to expectations, only 11% of sampled black iguana females produced litters consistent with being sired by multiple males. These data are the first evidence for the predominance of single paternity within an iguanid lizard, and suggest that black iguana may be more susceptible to loss of genetic variation in the face of gender-biased over-hunting pressure than previously thought.     

High levels of multiple paternity in a spermcast mating freshwater mussel

Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel (Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time.     

Implications of social dominance and multiple paternity for the genetic diversity of a captive-bred reptile population (tuatara)

Captive breeding is an integral part of many species recovery plans. Knowledge of the genetic mating system is essential for effective management of captive stocks and release groups, and can help to predict patterns of genetic diversity in reintroduced populations. Here we investigate the poorly understood mating system of a threatened, ancient reptile (tuatara) on Little Barrier Island, New Zealand and discuss its impact on the genetic diversity. This biologically significant population was thought to be extinct, due to introduced predators, until 8 adults (4 males, 4 females) were rediscovered in 1991/92. We genotyped these adults and their 121 captively-bred offspring, hatched between 1994 to 2005, at five microsatellite loci. Multiple paternity was found in 18.8% of clutches. Male variance in reproductive success was high with one male dominating mating (77.5% of offspring sired) and one male completely restricted from mating. Little Barrier Island tuatara, although clearly having undergone a demographic bottleneck, are retaining relatively high levels of remnant genetic diversity which may be complemented by the presence of multiple paternity. High variance in reproductive success has decreased the effective size of this population to approximately 4 individuals. Manipulation to equalize founder representation was not successful, and the mating system has thus had a large impact on the genetic diversity of this recovering population. Although population growth has been successful, in the absence of migrants this population is likely at risk of future inbreeding and genetic bottleneck.     

Multiple paternity in egg clutches of hawksbill turtles (Eretmochelys imbricata)

We present the first data collected on the genetic mating system of the hawksbill turtle Eretmochelys imbricata, the only marine turtle not studied to date. We examined paternity within 12 egg clutches from ten female hawksbill turtles from Sabah Turtle Islands, Malaysia. A total of 375 hatchlings were analysed using five microsatellite markers. Results demonstrated that clutches from two out of ten females were sired by multiple males (maximum of two). Although at a low frequency, observation of multiple paternity indicates that hawksbill turtles exhibit the same genetic mating system (polyandry) as observed for other species of marine turtles. Consistent paternity across multiple clutches laid by individual females in one breeding season supports the hypothesis that sperm are stored from mating prior to nesting and are then used to fertilize all subsequent clutches of eggs that season.     

Benefits of polyandry: Molecular evidence from field‐caught dung beetles

When females mate with multiple males, they set the stage for postcopulatory sexual selection via sperm competition and/or cryptic female choice. Surprisingly little is known about the rates of multiple mating by females in the wild, despite the importance of this information in understanding the potential for postcopulatory sexual selection to drive the evolution of reproductive behaviour, morphology and physiology. Dung beetles in the genus Onthophagus have become a laboratory model for studying pre‐ and postcopulatory sexual selection, yet we still lack information about the reproductive behaviour of female dung beetles in natural populations. Here, we develop microsatellite markers for Onthophagus taurus and use them to genotype the offspring of wild‐caught females and to estimate natural rates of multiple mating and patterns of sperm utilization. We found that O. taurus females are highly polyandrous: 88% of females produced clutches sired by at least two males, and 5% produced clutches with as many as five sires. Several females (23%) produced clutches with significant paternity skew, indicating the potential for strong postcopulatory sexual selection in natural populations. There were also strong positive correlations between the number of offspring produced and both number of fathers and paternity skew, which suggests that females benefit from mating polyandrously by inciting postcopulatory mechanisms that bias paternity towards males that can sire more viable offspring. This study evaluates the fitness consequences of polyandry for an insect in the wild and provides strong evidence that female dung beetles benefit from multiple mating under natural conditions.     

