Comparison of Biochemical Polymorphisms and Short Tandem Repeat (STR) DNA Markers for Paternity Testing in Rhesus Monkeys (Macaca mulatta)

Genetic markers are indispensable for molecular and statistical genetic research involving nonhuman primates. Genetic markers must be used to ascertain parentage and to confirm the accuracy of pedigrees based solely on housing or demographic records; otherwise, the results of pedigree, linkage, or quantitative genetic analyses may be unreliable. Until recently, most genetic markers used in nonhuman primates were plasma proteins or isozyme polymorphisms, which were required in large numbers, because levels of genetic variation revealed by these markers were rather low. We compared the newer, PCR-amplified short tandem repeat markers (STRs) with a panel of classical biochemical polymorphic markers, for paternity determination among captive-bred rhesus monkeys. The STR markers exhibited an average genetic diversity of 64% and an expected paternity exclusion probability of 0.443. Both of these were greater than the average 54.5% genetic diversity and 0.298 exclusion probability exhibited by the biochemical markers. The STRs were much more efficient than the biochemical markers for parentage determination, since they required only half the amount of genetic typing data to resolve an average paternity case. Thus, the results of applying these two classes of genetic markers in paternity tests were somewhat different than expected on the basis of theoretical exclusion probabilities. These differences were probably due to inbreeding and other genetic differences among breeding colonies. Because they are more informative and provide rapid and efficient genetic data, STRs are now the method of choice for parentage determination and pedigree corroboration among nonhuman primates.     

Molecular insight into patterns of colony composition and paternity in the common mole-rat Cryptomys hottentotus hottentotus

We report the discovery of intraspecific variation in both colony composition and patterns of paternity in two populations of the social common mole-rat Cryptomys hottentotus hottentotus. These two populations represent the mesic and arid habitat extremes of the species' broad ecological range in South Africa. Until recently colonies of the common mole-rat were thought to consist of familial groups whereby all colony members were the offspring of a monogamous reproductive pair. The remaining colony members were thought to forego reproduction until both social and ecological conditions favoured dispersal and opportunities for independent outbreeding. Results from genetic assignment tests using microsatellite markers indicate that while colony composition is dominated by familial groups, colonies within both populations included both adult and subadult foreign conspecifics. Analysis of parentage reveals that the social organization of C. h. hottentotus is not that of strict monogamy; paternity of offspring was not assigned consistently to the largest, most dominant male within the colony. Moreover, a number of significantly smaller males were found to sire offspring, suggesting a sneak-mating strategy by subordinate within-colony males. Extra-colony extra-pair paternity (ECP) was also found to characterize C. h. hottentotus colonies, occurring with similar frequencies in both habitats. Both dominant established breeding males and subordinate males were identified as siring young in nonsource colonies. Furthermore, established breeding males were found to sire extra-colony young in the same season as siring young within their source colonies. We discuss the significance of these results within the context of the divergent ecological regimes characterizing the two sites and observe that our results revisit the accuracy of using behavioural and morphological characters, which have structured the basis of our understanding of the behavioural ecology of this species, as indicators of breeding status in mark-recapture studies.     

Captive breeding, paternity determination, and genetic variation in chimpanzees (Pan troglodytes) in the Australasian region

DNA “fingerprinting” using polymorphic (CA)-repeat microsatellite markers was used to quantify the level of genetic variation present in chimpanzees (Pan troglodytes) in the Australasian region. These markers were also used to determine the paternity of chimpanzees born at Taronga Zoo over a 20-year period. The results suggested that the dominant male in the colony was responsible for siring most, but not all, of the offspring. Where the dominant male was excluded from paternity, the sire was identifiable if all candidate males were available for typing. This enabled us to prove the captive origin of offspring born in the colony during this period. Thus, microsatellite analysis was a useful tool for assignment of familial relationships and improving genetic management of breeding colonies.     

