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Crocodilians Are Promiscuous But Not to the Benefit of Heterozygosity
Abstract
Crocodilian mating systems are complex but molecular genetics is providing some of the tools necessary to begin their unravelling. The most significant unravelling has been the widespread occurrence of multiple paternity (more than one sire for a clutch of eggs) across the global crocodilian species. The advantages of this strategy are still being debated and may differ across the different species, being dependent on factors such as population density and habitat availability at the individual population level. This chapter reviews the available literature outlining the impact other complexities such as nest site selection, the potential for communal nesting and “alloprotection” (non-biological female exhibiting nest guarding of conspecific eggs) could have on defining the mating strategies of crocodilians. However, it is argued that any advantage of genetic gain, genetic variability or inbreeding avoidance that might achieved by multiple paternity is overcome by low embryo and offspring survival leading to low recruitment rates into the adult breeding population. The limitations of experimental design are also discussed which may be leading to upwardly biased estimates of hypothetical sire numbers.
... Multiple paternity is found across all vertebrate classes and shows a positive relationship with clutch/ litter size (Correia et al. 2021;Dobson et al. 2024). Additionally, it has been confirmed in several reptilian orders, including turtles, squamates, and crocodiles (Isberg 2021;Uller and Olsson 2008). However, whether this reproductive strategy has an evolutionary adaptive value, and the question of the proximate mechanisms involved, remain mostly unanswered (Griffith et al. 2002;Isberg 2021;Taylor et al. 2014;Uller and Olsson 2008). ...
... Additionally, it has been confirmed in several reptilian orders, including turtles, squamates, and crocodiles (Isberg 2021;Uller and Olsson 2008). However, whether this reproductive strategy has an evolutionary adaptive value, and the question of the proximate mechanisms involved, remain mostly unanswered (Griffith et al. 2002;Isberg 2021;Taylor et al. 2014;Uller and Olsson 2008). ...
... With the development of genetic technologies to infer genealogy and the decrease in laboratory costs, genetic mating systems can be inferred in a variety of species without the need for direct behavioral observations. Nowadays, we know that almost all the Old World and New World crocodylians that have been investigated show multiple paternity linked to polyandrous mating (Isberg 2021), which might be the ancestral state of crocodylian mating . Nevertheless, crocodylians' mating systems seem to be complex and influenced by environmental and social constraints. ...
The identification of genetic mating systems in a variety of species has challenged the previous view on animal mating patterns over the past decade, resulting in the identification of multiple paternity across all vertebrate classes. In crocodylians, all species that have been investigated demonstrate multiple paternity, which may represent the ancestral state of the clade. The smooth‐fronted caiman, Paleosuchus trigonatus, is one of the last species whose genetic mating system has yet to be investigated. In this study, we analyzed genetic samples of the smooth‐fronted caiman in French Guiana, a secretive species that is difficult to observe in the wild. Scute samples were taken from three populations and five groups of neonates that hatched shortly before. Microsatellite markers were used to infer the minimum number of fathers that sired each clutch. Our results clearly show that multiple paternity was common, with 60% of the sampled group of neonates showing a minimum of two sires. The potential ecological and behavioral implications of this finding are discussed, as well as recommendations for future research avenues to elucidate this cryptic species' mating behavior and environmental constraints.
... Copulation in crocodilians can precede ovulation by a month or more, and females may breed with multiple males during this time. Multiple paternity within clutches was first reported by Davis et al. (2001) and appears widespread among crocodilian species (Isberg, 2021;Muniz et al., 2011). Because a relatively small number of dominant males may be disproportionately represented in a large number of nests (Barragán-Contreras et al., 2021;Lance et al., 2009;Rossi Lafferriere et al., 2016;Zajdel et al., 2019), the promiscuous nature of crocodilian mating systems does not necessarily result in increased heterozygosity. ...
... Multiple paternity in crocodilian species is now well-established (Isberg, 2020). Twelve of the 25 extant crocodilians have shown the potential for multiple paternity, as summarised in Table 1, with paternity studies on the other species yet to be conducted but likely to yield the same conclusion. ...
Up to ten males have been reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single-sire saltwater crocodile pairs and their offspring which were originally confirmed using an 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single-locus minimum method and GERUD, which both require prior knowledge of family groupings (ie nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Consequently, crocodilian maternal genotypes should at least be obtained either directly (blood, tissue) or indirectly (eggshells, eDNA) in addition to a representative population sample of adult genotypes before embarking on multiple paternity determination. Running different scenarios using different analytical methods and reporting on the lowest number of inferred sires is encouraged remembering these sires are hypothetical until proven by true genotype.
... Multiple clutches (Larriera, 2002) and multiple paternity (i.e., polyandry) (Amavet et al., 2008(Amavet et al., , 2010(Amavet et al., , 2012(Amavet et al., , 2015Amavet, 2009) have been observed in wild populations in Argentina. Polyandry has also been observed in other crocodilians (Davis et al., 2001;McVay et al., 2008;Lance et al., 2009;Oliveira, 2010;Oliveira et al., 2010Oliveira et al., , 2014Muniz et al., 2011;Zajdel et al., 2019;Isberg, 2021). ...