Multiple Paternity Benefits Female Marbled Salamanders by Increasing Survival of Progeny to Metamorphosis

Multiple paternity occurs in most species and animal groups that have been studied. Because mating involves fitness costs to individual females, theory predicts that polyandrous females gain greater fitness benefits than costs, allowing the behavior to be maintained. Genetic, rather than material, benefits often occur in species where males provide females with little more than sperm and seminal fluid. We compared fitness correlates of single‐ and double‐sire clutches from female marbled salamanders (Ambystoma opacum) at the egg, hatchling, and metamorph stages of offspring development. Because clutches were collected from experimental breeding groups, strict paternity exclusion of offspring using microsatellite data allowed us to categorize each clutch as having either one or two fathers. Early offspring viability and size of hatchlings were not different between single‐ and multiple‐paternity clutches. Larvae from the two clutch types were allowed to develop together in field enclosures until metamorphosis. Although there was no difference in size at metamorphosis, survival to metamorphosis was significantly higher in multiple‐paternity clutches (44% vs. 40%) suggesting a benefit for females. The results were consistent with genetic benefits, although maternal effects could not be ruled out. The data did not support predictions of the genetic bet‐hedging and good sperm hypotheses for genetic benefits of polyandry.     

On the evolutionary consequences of increasing litter size with multiple paternity in wild boar (Sus scrofa scrofa)

Understanding how some species may be able to evolve quickly enough to deal with anthropogenic pressure is of prime interest in evolutionary biology, conservation, and management. Wild boar (Sus scrofa scrofa) populations keep growing all over Europe despite increasing hunting pressure. In wild boar populations subject to male‐selective harvesting, the initially described polygynous mating system may switch to a promiscuous/polyandrous one. Such a change in the mating system, where potentially more males sire a litter at one reproductive event, may be associated with the retention of high genetic diversity and an increase of litter size. We tested these hypotheses by estimating the number of sires per litter based on a six‐year long monitoring of a wild boar population subject to particularly high harvesting pressure. Our results show a high and stable genetic diversity and high rates of multiple paternity compared to other populations, thus depicting a promiscuous/polyandrous mating system in this population. We also show that litter size is positively linked to the number of sires, suggesting that multiple paternity increases fecundity. We finally discuss that multiple paternity may be one of the factors allowing rapid evolution of this population by maintaining both genetic and phenotypic diversity.     

Mating dynamics and multiple paternity in a long‐lived vertebrate

Multiple paternity is relatively common across diverse taxa; however, the drivers and implications related to paternal and maternal fitness are not well understood. Several hypotheses have been offered to explain the occurrence and frequency of multiple paternity. One set of hypotheses seeks to explain multiple paternity through direct and indirect benefits including increased genetic diversity or enhanced offspring fitness, whereas another set of hypotheses explains multiple paternity as a by‐product of sexual conflict and population‐specific parameters such as density. Here, we investigate mating system dynamics in a historically studied population of the American alligator (Alligator mississippiensis) in coastal South Carolina. We examine parentage in 151 nests across 6 years and find that 43% of nests were sired by multiple males and that male reproductive success is strongly influenced by male size. Whereas clutch size and hatchling size did not differ between singly sired and multiply sired nests, fertility rates were observed to be lower in multiply sired clutches. Our findings suggest that multiple paternity may exert cost in regard to female fitness, and raise the possibility that sexual conflict might influence the frequency of multiple paternity in wild alligator populations.     

She gets many and she chooses the best: polygynandry in Salamandrina perspicillata (Amphibia: Salamandridae)

Polyandry is a widespread mating strategy, found in a broad number of taxa. Among amphibians, polyandry, and multiple paternity as its direct consequence, is quite common in salamanders, especially within Ambystomatidae and Plethodontidae. In the suborder Salamadroidea the existence of two different types of spermatheca allows several kinds of polyandry strategies to appear. We used multilocus microsatellite genotyping to investigate the presence of polyandry and its effects on the paternity in a previously unstudied species with a terrestrial habit, Salamandrina perspicillata. We collected gravid females in their natural habitat and analysed the paternity of the offspring by using the software COLONY and GERUD. We found that all the analysed clutches had been fertilized by 2–4 males and that in every clutch one male had sired most of the offspring. Our results confirmed that polyandry is an important component of the mating system of this species, suggesting that females are able to recognize the sperm of the male that will provide a genetic benefit for their offspring. We found evidence of female cryptic choice based on males' genetic dissimilarity: (1) males who sire most of the offspring of a given female tend to be genetically different from their sexual partner; (2) a same male, when mated with two females, sired a proportion of the offspring inversely correlated with his genetic similarity to the female; (3) genetic dissimilarity between mating partners is positively correlated with offspring heterozygosity. According to the genetic compatibility model, we hypothesized that in the observed non resource‐based mating system the indirect benefit for the offspring should reflect interactions between paternal and maternal genomes rather than the inheritance of the so‐called ‘good genes’. This study suggests a polygynandrous mating system for the study species and provides the first report in a salamandrid species in natural condition that reproductive success of males is correlated with genetic dissimilarity between mates. Moreover, we found evidence of an offspring benefit (higher heterozygosity) derived from the most genetically dissimilar father.     

