Lineage Identification and Genealogical Relationships Among Captive Galápagos Tortoises

Genetic tools have become a critical complement to traditional approaches for meeting short‐ and long‐term goals of ex situ conservation programs. The San Diego Zoo (SDZ) harbors a collection of wild‐born and captive‐born Galápagos giant tortoises (n = 22) of uncertain species designation and unknown genealogical relationships. Here, we used mitochondrial DNA haplotypic data and nuclear microsatellite genotypic data to identify the evolutionary lineage of wild‐born and captive‐born tortoises of unknown ancestry, to infer levels of relatedness among founders and captive‐born tortoises, and assess putative pedigree relationships assigned by the SDZ studbook. Assignment tests revealed that 12 wild‐born and five captive‐born tortoises represent five different species from Isabela Island and one species from Santa Cruz Island, only five of which were consistent with current studbook designations. Three wild‐born and one captive‐born tortoise were of mixed ancestry. In addition, kinship analyses revealed two significant first‐order relationship pairs between wild‐born and captive‐born tortoises, four second‐order relationships (half‐sibling) between wild‐born and captive tortoises (full‐sibs or parent‐offspring), and one second‐order relationship between two captive‐born tortoises. Of particular note, we also reconstructed a first‐order relationship between two wild‐born individuals, violating the founder assumption. Overall, our results contribute to a worldwide effort in identifying genetically important Galápagos tortoises currently in captivity while revealing closely related founders, reconstructing genealogical relationships, and providing detailed management recommendations for the SDZ tortoises. Zoo Biol 31:107;–120, 2012. © 2011 Wiley Periodicals, Inc.     

Isolation and characterization of 23 polymorphic microsatellite loci for a West Indian iguana (Cyclura pinguis) from the British Virgin Islands

Twenty-three polymorphic microsatellite markers were identified and characterized for Cyclura pinguis, a critically endangered species of lizard (Sauria: Iguanidae) native to Anegada Island in the British Virgin Islands. We examined variation at these loci for 39 C. pinguis, finding up to five alleles per locus and an average expected heterozygosity of 0.55. Allele frequency estimates for these microsatellite loci will be used to characterize genetic diversity of captive and wild C. pinguis populations and to estimate relatedness among adult iguanas at the San Diego Zoo that form the nucleus of a captive breeding programme for this critically endangered species.     

The cryptic genetic structure of the North American captive gorilla population

Western lowland gorillas (Gorilla gorilla gorilla) were imported from across their geographical range to North American zoos from the late 1800s through 1974. The majority of these gorillas were imported with little or no information regarding their original provenance and no information on their genetic relatedness. Here, we analyze 32 microsatellite loci in 144 individuals using a Bayesian clustering method to delineate clusters of individuals among a sample of founders of the captive North American zoo gorilla collection. We infer that the majority of North American zoo founders sampled are distributed into two distinct clusters, and that some individuals are of admixed ancestry. This new information regarding the existence of ancestral genetic population structure in the North American zoo population lays the groundwork for enhanced efforts to conserve the evolutionary units of the western lowland gorilla gene pool. Our data also show that the genetic diversity estimates in the founder population were comparable to those in wild gorilla populations (Mondika and Cross River), and that pairwise relatedness among the founders is no different from that expected for a random mating population. However, the relatively high level of relatedness (R = 0.54) we discovered in a pair of known breeding pairs reveals the need for incorporating genetic relatedness estimates in the captive management of western lowland gorillas.     

Genetic management of captive prosimian populations,a report of experience withLoris tardigradus

Extinction of small, closed populations in captivity as well as in the wild is believed to be nearly inevitable, because inbreeding will adversely effect reproductive success, mortality, sex ratio and also the susceptibility to epidemic diseases and environmental stress. An ever increasing number of primate species exist only in small isolated populations, which contain only a part of the original genetic variability. In captive breeding programs research about genetic management strategies is, therefore, of essential importance. In 1980 we imported 9Loris tardigrdus nordicus (4 females, 5 males) from NE-Sri Lanka. The founders came from one natural breeding population. All sexual mature females are breeding. Up to now the colony contains 36 living individuals. The main goal of our long-term genetic management plan was to minimize inbreeding and to preserve the genetic diversity. Therefore, we try to pass the founder bottleneck rapidly by enlarging the population to a desired minimum population size of 25 pairs and to equalize the founder representation within any generation. The need to control the spread of sublethal genes, introduced by one of the founders, conflicts directly with the aim of equalizing the founder representation. A solution of this problem is discussed. To produce a sufficiently large population we intend to give animals to other institutions and to build up an exchange-system for offspring individuals, which should lead to an international studbook.     

