Implications of methodologies for integrating empirical kinships into ex situ population management using PMx: A case study of Baer's Pochard (Aythya baeri) in North America.

The application of molecular tools to population management can improve the long-term genetic viability of ex situ populations. In this study, we aimed to understand the implications of integrating empirical kinships into the genetic management of an ex situ population of the endangered waterfowl, Baer's pochard (Aythya baeri), in North America. Single nucleotide polymorphism data were generated for 141 Baer's pochard using double digest restriction site-associated DNA sequencing and empirical kinships were derived and integrated into the population management software PMx. Analyses suggested 37.7% of pairwise relationships previously assumed to be unrelated were first, second, or third-order relatives. We determined that most genetic summary statistics were impacted through the calculation of the population's mean kinship, which increased from MK¯=0.0772 to MK¯=0.2074 after empirical kinships were integrated into our analyses. Our results also revealed the importance of understanding how molecular kinships derived from a particular estimator are scaled, if the scale differs significantly from pedigree-based kinships. We describe the theory behind the genetic metrics impacted and provide general guidance on incorporating empirical kinships into ex situ population management as well as provide suggestions for sampling strategies to minimize the biases inherent in merging two types of kinship estimators.

Ex situ breeding programs benefit from science-based cooperative management.

Science-based management confers a variety of benefits to wildlife populations that are cooperatively managed by zoos and aquariums, including those managed through the Association of Zoos and Aquariums. Briefly, when management strategies are successful, they result in reproductively robust populations that better retain genetic diversity and limit inbreeding than unmanaged populations. Although the benefits of demographic and genetic management have been well documented throughout both the scientific and popular literature, it has also been established that the majority of managed populations in zoos and aquariums are not meeting the minimum criteria believed to convey long-term biological viability. For most of these populations, an inability to meet viability criteria is not an inherent failure of how cooperative management is implemented. Furthermore, in recent years, we have perceived that the need to meet specific viability goals sometimes has obscured the benefits that these populations receive from rigorous, science-based management. To better clarify the conversation surrounding population viability in zoos and aquariums, we seek to decouple viability measures and how they predict population persistence from the benefits conferred to populations through science-based management. A primary goal of population management is to facilitate the persistence of priority species for longer than would be expected if no such management were implemented. Although current viability measures and future projections of viability are important tools for assessing the likelihood of population persistence, they are not indicators of which populations may most benefit from science-based management. Here, we review the history and purpose of applying science-based management to zoo and aquarium populations, describe measures of population viability and caution against confusing those measures of viability with population management goals or long-term population sustainability, and clearly articulate the benefits conferred to zoo and aquarium populations by science-based management.

Strategies for establishing and using genome resource banks to protect genetic diversity in conservation breeding programs.

Genome resource banks (GRBs) have the potential to preserve the genetic diversity of a species over time, yet they are rarely utilized as effective components of conservation breeding programs. Advances have been made in reproductive biology, collection and storage techniques, and use of stored gametes for achieving successful reproduction, but there are few guidelines for integrating GRBs into established breeding programs. Here we present basic guidelines, focusing on strategies for the collection, maintenance, and use of semen GRBs for protecting genetic diversity. These guidelines should be applied in the context of the specific purposes and roles of a breeding program's GRB, which will differ among species depending on vulnerability to loss and the status of rescue and conservation efforts. We recommend establishing up to three types of collections: (1) a National Reserve to preserve a species' genetic diversity, to be used only as a last resort; (2) a Savings Account to be used periodically to invigorate a genetically depauperate population; and (3) a Checking Account to be used as a regular part of the breeding program. We present methods for identifying donors to maximize genetic diversity in a GRB, as well as strategies for maintaining and optimally using GRBs.

Use of molecular data in zoo and aquarium collection management: Benefits, challenges, and best practices.