No evidence of genetic benefits from extra-pair fertilisations in female sand martins (Riparia riparia)

Genetic parentage studies of socially monogamous birds reveal a widespread prevalence of extra-pair paternity. Variation in extra-pair paternity among individuals may depend on how different individuals benefit from extra-pair fertilisations and on the opportunity to pursue extra-pair copulations. A long-term study of sand martins (Riparia riparia) in Hungary allowed us to examine patterns of extra-pair fertilisations in a large colony of over 3,000 breeding pairs with many known age individuals. We used multi-locus DNA fingerprinting to determine whether extra-pair fertilisations occur when females are paired to (1) presumably low quality mates, or (2) genetically similar or dissimilar mates, and whether extra-pair fertilisations result in offspring of higher quality. Extra-paternal young were found in 38% of 47 broods and comprised 19% of 190 offspring. Males that lost paternity did not differ significantly from others in age or body condition. Social mates of broods containing extra-pair offspring did not differ in genetic similarity from pairs without extra-pair offspring. Furthermore, there was no significant difference in body condition between extra-pair young and their maternal half-siblings. We were unable to assign paternity and therefore cannot exclude the possibility that extra-pair males differed from the within-pair males they cuckolded, in age, body condition or genetic similarity with the female. We found a positive relationship between paternity losses and breeding density, suggesting that low breeding density may constrain opportunities for seeking extra-pair copulations.     

Low incidence of extra-pair paternity in the colonially nesting common swift Apus apus

The frequency of extra-pair paternity in a wild colony of swifts Apus apus was determined by multilocus DNA fingerprinting in two successive breeding seasons. The data were used to examine the expectation that extra-pair paternity is frequent in colonial-nesting species, either for proximal reasons such as the increased opportunity for extra-pair matings, or because extra-pair matings are important in the evolution and maintenance of coloniality. Forty-two broods containing 88 chicks were analysed. The genetic analysis revealed four cases of extra-pair paternity (4.5% of chicks) from four (9.5%) nests. Rapid mate-switching was considered unlikely to be the cause of extra-pair paternity since three of the cases were in the nests of previously established breeding pairs. Extra-pair copulations were not observed, but were assumed to be the cause of extra-pair paternity. The data show that high levels of extra-pair paternity are not an inevitable feature of high-density nesting.     

Copulation behaviour and paternity in shy albatrosses (Thalassarche cauta)

We investigated the mating system of shy albatrosses Thalassarche cauta by combining behavioural observations during the pre-laying period with genetic paternity analysis. Genetic data on the mating systems of several procellariiform seabirds have recently become available, but data on the reproductive behaviour of these species are rarely obtained. Our main aims were to describe the copulatory behaviour of this species and identify how males achieve within-pair and extra-pair paternity (EPP). Most copulations occurred on the nest, were unforced and were within-pair. Females controlled the success of copulations and were observed soliciting extra-pair matings. Within-pair and extra-pair copulations were behaviourally similar. A low frequency (7–10%, n =29 chicks) of EPP was detected despite male use of frequent copulation as a paternity guard. The pre-laying foraging exodus of female shy albatrosses differed from that in other albatrosses: it was relatively short in length, lasting c . 2 days, and within-pair copulations occurred after the female's return 2 days before laying. This may reflect the close proximity of feeding grounds to the breeding colony.     

The effect of breeding density and male quality on paternity-assurance behaviours in the house sparrow, <Emphasis Type="Italic">Passer domesticus</Emphasis>

Several factors can influence the risk of cuckoldry through extra-pair paternity for male birds. The number of neighbouring males is thought to affect the chance of females engaging in extra-pair copulations, and species which breed both socially (colonially) and solitarily provide an ideal opportunity to test the effect of close proximity on extra-pair behaviour and paternity guards. In this study, the extent to which male house sparrows, Passer domesticus, used two alternative strategies, namely frequent copulation and mate-guarding, to ensure paternity was investigated. We also examined how males vary the two paternity guards according to their breeding sociality. Pairs at the dense colony started to copulate at a higher rate at the beginning of the fertile period than those of the medium-sized colony and solitary breeding pairs. Male house sparrows appear to fine-tune their strategies according to the breeding density. Both strategies are alternatively used in the weak fertile period but are simultaneously used in the peak fertile period. Our results suggest that males modify their strategy according to their individual abilities: mate-guarding intensity was positively correlated with the black breast badge size.     

