Genetic analysis of the mating system and opportunity for sexual selection in northern water snakes (Nerodia sipedon)

We used data collected over 3 years at two study sites to quantifythe rates and consequences of multiple paternity and to determinethe opportunity for selection on male and female northern watersnakes (Nerodia sipedon). We sampled litters from 45 femalesthat gave birth to 811 offspring. Using eight microsatelliteDNA loci (probability of exclusion of nonparental males >0.99), we assigned paternity to 93% of neonates from one studypopulation and 69% of neonates from the other population. Observationsof participation in mating aggregations predicted individual reproductive success poorly for two reasons. First, males regularlycourted nonreproductive females. Second, more than half ofall sexually mature males obtained no reproductive successeach year, despite the fact that many of them participatedin mating aggregations. The number of sires per litter ranged from one to five, with 58% of all litters sired by more thanone male. Multiple paternity increased with female size, apparentlyboth because bigger females mated with more males and becausethe larger litters of big females provide paternity opportunitiesto more males. Multiple paternity was also more prevalent inyears with shorter mating seasons. We detected no advantage to multiple paternity in reducing either the number of unfertilizedovules or stillborn young. Despite the majority of males siringno young each year, some males fathered young with as manyas three different females in one year. Male reproductive successincreased by more than 10 offspring for each additional mate,whereas female success increased by fewer than 2 offspring foreach additional mate. The opportunity for sexual selectionwas more than five times higher in males than females.     

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.     

Sperm Competition in a Viviparous Fish

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

‘O father: where art thou?’— Paternity assessment in an open fission–fusion society of wild bottlenose dolphins (Tursiops sp.) in Shark Bay,Western Australia

Sexually mature male bottlenose dolphins in Shark Bay cooperate by pursuing distinct alliance strategies to monopolize females in reproductive condition. We present the results of a comprehensive study in a wild cetacean population to test whether male alliance membership is a prerequisite for reproductive success. We compared two methods for inferring paternity: both calculate a likelihood ratio, called the paternity index, between two opposing hypotheses, but they differ in the way that significance is applied to the data. The first method, a Bayesian approach commonly used in human paternity testing, appeared to be overly conservative for our data set, but would be less susceptible to assumptions if a larger number of microsatellite loci had been used. Using the second approach, the computer program cervus 2.0, we successfully assigned 11 paternities to nine males, and 17 paternities to 14 out of 139 sexually mature males at 95% and 80% confidence levels, respectively. It appears that being a member of a bottlenose dolphin alliance is not a prerequisite for paternity: two paternities were obtained by juvenile males (one at the 95%, the other at the 80% confidence level), suggesting that young males without alliance partners pursue different mating tactics to adults. Likelihood analyses showed that these two juvenile males were significantly more likely to be the true father of the offspring than to be their half-sibling (P < 0.05). Using paternity data at an 80% confidence level, we could show that reproductive success was significantly skewed within at least some stable first-order alliances (P < 0.01). Interestingly, there is powerful evidence that one mating was incestuous, with one calf apparently fathered by its mother's father (P < 0.01). Our study suggests that the reproductive success of both allied males, and of nonallied juveniles, needs to be incorporated into an adaptive framework that seeks to explain alliance formation in male bottlenose dolphins.     

The mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana)

This article reports the first genetic study of the mating system of the Sichuan snub‐nosed monkey (Rhinopithecus roxellana), an endemic and endangered species in China. The investigation was carried out in a population (WRT) in the Qinling Mountains using data from both field observation and paternity analysis through microsatellite DNA profiling. During a mating season, a male on an average copulated with 5.7 females. Approximately 18% of the females were observed to copulate with more than one male over the study period. The majority of copulations (94.5%) were initiated by females. Twenty‐eight of 430 observed matings were extra‐unit copulations. Eight polymorphic microsatellite loci were used for paternity analysis. The number of alleles at each locus ranged from 3 to 7 (mean=4.3). Observed heterozygosity ranged between 0.32 and 0.79. None of the loci showed significant deviation from Hardy–Weinberg equilibrium. Results from paternity exclusion showed that 12 of 21 (57.1%) immature individuals were sired by extra‐unit males. Although the basic social unit of snub‐nosed monkeys is consistent with a polygynous mating system, both field observation and genetic data suggests that their mating system is polygamous. Infanticide and inbreeding avoidance are the most likely explanations for the promiscuity of female snub‐nosed monkeys. Am. J. Primatol. 72:25–32, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Geographic variation of multiple paternity in the American lobster, Homarus americanus