Caiman latirostris has a large geographic distribution, that includes Argentina, Bolivia, Brazil, Paraguay, and Uruguay. In Brazil illegal hunting and land use change have caused population decline, relatively well documented in the last three decades. At such circumstances, the estimate of the species effective population size might help its viability analysis. Single-sample estimator has been developed to estimate current effective population size (Ne). Its main advantage over former methods is that it requires a single sampling, whereas temporal methods require at least two in distinct periods. Such method has been used to estimate Ne of broad-snouted caiman populations in the present study over representative areas of the species range in Brazil. As a result, we learned that in most populations only a relatively low number of adults effectively contribute to genetic variation. The following measures are proposed as management actions to mitigate such problem: (1) an update of population sampling on the main river basins where the species lives; (2) definition, delimitation and conservation of natural habitats for the conservation of the species in the wild, to guarantee its reproduction; and (3) new conservation genetic studies to determine current genetic diversity and its monitoring on the main river basins of the species range, with special attention to genetic diversity recovery.
... Multiple clutches (Larriera, 2002) and multiple paternity (i.e., polyandry) (Amavet et al., 2008(Amavet et al., , 2010(Amavet et al., , 2012(Amavet et al., , 2015Amavet, 2009) have been observed in wild populations in Argentina. Polyandry has also been observed in other crocodilians (Davis et al., 2001;McVay et al., 2008;Lance et al., 2009;Oliveira, 2010;Oliveira et al., 2010Oliveira et al., , 2014Muniz et al., 2011;Zajdel et al., 2019;Isberg, 2021). ...
This book aims to be a comprehensive review of the literature on the conservation genetics of the New World crocodilians, from the biological and demographical aspects of the living species to the application of molecular techniques for conservation purposes. It covers the current status of the molecular genetics applied to phylogenetics, phylogeography, diversity, kinship and mating system, and hybridization, as well its implications for decision making with regards to the conservation of these species at academic and governmental levels. This book can be used as a guide for graduate and undergraduate students to understand how conservation genetics techniques are carried out and how they can help preserve not only crocodilians but also other living species.
Up to 10 males were reported to sire clutches of crocodilian eggs but review of the underlying study designs raised questions of potential upward bias of inferred sire numbers. To test this premise, different scenarios were explored using a published dataset of 16 known single- sire saltwater crocodile pairs and their offspring which were originally confirmed using a 11 loci microsatellite panel in CERVUS. Varying the number of microsatellites, omitting one or both parental genotypes and using different parentage analysis techniques revealed that total allele number, rather than number of loci, determined inferred sire accuracy in two opposing ways. Using the single- locus minimum method and GERUD, which both require prior knowledge of family group-ings (i.e., nests), fewer alleles (and loci) accurately inferred only one father. In contrast, CERVUS and COLONY required all 11 loci (65 alleles) and both parental genotypes to (a) assign correct family groups and (b) infer the correct sire number, except in one family where two sires were equally assigned based on their number of homozygous loci. When less genotype information was provided, CERVUS and COLONY inferred up to six and seven sires, respectively. Given this data is from confirmed single-sire matings, and yet up to seven sires could be inferred, the significance of inappropriate study design is clearly demonstrated. Consideration should be carefully given to gen-otype data, particularly those collected specifically for population diversity studies, which are also used to infer multiple paternity because the underlying data collection assumptions are not equivalent between the two outcomes.
Sharks constitute one of the most threatened clades (Selachimorpha) of all marine fish, and substantial management efforts are required to help the recovery of their populations worldwide. Despite its significant impact on population dynamics and conservation, sharks’ reproductive and philopatric behavior has received little attention in fisheries management. The scalloped hammerhead shark (Sphyrna lewini), an endangered species listed on the IUCN’s Red List and on the CITES’ Appendix II, is an apex predator that potentially exhibits female philopatry to mating grounds. We reconstructed, for the first time in an open ocean species, the relationship among 166 juvenile individuals caught in a recently discovered aggregation of the Rewa Delta, Fiji, and determined the sample population’s mating system using 6,437 SNPs. Using two software packages, COLONY2 and SNP PEDIGREE, results revealed very high consistency in the identification of full and half sib. Moreover, COLONY2 allowed us to identify an equal breeding sex ratio for each cohort analyzed for this population (1.04:1; 1.02:1), as well as several cases of multiple paternity and numerous matings of the same male with different females suggesting polygynandry for this species. These findings reveal additional information about the complex life history of the scalloped hammerhead shark.
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. 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 multiple sired nests, fertility rates were observed to be lower in multiply sired clutches.
We isolated and characterized 10 new microsatellites loci for Paleosuchus trigonatus using ION TORRENT Sequencing Technology. We tested the transferability of these loci to three related species of the subfamily Caimaninae, and used these bi-parental markers to test population structure and genetic diversity of two populations of P. trigonatus impacted by hydroelectric dam construction on the Madeira (N = 16) and Xingu (N = 16) rivers. We also investigated the transferability of these markers to three related species: Paleosuchus palpebrosus (N = 5), Caiman crocodilus (N = 6) and Melanosuchus niger (N = 6). The genetic diversity of P. trigonatus was low in both the Madeira (He: 0.535 ± 0.148) and Xingu (He: 0.381 ± 0.222) populations, but the loci were sufficiently polymorphic to be used in system of mating and kinship studies in P. trigonatus. DAPC analysis with our set of microsatellites loci was able to separate the four species of Caimaninae studied and to detect a shallow genetic structure between Madeira and Xingu populations of P. trigonatus. AMOVA and STRUCTURE analyses using locprior model corroborate this shallow genetic structure. These novel molecular markers will be also useful in conservation genetics and phylogeographic studies of P. trigonatus, since they improve our ability to monitor the putative effects of dams on the loss of genetic diversity and allow us to investigate population dynamics and microevolutionary processes that occurred in the species.