The global zoo and aquarium community widely recognizes that its animal collections and cooperative breeding programs are facing a sustainability crisis. It has become commonly accepted that numerous priority species cannot be maintained unless new management strategies are adopted. While molecular data have the potential to greatly improve management across a range of scenarios, they have been generally underutilized by the zoo and aquarium community. This failure to effectively apply molecular data to collection management has been due, in part, to a paucity of resources within the community on which to base informed decisions about when the use of such data is appropriate and what steps are necessary to successfully integrate data into management. Here, we identify three broad areas of inquiry where molecular data can inform management: 1) taxonomic identification; 2) incomplete or unknown pedigrees; and 3) hereditary disease. Across these topics, we offer a discussion of the advantages, limitations, and considerations for applying molecular data to ex situ animal populations in a style accessible to zoo and aquarium professionals. Ultimately, we intend for this compiled information to serve as a resource for the community to help ensure that molecular projects directly and effectively benefit the long-term persistence of ex situ populations.

Demographic, environmental and genetic determinants of mating success in captive koalas (Phascolarctos cinereus).

Many factors have been shown to affect mating behavior. For instance, genes of the major histocompatibility complex (MHC) are known to influence mate choice in a wide variety of vertebrate species. The genetic management of captive populations can be confounded if intrinsic mate choice reduces or eliminates reproductive success between carefully chosen breeding pairs. For example, the San Diego Zoo koala colony only has a 45% copulation rate for matched individuals. Herein, we investigated determinants of koala mating success using breeding records (1984-2010) and genotypes for 52 individuals at four MHC markers. We quantified MHC diversity according to functional amino acids, heterozygosity, and the probability of producing a heterozygous offspring. We then used categorical analysis and logistic regression to investigate both copulation and parturition success. In addition, we also examined age, day length, and average pairwise kinship. Our post-hoc power analysis indicates that at a power level of 1-ß = 0.8, we should have been able to detect strong MHC preferences. However, we did not find a significant MHC effect on either copulation or parturition success with one exception: pairs with lower or no production of a joey had significantly lower MHC functional amino acid diversity in the categorical analysis. In contrast, day length and dam age (or age difference of the pair) consistently had an effect on mating success. These findings may be leveraged to improve the success of attempted pairs, conserve resources, and facilitate genetic management.

Applying SNP-Derived Molecular Coancestry Estimates to Captive Breeding Programs.

Captive breeding programs for wildlife species typically rely on pedigrees to inform genetic management. Although pedigree-based breeding strategies are quite effective at retaining long-term genetic variation, management of zoo-based breeding programs continues to be hampered when pedigrees are poorly known. The objective of this study was to evaluate 2 options for generating single nucleotide polymorphism (SNP) data to resolve unknown relationships within captive breeding programs. We generated SNP data for a zoo-based population of addax (Addax nasomasculatus) using both the Illumina BovineHD BeadChip and double digest restriction site-associated DNA (ddRAD) sequencing. Our results demonstrated that estimates of allele sharing (AS) between pairs of individuals exhibited low variances. Average AS variances were highest when using 50 loci (SNPchipall = 0.00159; ddRADall = 0.0249), but fell below 0.0003 for the SNP chip dataset when sampling ≥250 loci and below 0.0025 for the ddRAD dataset when sampling ≥500 loci. Furthermore, the correlation between the SNPchipall and ddRADall AS datasets was 0.88 (95%CI = 0.84-0.91) when subsampling 500 loci. Collectively, our results indicated that both SNP genotyping methods produced sufficient data for accurately estimating relationships, even within an extremely bottlenecked population. Our results also suggested that analytic assumptions historically integrated into the addax pedigree are not adversely impacting long-term pedigree-based management; kinships calculated from the analytic pedigree were significantly correlated (P << 0.001) with AS estimates. Overall, our conclusions are intended to serve as both a proof of concept and a model for applying molecular data to the genetic management of captive breeding programs.

Estrogenicity of captive southern white rhinoceros diets and their association with fertility.