Parentage in the cooperative breeding system of long-tailed tits, Aegithalos caudatus

In cooperatively breeding birds multiple maternity and paternity of broods is not uncommon, reproduction often being shared among group members as well as with extragroup members. We investigated the extent of extrapair paternity and intraspecific brood parasitism in a population of cooperatively breeding long-tailed tits. Our aim was to determine the frequency and cause of mixed parentage and to investigate whether shared maternity or paternity was associated with decisions made by helpers. Genetic analyses using eight microsatellite loci showed that extrapair paternity was low (2.4-6.9% of nestlings in 16-29% of broods), and that intraspecific brood parasitism was negligible. Mate switching and extrapair copulations were both observed, but mate switching was not responsible for the mixed paternity we recorded. Some extrapair offspring were assigned to males that became helpers at the nest containing their extrapair young, but these males were also close neighbours of the cuckolded males and so were the most likely males to gain extrapair paternity. There was no evidence that the existence of a direct reproductive stake in a brood played an important role in the helping decisions of either male or female helpers. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

DNA “fingerprinting” and the genetic management of a captive chimpanzee population (Pan troglodytes)

DNA fingerprinting probes are cloned sequences which simultaneously detect a large number of similar hypervariable loci in the target DNA. The resulting highly polymorphic pattern visualized on an autoradiograph allows resolution of questions concerning individual identification and parentage. M13 bacteriophage has been used as a DNA fingerprinting probe for paternity ascertainment among captive chimpanzees housed in multi-male groups as part of the National Chimpanzee Breeding and Research Program. In 31 cases of unknown paternity where DNA samples for mother, offspring, and all potential sires were available, DNA fingerprinting with M13 resulted in the unambiguous assignment of paternity for all 31 infants. Knowledge of pedigrees among the captive-born animals is used to address several issues important in the genetic management of captive breeding colonies, including estimation of effective population size and of the rate of decline in genetic variability, variance in male and female reproduction, and the effect of social dominance on male reproductive success. Our analysis demonstrates the beneficial effects of genetic management by comparing the managed dedicated cohort to the Bastrop colony as a whole.     

Extrapair paternity is influenced by breeding synchrony and density in the common yellowthroat

The effects of breeding synchrony and density on levels of extrapair paternity in birds are controversial. We used multilocus DNAfingerprinting and microsatellite analysis to examine the effectsof breeding synchrony and density on levels of extrapair paternityin the common yellowthroat (Geothlypis trichas). As in manyNeotropical migrants, breeding synchrony was greatest at thebeginning of the breeding season. Levels of extrapair paternitywere higher after the peak in synchrony, leading to an overallnegative relationship between extrapair paternity and breeding synchrony. However, there was a significant interaction betweenbreeding synchrony and density, as levels of extrapair paternitywere higher only for males breeding when both synchrony anddensity were low. We discuss several possible explanationsfor this interaction, including lower quality males or territoriesin low density areas and greater demands on mate guarding among males with larger territories. Most studies have not consideredsimultaneously the effects of breeding synchrony and densityon extrapair paternity. Our results suggest that ecologicalcorrelates of paternity may be revealed only after testingfor interactions in multivariate analyses.     

Development of a microsatellite set for paternity assignment of captive rhesus macaques (Macaca mulatta) from Anhui Province, China

Microsatellites are playing an important role in paternity assignment of animals. Given the cost and effort, it would be optimal to develop a minimal microsatellite marker set for paternity testing. This study was the first to assess paternity in a captive colony of rhesus macaques (Macaca mulatta) from the Chinese province of Anhui using 10 polymorphic microsatellites. Results indicated that if at least 6 loci were genotyped, the probability of paternity assignment success was nearly 100%. Our results provide a panel of 6 markers that is effective for assessing paternity of subspecies M. m. siamica of Anhui origin.     

