Comparison of genomic diversity and structure of sable antelope (Hippotragus niger) in zoos, conservation centers, and private ranches in North America

Abstract As we enter the sixth mass extinction, many species that are no longer self‐sustaining in their natural habitat will require ex situ management. Zoos have finite resources for ex situ management, and there is a need for holistic conservation programs between the public and private sector. Ex situ populations of sable antelope, Hippotragus niger, have existed in zoos and privately owned ranches in North America since the 1910s. Unknown founder representation and relatedness has made the genetic management of this species challenging within zoos, while populations on privately owned ranches are managed independently and retain minimal‐to‐no pedigree history. Consequences of such challenges include an increased risk of inbreeding and a loss of genetic diversity. Here, we developed and applied a customized targeted sequence capture panel based on 5,000 genomewide single‐nucleotide polymorphisms to investigate the genomic diversity present in these uniquely managed populations. We genotyped 111 sable antelope: 23 from zoos, 43 from a single conservation center, and 45 from ranches. We found significantly higher genetic diversity and significantly lower inbreeding in herds housed in zoos and conservation centers, when compared to those in privately owned ranches, likely due to genetic‐based breeding recommendations implemented in the former populations. Genetic clustering was strong among all three populations, possibly as a result of genetic drift. We propose that the North American ex situ population of sable antelope would benefit from a metapopulation management system, to halt genetic drift, reduce the occurrence of inbreeding, and enable sustainable population sizes to be managed ex situ.


| INTRODUC TI ON
Ex situ conservation breeding mitigates the probability of extinction for many endangered species that are declining or no longer self-sustaining in their natural habitat (Conde, Flesness, Colchero, Jones, & Scheuerlein, 2011). The goal of ex situ conservation breeding is to increase the number of individuals to ensure a stable population and manage genetic diversity through selective breeding and inbreeding avoidance (Hoffmann, Sgro, & Kristensen, 2017). Various factors including founder effects, selection, random genetic drift, and inbreeding can alter the fitness and genetic diversity of a population (Frankham, 2008;Gooley, Hogg, Belov, & Grueber, 2017;Lacy, 1987;Wright, 1931), and in the absence of appropriate management, such processes can result in adaptation to captivity, genetic erosion, and inbreeding depression (Frankham, Ballou, & Briscoe, 2002;Jule, Leaver, & Lea, 2008;Williams & Hoffman, 2009). These processes have been identified as possible contributors to declines in reproductive fitness and neonatal survival in captivity as well as the historically limited success of species reintroductions (Christie, Marine, French, & Blouin, 2012;Farquharson, Hogg, & Grueber, 2018;Fischer & Lindenmayer, 2000;Willoughby & Christie, 2019). Gaining an understanding of genetic diversity within ex situ populations and providing best practice guidelines to retain this diversity is common among breeding programs (e.g., Gautschi, Muller, Schmid, & Shykoff, 2003;McLennan et al., 2018).
The World Association of Zoos and Aquariums (WAZA) provides global guidelines to help manage species in captivity, some of which aim to combat the above-mentioned challenges through the maintenance of genetic health and sufficient population sizes (WAZA, 2005). Mean kinship (MK) pairing is the most widely used genetic management strategy within zoo populations. Using pedigree-derived MK values (the average kinship of an individual to the entire captive population, including itself), the least related and under-represented individuals are prioritized and recommended for breeding (Ballou & Lacy, 1995). Minimizing the MK of each generation in ex situ populations, in theory, should retain greater genetic diversity when compared to random mating (Ballou & Lacy, 1995), as each individual contributes equally to the population. However, while minimizing MK has been shown to effectively retain founder genetic diversity in both laboratory and simulated populations (Ivy & Lacy, 2012;Willoughby et al., 2015), it can be challenging to implement such a strategy in certain ex situ management conditions. For example, in group housing enclosures where the pedigree of offspring is unknown (e.g., Farquharson, Hogg, & Grueber, 2019; or in polygamous species such as antelope, it may be difficult to equalize male reproductive success (e.g., Mucha & Komen, 2016).
Additionally, maintaining sufficient population sizes ex situ, especially for large animals that require spacious enclosures, can be challenging in a traditional urban zoo setting . Center for Species Survival (C2S2; www.conse rvati oncen ters.org), aims to tackle this challenge by bringing together urban zoos, conservation centers, and privately owned ranches to manage select species collectively as metapopulations. The SPA's initial focus is on four ungulate species (the scimitar-horned oryx, Oryx dammah; the dama gazelle, Nanger dama; the addax, Addax nasomaculatus; and the sable antelope, Hippotragus niger) that are not only of high conservation value, but also have large population sizes in the North American private sector relative to the number of animals in the AZA's Species Survival Plan (SSP ® ) programs for these species .
The SPA brings together institutions that have varying degrees of genetic management, ranging from AZA-accredited zoos implementing pedigree-based breeding recommendations, to privately owned ranches of isolated, nonpedigreed populations. However, even within AZA populations, solely relying on pedigree-based genetic management has been difficult for ungulates (see for example the sable antelope SSP ® [Piltz, Sorensen, & Ferrie, 2015], the addra gazelle (N. d. ruficollis) SSP ® [Thier & Spevak, 2017]  . Our study aimed to (a) quantify the genetic diversity and structure present in AZA zoo populations, conservation centers, and privately managed ranch populations in North America and make inferences between captive management strategies and retention of genetic diversity and, (b) for the first time, provide management teams with new genomic information to make better breeding recommendations for ex situ populations of sable antelopes.