The captive southern white rhinoceros (SWR) population is not currently self-sustaining, primarily due to poor or absent reproduction of captive-born (F1+) females. In this study, we investigate the role of dietary phytoestrogens in this reproductive phenomenon by characterizing activation of SWR estrogen receptors (ESRs) 1 and 2 by diet items from nine North American institutions and comparing female SWR fertility to total diet estrogenicity. Of the diet items tested, alfalfa hay and soy and alfalfa-based commercial pellets were found to be the most potent activators of SWR ESRs. In contrast, most grass hays tested were not estrogenic. The estrogenicity of total diets varied across the institutions surveyed and the degree of diet estrogenicity was positively associated with the percentage of the total diet comprised by pellets. Comparisons of fertility records of the institutions surveyed showed no significant relationship between diet estrogenicity and fertility for female SWR conceived or born in the wild (F0). However, for F1+ females, there was a significant negative relationship between institutional diet estrogenicity and fertility. Taken together, these data suggest that developmental exposure to phytoestrogens may be the cause of poor fertility in captive-born female SWR. Whether the low fertility of the current population of captive-born female SWR is permanent or can be reversed by removing phytoestrogens from the diet remains unclear. However, our findings suggest that in order for the SWR population to become self-sustaining, the development and feeding of low phytoestrogen diets should be strongly considered.

Immunomics of the koala (Phascolarctos cinereus).

The study of the koala transcriptome has the potential to advance our understanding of its immunome--immunological reaction of a given host to foreign antigens--and to help combat infectious diseases (e.g., chlamydiosis) that impede ongoing conservation efforts. We used Illumina sequencing of cDNA to characterize genes expressed in two different koala tissues of immunological importance, blood and spleen. We generated nearly 600 million raw sequence reads, and about 285 million of these were subsequently assembled and condensed into ~70,000 subcomponents that represent putative transcripts. We annotated ~16% of these subcomponents and identified those related to infection and the immune response, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), major histocompatibility complex (MHC) genes, and koala retrovirus (KoRV). Using phylogenetic analyses, we identified 29 koala genes in these target categories and report their concordance with currently accepted gene groups. By mapping multiple sequencing reads to transcripts, we identified 56 putative SNPs in genes of interest. The distribution of these SNPs indicates that MHC genes (34 SNPs) are more diverse than KoRV (12 SNPs), TLRs (8 SNPs), or RLRs (2 SNPs). Our sequence data also indicate that KoRV sequences are highly expressed in the transcriptome. Our efforts have produced full-length sequences for potentially important immune genes in koala, which should serve as targets for future investigations that aim to conserve koala populations.

Impacts of early viability selection on management of inbreeding and genetic diversity in conservation.

Maintaining genetic diversity is a crucial goal of intensive management of threatened species, particularly for those populations that act as sources for translocation or re-introduction programmes. Most captive genetic management is based on pedigrees and a neutral theory of inheritance, an assumption that may be violated by selective forces operating in captivity. Here, we explore the conservation consequences of early viability selection: differential offspring survival that occurs prior to management or research observations, such as embryo deaths in utero. If early viability selection produces genotypic deviations from Mendelian predictions, it may undermine management strategies intended to minimize inbreeding and maintain genetic diversity. We use empirical examples to demonstrate that straightforward approaches, such as comparing litter sizes of inbred vs. noninbred breeding pairs, can be used to test whether early viability selection likely impacts estimates of inbreeding depression. We also show that comparing multilocus genotype data to pedigree predictions can reveal whether early viability selection drives systematic biases in genetic diversity, patterns that would not be detected using pedigree-based statistics alone. More sophisticated analysis combining genomewide molecular data with pedigree information will enable conservation scientists to test whether early viability selection drives deviations from neutrality across wide stretches of the genome, revealing whether this form of selection biases the pedigree-based statistics and inference upon which intensive management is based.

The impacts of inbreeding, drift and selection on genetic diversity in captive breeding populations.