Impact of candidate sire number and sire relatedness on DNA polymorphism-based measures of exclusion probability and probability of unambiguous parentage

Genetic paternity testing can provide sire identity data for offspring when females have been exposed to multiple males. However, correct paternity assignment can be influenced by factors determined in the laboratory and by size and genetic composition of breeding groups. In the present study, DNA samples from 26 commingled beef bulls and their calves from the Nebraska Reference Herd-1 (NRH1), along with previously reported Illinois Reference/Resource Families data, were used to estimate the impact of sire number and sire relatedness on microsatellite-based paternity testing. Assay performance was measured by exclusion probabilities and probabilities of unambiguous parentage (PUP) were derived. Proportion of calves with unambiguous parentage (PCUP) was also calculated to provide a readily understandable whole-herd measure of unambiguous paternity assignment. For NRH1, theoretical and observed PCUP values were in close agreement (85.3 and 85.8%, respectively) indicating good predictive value. While the qualitative effects on PUP values of altering sire number and sire relatedness were generally predictable, we demonstrate that the impacts of these variables, and their interaction effects, can be large, are non-linear, and are quantitatively distinct for different combinations of sire number and degree of sire relatedness. In view of the potentially complex dynamics and practical consequences of these relationships in both research and animal production settings, we suggest that a priori estimation of the quantitative impact of a given set of interacting breeding group-specific and assay-specific parameters on PUP may be indicated, particularly when candidate sire pools are large, sire relatedness may be high, and/or loci numbers or heterozygosity values may be limiting.     

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.     

Geographical variation in patterns of parentage and relatedness in the co‐operatively breeding Ground Tit Parus humilis

The Ground Tit is a large parid species endemic to the Tibetan Plateau. Here we describe its genetic breeding system based on 2 years of fieldwork on a population from Damshung, Tibet. Genetic relatedness and parentage were analysed using 16 microsatellite markers and sex was determined with a marker on the Z and W chromosomes. We established that 16 of 75 families (21%) were assisted by one or occasionally two male helpers, which in most cases were young from a previous brood of one or both of the breeding adults. The helpers typically stayed with their families throughout the breeding season. Helpers never obtained any paternity in their own families, but one helper was identified as the sire in the only instance of extra‐group (and extra‐pair) parentage detected. Thus, the level of extra‐group/extra‐pair parentage appears to be very low in this Ground Tit population, and sharing of reproduction within family groups is apparently absent. Our results contrast with the findings from another Ground Tit population in Gansu, further northeast in China, with respect to both the number and sex of helpers and the division of parentage within and among family groups. In Gansu, helpers regularly produced offspring and both extra‐pair and extra‐group paternity as well as maternity (egg dumping) was common. Differences in family structure, philopatry, territoriality and potential inclusive fitness benefits can probably explain this contrast, and are likely to reflect the relative costs and benefits of co‐operative breeding.     

Male-Biased Sexual Size Dimorphism,Resource Defense Polygyny,and Multiple Paternity in the Emei Moustache Toad (Leptobrachium boringii)

We tested the hypotheses that the Emei moustache toad (Leptobrachium boringii) exhibits resource defense polygyny and that combat led to the evolution of male-biased sexual size dimorphism. Between February and March of 2011 and 2012, 26 female and 55 male L. boringii from Mount Emei UNESCO World Heritage Site, Sichuan, China, were observed throughout the breeding season. Prior to the breeding season, males grow 10–16 keratinized maxillary nuptial spines, which fall off once the season has ended. Throughout this time, males construct and defend aquatic nests where they produce advertisement calls to attract females. In a natural setting, we documented 14 cases involving a total of 22 males where males used their moustaches for aggressive interaction, and nest takeover was observed on seven occasions. Males were also observed to possess injuries resulting from combat. Genetic analysis using microsatellite DNA markers revealed several cases of multiple paternity, both within nest and within clutch. This observation indicated that some alternative male reproductive strategy, such as satellite behaviour, is occurring, which may have led to the multiple paternity. Larger males were observed to mate more frequently, and in multiple nests, suggesting that females are selecting for larger males, or that larger males are more capable of defending high quality territories.     