Unravelling first-generation pedigrees in wild endangered salmon populations using molecular genetic markers

There is increasing interest in the use of molecular genetic data to infer genealogical relationships among individuals in the absence of parental information. Such analyses can provide insight into mating systems and estimations of heritability in the wild. In addition, accurate pedigree reconstruction among the founders of endangered populations being reared in captivity would be invaluable. Many breeding programs for endangered species attempt to minimize loss of genetic variation and inbreeding through strategies designed to minimize global co-ancestry, but they assume a lack of relatedness among the founders. Yet populations that are the target of such programs are generally in serious demographic decline, and many of the available founders may be closely related. Here we demonstrate determination of full and half-sib relationships among the wild founders of a captive breeding program involving two endangered Atlantic salmon populations using two different approaches and associated software, pedigree and colony. A large portion of the juveniles collected in these two rivers appear to be derived from surprisingly few females mating with a large number of males, probably small precocious parr. Another group of potential founders, obtained from a local hatchery, clearly originated from a small number of full-sib crosses. These results allowed us to prioritize individuals on the basis of conservation value, and are expected to help minimize loss of genetic variation through time. In addition, insight is provided into the number of contributing parents and the mating systems that produced this last generation of endangered wild Atlantic salmon.     

How many came home? Evaluating ex situ conservation of green turtles in the Cayman Islands

Ex situ management is an important conservation tool that allows the preservation of biological diversity outside natural habitats while supporting survival in the wild. Captive breeding followed by re‐introduction is a possible approach for endangered species conservation and preservation of genetic variability. The Cayman Turtle Centre Ltd was established in 1968 to market green turtle (Chelonia mydas) meat and other products and replenish wild populations, thought to be locally extirpated, through captive breeding. We evaluated the effects of this re‐introduction programmme using molecular markers (13 microsatellites, 800‐bp D‐loop and simple tandem repeat mitochondrial DNA sequences) from captive breeders (N = 257) and wild nesting females (N = 57) (sampling period: 2013–2015). We divided the captive breeders into three groups: founders (from the original stock), and then two subdivisions of F₁ individuals corresponding to two different management strategies, cohort 1995 (“C1995”) and multicohort F₁ (“MCF1”). Loss of genetic variability and increased relatedness was observed in the captive stock over time. We found no significant differences in diversity among captive and wild groups, and similar or higher levels of haplotype variability when compared to other natural populations. Using parentage and sibship assignment, we determined that 90% of the wild individuals were related to the captive stock. Our results suggest a strong impact of the re‐introduction programmme on the present recovery of the wild green turtle population nesting in the Cayman Islands. Moreover, genetic relatedness analyses of captive populations are necessary to improve future management actions to maintain genetic diversity in the long term and avoid inbreeding depression.     

Using molecular markers for pedigree reconstruction of the greater amberjack (Seriola dumerili) in the absence of parental information

Ensuring appropriate levels of genetic diversity in captive populations is essential to avoid inbreeding and loss of rare alleles by genetic drift. Pedigree reconstruction and parentage analysis in the absence of parental genotypes can be a challenging task that relies in the assignment of sibship relationships among the offspring. Here, we used eight highly variable microsatellite markers and three different assignment methods to reconstruct the most likely genotypes of a parental group of wild Seriola dumerili fish based on the genotypes of six cohorts of their offspring, to assess their relative contributions to the offspring. We found that a combination of the four most variable microsatellites was enough to identify the number of parents and their contribution to the offspring, suggesting that the variability of the markers can be more critical than the number of markers. Estimated effective population sizes were lower than the number of breeders and variable among years. The results suggest unequal parental contribution that should be accounted for breeding programs in the future.     