| Study species and sample acquisition
The sable antelope consists of five genetically distinct and geographically coherent populations distributed across eastern and southern Africa in woodland savanna habitat (Vaz Pinto, 2018 Group, 2017). However, recent population assessments indicate their numbers in the wild may be declining, as the species has lost at least 51% of its former range (Ripple et al., 2015) and once formerly contiguous populations have become fragmented and isolated.
The current estimated population of sable antelope in the wild is between 50,000 and 60,000 (IUCN/SSC Antelope Specialist Group, 2017). For the southern sable antelope subspecies, populations across South Africa, Zimbabwe, Mozambique, Botswana, and Namibia totaled ~37,000 animals, based on estimates collected in the late 1990s (East, 1999). In the North American public sector, including both accredited AZA zoos and conservation centers and some non-AZA zoos, the ex situ population numbers 129 individuals (38 males, 90 females, 1 unknown) located in 15 institutions, with herd sizes ranging from 3 to 39 animals (Piltz et al., 2015). The private ex situ population in North America, however, is estimated to be over 3,000 (Mungall, 2018). These North American populations are almost entirely comprised of animals representing the southern sable antelope subspecies.
Presently, only 35% of the pedigree is considered to be known for the AZA managed population of sable antelope, with several parental assumptions (hypothetical parents to group family lineages) and exclusions (individuals which are removed due to sterility or age) (Piltz et al., 2015). This decreases to 27% prior to parental assumptions. Including pedigree assumptions, 39 founders have contributed to the AZA population, but their original relatedness and their representation is largely unknown. As the majority of the pedigree remains incomplete, MK values are unable to be calculated; as a result, current breeding recommendations achieve inbreeding avoidance via breeding males being rotated among AZA facilities every two to three years.
For the purpose of this study, three urban zoo AZA facilities were analyzed together as a population referred to as "AZA" (details about origins and sample sizes are summarized in Table 1). The "AZA" population is intensively managed with small herds or breeding pairs. Sable antelope from Fossil Rim Wildlife Center (an AZAaccredited facility) were analyzed as a separate population, due to the considerably larger herd size and less intensive management relative to urban AZA zoos. Samples were also obtained from four privately owned sable antelope populations that are participants in sampling list were also genotyped, as samples were readily available (see Table 1 for sample distribution across AZA facilities).
In total, whole blood samples (1-4 ml) or skin biopsies via biopsy darts were acquired from 111 sable antelope (66 from four AZA zoos and 45 from four privately owned ranches). All samples were collected opportunistically in conjunction with routine neonate examination/ear tagging/medical procedures. Therefore, no animal care and use approval was required by the Smithsonian Institution or the various collaborating institutions.