Multiple paternities increase genetic diversity of offspring in Brandt's voles

Mating system and philopatry influence the genetic structure of a social group in mammals. Brandt's vole (Lasiopodomys brandtii) lives in social groups year-round and has male biased dispersal, which makes the vole a model system for studies of genetic consequences of mating system and philopatry. This study aimed to test the hypotheses that: (1) multiple paternity (MP) would exist in Brandt's voles, enhance offspring genetic diversity and reduce genetic relatedness between littermates; (2) promiscuity would occur in this species in that males and females mate with multiple partners; and (3) plural breeders of a social group would be genetically related because of philopatry of female juveniles in Brandt's voles. Paternity analysis indicated that MP occurred in 11 (46%) of 24 social groups examined and that promiscuity existed in this species. Multiple paternity litters had twice the offspring genetic diversity and half the average within-litter genetic relatedness of single paternity litters. We also found plural breeding females in six social groups. Average pairwise genetic relatedness of plural breeders ranged from 0.41 to 0.72 in four social groups, suggesting first-order kinship. Future studies need to investigate effects of reproductive skew and MP on population genetic structure of Brandt's voles.     

Mating system, multiple paternity and effective population size in the endemic flatback turtle (Natator depressus) in Australia

In recent years, genetic studies have been used to investigate mating systems of marine turtles, but to date no such research has been conducted on the flatback turtle (Natator depressus). This study investigates paternity of flatback turtle clutches at two rookeries in Queensland, Australia; Peak Island (Keppel Bay), and Mon Repos (Bundaberg). In the 2004–2005 nesting season, tissue samples were taken from either single or multiple clutches (n = 16) of nesting females (n = 8) representing a sampling effort ranging from 25% to 50% offspring per nest. Determination of the extent of multiple paternity was done using a comparative approach that included initial inferences based on observed alleles, Chi-square tests for deviations from Mendelian expectations, and three software programs (PARENTAGE1.0, GERUD2.0 and MER3.0). Results varied depending on the approach, but by calculating a consensus value of the output from these different methods, the null hypothesis of single paternity could be rejected in at least 11 of the 16 clutches (69%). Multiple paternity was thus observed in the clutches of six of nine females (67%), with two or three fathers being the most likely outcome. Analyses of successive clutches illustrated that paternal contribution to clutch fertilization can vary through time, as observed for two females. This first evidence regarding the mating system of flatback turtles indicates that multiple paternity is common in this species and that the observed frequency of multiple paternity is among the higher values reported in marine turtle species. Application of these results to estimates of effective population size (N _e) suggests that population size may have been relatively stable over long periods. Continued monitoring of population dynamics is recommended to ensure that future changes in the east coast can be detected.     

Do Genetic Diversity Effects Drive the Benefits Associated with Multiple Mating? A Test in a Marine Invertebrate

Background

Mothers that mate with multiple males often produce higher quality offspring than mothers that mate with a single male. By engaging in polyandry, mothers may increase their chances of mating with a compatible male or promote sperm competition - both of which act to increase maternal fitness via the biasing of the paternity of offspring. Surprisingly, mating with multiple males, can carry benefits without biasing paternity and may be due simply to differences in genetic diversity between monandrous and polyandrous clutches but role of genetic diversity effects in driving the benefits of polyandry remains poorly tested. Disentangling indirect, genetic benefits from genetic diversity effects is challenging but crucial if we are to understand the selection pressures acting to promote polyandry.

Methodology/Principal Findings

Here, we examine the post-fertilisation benefits of accessing the sperm of multiple males in an externally fertilising polychaete worm. Accessing the sperm of multiple males increases offspring performance but this benefit was driven entirely by genetic diversity effects and not by the biasing of paternity at fertilisation.

Conclusions/Significance

Previous studies on polyandry should be interpreted cautiously as genetic diversity effects alone can explain the benefits of polyandry yet these diversity effects may be difficult to disentangle from other mechanisms. We suggest that future studies use a modified experimental design in order to discriminate between genetic diversity effects and indirect, genetic benefits.     