Evidence of multiple paternity and mate selection for inbreeding avoidance in wild eastern chipmunks

Mate selection for inbreeding avoidance is documented in several taxa. In mammals, most conclusive evidence comes from captive experiments that control for the availability of mates and for the level of genetic relatedness between mating partners. However, the importance of mate selection for inbreeding avoidance as a determinant of siring success in the wild has rarely been addressed. We followed the reproduction of a wild population of eastern chipmunks (Tamias striatus) during five breeding seasons between 2006 and 2009. Using molecular tools and parentage assignment methods, we found that multiple paternity (among polytocous litters) varied from 25% in an early-spring breeding season when less than a quarter of females in the population were reproductively active to 100% across three summer breeding seasons and one spring breeding season when more than 85% of females were reproductively active. Genetically related parents were common in this population and produced less heterozygous offspring. Furthermore, litters with multiple sires showed a higher average relatedness among partners than litters with only a single sire. In multiply sired litters, however, males that were more closely related to their partners sired fewer offspring. Our results corroborate findings from captive experiments and suggest that selection for inbreeding avoidance can be an important determinant of reproductive success in wild mammals.     

Analysis of relatedness and determination of the source of founders in the captive bearded vulture, Gypaetus barbatus, population

Genetic relatedness among founders is a vitalparameter in the management of captivepopulations as kin structure can have asignificant effect on subsequent populationstructure. Methods for inferring relatednessfrom microsatellite markers have all beendeveloped for natural populations; theirapplicability to captive populations withunknown founder origins needs thereforetesting. We used information derived from 14microsatellites in 177 individuals and Quellerand Goodnight's approach, to estimaterelatedness in the captive bearded vulturepopulation and to test the assumption ofunrelated founders. Mean relatedness of knownparent–offspring, full-sib and half-sib pairswithin the captive population were in agreementwith theoretical distributions. Pairwiserelatedness values among the founders had amean of –0.051 (SE ± 0.007) and theirdistribution did only differ marginally fromthe one found in the natural Pyreneanpopulation. A maximum likelihood approach wasused to determine the likelihood of founderpairs to be as closely related as full-sibs orparent–offspring. These results were combinedwith data from 268 bp mitochondrial DNA controlregion sequences and studbook information. Wecould exclude a close relationship among themajority of the 36 successfully reproducingfounders. Our study therefore removesmanagement concerns about hidden problems ofinbreeding and inbreeding depression. Itdemonstrates the applicability of relatednessestimates based on microsatellite allelefrequency data even in captive populations.Furthermore, we verified studbook informationon the origin of two founders from thePyrenees, and show the value of assignmenttests based on microsatellites for deducingfounder origins and their important role infuture monitoring projects.     

Increasing breeding behaviors in a captive colony of Northern Bald Ibis through conspecific acoustic enrichment

The goal of this study was to increase reproductive behaviors in a captive colony of Northern Bald Ibis (Geronticus eremita) at the Bronx Zoo. The Northern Bald Ibis is classified as critically endangered by the IUCN, with only about 100 known breeding pairs in the wild. Our analysis of zoo breeding and colony size data confirmed earlier observations that small captive colonies of this species do not breed as well as larger colonies, possibly because of insufficient social cues. Using principles from social facilitation theory, we attempted to provide such social cues by presenting acoustic enrichment in the form of conspecific breeding vocalization playbacks. In May 2009, we tracked multiple breeding behaviors during playbacks and control periods. Although breeding behaviors increased in response to playbacks, the colony produced no eggs. We repeated this experiment in April 2010 using playbacks of higher quality recordings acquired from a semi‐wild breeding colony of Northern Bald Ibis in Austria. Breeding behaviors again increased during playbacks. In addition, five pairs in this colony of 15 birds produced 13 eggs, and six chicks successfully fledged—the first reproductive success in this colony since 2007. The acoustic enrichment techniques we employed may encourage increased breeding activity in captive populations of colonial species as well as wild colonies with small populations. Zoo Biol 31:71;–81, 2012. © 2011 Wiley Periodicals, Inc.     