We studied the frequency of multiple paternity for American lobster (Homarus americanus) at three Canadian sites differing in exploitation rate and mean adult size. The probability of detecting multiple paternity using four microsatellite loci and 100 eggs per female was in excess of 99% under various scenarios of paternal contribution. Overall, 13% of the 108 examined females carried a clutch sired by two or three males. Multiple paternity was observed at the two most exploited sites (11% at Magdalen Islands and 28% at Grand Manan Island), whereas single paternity only was observed at the least exploited site (Anticosti Island). Within populations females with a clutch sired by more than one male tended to be smaller than females with a clutch sired by a single male. Based on these and other findings, we postulate a link between female promiscuity and sperm limitation in the American lobster.     

Mating system and reproductive success in eastern Pacific harbour seals

Harbour seals sometimes breed along inland travel corridors where females become clustered in space and time and males establish underwater acoustic display territories similar to terrestrial arenas known as resource-based leks. Under these conditions, we predicted that higher levels of polygyny would be observed than has been previously reported for this species mating in open coast environments without travel corridors. Reproductive success (RS) of 70 males was measured using 20 microsatellite DNA loci and likelihood-based paternity analysis of 136 offspring collected over 3 years. Most males were assigned either zero or one paternity with 80% confidence. The greatest number of pups assigned to one male in a season was two. Variance in RS was higher for males than females (which are biologically limited to one offspring per year) indicating low to mild polygyny. In addition, distributions of relatedness values among pups within year classes did not differ significantly from a simulated distribution with R = 0, indicating that half-siblings were uncommon. Overall, polygyny levels were low relative to terrestrial pinniped mating systems and similar to observations from a harbour seal population along an open coast. Due to large confidence intervals associated with our results, we cannot rule out the hypothesis that a travel corridor might increase the degree of polygyny skew relative to that observed in open coast environments. Habitat appeared to influence male strategies as the most successful males in open coast environments patrolled offshore, while the most successful male in this study defended a territory along the travel corridor.     

Genetic analysis of the mating system of the common brushtail possum (Trichosurus vulpecula) in New Zealand farmland

We examined male reproductive success in a common brushtail possum population in New Zealand farmland. Paternity was assigned to 66 of 91 pouch young (maternity known), using a likelihood approach applied to genotypes at six microsatellite loci having an overall average exclusion probability of around 99%. The distribution of number of offspring per male was L-shaped with a standardized variance of 1.52. At least 46% of the 76 sampled reproductively mature males, bred, siring between one and four offspring each. Although breeding males were on average older and larger than nonbreeding males, the small differences did not result in a significant overall difference between the two groups in a multivariate permutation test analagous to a t-test. Paternity analysis of 22 sibling pairs (resulting from experimental removal of pouch young early in the breeding season, inducing a second oestrous) suggested that sequential mating of females with the same male was uncommon ( approximately 16-27%). Whilst there was a tendency for female possums to mate with nearby males, consistent with previous observations of territorial mating behaviour in Australian populations, some interhabitat matings were also inferred. The study population displayed only a low degree of polygyny, which may in part reflect population and habitat characteristics of the study site. A comprehensive understanding of the mating system of Trichosurus vulpecula awaits genetic paternity analysis in additional populations from both Australia and New Zealand, using quantitative approaches undertaken in this study.     