Paternity and kinship patterns in polyandrous moustached tamarins (Saguinus mystax)

We studied patterns of genetic relatedness and paternity in moustached tamarins, small Neotropical primates living in groups of 1–4 adult males and 1–4 adult females. Generally only one female per group breeds, mating with more than one male. Twin birth are the norm. In order to examine the genetic consequences of this mating pattern, DNA was extracted from fecal samples collected from two principal and six neighboring groups. DNA was characterized at twelve microsatellite loci (average: seven alleles/locus). We addressed the following questions: Do all adult males have mating access to the reproductive female of the group? How is paternity distributed across males in a group? Can polyandrous mating lead to multiple paternity? Are nonparental animals more closely related to the breeders than to the population mean? And, are mating partners unrelated? Breeding females mated with all nonrelated males. In at least one group the father of the older offspring did not sire the youngest infant although he was still resident in the group. We also found evidence for multiple paternity in a supposed twin pair. Yet, within each group the majority (67–100%) of infants had the same father, suggesting reproductive skew. Relatedness within groups was generally high (average R = 0.31), although both nonrelated males and females occurred, i.e., immigrations of both sexes are possible. Mating partners were never found to be related, hence inbreeding seems to be uncommon. The results suggest that while the social mating system is polyandry, paternity is often monopolized by a single male per group. Am J Phys Anthropol, 2005. © 2004 Wiley-Liss, Inc.     

Multiple Paternity in a Reintroduced Population of the Orinoco Crocodile (Crocodylus intermedius) at the El Frío Biological Station,Venezuela

The success of a reintroduction program is determined by the ability of individuals to reproduce and thrive. Hence, an understanding of the mating system and breeding strategies of reintroduced species can be critical to the success, evaluation and effective management of reintroduction programs. As one of the most threatened crocodile species in the world, the Orinoco crocodile (Crocodylus intermedius) has been reduced to only a few wild populations in the Llanos of Venezuela and Colombia. One of these populations was founded by reintroduction at Caño Macanillal and La Ramera lagoon within the El Frío Biological Station, Venezuela. Twenty egg clutches of C. intermedius were collected at the El Frío Biological Station for incubation in the lab and release of juveniles after one year. Analyzing 17 polymorphic microsatellite loci from 335 hatchlings we found multiple paternity in C. intermedius, with half of the 20 clutches fathered by two or three males. Sixteen mothers and 14 fathers were inferred by reconstruction of multilocus parental genotypes. Our findings showed skewed paternal contributions to multiple-sired clutches in four of the clutches (40%), leading to an overall unequal contribution of offspring among fathers with six of the 14 inferred males fathering 90% of the total offspring, and three of those six males fathering more than 70% of the total offspring. Our results provide the first evidence of multiple paternity occurring in the Orinoco crocodile and confirm the success of reintroduction efforts of this critically endangered species in the El Frío Biological Station, Venezuela.     

High rates of extra‐pair paternity in a socially monogamous beetle with biparental care

1. Nest construction and paternity assurance are predicted to favour biparental care in insects. The horned passalus (Odontotaenius disjunctus) is a socially monogamous beetle with biparental care that breeds in decaying logs. The genetic mating system of the horned passalus was investigated to determine if paternity assurance is likely to drive the evolution or maintenance of paternal care in this system. Parental time budgets were also examined to better understand the types and frequencies of behaviours performed by parents. 2. Genotyping‐by‐sequencing revealed high levels of extra‐pair paternity, with 54.8% of offspring sired by extra‐pair males and 70% of nests containing extra‐pair young. 3. More heterozygous social males were cuckolded less than more homozygous social males. Extra‐pair mating, however, seems unlikely to increase offspring genetic diversity as extra‐pair offspring were not more heterozygous than within‐pair offspring, and average brood heterozygosity did not increase with higher rates of extra‐pair paternity. 4. Behavioural observations demonstrated that parents spent on average 46.5% of their time processing the decaying wood resource for larval offspring. Because resource processing is a by‐product of feeding and provides shareable benefits for all larvae in the brood, this form of paternal care could be favoured despite low paternity.     

