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.     

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 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.     

MOLECULAR GENETIC DISSECTION OF SPAWNING,PARENTAGE, AND REPRODUCTIVE TACTICS IN A POPULATION OF REDBREAST SUNFISH,LEPOMIS AURITUS

Despite a great diversity of reproductive behaviors in fishes, few studies have examined the genetic consequences of alternative reproductive tactics. Here we develop and employ microsatellite markers to assess genetic paternity and maternity of progeny cohorts in a population of redbreast sunfish (Lepomis auritus), a species in which males build and tend nests. Nearly 1000 progeny from 25 nests, plus nest-attendant males and nearby adults, were genotyped at microsatellite loci that displayed more than 18 alleles each. The genetic data demonstrate that multiple females (at least two to six) spawned in each nest, their offspring were spatially dispersed across a nest, and more than 90% of the young were sired by the attendant male. However, about 40% of the nests also showed genetic evidence of low-level reproductive parasitism, and two nests were tended by males that had fathered none of the sampled offspring. Genetically deduced reproductive behaviors in this population of redbreast sunfish contrast with those reported previously in bluegill sunfish (L. macrochirus) wherein heteromorphic males specialized for parasitism or for parental care coexist in high frequency. Thus, nest-parasitic reproductive behaviors in fishes appear to be evolutionary labile.     

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.     

Microsatellite markers and multiple paternity in the garter snake thamnophis sirtalis

Data from four microsatellite loci developed for the common garter snake, Thamnophis sirtalis, show that multiple paternity is common in a natural population on Beaver Island, Michigan. Six of eight litters tested, and all litters with five or more neonates, were multiply sired. At least triple paternity was documented in the largest litter examined (n = 13 neonates). Inheritance patterns and genotype frequencies in the wild population indicate the presence of null allele(s) at one of the microsatellite loci. Garter snakes are widely used in quantitative genetics research, and paternity testing is essential in studies that rely on sibling analysis.     

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.     

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.     

High prevalence of multiple paternity within fruits in natural populations of Silene latifolia, as revealed by microsatellite DNA analysis

Data on multiple paternity within broods has been gathered in several animal species, and comparable data in plants would be of great importance to understand the evolution of reproductive traits in a common framework. In this study, we first isolated and characterized six microsatellite loci from the dioecious plant Silene latifolia (Caryophyllaceae). The polymorphism of the loci was assessed in 60 individual females from four different populations. Two of the investigated loci showed a pattern of inheritance consistent with X-linkage. These microsatellite loci were highly polymorphic and therefore useful tools for parentage analysis. We then used four of the markers to determine paternity within naturally pollinated fruits in four European populations. This study revealed widespread multiple paternity in all populations investigated. The minimum number of fathers per fruit varied from one to nine, with population means ranging from 3.4 to 4.9. The number of fathers per fruit was not significantly correlated with offspring sex ratios. High prevalence of multiple paternity within fruits strongly suggest that pollen competition is likely to occur in this species. This may substantially impact male reproductive success and possibly contribute to increase female and offspring fitness, either through postpollination selection or increased genetic diversity. Wide variation in outcrossing rates may be an overlooked aspect of plant mating systems.     

Microsatellites provide insight into contrasting mating patterns in arribada vs. non-arribada olive ridley sea turtle rookeries

Molecular studies of sea turtles have shown that the frequency of multiple paternity (MP) varies between species, and between rookeries of the same species. This study uses nuclear microsatellite markers to compare the incidence of MP in two neighbouring olive ridley rookeries on the Pacific coast of Costa Rica, with contrasting nesting behaviours -- the 'arribada' population nesting at Ostional and the solitary nesters of Playa Hermosa. Using two highly polymorphic microsatellite markers, we tested 13 nests from each location and found a significant difference (P < 0.001) between the level of MP of the arribada rookery (92%- the highest found for marine turtles) and that of the solitary nesting rookery (30%). Additional analyses based on six microsatellite loci revealed no genetic differentiation between nesting females from the two locations, or between nesting females and attendant males from the Ostional breeding area. Sixty-nine per cent of the nests with MP were fathered by a minimum of three different males, and three nests showed evidence of at least four fathers. The results suggest that the differences observed in levels of MP between arribada and solitary rookeries are due to an effect of abundance of individuals on the mating system. This is supported by a regression analysis combining other paternity studies on sea turtles which shows that levels of MP increase with increasing abundance of nesting females.     

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.     

Polyandry and multiple paternities in the threatened Agassiz’s desert tortoise, <Emphasis Type="Italic">Gopherus agassizii</Emphasis>

We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.     

Identification of novel microsatellite loci in the sand martin, Riparia riparia, and cross-amplification of loci from other bird species

We isolated and characterised six novel microsatellite loci for paternity analysis in the sand martin Riparia riparia, by screening an enriched genomic library. In addition, we tested 16 already published microsatellite markers, five of which were also polymorphic in the sand martin. Only one of these 11 loci exhibited evidence of null alleles, and all were polymorphic (mean H _o = 0.68, range of number of alleles per locus = 4–24), making them suitable for individual heterozygosity quantification and paternity assessment in this species (exclusion probability of 11 unlinked loci = 0.999997).     