The genetic mating system and tests for cuckoldry in a pipefish species in which males fertilize eggs and brood offspring externally

Highly variable microsatellite loci were used to study the mating system of Nerophis ophidion, a species of pipefish in which pregnant males carry embryos on the outside of their body rather than in an enclosed brood pouch. Despite this mode of external fertilization and brooding, otherwise rare in the family Syngnathidae, the genotypes of all embryos proved to be consistent with paternity by the tending male, thus indicating that cuckoldry by sneaker males is rare or nonexistent in this species. N. ophidion is a phylogenetic outlier within the Syngnathidae and its reproductive morphology is thought to be close to the presumed ancestral condition for pipefishes and seahorses. Thus, our genetic results suggest that the evolutionary elaboration of the enclosed brood pouch elsewhere in the family was probably not in response to selection pressures on pregnant males to avoid fertilization thievery. With regard to maternity assignments, our genotypic data are consistent with behavioural observations indicating that females sometimes mate with more than one male during a breeding episode, and that each male carries eggs from a single female. Thus, the polyandrous genetic mating system in this species parallels the social mating system, and both are consistent with a more intense sexual selection operating on females, and the elaboration of secondary sexual characters in that gender.     

Molecular determination of paternity in a natural population of the multiply mating polygynous lizard Eulamprus heatwolei

We studied the mating system of the southern water skink, Eulamprus heatwolei, during spring and summer (encompassing the breeding season) in a population in southeastern Australia. We examined potential attributes that might influence the mating system and male reproductive success including home range size, physical proximity of adults and body size, and then genotyped all mothers, offspring and potential sires. Home range overlap of both sexes was extensive, with adult females sharing the greatest amount of space with each other and adult males the least amount of space with each other. However, not all adults hold home ranges. We classified approximately one quarter of adult males as home range holders and the rest as 'floaters'. Adult females occupy home ranges more than males, with approximately three-quarters classified as home range holders. Home range ownership is not correlated with body size for either sex, however, male body size is positively correlated with the number of adult female home ranges that his home range overlaps and adult male home ranges are larger than those of females. We used microsatellite genotyping to assign paternities to 55 offspring from 17 litters and then compared this data with our home range and behavioural observations. This species displays extreme levels of multiple paternity given the small mean clutch size of three. Multiple paternity was confirmed in 11 (64.7%) of 17 clutches but three other clutches (for a total of 82.4%) also may display multiple paternity. A total of 30 offspring from 12 litters were assigned to 10 of the 32 genotyped adult males from our study site. Of these 10 adult males, half were home range holders. Five complete clutches and a total of 25 out of the 55 offspring could not be positively assigned to any male surveyed as part of the study and were attributed to floater males or resident males adjacent to our study site that had not been genotyped. While sample sizes are small, neither male home range ownership nor body size is significantly correlated with the number of paternities a male obtained. Our study suggests a polygynous mating system for this species.     

High male reproductive success of hermaphrodites in the androdioecious Phillyrea angustifolia

Androdioecy, the coexistence of males and hermaphrodites within a population, is a rare breeding system, often considered as unlikely to evolve because of restrictive conditions for its maintenance. Phillyrea angustifolia, a wind-pollinated shrub, is one of the handful species reported to be androdioecious. Our previous studies have shown that natural populations of this species in southern France exhibit higher male frequencies (approximately 50%) than predicted on theoretical grounds. Thus, the male functionality of hermaphrodites is still debated. To assess the functional breeding system of this species in the wild, a paternity analysis was performed with two highly polymorphic microsatellite loci on 729 seeds collected on 10 maternal shrubs in a natural population of 24 mature individuals of P. angustifolia. A large proportion of seeds were found to have been sired by pollen from outside the population. Analysis of seeds sired by individuals within the study population revealed a high male fertility of hermaphrodites resulting in a low male advantage in fertility for male plants. Intermate distances were found to have a strong impact on male reproductive success, whereas sexual morph had no effect, with males and hermaphrodites performing equivalently. This study is the first to unequivocally document the occurrence of a male function of hermaphrodites in a natural population of an androdioecious species.     