Effectiveness of human microsatellite loci for assessing paternity in a captive colony of vervets (Chlorocebus aethiops sabaeus)

Microsatellite polymorphisms are playing an increasingly vital role in primatological research, and are particularly useful for paternity exclusion in both wild and captive populations. Although vervet monkeys (Chlorocebus aethiops) are commonly studied in both settings, few previous studies have utilized microsatellite markers for assessing genetic variation in this species. In a pilot project to assess paternity in the UCLA-VA Vervet Monkey Research Colony (VMRC), we screened 55 commercially available human microsatellite markers chosen from a panel of 370 that have been shown to be polymorphic in baboons (Papio hamadryas). Using a standard PCR protocol, 43 (78%) loci produced amplifiable product. Of these, 14 were polymorphic and 11 were genotyped in 51 individuals, including 19 offspring and 14 potential sires. The average heterozygosity across the 11 loci was.719. In all 19 paternity cases all but one male was excluded as the true sire at two or more loci. This includes successfully distinguishing between two maternal half-sib brothers who were potential sires in most of the paternity cases. Given that the colony is descended from 54 wild-caught founders trapped between 1975 and 1987 from an introduced population on St. Kitts, West Indies, these values imply high microsatellite variability in natural vervet populations. Our results provide a panel of markers derived from the human genome that is suitable for assessing genetic variation and paternity in vervets.     

Same school,different conduct: rates of multiple paternity vary within a mixed‐species breeding school of semi‐pelagic cichlid fish (Cyprichromis spp.)

Mating system variability is known to exist between and within species, often due to environmental influences. An open question is whether, vice versa, similar environmental conditions entail congruent mating behavior, for example in terms of multiple paternity, in species or populations sharing largely comparable breeding modes. This study employed microsatellite markers to investigate the incidence of multiple paternity in Cyprichromis coloratus and Cyprichromis leptosoma, two sympatric, closely related, mouthbrooding Lake Tanganyika cichlids with similar ecological and behavioral characteristics including the formation of open‐water schools. Mouthbrooding females of both species were collected from the same mixed‐species breeding school at the same time, minimizing environmental variation during courtship and mating. In C. coloratus, four of 12 broods had more than one sire, with a mean of 1.33 reconstructed sires per brood. C. leptosoma exhibited multiple paternity in 18 of 22 broods, with a mean of 2.59 or 2.86 reconstructed sires per brood according to the programs gerud and colony , respectively. In addition, two broods were found to contain offspring transplanted from another brood. There was no significant difference in brood size between species, but mean sire number did differ significantly. Hence, substantial similarity in reproductive behavior along with shared environmental conditions during courtship and spawning did not lead to equal rates of polyandry or sneaking in the two species.     

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens)

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment- length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist.      

Analysis of microsatellites and parentage testing in saltwater crocodiles

Fifteen microsatellite loci were evaluated in farmed saltwater crocodiles for use in parentage testing. One marker (C391) could not be amplied. For the remaining 14, the number of alleles per locus ranged from two to 16, and the observed heterozygosities ranged from 0.219 to 0.875. The cumulative exclusion probability for all 14 loci was .9988. the 11 loci that showed the greatest level of polymorphism were used for parentage testing, with an exclusion probability of .9980. With these 11 markers on 107 juveniles from 16 known breeding pairs, a 5.6% pedigree error rate was detected. This level of pedigree error, if consistent, could have an impact on the accuracy of gentic parameter and breeding value estimation. The usefulness of these markers was also evaluated for assigning parentage in situations where maternity, paternity, or both may not be known. In these situations, a 2% error in parentage assignment was predicted. It is therefore recommended that more micro-satellite markers be used in these situations. The use of these microsatellite markers will broaden the scope of a breeding program, allowing progeny to be tested from adults maintained in large breeding lagoons for selection as future breeding animals.     

Chimpanzee microsatellite PCR primers applied to paternity testing in a captive colony

Previously designed primers for the polymerase chain reaction (PCR) amplifying microsatellite DNA segments containing GT/AC dinucleotide repeats in the chimpanzee (Pan troglodytes) genome were used for paternity testing in a breeding colony in captivity. Combinations of three PCR primers identified the fathers of all the tested 40 chimpanzees born in an eight-year period. The results suggested: (1) a positive (though not conclusive) correlation between male rank and number of offspring; (2) choice of mating partners by the female rather than by the male; and (3) absence of stable mating pairs over the years. For studies of chimpanzees in captivity and in the wild, these primers should be useful for paternity testing, for investigating genetic variations, and for improving genetic maintenance of breeding colonies.