The goal of captive breeding programmes is often to maintain genetic diversity until re-introductions can occur. However, due in part to changes that occur in captive populations, approximately one-third of re-introductions fail. We evaluated genetic changes in captive populations using microsatellites and mtDNA. We analysed six populations of white-footed mice that were propagated for 20 generations using two replicates of three protocols: random mating (RAN), minimizing mean kinship (MK) and selection for docility (DOC). We found that MK resulted in the slowest loss of microsatellite genetic diversity compared to RAN and DOC. However, the loss of mtDNA haplotypes was not consistent among replicate lines. We compared our empirical data to simulated data and found no evidence of selection. Our results suggest that although the effects of drift may not be fully mitigated, MK reduces the loss of alleles due to inbreeding more effectively than random mating or docility selection. Therefore, MK should be preferred for captive breeding. Furthermore, our simulations show that incorporating microsatellite data into the MK framework reduced the magnitude of drift, which may have applications in long-term or extremely genetically depauperate captive populations.

Kinship-based management strategies for captive breeding programs when pedigrees are unknown or uncertain.

Zoo-based captive breeding programs typically rely on accurate pedigrees to maintain long-term population genetic diversity and prevent close inbreeding. For many mixed-sex captive populations, it is difficult to assign parentage of offspring with certainty without conducting DNA-based parentage analyses. Using the demographic parameters of a North American captive population of Arabian oryx (Oryx leucoryx), 2 kinship-based breeding-pair selection strategies were modeled for their performance in handling pedigrees with varying degrees of parentage uncertainty. We also compared these strategies with 2 nonkinship-based methods. Pedigrees simulated under different management strategies were compared for their long-term ability to maintain gene diversity (GD) and avoid inbreeding. For the Arabian oryx, results indicate that recording multiple possible parents instead of removing the unknown genomic portion of the pedigree can more efficiently utilize all animals available for breeding without compromising GD and inbreeding avoidance. Both kinship-based breeding-pair selection strategies significantly outperformed the nonkinship-based strategies.

Evaluating the performance of captive breeding techniques for conservation hatcheries: a case study of the delta smelt captive breeding program.

The delta smelt, an endangered fish species endemic to the San Francisco Bay-Delta, California, United States, was recently brought into captivity for species preservation. This study retrospectively evaluates the implementation of a genetic management plan for the captive delta smelt population. The captive genetic management plan entails tagging fish, molecular data collection, pedigree reconstruction, relatedness estimation, and recommending fish crosses annually in an effort to minimize the average coancestry in the population and limit inbreeding. We employed 12 microsatellite DNA markers to examine temporal genetic diversity in consecutive, discrete generations to determine the effects of intensive genetic management on the population and to quantify the amount of wild genetic diversity present within each captive generation. Wild fish are incorporated into the captive population each generation to minimize genetic drift, and 91% of the original founders are still represented in the F(3) generation. The average mean kinship in the third generation in captivity was 0.0035. There was no evidence of significant genetic divergence of the captive population from the wild population. The results of this study yield management insights into the practical application of genetic management plans for captive populations and conservation hatcheries, in an attempt to preserve the genetic integrity of endangered species.

Using rattlesnake microsatellites to determine paternity in captive bushmasters (Lachesis muta).

We used 10 microsatellite DNA markers originally described for two Crotalus and one Sistrurus species to infer paternity in a captive-hatched clutch of Lachesis muta. Although the dam was known, records listed two potential sires, which prevented the inclusion of those offspring in a captive breeding program. Samples were collected from both possible sires, the presumed dam and two offspring. Five of the ten markers were paternity informative and either unambiguously identified one of the two males as the sire of the offspring or excluded the other male. A sixth marker identified the sire in one of the two offspring. These results reveal rattlesnake DNA markers as a useful tool in paternity testing and captive breeding management of Lachesis muta. It also indicates that, within the American crotalid radiation, a selection of microsatellite DNA markers cross-amplify and provide useful genotypic information across species and genera.

Genetic diversity in global populations of the critically endangered addax (Addax nasomaculatus) and its implications for conservation.

