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.     

Strong male‐biased operational sex ratio in a breeding population of loggerhead turtles (Caretta caretta) inferred by paternal genotype reconstruction analysis

Characterization of a species mating systems is fundamental for understanding the natural history and evolution of that species. Polyandry can result in the multiple paternity of progeny arrays. The only previous study of the loggerhead turtle (Caretta caretta) in the USA showed that within the large peninsular Florida subpopulation, multiple paternity occurs in approximately 30% of clutches. Our study tested clutches from the smaller northern subpopulation for the presence of multiple paternal contributions. We examined mothers and up to 20 offspring from 19.5% of clutches laid across three nesting seasons (2008–2010) on the small nesting beach on Wassaw Island, Georgia, USA. We found that 75% of clutches sampled had multiple fathers with an average of 2.65 fathers per nest (1–7 fathers found). The average number of fathers per clutch varied among years and increased with female size. There was no relationship between number of fathers and hatching success. Finally, we found 195 individual paternal genotypes and determined that each male contributed to no more than a single clutch over the 3‐year sampling period. Together these results suggest that the operational sex ratio is male‐biased at this site.     

Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea)

Multiple paternity in the olive ridley sea turtle (Lepidochelys olivacea) population nesting in Suriname was demonstrated using two microsatellite loci, viz., Ei8 and Cm84. The large number of offspring sampled per clutch (70 on average, ranging from 15 to 103) and the number of alleles found at the two loci (18 and eight alleles, respectively) enabled unambiguous assessment of the occurrence of multiple paternity. In two out of 10 clutches analysed, the offspring had been sired by at least two males, which was confirmed at both loci. In both clutches, unequal paternity occurred: 73% and 92% of the offspring had been sired by the primary male. The probability of detecting multiple paternity was 0.903, and therefore there is a small chance that multiple paternity occurred but remained undetected in some of the eight clutches that appeared to be singly sired. Analysis of 703 offspring revealed a high mutation rate for locus Ei8 (micro = 2.3 x 10(-2)) with all 33 mutations occurring in maternal alleles. In particular, one allele of 274 bp mutated at a high frequency in a clutch to which the mother contributed the allele, but in another clutch where the father contributed the same allele, no such mutations were observed. Inferred allele-specific mutation rates for Ei8 and expected numbers of mutations per clutch confirmed that maternal alleles for Ei8 are more likely to mutate in the olive ridley sea turtle than paternal alleles. Possible explanations are discussed.     

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.     

Detection of multiple paternity in the Kemp's ridley sea turtle with limited sampling

An important question in sea turtle biology is the number of males that contribute to the fertilization of a clutch of eggs. Previous studies on other sea turtle species have indicated little to no multiple paternity. We conclude here that female Kemp's ridleys, Lepidochelys kempi , are polyandrous. DNA from 26 mother and offspring groups was analysed at three microsatellite loci to identify paternal alleles. Three paternal alleles were observed among 14 of the clutches; four paternal alleles were observed among the offspring of an additional female. A maximum likelihood analysis not only rejects the model of single paternity, but also rejects the model of equal paternal contribution to the clutch. By explicitly addressing the high mutation rate of microsatellite markers, our analysis rejected mutation as the sole cause of multiple paternal alleles.     

Tetranucleotide markers from the loggerhead sea turtle (Caretta caretta) and their cross-amplification in other marine turtle species

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The leatherback turtle,Dermochelys coriacea,exhibits both polyandry and polygyny

The leatherback turtle (Dermochelys coriacea) is an endangered species, and world-wide populations are declining. To understand better the mating structure of this pelagic and fragile species, we investigated paternity in nearly 1000 hatchlings from Playa Grande in Parque Marino Nacional Las Baulas, Costa Rica. We collected DNA samples from 36 adult female leatherbacks and assessed allele frequency distributions for three microsatellite loci. For 20 of these 36 females, we examined DNA from hatchlings representing multiple clutches, and in some cases assessed up to four successive clutches from the same female. We inferred paternal alleles by comparing maternal and hatchling genotypes. We could not reject the null hypothesis of single paternity in 12 of 20 families (31 of 50 clutches), but we did reject the null hypothesis in two families (eight of 50 clutches). In the remaining six families, the null hypothesis could not be accepted or rejected with certainty because the number of hatchlings exhibiting extra nonmaternal alleles was small, and could thus be a result of mutation or sample error. Successive clutches laid by the same female had the same paternal allelic contribution, indicating sperm storage or possibly monogamy. None of 20 females shared the same three-locus genotype whereas there were two instances of shared genotypes among 17 inferred paternal three-locus genotypes. We conclude that both polyandry and polygyny are part of the mating structure of this leatherback sea turtle population.     

Multiple paternity in the loggerhead turtle (Caretta caretta)

Genotypic ratios within clutches of loggerhead turtle (Caretta caretta) embryos, from the Mon Repos rookery (Queensland), deviate significantly from the Mendelian ratios expected on the null hypothesis of single paternity. One-third of all clutches provide evidence for multiple insemination, indicating that multiple mating constitutes the major breeding pattern for C. caretta. Clutches from two females indicate that C. caretta females may mate between nestings.     

Effects of hybridization on sea turtle fitness

Sea turtle hybridization is a common phenomenon in Brazil between loggerheads (Caretta caretta) and hawksbills (Eretmochelys imbricata) as well as between loggerheads and olive ridleys (Lepidochelys olivacea). In a previous study we showed that the reproductive output of loggerhead/hawksbill hybrids is similar to that of parental species, suggesting no negative effect of hybridization at this life stage. In this study, we used pooled amplicon sequencing to assign species identity to dams and their progeny, and to investigate the fitness consequences of hybridization, using hatchling viability as a proxy for fitness. We genotyped 4829 hatchlings from egg clutches laid by 78 loggerheads, 13 hawksbills, seven loggerhead/hawksbill hybrids, and three loggerhead/olive ridley hybrids. The proportion of viable hybrid (heterozygous) hatchlings was similar to that of homozygous hatchlings (based on data at two loci), independent of the dam’s genotype. Multiple species paternity was observed in 35.7% of the nests. Both hybrid males and females were fertile and produced viable offspring, and we found no evidence for hybrid breakdown. We suggest a genome-wide study of the hybrids and parental species to better characterize hybrids, as well as studies on additional demographic and ecological parameters to further assess the effects of hybridization and its consequences for sea turtles and their environment.     

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.