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.     

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

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

Paternal 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.     

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 in the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

The reproductive strategies and variation in reproductive success of ticks are poorly understood. We determined variation in multiple paternity in the American dog tick Dermancentor variabilis . In total, 48 blood-engorged female ticks and 22 male companion ticks were collected from 13 raccoon ( Procyon lotor ) hosts. In the laboratory, 56.3% of blood-engorged females laid eggs, of which 37.0% hatched or showed signs of development. We examined the presence of multiple paternity in the ensuing clutches by genotyping groups of eggs and larvae at 5 microsatellite loci and subtracting the known maternal alleles, thereby identifying male-contributed alleles. Seventy-five percent of the clutches presented multiple paternity, with a mode of 2 fathers siring the clutch. Males associated with the females on the host always sired some offspring. In 1 case, a male was the sire of clutches derived from 2 females, indicating both polygyny and polyandry may occur for this species. These results, combined with those of several other recent studies, suggest that multiple paternity might be frequent for ixodid ticks.     

Mating system variation and morph fluctuations in a polymorphic lizard

In polymorphic male painted dragon lizards (Ctenophorus pictus), red males win staged contests for females over yellow males, and yellow males have greater success in staged sperm competition trials than red males. This predicts different reproductive strategies in the wild with red males being more coercive or better mate guarders than yellow males. Yellow males would be expected to sire more offspring per copulation and have a greater proportion of offspring from clutches with mixed paternity. However, here we show using microsatellites that the frequency of mixed paternity in the wild is low (< 20% on average across years), that all morphs on average have the same number of offspring sired per year, and that mating system variation (polyandry vs. monandry) is strongly correlated with perch density on male territories. Furthermore, a logistic regression on male successful vs. unsuccessful mate acquisition showed that red males were under negative selection when they dominated the population, which suggests ongoing frequency dependent selection on male colouration.     

Conflict over multiple-partner mating between males and females of the polygynandrous common lizards

The optimal number of mate partners for females rarely coincides with that for males, leading to a potential sexual conflict over multiple-partner mating. This suggests that the population sex ratio may affect multiple-partner mating and thus multiple paternity. We investigate the relationship between multiple paternity and the population sex ratio in the polygynandrous common lizard (Lacerta vivipara). In six populations the adult sex ratio was biased toward males, and in another six populations the adult sex ratio was biased toward females, the latter corresponding to the average adult sex ratio encountered in natural populations. In males the frequency and the degree of polygyny were lower in male-biased populations, as expected if competition among males determines polygyny. In females the frequency of polyandry was not different between treatments, and polyandrous females produced larger clutches, suggesting that polyandry might be adaptive. However, in male-biased populations females suffered from reduced reproductive success compared to female-biased populations, and the number of mate partners increased with female body size in polyandrous females. Polyandrous females of male-biased populations showed disproportionately more mating scars, indicating that polyandrous females of male-biased populations had more interactions with males and suggesting that the degree of multiple paternity is controlled by male sexual harassment. Our results thus imply that polyandry may be hierarchically controlled, with females controlling when to mate with multiple partners and male sexual harassment being a proximate determinant of the degree of multiple paternity. The results are also consistent with a sexual conflict in which male behaviors are harmful to females.     

Cross‐fostering eggs reveals that female collared flycatchers adjust clutch sex ratios according to parental ability to invest in offspring

Across animal taxa, reproductive success is generally more variable and more strongly dependent upon body condition for males than for females; in such cases, parents able to produce offspring in above‐average condition are predicted to produce sons, whereas parents unable to produce offspring in good condition should produce daughters. We tested this hypothesis in the collared flycatcher (Ficedula albicollis) by cross‐fostering eggs among nests and using the condition of foster young that parents raised to fledging as a functional measure of their ability to produce fit offspring. As predicted, females raising heavier‐than‐average foster fledglings with their social mate initially produced male‐biased primary sex ratios, whereas those raising lighter‐than‐average foster fledglings produced female‐biased primary sex ratios. Females also produced male‐biased clutches when mated to males with large secondary sexual characters (wing patches), and tended to produce male‐biased clutches earlier within breeding seasons relative to females breeding later. However, females did not adjust the sex of individuals within their clutches; sex was distributed randomly with respect to egg size, laying order and paternity. Future research investigating the proximate mechanisms linking ecological contexts and the quality of offspring parents are able to produce with primary sex‐ratio variation could provide fundamental insight into the evolution of context‐dependent sex‐ratio adjustment.     