The yacare caiman (Caiman yacare) is a reptile from South America and one of the two crocodilian species present in Argentina. The degradation of their natural habitat and strong hunting pressure led to a sharp numerical decline of wild populations. Nowadays C. yacare is included in the Appendix II of CITES, and at the same time it has been subjected to ranching practices in some areas in Argentina. In this context, it is important to better understand the population structure and mating system of the species. To do this, we amplified ten microsatellite markers (SSRs) in 148 individuals of 13 C. yacare nests. All of markers were polymorphic with 2-12 alleles per locus, with allelic sizes ranging between 154 and 400 bp and medium levels of polymorphism (Ho = 0.152-0.551 and He = 0.221-0.621). We were able to determine the maternal genotype in 9 out of 13 nests. In 6 of them we found more than one paternal genotype, being the maximum 3 fathers for a single nest. This study is the first to provide evidence of multiple paternity behavior. These findings will be useful to improve management and conservation strategies for the species.
Inside “El Fisco” reserve (S 30 ° 15W 60 ° 55 ') located in San Cristobal department, northwest of Santa Fe province – Argentina, nests have been harvested for the sustainable use program "Proyecto Yacaré", since 1992. For many years, the effectiveness of reintroduction of young crocodilians to wild population from ranching programs has been questioned, arguing that individuals bred in captivity and released in the wild could not survive, possibly as a result of having to learn to hunt for food and having to adapt to a new environment. However, not only survival but also the reproduction of individuals who were released by Proyecto Yacaré had been confirmed. This work is a preview of an ongoing research, entitled "Dynamic Population of released and wild Caiman latirostris in El Fisco". We analyzed data recorded using night counts during 2015, in months before nest eclosion (October to February). From these data, preliminary results are presented, obtained by two methods. On one hand, the population size was analyzed by the method of Messel et al. (1981), which showed an estimate between 357 and 406 animals. Furthermore, the method of Maximum Value (King and Messel in Cerrato, 1991) was used, and it was obtained a result of 497 animals. Historical records for 1992 recorded an estimate of less than 1 ind / km population density, then increased to 10 ind / km in 1996 and according to our current study the density would be 41 ind / km. After over 20 years of the management program in the reserve, can be observe an increase in the population of C. latirostris occurred, demonstrating its sustainability.
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.
The reproductive success of the American alligator in the Everglades is constrained primarily by egg mortality, due to flooding by seasonally fluctuating water levels. The timing of nesting is positively correlated with spring air temperatures, and nesting during the summer rainy season places eggs at risk to flooding. Water level at the time of nesting is positively correlated with the elevation at which eggs are deposited above the marsh bottom, which involves nest size, egg placement, and site elevation. Alligators respond and to some extent accommodate to higher water levels at the time of nesting. The percent of nests lost to flooding depended on clutch elevation and on the maximum water depth during the incubation period. Clutch elevation frequencies were normally distributed. Regression models permit estimation of the extent of nest flooding over a 30-yr period. Flooding has increased in recent years, coincident with the institution of water management in the southern Everglades. Prior to water management, the increment of water level rise during the incubation period was predictably correlated with water level at the time of nest construction. This predictability disappeared during the current period of water management, thereby altering the pattern of environmental variation. These results suggest that conservation of alligators in the Everglades, and by extension of the Everglades wetland ecosystem, depends on restoration of more predictable hydrological fluctuations.
Studying the mating system of wild populations of American crocodiles, Crocodylus acutus, has important conservation implications. We conducted a preliminary analysis of the mating system of C. acutus in Las Baulas (2007 and 2008), Santa Rosa (2007) and Palo Verde (2008 and 2009) National Parks in Guanacaste, Costa Rica. We captured hatchlings during crocodile surveys and analyzed them with nine polymorphic microsatellite loci to determine relatedness values. High relatedness values indicated that full and half siblings were sampled in a single locality and season. We found full siblings between the years that hatchlings were collected in Las Baulas and Palo Verde National Parks, which suggested mate fidelity. The mate fidelity and high relatedness values could be a consequence of the smaller number of adult crocodiles found within these areas or indicative of a small number of dominant males in the populations. Our results support the need to conduct future studies describing the mating system and nesting success within populations of C. acutus. Understanding of these population factors is crucial to the continued success and maintenance of viable populations of C. acutus.
Reports nest location and dimensions, clutch size, egg size, and temperature relations of nest chamber; describes reproductive behavior and length of nesting season; and estimates the influence of flooding and predation on hatching success under natural conditions. -from Author
Microsatellite loci of American alligators were identified from small insert DNA libraries. The average length of microsatellites (18.4 repeats) was similar to that observed in mammals. Polymerase chain reaction (PCR) primers were developed and tested for 20 microsatellite loci that contained at least 10 uninterrupted AC or AG repeats. Genotypes for the 15 loci that could be scored readily were obtained for alligators from Louisiana and Florida. Eleven of the 15 loci were polymorphic. For the polymorphic loci, the number of alleles per locus ranged from 4-17 (average = 8.5), and observed heterozygosity within populations ranged from 0.231-0.865 (average = 0.466). Heterozygosity of these loci is almost 20 times higher than values obtained using isozymes. Populations from Louisiana and Florida differed substantially at these loci (overall F ST=0.137,θ p=0.239, and R ST=0.387). The PCR primers also produced single amplicons from species of Alligatoridae, Gavialidae, and Crocodilidae, indicating that they may be useful for genetic studies in other crocodilians.