An Analysis of Single Clutch Paternity in the Burrower Bug Sehirus cinctus Using Microsatellites

Recent studies of the burrower bug, Sehirus cinctus, have examined the genetic basis of parental care. An understanding of the burrower bug mating system, and the subsequent pattern of offspring relatedness that this system generates, is critical to further interpret genetic data. To this end, we developed three consistently amplifiable highly polymorphic microsatellite loci and used them to determine genotypic patterns at the level of both the population and the single clutch. We found that all clutches were sired by single males. Further, we find no evidence for inbreeding. We hypothesize that single paternity within a clutch may play an important role in reducing the potential for sibling rivalry, by increasing the relatedness among clutchmates.     

High frequency of multiple paternity in the largest rookery of Mediterranean loggerhead sea turtles

Mating systems are a central component in the evolution of animal life histories and in conservation genetics. The patterns of male reproductive skew and of paternal shares in batches of offspring, for example, affect genetic effective population size. A prominent characteristic of mating systems of sea turtles seem to be a considerable intra- and interspecific variability in the degree of polyandry. Because of the difficulty of observing the mating behaviour of sea turtles directly in the open sea, genetic paternity analysis is particularly useful for gaining insights into this aspect of their reproductive behaviour. We investigated patterns of multiple paternity in clutches of loggerhead sea turtles in the largest Mediterranean rookery using four highly variable microsatellite loci. Furthermore, we tested for a relationship between the number of fathers detected in clutches and body size of females. More than one father was detected in the clutches of 14 out of 15 females, with two clutches revealing the contribution of at least five males. In more than half the cases, the contributions of different fathers to a clutch did not depart from equality. The number of detected fathers significantly increased with increasing female body size. This relationship indicates that males may prefer to mate with large, and therefore productive, females. Our results suggest that polyandry is likely to increase effective population size compared to a population in which females would mate with only one male; male reproductive contributions being equal.     

Does restored plant diversity play a role in the reproductive functionality of Banksia populations?

Vegetation structure and plant species diversity of restoration sites are predicted to directly affect pollinator attraction, with potential impacts on gene flow, reproduction, genetic diversity of future generations, and ultimately restoration success. We compared Banksia attenuata R.Br. (Proteaceae) in a low species diversity restoration site and an adjacent natural remnant. We assessed fecundity genetic diversity in adult plants and their offspring, mating system parameters and pollen dispersal using paternity assignment. Results were compared to an earlier study of reproductive functionality within a high species diversity restoration site that was restored in a similar manner, enabling us to investigate any association between plant species diversity and fecundity. Seed set data indicated no significant differences between restored and adjacent natural sites; however, seed set data between restoration sites was significantly different (2.08 ± 0.39 and 6.89 ± 1.12, respectively). The mean number of fruits (follicles) per inflorescence was not significantly different between restoration sites. Genetic diversity of adult plants and their offspring were comparable in all sites. Higher allelic richness and genetic differentiation in one restored site reflected sourcing beyond local provenance. Low correlated paternity indicated high levels of multiple siring of seeds and paternity assignment demonstrated strong genetic connectivity between sites. Reproductive functionality, as measured by fecundity and genetic diversity in the offspring of B. attenuata, is resilient to low species diversity within a restored plant community. We consider our results in the context of establishing seed production areas (SPAs) that maximize the quantity and genetic quality of Banksia seeds for restoration.     

Variation in multiple paternity and sperm utilization patterns in natural populations of a simultaneous hermaphrodite land snail

Mating frequency has important implications for patterns of sexual selection and sexual conflict, and hence for issues such as the maintenance of genetic diversity and speciation. We assessed the level of multiple paternity and sperm utilization patterns in four natural populations of the simultaneous hermaphrodite land snail Arianta arbustorum using four polymorphic microsatellite loci. A total of 1088 offspring from 26 wild‐caught snails were genotyped to determine the number of fathers siring each brood and paternity skew in succeeding clutches. Multiple paternity was detected in the offspring of all 26 mother snails examined with the contribution of two to six fathers. The four populations examined differed in the level of multiple paternity. Snails in the population with the highest density of adults showed the highest level of multiple paternity, whereas snails in the population with the lowest density exhibited the lowest value of multiple paternity. Highly skewed paternity patterns were found in the progeny of 15 (57.7%) of the 26 mother snails. The number and identity of fathers siring the offspring of single mothers also varied among successive clutches. Furthermore, genetic analyses indicate a low level of self‐fertilization in one of the four populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 350–361.