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

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

Multiple paternity in loggerhead turtle (Caretta caretta) nests on Melbourne Beach, Florida: a microsatellite analysis

Many aspects of sea turtle biology are difficult to measure in these enigmatic migratory species, and this lack of knowledge continues to hamper conservation efforts. The first study of paternity in a sea turtle species used allozyme analysis to suggest multiple paternity in loggerhead turtle (Caretta caretta) clutches in Australia. Subsequent studies indicated that the frequency of multiple paternity varies from species to species and perhaps location to location. This study examined fine-scale population structure and paternal contribution to loggerhead clutches on Melbourne Beach, FL, USA using microsatellite markers. Mothers and offspring from 70 nests collected at two locations were analysed using two to four polymorphic microsatellite loci. Fine-scale population differentiation was not evident between the sampled locations, separated by 8 km. Multiple paternity was common in loggerhead nests on Melbourne Beach; 22 of 70 clutches had more than one father, and six had more than two fathers. This is the first time that more than two fathers have been detected for offspring in individual sea turtle nests. Paternal genotypes could not be assigned with confidence in clutches with more than two fathers, leaving the question of male philopatry unanswered. Given the high incidence of multiple paternity, we conclude that males are not a limiting resource for this central Florida nesting aggregate.     

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.     

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.     

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.     

Double nesting of the Red-legged Partridge Alectoris rufa

Some Red-legged Partridge females lay two clutches in separate nests, one immediately after the other, which are incubated separately by the male and female of the pair. Pairs remained together during the laying of both clutches so that there were delays between the end of laving and incubation at the first nests. Incubation of the two nests began at approximately the same time but discrepancies of up to ten days occurred. Males incubated first clutches and females usually the second, but probably the first if the second was destroyed during laying. The proportion of surviving eggs which hatched was similar in first and second clutches but declined if the delay between laying and hatching was exceptionally long.
Yearling females began laying late and few seemed to attempt two clutches compared with older females of which 60–80% showed the double nesting habit. A model predicting reproductive success for both sexes in relation to the rate of nest predation during laying, suggested that attempting two clutches rather than one would be disadvantageous at high predation rates. Females would produce more young if their mates incubated their first clutch immediately it was complete rather than accompanying them during the laying of the second. However, males may benefit by this delay, even though it exposes the nest to predators for a longer time, because they are able to guard their mate and prevent other males from mating with her and fertilizing eggs of her second clutch.     

A molecular genetic analysis of the communal nesting of the ostrich (Struthio camelus)

The ostrich breeding system is complex and unique; communal clutches are laid by several females, although only one female, the major female, and the resident territorial male provide parental care. More eggs are laid in the nest than can be incubated and the major female ejects surplus eggs from the incubated central clutch. Microsatellite markers were used to analyse the parentage of communal nests in Nairobi National Park. This revealed that major females contributed a disproportionate number of fertile eggs to the central, incubated clutch and that multiple paternity and maternity within a nest were common; 68.9% of all incubated eggs on a nest were not parented by both the resident territorial male and the major female of that nest. All the males fertilized eggs on the clutches of neighbouring males. Unexpectedly, every major female with her own nest was also simultaneously a minor female with incubated eggs on neighbouring clutches. The relatedness between females laying in the same nest was not significantly different from the population average and significantly less than that between chicks hatched from the same nest.     

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.     

Effect of clutch size on male egg-fanning behavior and hatching success in the goby, Eviota prasina (Klunzinger)

In fish that exhibit paternal care, the females often choose their mates on the basis of male traits that are indicative of the parental ability of the males. In a marine goby, Eviota prasina, males tend their eggs within their nests until hatching, and females prefer males that have longer dorsal fins and exhibit courtship behavior with a higher frequency as their mates. In order to clarify the relationship between these sexually selected traits and the parental ability of males of E. prasina, the factors affecting the hatching success of eggs within male nests and the male parental care behavior were examined in an aquarium experiment. Females spawned their eggs in male nests and the clutch size of females showed a high individual variation (range = 88-833 eggs). The hatching success of eggs within male nests showed a positive correlation with the time spent by males in fanning eggs and the clutch size. In contrast to the prediction, however, the hatching success did not show a significant correlation with the sexually selected traits, i.e., the male dorsal fin length and the frequency of courtship displays. Moreover, multiple regression analysis indicated that the time spent by the males in fanning was the most important factor affecting the survival rate of the eggs. The time spent by males in fanning behavior was influenced by the clutch size within their nests; the fanning behavior of males occurred with a higher frequency when they tended larger clutches. Males are required to invest a greater effort in egg-tending behavior to achieve a higher hatching success when they receive larger clutches, probably due to the greater reward for their parental behavior. Based on their mate choice, females may obtain other benefits such as high quality offspring.     

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.     

Double-nesting behaviour and sexual differences in breeding success in wild Red-legged Partridges Alectoris rufa

Double-nesting behaviour, a rare breeding system in which females lay in two nests, one incubated by herself and the other one by her mate, could be considered an intermediate stage in the evolutionary trend from biparental to uniparental care of single clutches. We examined the occurrence and success of double-nesting behaviour in Red-legged Partridges Alectoris rufa in Central Spain. Clutch size and hatching success were recorded, as well as the variation in these between years and between incubating sexes. Participation in incubation was higher for females (94.76%) than males (41.0%), and the proportion of incubating males varied markedly between years, with no incubating males in one dry year and approximately 50% of males incubating in other years. There was significant variation among years and between sexes in laying date, clutch size and hatching success. Clutch size decreased with later laying date in males and females. The probability of clutch loss to predation differed between sexes, being much higher for nests incubated by females. Our results suggest that both rainfall and predation influence the occurrence and success of double-nesting.