Flooding of nests and predation were identified as the major mortality agents of eggs of Caiman crocodilus yacare in Brazil. Nests on floating grass mats and in forests were flooded in 1989, but only floating grass mats nests were flooded in 1990. Predation on eggs was high in forest nests, but my presence may have increased predation. No nest on floating grass mats was attacked by predators. The period of sex determination of C. c. yacare can extend to 40 d of incubation. I predicted nest temperature with a multiple regression model incorporating weather variables during incubation. The predicted nest temperatures for nests on floating grass mats and nests in forest were correlated with the sex ratio of the clutch. High nest temperatures (> 31.5 C) produced mostly males, and low nest temperatures (<30.5 C) produced only females. The estimated sex ratio varied between years in nests on floating grass mats, but not in forest nests. Estimated nest temperatures significantly affected the snout-vent lengths of hatchlings but not their masses. Fifty percent of nesting occurred on floating grass mats, so the destruction of this habitat by introduced animals such as the water buffalo (Bubalus bubalis) will adversely affect the productivity and hence long-term density of caimans in the Pantanal.
To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.
Hatchling fitness in crocodilians is affected by "runtism" or failure to thrive syndrome (FTT) in captivity. In this study, 300 hatchling C. porosus, artificially incubated at 32°C for most of their embryonic development, were raised in semi-controlled conditions, with growth criteria derived for the early detection of FTT (within 24 days). Body mass, four days after hatching (BM4d), was correlated with egg size and was highly clutch specific, while snout-vent length (SVL4d) was much more variable within and between clutches. For the majority of hatchlings growth trajectories within the first 24 days continued to 90 days and could be used to predict FTT affliction up to 300 days, highlighting the importance of early growth. Growth and survival of hatchling C. porosus in captivity was not influenced by initial size (BM4d), with a slight tendency for smaller hatchlings to grow faster in the immediate post-hatching period. Strong clutch effects (12 clutches) on affliction with FTT were apparent, but could not be explained by measured clutch variables or other factors. Among individuals not afflicted by FTT (N = 245), mean growth was highly clutch specific, and the variation could be explained by an interaction between clutch and season. FTT affliction was 2.5 times higher among clutches (N = 7) that hatched later in the year when mean minimum air temperatures were lower, compared with those clutches (N = 5) that hatched early in the year. The results of this study highlight the importance of early growth in hatchling C. porosus, which has implications for the captive management of this species.
Although adult crocodilians have few predators (mostly humans and other crocodilians), hatchlings and eggs are killed and consumed by a diverse array of invertebrates, fishes, anurans, reptiles, birds, and mammals. We review published literature to evaluate the incidence of predation in crocodilian populations, and the implications of that mortality for crocodilian life-history evolution. Presumably because predation is size-dependent, small-bodied crocodilian taxa appear to be more vulnerable to predation (across a range of life stages) than are larger-bodied species. Several features of crocodilian biology likely reflect adaptations to reducing vulnerability to predation. For example, the threat of predation may have influenced the evolution of traits such as nest-site selection, maternal care of eggs and hatchlings, cre`che behavior in hatchlings, and cryptic coloration and patterning. Even for such large and superficially invulnerable taxa such as crocodilians, the avoidance of predation appears to have been a significant selective force on behavior, morphology, and ecology.
Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.Cell Research advance online publication 6 August 2013; doi:10.1038/cr.2013.104.
This study assesses the properties of 10 microsatellite loci and two popular computer programs for likelihood-based parentage identification in commercial strains of the giant freshwater prawn Macrobrachium rosenbergii. It was our interest to determine the number of markers needed to optimize between genotyping cost and power of the test. Nine loci were informative, with average expected heterozygosity of 0.80 and PIC score of 0.76. Low and non-significant frequencies of null alleles (<0.05) were detected at the nine loci. Simulations implemented in the CERVUS program were performed to determine the number of informative loci needed to maximize assignment success. The accuracy in assignment was determined in 21 full-sib and two paternal half-sib families using two contrasting methods, a pair-wise likelihood comparison approach in CERVUS and a full-pedigree likelihood method in the COLONY program. When genotyping error was set at 0.05, use of four highly informative loci was sufficient for COLONY to resolve the genetic structure of this population, while seven loci would be required to obtain 94-99% correct assignment with CERVUS. As typing rate of error increased to 0.2, COLONY required only four loci to assign parentage to 85-90% of the offspring, while CERVUS required five to seven loci to obtain the similar proportions of assignment. Results suggest that this set of microsatellites, used in conjunction with COLONY would be an effective tool for parentage and sibship identification, testing performance of families and estimating genetic parameters in selective breeding programs of the giant freshwater prawn.