Effects of inbreeding on reproductive success,performance, litter size,and survival in captive red wolves (Canis rufus)

Captive‐breeding programs have been widely used in the conservation of imperiled species, but the effects of inbreeding, frequently expressed in traits related to fitness, are nearly unavoidable in small populations with few founders. Following its planned extirpation in the wild, the endangered red wolf (Canis rufus) was preserved in captivity with just 14 founders. In this study, we evaluated the captive red wolf population for relationships between inbreeding and reproductive performance and fitness. Over 30 years of managed breeding, the level of inbreeding in the captive population has increased, and litter size has declined. Inbreeding levels were lower in sire and dam wolves that reproduced than in those that did not reproduce. However, there was no difference in the inbreeding level of actual litters and predicted litters. Litter size was negatively affected by offspring and paternal levels of inbreeding, but the effect of inbreeding on offspring survival was restricted to a positive influence. There was no apparent relationship between inbreeding and method of rearing offspring. The observable effects of inbreeding in the captive red wolf population currently do not appear to be a limiting factor in the conservation of the red wolf population. Additional studies exploring the extent of the effects of inbreeding will be required as inbreeding levels increase in the captive population. Zoo Biol 29:36–49, 2010. © 2009 Wiley‐Liss, Inc.     

Genetic variation in Przewalski's horses, with special focus on the last wild caught mare, 231 Orlitza III

In our continuing efforts to document genetic diversity in Przewalski's horses and relatedness with domestic horses, we report genetic variation at 22 loci of blood group and protein polymorphisms and 29 loci of DNA (microsatellite) polymorphisms. The loci have been assigned by linkage or synteny mapping to 20 autosomes and the X chromosome of the domestic horse (plus four loci unassigned to a chromosome). With cumulative data from tests of 568 Przewalski's horses using blood, hair or tooth samples, no species-defining markers were identified, however a few markers were present in the wild species but not in domestic horses. Inheritance patterns and linkage relationships reported in domestic horses appeared to be conserved in Przewalski's horses. A derived type for the last wild caught mare 231 Orlitza III provided evidence for markers apparently not found in (or not currently available by descent from) the other species founders that were captured at the end of the nineteenth century. This information has been critical to the development of parentage analyses in the studbook population of Przewalski's horses at Askania Nova, at one time the largest herd of captive animals and the source of stock for reintroduction efforts. Some horses in the study showed genetic incompatibilities with their sire or dam, contradicting published studbook information. In many cases alternative parentage could be assigned from living animals. To assist in identification of correct parentage, DNA marker types for deceased horses were established from archived materials (teeth) or derived from offspring. Genetic markers were present in pedigreed animals whose origin could not be accounted for from founders. Genetic distance analysis of erythrocyte protein, electrophoretic and microsatellite markers in Przewlaski's horses and ten breeds of domestic horse place the Przewalski's horse as an outgroup to domestic horses, introgression events from domestic horses not withstanding.     

Conservation Genetics of Threatened Hippocampus guttulatus in Vulnerable Habitats in NW Spain: Temporal and Spatial Stability of Wild Populations with Flexible Polygamous Mating System in Captivity

This study was focused on conservation genetics of threatened Hippocampus guttulatus on the Atlantic coast of NW Iberian Peninsula. Information about spatial structure and temporal stability of wild populations was obtained based on microsatellite markers, and used for monitoring a captive breeding program firstly initiated in this zone at the facilities of the Institute of Marine Research (Vigo, Spain). No significant major genetic structure was observed regarding the biogeographical barrier of Cape Finisterre. However, two management units under continuous gene flow are proposed based on the allelic differentiation between South-Atlantic and Cantabrian subpopulations, with small to moderate contemporary effective size based on single-sample methods. Temporal stability was observed in South-Atlantic population samples of H. guttulatus for the six-year period studied, suggesting large enough effective population size to buffer the effects of genetic drift within the time frame of three generations. Genetic analysis of wild breeders and offspring in captivity since 2009 allowed us to monitor the breeding program founded in 2006 in NW Spain for this species. Similar genetic diversity in the renewed and founder broodstock, regarding the wild population of origin, supports suitable renewal and rearing processes to maintain genetic variation in captivity. Genetic parentage proved single-brood monogamy in the wild and in captivity, but flexible short- and long-term mating system under captive conditions, from strict monogamy to polygamy within and/or among breeding seasons. Family analysis showed high reproductive success in captivity under genetic management assisted by molecular relatedness estimates to avoid inbreeding. This study provides genetic information about H. guttulatus in the wild and captivity within an uncovered geographical range for this data deficient species, to be taken into account for management and conservation purposes.     