| Design of the sable antelope targeted sequence capture SNP panel
We used the SNPs previously identified from a reference southern sable antelope genome assembly and the re-sequenced whole genomes of four southern and one Zambian sable antelope derived from ex situ populations  to custom design a sequence capture panel containing 5,000 SNPs distributed across the sable antelope genome. Putatively neutral SNPs were selected from nonrepetitive (repeat-masked) and noncoding (outside exons based on gene annotation) regions and filtered according to the GC content skew. SNPs containing gaps in their flanking sequences (100 bp upstream and downstream) were excluded. To minimize linkage, we selected SNPs that were located at least 10 kbp apart in the scaffolds of the sable antelope genome assembly reported in Koepfli et al. (2019). Using these filtering criteria, we randomly selected 5,000 SNPs distributed across the genome.
To visualize the distribution pattern of the 5,000 selected SNPs across each of the 29 autosomes and X chromosome of the sable antelope genome, we mapped the SNP positions from the 16,927 scaffolds of the sable antelope genome assembly reported in  to scaffolds of the sable antelope chromosome-length assembly downloaded from the DNA Zoo (https://www.dnazoo. org/assem blies /Hippo tragus_niger) using a custom Python script.
We then visualized the density and distribution of SNPs across the 30 largest chromosome-length scaffolds by splitting the scaffolds into 100 kbp nonoverlapping windows and highlighting the windows with the selected SNPs.

| Construction of the sable antelope SNP panel
The 5

| Genomic DNA isolation
For each individual in our study, genomic DNA was extracted from either 100 or 300 μl of whole blood or a skin biopsy punch. For blood samples, DNA was extracted using either the DNeasy Blood and Tissue Kit (Qiagen) or FlexiGene DNA kit (Qiagen) following the manufacturer's protocol and eluted in 200 μl 10 mM Tris, pH 8.0.
Genomic DNA was extracted from biopsy punches using the GENE PREP system (AutoGen) and eluted in 100 μl of R9 buffer (AutoGen).

| Variant calling
Sequencing reads from 111 sable antelope were aligned to the sable antelope reference genome  using the Burrows-Wheeler Aligner (BWA; version 0.7.17 [Li & Durbin, 2009]

| Genetic diversity analyses
Individual observed heterozygosity was calculated using PLINK (Purcell et al., 2007, http

| Genetic structure analyses
To detect genetic clustering among the three designated North American populations, we performed a principle component analyses (PCA) using the R package SNPRelate (Zheng et al., 2012).
Coordinates for principle component 1 and principle component 2 were plotted using the R package ggplot2 (Wickham, 2009).

| Distribution of SNPs across the sable antelope genome
Out of the 5,000 SNPs selected for the design of the myBaits ® se-  Zimin et al., 2009), which was performed using LAST (Kiełbasa, Wan, Sato, Horton, & Frith, 2011). Numbers shown on the left of scaffolds are chromosome numbers or sex chromosomes of the domestic cow, while numbers shown on the right are the corresponding scaffolds of the sable antelope genome assembly (see: https://www.dnazoo.org/assem blies /Hippo tragus_niger). Only the 30 chromosome-length scaffolds are shown. Length of scaffolds in Mbp (x-axis) range = 396-10,757,538 bp, and 5%-95% percentile range = 12,387-227,434 bp ( Figure S1).