Yearling male great tits, Parus major, suffer more strongly from cuckoldry than older males

In the majority of socially monogamous bird species, females have offspring sired by males other than their social mate as the result of extra-pair copulations. While it is widely recognised that there is considerable variation in the frequency of extra-pair paternity between species, between populations of a species and between individuals of a population, determinants of this variation are surprisingly difficult to establish. With respect to individual variation within a population, it is an important step to test for male and female correlates of cuckoldry to better understand the patterns as well as the adaptive significance of extra-pair mating behaviour. Here, we analysed patterns of extra-pair paternity in relation to male age, female age and their interaction in the great tit Parus major, a socially monogamous passerine with a moderate frequency of extra-pair paternity. Based on a large sample of 316 genotyped first broods from five successive years, we failed to demonstrate interaction effects of male and female age on both the proportion of extra-pair offspring and the likelihood that at least one extra-pair offspring is present within a brood. However, both the proportion of extra-pair offspring and the likelihood of paternity loss were higher for yearling as compared to older males, while this was not the case for yearling vs. older females. Furthermore, the proportion of extra-pair offspring within a brood decreased with increasing age of the attending male in within-individual analyses. We found a comparable effect also for attending females in within-individual analyses, but only when excluding two individuals with 100% extra-pair paternity. A female (extra-pair) mating preference for older males and/or a limited ability of yearling males to prevent cuckoldry in their broods could explain these age-related patterns of paternity loss. Effect sizes, however, were not particularly large and substantial residual variation within age categories suggests the importance of further yet unidentified determinants of variation in paternity loss in the study population.     

Paternity assessment in wild groups of toque macaques Macaca sinica at Polonnaruwa, Sri Lanka using molecular markers

Genetic variation at four microsatellite loci in conjunction with that at a highly variable allozyme locus was used to analyse paternity over a 12-year period in 13 social groups of toque macaques Macaca sinica inhabiting a natural forest in Polonnaruwa, Sri Lanka. Paternity exclusion analysis revealed that the set of offspring produced by a female usually consists of half-siblings because few males father more than one offspring with a particular female. No evidence of offspring produced by matings between first degree relatives was found. The social unit in toque macaques was not identical to the reproductive unit and the possibility of paternity by males outside the social group should be considered when estimating male reproductive output. Although it was common for multiple males to father offspring in a social group each year, reproduction within a group during a breeding season tended to be limited to a few males. The mean number of males reproducing per group per year was independent of the number of males in a group. The paternity data suggests that many males may father relatively few offspring during their entire lives and that the effective population size for toque macaques may be much smaller than indicated by demographic data.     

Extra-pair paternity in relation to male age in Bullock's orioles

Single-locus minisatellite DNA profiling was used to assign paternity in a population of Bullock's orioles, Icterus galbula bullockii, and to determine the contribution of age to a male's success in obtaining extra-pair paternity. There was a very low rate of intraspecific brood parasitism (2/202 = 1.0% of chicks). Older adult males lost less within-pair paternity and gained more extra-pair fertilizations than did yearling subadult males. This resulted in adult males benefiting from an annual reproductive success more than double that of subadult males. Behavioural observations, used to determine the role of female choice in extra-pair copulations (EPCs), indicated that females actively participate in EPCs and that they prefer to obtain them from older males. While it was possible that females obtained EPCs as an insurance against the possible infertility of their social mate, the results of this study fit best with the hypothesis that females were attempting to obtain better-quality genes for their offspring by obtaining EPCs with older, better-quality males.     