Assimilation of new founders into existing captive populations

When new founders are added to an existing captive population, it is useful to establish a target number of offspring from each of these new founders that will maximize the amount of gene diversity retained in the captive population. This article presents a method for calculating an optimal number of offspring that should be produced from each new founder by considering the retention of founder genomes from dead and non-reproductive founders. © 1994 Wiley-Liss, Inc.     

Identification of kin structure among Guam rail founders: a comparison of pedigrees and DNA profiles

Kin structure among founders can have a significant effect on subsequent population structure. Here we use the correlation between DNA profile similarity and relatedness calculated from pedigrees to test hypotheses regarding kin structure among founders to the captive Guam rail (Rallus owstoni) population. Five different pedigrees were generated under the following hypotheses: (i) founders are unrelated; (ii) founders are unrelated except for same-nest chicks; (iii) founders from the same major site are siblings; (iv) founders from the same local site are siblings; and (v) founders are related as defined by a UPGMA cluster analysis of DNA similarity data. Relatedness values from pedigrees 1, 2 and 5 had the highest correlation with DNA similarity but the correlation between relatedness and similarity were not significantly different among pedigrees. Pedigree 5 resulted in the highest correlation overall when using only relatedness values that changed as a result of different founder hypotheses. Thus, founders were assigned relatedness based on pedigree 5 because it had the highest correlations with DNA similarity, was the most conservative approach, and incorporated all field data. The analyses indicated that estimating relatedness using DNA profiles remains problematic, therefore we compared mean kinship, a measure of genetic importance, with mean DNA profile similarity to determine if genetic importance among individuals could be determined via use of DNA profiles alone. The significant correlation suggests this method may provide more information about population structure than was previously thought. Thus, DNA profiles can provide a reasonable explanation for founder relatedness and mean DNA profile similarity may be helpful in determining relative genetic importance of individuals when detailed pedigrees are absent.     

Status of a Relocated Population of Endangered Iguana pinguis on Guana Island, British Virgin Islands

Eight individual rock iguanas (Iguana pinguis) from Anegada Island were relocated to Guana Island by Lazell, 1984–1987, in order to establish a second population reservoir for this endangered species. The species may have originally occupied the entire Puerto Rico Bank. The relocation has been successful and, in the area currently providing the best habitat, we estimate a density of 9 or 12 animals of various age classes per 19 ha. The optimal area contains a sheep exclosure with relatively dense understory vegetation and numerous exotic as well as native species of plants. Iguana activity is concentrated on east facing slopes and ridge-tops that get morning sun. Outside the exclosure most edible ground cover and shrubs have been eaten by sheep, leaving toxic or noxious species, such as Croton or Lantana, in the understory where I. pinguis adults generally forage. Removal of sheep may be critical to continued population growth of these reptiles. Views on relocation or repatriation of other endangered Antillean Iguana species are advanced, with some ideas on minimum viable population sizes and a possible explanation for the extirpation of I. pinguis from much of its former range.     

Rodrigues fruit bats (<Emphasis Type="Italic">Pteropus rodricensis</Emphasis>, Megachiroptera: Pteropodidae) retain genetic diversity despite population declines and founder events

Fruit bats of the genus Pteropus are important contributors to ecosystem maintenance on islands through their roles as pollinators and seed dispersers. However, island faunas are the most prone to extinction and there is a real need to assess the possible genetic implications of population reductions in terms of extinction risk. An effective method of ameliorating extinction risk in endangered species is the establishment of captive populations ex situ. The effectiveness of captive breeding programmes may be assessed by comparing the genetic variability of captive colonies to that of wild counterparts. Here, we use polymorphic microsatellite loci to assess genetic variability in wild, critically endangered Rodrigues fruit bats (Pteropus rodricensis, Dobson 1878) and we compare this variability to that in a captive colony. We document remarkable conservation of genetic variability in both the wild and captive populations, despite population declines and founder events. Our results demonstrate that the wild population has withstood the negative effects of population reductions and that captive breeding programmes can fulfil the goals of retaining genetic diversity and limiting inbreeding.     