| Alignment rate of targeted sequence capture reads
We analyzed the aligned reads using the samtools flagstat tool from the SAMtools package . The alignment rate of reads generated from the two targeted sequence capture protocols (1 and 2) to the sable antelope genome had a mean value of 98.8% (4.2 million reads) across the 111 sable antelope samples ( Figure S2)

| Genetic structure analyses
Our principal component analysis (PCA) comparing North American sable antelope populations showed that each population formed an obvious cluster ( Figure 4). However, sable antelope that originated from Texas (Fossil Rim and the Ranch populations) showed overlapping clusters. Sable antelope from AZA zoos comprised a largely distinct cluster along both axes of the plot.

| D ISCUSS I ON
This study is the first to compare genetic diversity, genetic clustering, and genetic drift among a subset of sable antelope managed in zoos, conservation centers, and ranches, and to use this information to propose and support metapopulation management between traditionally isolated populations.  significantly greater genetic diversity and have significantly lower inbreeding coefficients than populations managed in the privately owned ranches. Southern sable antelope were brought in to captivity in North America during the 1910s to 1950s (Piltz et al., 2015). In the absence of a complete pedigree since founding, we do not know which founding lines formed the current AZA populations nor the private ranch populations, and only through using genomic tools can we begin to understand some of the genetic structuring between sable antelope populations in North America and make breeding and management recommendations. The overarching aim of this study was to empirically assess how traditional zoo population management methods influence genetic diversity retention and differentiation between populations F I G U R E 3 Realized inbreeding coefficients calculated from 3,954 SNPs using the RZooRoH model in three North American populations of sable antelope. Each bar represents an individual, displaying overall individual inbreeding coefficients (y-axis) and the proportion of genome assigned to specific homozygosity by descent (HBD) classes. HDB class values are assigned in concordance with the length of the run of homozygosity, where longer runs of homozygosity indicate more recent inbreeding events. The color legend indicates the HBD class, with class numerical values corresponding to approximating double the generation value of an inbreeding event (e.g., class 2 [color black] corresponds to inbreeding events occurring between ancestors 1 generation past, and class 16 [color blue] corresponds to inbreeding events occurring between ancestors 8 generations past

| Genetic diversity
Ex situ conservation programs aim to conserve existing genetic diversity, under the assumption that high and variable genetic diversity correlates with adaptive potential, and is key in ensuring long-term species survival (Russello & Jensen, 2018). We found significantly higher heterozygosity and significantly lower inbreeding occurring in the AZA zoo managed populations (this includes the Fossil Rim population; a larger conservation center managed by the AZA), relative to the privately managed herds (Figure 2a, (Willoughby et al., 2015). Even though we found a range of inbreeding levels across all study populations, higher inbreeding coefficients and more recent inbreeding events were observed in the private ranches ( Figure 3). This is in direct com- Genotyping complete herds, from more ranches, will allow us to better understand the genetic diversity present in the private sector.

| Genetic structure
We found evidence of genetic differentiation and clustering between all three study populations (Figure 4). There are several nonmutually exclusive explanations for the observed genetic clustering and differentiation between our North American sable populations: (a) initial founder effects during the establishment of the North American captive populations combined with (b) several of the North American populations historically being managed in isolation may have resulted in genetic drift that has led to genetic differentiation between captive populations. Moreover, novel selection pressures experienced in captive environments may have contributed in part to the observed genetic differentiation through adaptation to captivity, and the small effective breeding size (common to most ungulate species) in captive sable antelope populations will not only accelerate potential genetic drift, but also increase the overall kinship of the entire population.
Small populations such as ex situ managed collections in zoos and conservation centers are most at risk of genetic drift, and this process is exacerbated in polygamous species that experience high reproductive skews Gustafson, Vickers, Boyce, & Ernest, 2017;Lacy, 1987;Willoughby et al., 2015). However, this is primarily managed through kinship and guided bull rotations for inbreeding avoidance, equalizing male reproductive outputs as best as possible. On the private ranches, bull rotations can and do occur, though it is not as structured as the AZA management system nor is it based on pedigree-derived kinship values and recent pedigree history. Genetic drift occurring independently in the public and private facilities may explain the genetic clustering observed among ex situ populations of sable antelope in North America (Figure 4). These findings support the proposal of a metapopulation management system, whereby the loss of genetic diversity resulting from genetic drift can be reduced through the augmented immigration of as little as one individual per generation (Gustafson et al., 2017;Lacy, 1987).