The formation of dominance hierarchies in which the female mates with a large dominant male is common among crocodilians. However, there is the possibility of polyandry, in which females mate with multiple partners during a single breeding season and generate offspring with multiple paternity. In the present study, eight pairs of heterologous primers developed for Alligator mississippiensis and Caiman latirostris were used to determine whether multiple paternity exists in the Black Caiman, Melanosuchus niger. For such, we analyzed 34 Black Caiman offspring from the Anavilhanas Archipelago in the Negro River (state of Amazonas, Brazil). The specimens came from six groups, each containing five or six hatchlings. Paternity exclusion and genetic identity indices were calculated to test the robustness of the microsatellite loci. Simple allele counts and maximum likelihood estimation of family clusters were used to determine the likelihood of occurrence of multiple paternity. Among the eight loci tested, five were effective at determining paternity, with paternity exclusion values close to 1.0 (QC = 0.92) and genetic identity values close to zero (IC < 0.01). Using the simple allele count, six cases of multiple paternity were detected and confirmed in three hatchling groups by four different microsatellite loci. However, maximum likelihood analysis indicated multiple paternity in all the groups analyzed, with five family clusters identified in one hatchling group alone. Considering that this species is listed according to IUCN as Lower Risk/Conservation Dependent, our results have direct conservation implications. Multiple paternity increases effective population size by maintaining genetic variation, and thus could be an important mechanism to maintain genetic diversity in isolated local populations.
Seven Black Caiman (Melanosuchus niger) nests were located and monitored at Limoncocha
and Anangu lagoons (northeastern Ecuador), between October 2002 and March 2003. Melanosuchus niger nesting coincided with the annual low water level season in the EcuadorianAmazon. Mean number of eggs per nest was 28 and 34 eggs at Limoncocha and Anangu, respectively. Egg chamber temperature was monitored in two nests during the incubation period and showed no relationship with external air temperature, suggesting that the nests have their own heating sources. Mean hatching success was 42.4%, and flooding of the nests was identified as the main cause of egg mortality (29%of all the eggs).
In this study, microsatellite markers, developed for Alligator mississipiensis and Caiman latirostris, were used to assess parentage among individuals from the captive colony of Caiman latirostris at the University of São Paulo, in Piracicaba, São Paulo, Brazil. Many of the females in the colony were full siblings, which made maternal identification difficult due to genotypic similarity. Even so, the most likely mother could be identified unambiguously among offspring in most of the clutches studied. Two non-parental females displayed maternal behavior which would have misled managers in assigning maternity based on behavior alone. This set of variable loci demonstrates the utility of parentage testing in captive propagation programs.
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.
In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.
Parentage analysis is a cornerstone of molecular ecology that has delivered fundamental insights into behavior, ecology, and evolution. Microsatellite markers have long been the king of parentage, their hypervariable nature conferring sufficient power to correctly assign offspring to parents. However, microsatellite markers have seen a sharp decline in use with the rise of next‐generation sequencing technologies, especially in the study of population genetics and local adaptation. The time is ripe to review the current state of parentage analysis and see how it stands to be affected by the emergence of next‐generation sequencing approaches. We find that single‐nucleotide polymorphisms (SNPs), the typical next‐generation sequencing marker, remain underutilized in parentage analysis but are gaining momentum, with 58 SNP‐based parentage analyses published thus far. Many of these papers, particularly the earlier ones, compare the power of SNPs and microsatellites in a parentage context. In virtually every case, SNPs are at least as powerful as microsatellite markers. As few as 100‐500 SNPs are sufficient to resolve parentage completely in most cases. We also provide an overview of the analytical programs that are commonly used and compatible with SNP data. As the next‐generation parentage enterprise grows, a reliance on likelihood and Bayesian approaches, as opposed to strict exclusion, will become increasingly important. We discuss some of the caveats surrounding the use of next‐generation sequencing data for parentage analysis and conclude that the future is bright for this important realm of molecular ecology.
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Conservation strategies can be most effective when factors influencing the persistence of populations are wellunderstood, including aspects of reproductive biology such as mating system. Crocodylians have been traditionally associated with a polygynous mating system, with genetic studies revealingmultiple paternity of clutches in several species. The endemic Cuban crocodile, Crocodylus rhombifer, is currently listed as Critically Endangered, and is one of the least understood crocodylian species in terms of its mating behavior. Here, we tested a hypothesis of multiple paternity in the Cuban crocodile by collecting genotypic data at nine microsatellite loci for 102 hatchlings from five nests sampled at the Zapata Swamp captive breeding facility and analyzing them in relation to data previously collected for 137 putative parents. All five nests showed evidence of multiple paternity based on the numbers of alleles per locus, with sibship analyses reconstructing all nests as having four to six full-sib family groups. Accordingly, mean pairwise relatedness values per nest ranged from 0.21 to 0.39, largely intermediate between theoretical expected values for half-siblings (0.25) and full-siblings (0.50). It is not possible to differentiate whether the multiple paternity of a nest was due to multiple matings during the same breeding season, or a result of sperm storage. Our results reveal that the C. rhombifer mating system is likely best characterized as promiscuous and suggest that the standard practice of enforcing a 1:2 sex ratio at the captive breeding facility should be altered in order to better maintain a demographically and genetically healthy ex situ population.