Threatened species are frequently patchily distributed across small wild populations, ex situ populations managed with varying levels of intensity and reintroduced populations. Best practice advocates for integrated management across in situ and ex situ populations. Wild addax (Addax nasomaculatus) now number fewer than 100 individuals, yet 1000 of addax remain in ex situ populations, which can provide addax for reintroductions, as has been the case in Tunisia since the mid-1980s. However, integrated management requires genetic data to ascertain the relationships between wild and ex situ populations that have incomplete knowledge of founder origins, management histories, and pedigrees. We undertook a global assessment of genetic diversity across wild, ex situ and reintroduced populations in Tunisia to assist conservation planning for this Critically Endangered species. We show that the remnant wild populations retain more mitochondrial haplotypes that are more diverse than the entirety of the ex situ populations across Europe, North America and the United Arab Emirates, and the reintroduced Tunisian population. Additionally, 1704 SNPs revealed that whilst population structure within the ex situ population is minimal, each population carries unique diversity. Finally, we show that careful selection of founders and subsequent genetic management is vital to ensure genetic diversity is provided to, and minimize drift and inbreeding within reintroductions. Our results highlight a vital need to conserve the last remaining wild addax population, and we provide a genetic foundation for determining integrated conservation strategies to prevent extinction and optimize future reintroductions.

A comparison of strategies for selecting breeding pairs to maximize genetic diversity retention in managed populations.

Captive breeding programs aim to maintain populations that are demographically self-sustaining and genetically healthy. It has been well documented that the best way for managed breeding programs to retain gene diversity (GD) and limit inbreeding is to select breeding pairs that minimize a population's average kinship. We used a series of computer simulations to test 4 methods of minimizing average kinship across a variety of scenarios with varying generation lengths, mortality rates, reproductive rates, and rates of breeding pair success. "Static MK Selection" and "Dynamic MK Selection" are 2 methods for iteratively selecting genetically underrepresented individuals for breeding, whereas "Ranked MK Selection" and "Simultaneous MK Selection" are 2 methods for concurrently selecting the group of breeding individuals that produce offspring with the lowest average kinship. For populations with discrete generations (24 tested scenarios), we found that the Simultaneous and Ranked MK Selection methods were generally the best, nearly equivalent methods for selecting breeding pairs that retained GD and limited inbreeding. For populations with overlapping generations (198 tested scenarios), we found that Dynamic MK Selection was the most robust method for selecting breeding pairs. We used these results to provide guidelines for identifying which method of minimizing average kinship was most appropriate for various breeding program scenarios.

A suite of microsatellite markers optimized for amplification of DNA from Addax (Addax nasomaculatus) blood preserved on FTA cards.

The addax (Addax nasomaculatus) is a critically endangered antelope that is currently maintained in zoos through regional, conservation breeding programs. As for many captive species, incomplete pedigree data currently impedes the ability of addax breeding programs to confidently manage the genetics of captive populations and to select appropriate animals for reintroduction. Molecular markers are often used to improve pedigree resolution, thereby improving the long-term effectiveness of genetic management. When developing a suite of molecular markers, it is important to consider the source of DNA, as the utility of markers may vary across DNA sources. In this study, we optimized a suite of microsatellite markers for use in genotyping captive addax blood samples collected on FTA cards. We amplified 66 microsatellite loci previously described in other Artiodactyls. Sixteen markers amplified a single product in addax, but only 5 of these were found to be polymorphic in a sample of 37 addax sampled from a captive herd at Fossil Rim Wildlife Center in the US. The suite of microsatellite markers developed in this study provides a new tool for the genetic management of captive addax, and demonstrates that FTA cards can be a useful means of sample storage, provided appropriate loci are used in downstream analyses.

A retrospective and prospective study of megaesophagus in the parma wallaby (Macropus parma) at the San Diego Zoo, California, USA.