Exclusion probabilities for single-locus paternity analysis when related males compete for matings

The statistical power of single-locus paternity analyses has previously been assessed by calculating an expected exclusion probability ( E ), the probability of excluding a randomly chosen nonfather. This E -statistic assumes that putative sires are a random selection of individuals from a panmictic study population. In species that display male natal philopatry, closely related individuals may be the principal competitors for paternity. In such structured populations, the E statistic will overestimate exclusion probability because males competing for paternity are more closely related than males chosen randomly from the population. A suite of loci thought to be sufficient for a panmictic population may frequently incorrectly assign paternity to close relatives of true sires. This study provides equations for calculating the expected probability of excluding a close male relative of the genetic sire ( Erel ) for any genotyping system that uses codominant markers. We also describe the use of Monte Carlo modelling to estimate exclusion probabilities when multiple male relatives compete for paternity. We show that the utility of a set of codominant markers will depend on the breeding behaviour and social system of the species in question.     

Reconstructing paternal genotypes to infer patterns of sperm storage and sexual selection in the hawksbill turtle

Postcopulatory sperm storage can serve a range of functions, including ensuring fertility, allowing delayed fertilization and facilitating sexual selection. Sperm storage is likely to be particularly important in wide‐ranging animals with low population densities, but its prevalence and importance in such taxa, and its role in promoting sexual selection, are poorly known. Here, we use a powerful microsatellite array and paternal genotype reconstruction to assess the prevalence of sperm storage and test sexual selection hypotheses of genetic biases to paternity in one such species, the critically endangered hawksbill turtle, Eretmochelys imbricata. In the majority of females (90.7%, N = 43), all offspring were sired by a single male. In the few cases of multiple paternity (9.3%), two males fertilized each female. Importantly, the identity and proportional fertilization success of males were consistent across all sequential nests laid by individual females over the breeding season (up to five nests over 75 days). No males were identified as having fertilized more than one female, suggesting that a large number of males are available to females. No evidence for biases to paternity based on heterozygosity or relatedness was found. These results indicate that female hawksbill turtles are predominantly monogamous within a season, store sperm for the duration of the nesting season and do not re‐mate between nests. Furthermore, females do not appear to be using sperm storage to facilitate sexual selection. Consequently, the primary value of storing sperm in marine turtles may be to uncouple mating and fertilization in time and avoid costly re‐mating.     

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.     

Male age mediates reproductive investment and response to paternity assurance

Theory predicts that male response to reduced paternity will depend on male state and interactions between the sexes. If there is little chance of reproducing again, then males should invest heavily in current offspring, regardless of their share in paternity. We tested this by manipulating male age and paternity assurance in the burying beetle Nicrophorus vespilloides. We found older males invested more in both mating effort and parental effort than younger males. Furthermore, male age, a component of male state, mediated male response to perceived paternity. Older males provided more prenatal care, whereas younger males provided less prenatal care, when perceived paternity was low. Adjustments in male care, however, did not influence selection acting indirectly on parents, through offspring performance. This is because females adjusted their care in response to the age of their partner, providing less care when paired with older males than younger males. As a result offspring, performance did not differ between treatments. Our study shows, for the first time, that a male state variable is an important modifier of paternity–parental care trade-offs and highlights the importance of social interactions between males and females during care in determining male response to perceived paternity.     

Multiple paternity in a natural population of a wild tobacco fly, Bactrocera cacuminata (Diptera: Tephritidae), assessed by microsatellite DNA markers

Mating frequency 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 patterns can also lead to more effective control of pest tephritid species, in which suppression programmes, such as the sterile insect technique (SIT) are employed. Multiple mating by females may compromise the success of SIT. We investigated the level of polyandry and sperm utilization in a Brisbane field population of the tropical fruit fly, Bactrocera cacuminata (Hering), using seven polymorphic microsatellite loci. The offspring of 22 wild-caught gravid females were genotyped to determine the number of males siring each brood and paternity skew, using the programs gerud and scare. Our data showed that 22.7% of females produced offspring sired by at least two males. The mean number of mates per female was 1.72. Paternal contributions of double-sired broods were skewed with the most successful male having sired between 76.9% and 87.5% of the offspring. These results have implications for SIT, because the level of remating we have identified would indicate that wild females could mate with one or more resident fertile males.