Genetic marker based identification of distinct individuals and recognition of duplicated individuals has important applications in many research areas in ecology, evolutionary biology, conservation biology and forensics. The widely applied genotype mismatch (MM) method, however, is inaccurate because it relies on a fixed and suboptimal threshold number (TM ) of mismatches, and often yields self-inconsistent pairwise inferences. In this paper I improved MM method by calculating an optimal TM to accommodate the number, mistyping rates, missing data and allele frequencies of the markers. I also developed a pairwise likelihood relationship (LR) method and a likelihood clustering (LC) method for individual identification, using poor-quality data that may have high and variable rates of allelic dropouts and false alleles at genotyped loci. The 3 methods together with the relatedness (RL) method were then compared in accuracy by analysing an empirical frog dataset and many simulated datasets generated under different parameter combinations. The analysis results showed that LC is generally one or two orders more accurate for individual identification than the other methods. Its accuracy is especially superior when the sampled multilocus genotypes have poor quality (i.e. teemed with genotyping errors and missing data) and highly replicated, a situation typical of noninvasive sampling used in estimating population size. Importantly, LC is the only method that guarantees to produce self-consistent results by partitioning the entire set of multilocus genotypes into distinct clusters, each cluster containing one or more genotypes that all represent the same individual. The LC and LR methods were implemented in a computer program COLONY for free download from the internet. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Microsatellite markers were used to investigate the mating system of the Estuarine Crocodile (Crocodylus porosus). Three-hundred and eighty-six hatchlings from 13 clutches from a wild Northern Territory population, and 364 hatchlings from 21 clutches from a captive North Queensland population, were sampled. All samples were genotyped across five microsatellite loci. Multiple paternity was found in 69% of clutches in the wild population compared to 38% of clutches in the captive population. Up to three possible fathers were indicated in some clutches. Shared paternity was suggested by the presence of a common paternal genotype within two clutches in the wild population and among up to three clutches from a large shared pen in the captive population. The probability of detecting multiple paternity at all loci was high; 95% in the wild population and 98% in the captive population. There was no evidence of increased hatching success in the clutches that indicated multiple paternity compared to single paternity clutches in either population (P = 0.43 to P = 0.67). It is unknown whether the occurrence of multiple paternity in C. porosus is a result of multiple mating within the same breeding season or of sperm stored from matings in a previous season. These results suggest the genetic mating system for C. porosus is not polygynous but more likely promiscuous, and there is no evidence of dominant alpha males who control paternity in large areas.
Despite the widespread distribution of Caiman crocodilus fuscus (=chiapasius) in Central America, few detailed studies of its reproduction are available. In northern Costa Rica, brown caimans nested in riverine forest adjacent to rivers or canals during the early wet season. Thirty-five nests were located and measured in 1988-1989; detailed egg parameters were measured from 17 clutches. Mean incubation temperature of nine nests averaged 31.8 C (30.5-32.8 C). Individual nest temperatures ranged from 29.6 to 36.0 C. Egg temperatures increased by 3 C in five nests during the nesting season, but temperatures in four other nests remained nearly constant. Flooding accounted for high embryonic mortality by destroying entire nests; egg predation was minimal. Hatching occurred in September and October during high water levels when aquatic habitat was abundant. Delayed hatching of some nests suggests that a stimulus may be useful to initiate hatching. Typically, parent caimans opened nests to release the young. Hatchlings formed sibling groups near the nest site and often remained together for over 1.5 years. Mound nesting was favored by the optimal conditions of the wet season in Cano Negro. Wet season nesting increases the availability of resources and suitable habitat to hatchling caimans. Abundant habitat during the wet season enables hatchling to avoid predation and cannibalism. The predominant use of riverine forests for nesting by caimans in Cano Negro has important ecological consequences and management implications.
The knowledge about reproductive strategies of a species contributes to their conservation. Multiple paternity is a reproductive behavior increasing effective population size, which could increase genetic diversity particularly in populations submitted to bottlenecks events. In Argentina, wild populations of Caiman latirostris are subject of a management plan devoted to their preservation and sustainable utilization based on its commercial interest. This program started in response to the evident numeric reduction of the populations, as a consequence of hunting pressure and habitat modification; it had a remarkable success in population recovery allowing the commercial use of C. latirostris. Data on reproductive behavior of C. latirostris are limited because mating occurs in the water and the information about their genetic diversity is scarce too. Our specific aims were to study the mating system and population genetic structure applying microsatellite markers in twelve C. latirostris families. The obtained results showed highly significant difference among populations and a lack of correspondence between geographical distance and genetic differentiation suggesting that populations of C. latirostris represent unstable metapopulations. In the paternity analysis was detected more than one father in two nests, which could be explained by capacity of storage sperm, proposed in females of a related species. The behavior of multipaternity could contribute to maintain viable populations of C. latirostris, since the maintenance of genetic variability within populations could help increase their capacity to respond to selective pressure. Further studies employing genetic and behavioral framework are needed to better understand the reproductive biology of C. latirostris.
The mating system of the spectacled caiman (Caiman crocodilus) was investigated in the Piagaçu-Purus Sustainable Development Reserve, Amazonas, Brazil. We used 6 polymorphic microsatellite
loci to genotype 13 females and 174 hatchlings representing 20 nests sampled over 4 consecutive reproductive seasons (2007–2010).