Isolation and characterization of microsatellite markers in the Atlantic slipper shell Crepidula fornicata for use in paternity analysis

Crepidula fornicata is an ideal species in which to study the evolution and timing of sex change. In order to test current theory on the timing of sex change, male reproductive success must be quantified. Because C. fornicata is polygamous, this can only be achieved through the development of molecular markers and paternity analysis. Here, I report the development of five polymorphic microsatellite loci that are inherited in a Mendelian fashion. The levels of polymorphism and the inheritance patterns of these loci make them suitable for paternity analysis despite the presence of null alleles.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Assessment of multiple paternity in single litters from three species of carcharhinid sharks in Hawaii

We tested for presence or absence of multiple paternity in single litters from each of three congeneric shark species in Hawaii: the sandbar shark, Carcharhinus plumbeus, bignose shark, Carcharhinus altimus, and Galapagos shark, Carcharhinus galapagensis. Based on eight polymorphic microsatellite loci, we excluded paternity by a single sire in sandbar and bignose sharks, but could not exclude a single sire for the litter from the Galapagos shark. This study doubles the number of shark species tested for multiple paternity, and is the first demonstration of multiple paternity in sandbar and bignose sharks.     

大山雀的婚外父权调查

婚配制度作为一种进化稳定对策是动物对某一环境包括种群内部环境适应的结果,在动物生殖过程中具有重要意义.大山雀(Parus major)是一种广域分布物种,研究显示广域分布物种的形态、生理、行为、生态特征及婚配制度在不同地区或种群间存在显著差异,因此,该文选择了分布于中国辽宁仙人洞国家级自然保护区的大山雀(P.m.minor)种群开展其婚配制度研究.野外共采集了22巢大山雀亲代和子代血液样本.结果如下:(1)从11个微卫星位点中筛选出了8个多态性较好的微卫星位点用于大山雀父权鉴定,在母本已知的情况下确定父权的准确率可达99.98％;(2)巢内父权鉴定结果显示,31.8％ (7/22)的巢包含婚外父权,8.12％ (16/197)的子代为婚外子代.与其他森林雀形目鸟类相比,大山雀婚外子代的比例明显偏低(＜10％),每个存在婚外父权巢的婚外子代比例各异(55.6％～9.1％),且无明显规律.     

Microsatellite assessment of multiple paternity in natural populations of a live-bearing fish,Gambusia holbrooki

Three polymorphic microsatellite loci were isolated and employed to examine paternity patterns in two natural populations of live-bearing mosquitofish, Gambusia holbrooki. Each locus displayed four to five alleles per population in samples of presumably unrelated adults. Nearly 900 embryos from a total of 50 pregnant females were assayed individually, and paternal alleles in each embryo were identified. Counts of paternal alleles, Mendelian segregation patterns, multilocus allelic associations and genetic relatedness coefficients were employed to estimate the minimum and effective numbers of fathers per brood. At least 90% of the assayed broods were shown to have been fathered by multiple males, a figure substantially higher than previous estimates based on less polymorphic genetic loci. However, the genetic data yield a face-value estimate of only about 2.2 fathers per brood, a number that seems perhaps surprisingly low based on frequencies of attempted copulations by males. Both biological and sampling factors that might bias mean sire counts downward are considered. Although higher sire counts per brood might be obtained from loci with even greater numbers of alleles, little statistical room remains for higher frequency estimates of multiple paternity in Gambusia.     

High levels of extra-pair paternity in an isolated, low-density, island population of tree swallows (Tachycineta bicolor)

Molecular genetic studies have suggested that apparently nonbreeding males ('floaters') may account for a significant proportion of extra-pair paternity (EPP) in avian populations. Attempts to determine the influence of breeding density on EPP are therefore confounded by the presence of a subpopulation of floaters whose numbers are difficult to estimate. To study EPP in a tree swallow (Tachycineta bicolor) population with few floaters, we chose a nestbox grid on an island with an excess of available breeding sites and very few floaters. We obtained DNA samples from 13 complete families and performed DNA profiling on them using four microsatellite loci. For comparison, we also obtained a sample of 58 extra-pair young (EPY) from a mainland population typed at five microsatellite loci. Paternity assignments among resident males in both populations were made using the microsatellite profiles and a likelihood-based statistical method. Of the 67 island nestlings typed, we found 37 (55%) nestlings from 11 (85%) different nests that were EPY. The proportion of nestlings that were EPY and the proportion of broods containing EPY did not differ significantly between island and mainland populations studied previously. There was no significant difference between island and mainland populations in the proportion of extra-pair paternities assigned among neighbouring resident males. Male breeding density does not appear to affect the ability of female tree swallows to obtain extra-pair fertilizations, at least over the range of densities studied so far. The rate of EPP has remained remarkably consistent over many years, studies and populations implying an important role of active female choice in determining EPP.     

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.