| Metapopulation management
In order to enable our results to be used in applied conservation has transferred bulls to the privately owned ranch populations studied herein, and this is reflected in our principal component analysis (Figure 4), where we see overlap between the two population clusters. In comparison, our collectively termed AZA population has mostly been managed in isolation from the privately owned ranch populations; this too is reflected in our principal component analysis ( Figure 4) where we see minimal-to-no overlap in the two population clusters. As a proof of concept, we advised a bull from Fossil Rim to be transferred to The Wilds, which, during our study period, successfully sired two offspring, which we genotyped using our targeted sequence capture panel. The two offspring can be observed in Figure 4, located directly between our AZA cluster and Fossil Rim cluster. These individuals not only demonstrate the relatedness between offspring and parents originating from two different "sources," but also how metapopulation management can reduce the genetic differentiation between facilities. The value of managing captive sable antelope as a metapopulation including public and private breeding facilities, as proposed by the SPA, is exemplified here. We acknowledge that only a subset of North American sable antelope were genotyped herein, and therefore, our dataset does not represent a full characterization of the genetic diversity present in all North American sable antelope. This may indeed result in either underestimating or overestimating the genetic differentiation between facilities, and thus, further data collection would be required to gain a complete understanding of how best to empirically manage this species.
Nevertheless, our results demonstrate that transfers of individuals between the different breeding facilities may act to reduce the occurrence of genetic drift in captivity, better maintain population-wide genetic diversity and reduce the occurrence of inbreeding (Weeks, Stoklosa, & Hoffmann, 2016). Similar results have been observed in the forest musk deer, wherein of three captive populations, the highest genetic diversity was observed in the only population to exchange individuals with surrounding musk deer farms (Fan et al., 2019). As acquiring wild founders is presently not achievable for the North American sable antelope populations, we suggest that the larger managed populations (e.g., Fossil Rim) can act as gene pools for transfers to smaller and closer proximity populations within both the public and private breeding facilities. Male rotations, based on molecular mean kinship, inbreeding avoidance, and molecular relatedness, will be the most effective way to increase the effective population size and reduce the impact of genetic drift.

| Future directions
Using the species-specific sequence capture genotyping tool developed herein, our next goal is to integrate genomic data into metapopulation management. Creating a framework for molecular kinship-based breeding recommendations and exchanging novel genetic diversity between the different public and private breeding facilities is a future goal and currently underway in our research team.
Furthermore, we plan to compare genomic diversity of sable antelope from Africa (both wild and captive), to our North America sable antelope, as to understand how genomic diversity has differentiated since the founding of the North American ex situ populations.
Point Ranch), and anonymously participating Conservation Centers for Species Survival's Source Population Alliance members for providing samples. We thank Alison Devault from Arbor Biosciences for the construction and quality checking of the sable antelope my-Baits array and for comments on the manuscript. We also thank Dr. Janine Brown, Smithsonian Conservation Biology Institute for substantial discussions during early stages of this project and funding support and Ms. Jill Piltz, Co-Ordinator -AZA Sable Antelope Species Survival Plan for regular feedback and guidance throughout this project. K.P.K. and the research reported herein was supported by the Smithsonian Institution Competitive Grants Program, the Sichel Endowment, and the Phil Reed Fund. G.T. was supported in part by funding provided through the Smithsonian Institution's Short-Term Visitor Fellowship program.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Unfiltered.vcf files, sample ID information, and R codes used for analyses are available from the following data repository: https:// figsh are.com/proje cts/Compa rison_of_genom ic_diver sity_and_ struc ture_of_sable_antel ope_Hippo tragus_niger_in_zoos_breed ing_cente rs_and_priva te_ranch es_in_North_Ameri ca/74874