Paternity was determined by 2 methods: simple counts, and statistical analysis using Gerud 2.0. Results were congruent between
the 2 approaches, and the null hypothesis of single paternity was rejected in 19 of the 20 nests analyzed, thereby demonstrating
that C. crocodilus females are polyandrous and that the breeding system of the species can be characterized as polygamous. The data suggest
that 1–4 fathers were responsible for the paternity of the offspring, and that the males contributed differently (9–100%)
to each of the 20 nests. A total of 53 males copulated with the 20 females mothering the nests. This is the first study on
the mating system of C. crocodilus and the frequency of multiple paternity is among the highest values (95%) reported thus far for crocodilians.
Abstract Large groups of related progeny, as can be collected from discrete egg masses or fruits, present excellent opportunities for parentage analysis by allowing the reconstruction of parental genotypes. Current techniques of parental genotypic reconstruction require the knowledge of at least one parental genotype. Here, I present a new computer program that reconstructs parental genotypes when no parents are known, provided that the progeny array contains only full and half siblings. The prospects for successfully reconstructing parental genotypes for such progeny arrays with unknown parents are nearly as good as those for the case when one parent is known with certainty.
Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, NY 10460, U.S.A., email jcaiman@aol.com
Ambient temperatures were significantly correlated with alligator (Alligator mississippiensis) nesting activity. Nesting occurred earliest when March-April-May ambient temperatures were highest. Rainfall had no significant relationship with time of nesting activity although water levels did affect the degree of nesting. The bulk of the nesting took place within a 2-week period each year.
Nesting chronology and factors affecting nesting were investigated at Rockefeller Refuge from 1964 through 1987. Courtship activities generally began in early April and progressively intensified until early June. From late May through the first week of June courtship and copulation were intense, the females ovulated, and the high point of spermatogenesis occurred. Factors which seriously affected nesting success were floods, droughts, and predation. Salinity levels affected nesting densities along the coast. Nesting success (% of nests which produced live hatchlings) averaged 68.3% for 266 nests followed during a 4-yr study. Hatching success (% of eggs which hatched) averaged 58.2% for 154 nests followed during two nesting seasons.
Habitat selection, home ranges, and daily movements were delineated for adult males and females. Courtship occurs in deep open water during springtime. Adult females selectively seek out dense vegetation adjacent to isolated ponds for nesting during the summer. Adult and sub-adult males tend to prefer open water all year round and only occasionally venture into the more secluded and heavily vegetated areas used by the females. Home range sizes and daily movements were much larger for adult males than for adult females. Laboratory studies evaluated the effects of four different egg incubation temperatures on hatching success, sex determination, size at hatching, and growth rates to 18 mo of age. The results indicate that incubation temperature can have a profound effect on sex ratios, hatchling size, and post-hatching growth. No significant differences were observed in hatching success at the four different temperatures.
Paternity testing was determined in Chinese alligator (Alligator sinensis) clutches during a reproductive season at Xuanzhou Nature Reserve, using five microsatellite loci. DNA from ten mother and offspring clutches was analysed to identify paternal alleles. Three or four paternal alleles were observed among three of ten clutches. These clutches were sired by at least two different males. This present study confirmed the effectiveness of microsatellite DNA markers in detecting multiple paternity within natural populations of Chinese alligator. However, to reduce the confounding effects of mutations and null alleles on allele assignment and to increase power to monitor individual's genetic contribution, we need additional variable genetic markers.
Little is known about nest-site fidelity in wild Alligator mississippiensis (American Alligator). As part of a long-term study on multiple paternity and mate fidelity that required capture and marking of nesting female alligators, we documented several instances of nest-site reuse by individual females. In one case, the female nested at the same site seven years after initial capture, demonstrating long-term nest-site fidelity. Another female used one nest site in 2000, 2002, and 2004. Herein, we provide data on minimum nesting intervals for recaptured alligators, including three cases of females nesting in two consecutive years. Despite tremendous habitat alteration and storm surge from Hurricane Rita in 2005 and a catastrophic drought precluding nesting in 2006, we found female alligators in 2007 at nests within 20 m and 170 m of their nest sites from seven years and three years, respectively, prior to those events.
Molecular techniques are making ever more genetic markers available for use in parentage assignment and measures of relatedness. We present a program, Kinship, designed to use likelihood techniques to test for any non-inbred pedigree relationship between pairs of individuals, using single-locus codominant genetic markers. Kinship calculates the likelihood that each pair of individuals in a data set are related by a given pedigree hypothesis, and likelihood ratios for any pair of hypotheses. The program also uses a simulation routine to attach statistical significance to its results.
Many avian studies, aimed at collecting samples for genetic analysis, rely upon invasive procedures involving the capture and handling of parents and their offspring. Our goal was to develop a nondestructive method for sampling maternal DNA that would not require blood collection from the mother. Herein, we describe a method for isolating genomic DNA from eggshell powder, obtained by filing the outer shell of an avian egg. Comparison of microsatellite profiles, obtained from genomic DNA found within eggshell matrices and their corresponding parents, verified the presence of maternal DNA in the eggshell matrix in 100% of the herring gull nests assessed (n= 11). In addition, the microsatellite profiles of eggshell DNA were identical among eggs from the same clutch. The ability to rapidly obtain a DNA sample from an avian eggshell in a noninvasive manner could aid in a wide range of genetic sampling studies, and in this study, we provide one potential application of this finding: assessing the fertilization status of nonviable herring gull (Larus argentatus) eggs from the Laurentian Great Lakes. Detection of fertilization was successful as the microsatellite profiles of eggshell powder (maternal only) and the fertilized embryonic contents of those eggs did not match. Ideally, the application of such an approach will help to discriminate unfertilized eggs from embryos aborted early in development and provide insights into avian reproductive health.