Testing alternative captive breeding strategies with the subsequent release into the wild

We used the housefly (Musca domestica L.) as an experimental model to compare two strategies for the captive breeding of an endangered species: a strategy to minimize inbreeding and balance founder contributions (termed “MAI” for “maximum avoidance of inbreeding”) versus a scheme to select against less fit individuals (disregarding relatedness). By balancing the initial founder contributions, the MAI protocol was analogous to methods for minimizing kinship. In both breeding strategies, the population growth rate was limited to a maximum increase of 50% per generation. Five replicate populations, each starting with five male–female pairs, were subjected to five generations of captive breeding. Six generations of simulated “release into the wild” allowed ad lib breeding with less restrictive population growth potential, in either a benign or stressful environment (i.e., constant or variable temperature). Population size, fecundity, and fertility were assayed throughout the experiment, with juvenile‐to‐adult survival assayed in the second phase of the project. Allozyme assays determined the resultant inbreeding coefficients from the captive breeding schemes. The MAI breeding scheme resulted in significantly lower inbreeding coefficients and higher fitness, with qualitatively reduced extinction potential, most notable in the stressful environment. Spontaneous fitness rebounds suggested that the MAI strategy facilitated some form of purging of inbreeding depression effects. Importantly, the advantages of the MAI strategy were difficult to detect during the captive breeding phase, suggesting that the long‐term advantages of the MAI approach could be underestimated in actual breeding programs. We concur with the common recommendation of maximum avoidance of inbreeding at least for systems with low reproductive potential. Zoo Biol 0:1–18, 2005. © 2005 Wiley‐Liss, Inc.     

DNA variability and parentage testing in captive Waldrapp ibises

The Waldrapp ibis ( Geronticus eretnita ) is one of the most endangered birds in the wild. The only real hope for its survival seems to rest with captive propagation, which has fortunately proved straightforward. Preservation of genetic diversity for maintaining viable populations is a major goal in any species conservation programme. Therefore sensitive tools have to be developed for the detection and regular monitoring of population variability. Individual identity has to be verified and each individual's pedigree defined in order to enable effective selective breeding. In this study, extensive DNA fingerprinting technology was applied to a group of 39 captive Waldrapp ibises. Five minisatellite probes were used. Despite the fact that the colony was founded by only six birds and that some of the founders were apparently related, highly polymorphic banding patterns were obtained. The human- and pig-derived probes MS1, MS31, pλg3 and pCMS12 each detected multiple distinct loci suitable for individual identification and resolution of parentage. Probe MS51 was locus-specific and revealed six different alleles. In addition, MS31 and pλg3 co-detected a band present in all females but none of the males and thus could be used for sex determination. For all but four individuals both parents could be identified unambiguously. The exceptions were offspring derived from a full sib mating in which DNA results allowed no distinction between the parents and the grandparents. Comparison of the parentage assigned by DNA analysis and by observation of breeding behaviour, respectively, showed agreement in all but five instances. Here, DNA data uncovered interchange of individuals in the stud book. Application of these minisatellite probes therefore offers an extremely sensitive means for individual identification and verification and completion of pedigree records.     

Breeding orange-winged Amazon parrots in captivity

Captive propagation of parrots for the companion bird trade provides a potential means of reducing the economic incentive to capture these birds from the wild. To test whether environmental and social manipulations might stimulate reproduction in captive, wild-caught orange-winged Amazon parrots (Amazona amazonica), we compared reproductive performance in pairs presented with nest boxes (controls) or nest boxes plus additional environmental and social manipulations (enriched group) consisting of misting, fruit supplementation, nest hole restriction, enlarged nest boxes, and pair separation/reunification. In the first breeding trial, in 1990, enriched pairs were more likely to lay eggs (six of seven enriched pairs vs. one of eight control pairs; P < 0.09). When treatments were reversed the following years (e.g., 1990 controls were exposed to enriched conditions in 1991), reproductive performance was not different between the groups (four of seven enriched pairs laid eggs vs. five of seven control pairs), although three pairs in the 1991 enriched group laid eggs which had not laid eggs before. These results are consistent with the idea that while enriched environments may enhance the probability of a first episode of egg laying in captivity, once pairs have laid eggs in captivity, little stimulation beyond that provided by nest box presentation is required to reinitiate egg laying. The environmental and social manipulations of the enriched conditions could be useful in increasing reproductive performance of captive Amazon parrots for the companion bird trade. © 1995 Wiley-Liss, Inc.