At the San Diego Zoo (California, USA), 22 cases of megaesophagus were diagnosed in the parma wallaby (Macropus parma); a prevalence of 21.1%. Parma wallabies often have no clinical signs until severe and chronic dilation of the esophagus is present. Clinical signs of advanced disease include weight loss, swelling of the cervical region, regurgitation without reswallowing of ingesta, short flight distance, depression, collapse, dyspnea, and sudden death. Retrospective and prospective studies at the San Diego Zoo and a multi-institutional survey in the United States were used to try to determine the cause of megaesophagus. The retrospective study did not identify an etiology. The prospective study revealed megaesophagus and severely delayed esophageal transit time in eight of eight animals. Myasthenia gravis, lead toxicosis, toxoplasmosis, and thyroid disease were eliminated as possible causes. Of 286 living and dead parma wallabies surveyed at other institutions, three cases of esophageal diverticulum and one case of megaesophagus were reported. The cause of megaesophagus in parma wallabies was not determined.

A retrospective and prospective study of megaesophagus in the parma wallaby (Macropus parma) at the San Diego Zoo, California, USA.

At the San Diego Zoo (California, USA), 22 cases of megaesophagus were diagnosed in the parma wallaby (Macropus parma), yielding a prevalence of 21.1%. Parma wallabies often have no clinical signs until severe and chronic dilation of the esophagus is present. Clinical signs of advanced disease include weight loss, swelling of the cervical region, regurgitation without reswallowing of ingesta, short flight distance, depression, collapse, dyspnea, and sudden death. Retrospective and prospective studies at the San Diego Zoo and a multi-institutional survey in the United States were used to try to determine the cause of megaesophagus. The retrospective study did not identify an etiology. The prospective study revealed megaesophagus and severely delayed esophageal transit time in eight of eight animals. Myasthenia gravis, lead toxicosis, toxoplasmosis, and thyroid disease were eliminated as possible causes. Of 286 living and dead parma wallabies surveyed at other institutions, three cases of esophageal diverticulum and one case of megaesophagus were reported. The cause of megaesophagus in parma wallabies was not determined.

Methods and prospects for using molecular data in captive breeding programs: an empirical example using parma wallabies (Macropus parma).

Zoo and aquarium breeding programs rely on accurate pedigrees to manage the genetics and demographics of captive populations. Breeding recommendations are often encumbered, however, by unknown parentage. If an individual has any amount of unknown ancestry, the relationships between that individual and all other individuals in a population are ambiguous, and breeding recommendations cannot be tailored to maximize genetic diversity and minimize inbreeding. In those situations, breeding program management might be improved by the incorporation of molecular data. We developed microsatellite markers for the parma wallaby (Macropus parma) and investigated how genetic data might be used to improve the management of the captive population. The parma wallaby is a small marsupial found in fragmented forests near the coast of New South Wales, Australia. Because the species is of conservation concern, the captive population in North America is managed by recurring breeding recommendations. The effectiveness of the population's management is hampered, however, because over half of the individuals have some amount of unknown ancestry. We used microsatellite data to resolve unknown parentage, described how molecular estimates of relatedness might inform future breeding recommendations, and used computer simulations to investigate how molecular estimates of relatedness among founders might contribute to the genetic management of the population. Our results indicated that microsatellite appraisals of parentage were useful with respect to clarifying pedigrees but that molecular assessments of founder relatedness provided very marginal benefits with regard to the preservation of genetic diversity and the avoidance of inbreeding.

Impacts of natural history and exhibit factors on carnivore welfare.

To improve the welfare of nonhuman animals under professional care, zoological institutions are continuously utilizing new methods to identify factors that lead to optimal welfare. Comparative methods have historically been used in the field of evolutionary biology but are increasingly being applied in the field of animal welfare. In the current study, data were obtained from direct behavioral observation and institutional records representing 80 individual animals from 34 different species of the order Carnivora. Data were examined to determine if a variety of natural history and animal management factors impacted the welfare of animals in zoological institutions. Output variables indicating welfare status included behavioral diversity, pacing, offspring production, and infant mortality. Results suggested that generalist species have higher behavioral diversity and offspring production in zoos compared with their specialist counterparts. In addition, increased minimum distance from the public decreased pacing and increased offspring production, while increased maximum distance from the public and large enclosure size decreased infant mortality. These results have implications for future exhibit design or renovation, as well as management practices and priorities for future research.