The prevalence of female multiple mating in natural populations is important for many questions in mating system evolution. Several statistical techniques use genetic data to estimate the number of fathers that contribute gametes to broods, but they have not been widely compared to assess the magnitude of differences in their performance. With a combination of new data and reanalysis of previously published data, we compared five analytical approaches: (1) allele-counting, (2) parental reconstruction in GERUD, (3) a Bayesian probability model to estimate the frequency of multiple mating (FMM), (4) computer simulations based on population allele frequencies in HAPLOTYPES and (5) Bayesian parental reconstruction in PARENTAGE. The results show that choice of analysis technique can significantly affect estimates of sire number. Estimates from GERUD conformed exactly to results obtained from strict exclusion of potential sires in an experimental context. However, estimates yielded by HAPLOTYPES and PARENTAGE sometimes exceeded the numbers from GERUD by as much as 120 and 55%, respectively. We recommend GERUD over these other approaches for most purposes because of its accuracy and consistency in this analysis. Our novel genetic data set allowed us to investigate the extent and frequency of multiple paternity in a marbled salamander (Ambystoma opacum) population in South Carolina, USA. A. opacum contrasted with other salamander species by having relatively low levels of multiple paternity (only 31-54% compared with 71-96%). Although A. opacum had the lowest level of multiple paternity under all analytical approaches used here, the magnitude of differences among species varied.
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.
The annual oviductal cycle of the American alligator, Alligator mississippiensis, is described using light and electron microscopy. Previous work done by Palmer and Guillette ([1992] Biol Reprod 46:39-47) shed some light on the reproductive morphology of the female alligator oviduct; however, their study was limited and did not report details relating to variation across the reproductive season. We recognize six variable regions of the oviduct: infundibulum, tube, isthmus, anterior uterus, posterior uterus, and vagina. Each area shows variation, to some degree, in the histochemistry and ultrastructure of oviductal secretions. Peak secretory activity occurs during the months of May and June, with the greatest variation occurring in the tube and anterior uterus. During the month of May, high densities of neutral carbohydrates and proteins are found within the tubal and anterior uterine glands. The epithelium of the entire oviduct secretes neutral carbohydrates throughout the year, but many regions lack protein secretions, and the posterior uterine glands show little secretory activity of any type throughout the year. After oviposition, secretory activity decreases drastically, andthe oviduct resembles that of the premating season. This study also provides evidence to support the homology between alligator and bird oviducts. Sperm were observed in glands at the tubal-isthmus and utero-vaginal junctions in preovulatory, postovulatory and postovipository females.
Microsatellite data were generated from hatchlings collected from ten nests of Morelet's Crocodile (Crocodylus moreletii) from New River Lagoon and Gold Button Lagoon in Belize to test for evidence of multiple paternity. Nine microsatellite loci were genotyped for 188 individuals from the 10 nests, alongside 42 nonhatchlings from Gold Button Lagoon. Then mitochondrial control region sequences were generated for the nonhatchlings and for one individual from each nest to test for presence of C. acutus-like haplotypes. Analyses of five of the nine microsatellite loci revealed evidence that progeny from five of the ten nests were sired by at least two males. These data suggest the presence of multiple paternity as a mating strategy in the true crocodiles. This information may be useful in the application of conservation and management techniques to the 12 species in this genus, most of which are threatened or endangered.
Detecting multiple paternity in wild populations of the broad-snouted caiman (Caiman latirostris) has important implications for conservation efforts. We have applied microsatellite markers to examine genetic variation in C. latirostris and also have provided the first data concerning detection of multiple paternity in wild populations of this species. Blood samples from four nest-guarding C. latirostris females and their hatchlings were obtained from Santa Fe Province, Argentina. Amplified products were analyzed by electrophoresis on 10% polyacrylamide gels and visualized with silver staining. Four out of the eight markers tested reliably amplified and yielded useful data. Using polyacrylamide gels with silver staining provides high enough resolution to obtain individual genotypes. In order to assess the presence or absence of more than two parents in each nest, we used the single locus Minimum Method, and applied Cervus 3.0 and Gerud 2.0 software in parentage analyses. Our results indicate more than one father in at least two families. This behavior could be the consequence of high habitat variability in the area where our population was sampled. The ability to understand mating systems is important for maintaining viable populations of exploited taxa like C. latirostris.
We address various statistical aspects of biological parentage in multi-offspring broods that arise via multiple paternity or multiple maternity and, hence, consist of mixtures of full- and half-sibs. Conditioned on population genetic parameters, computer simulations described herein permit estimation of: (1) the mean number of offspring needed to detect all parental gametes in a brood and (2) the relationship between the number of distinct parental gametes found in a brood and the number of parents. Results are relevant to the design of empirical studies employing molecular markers to assess genetic parentage in polygynous or polyandrous species with large broods, such as are found in many fishes, amphibians, insects, plants and other groups. The utility of this approach is illustrated using two empirical data sets.
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.
