Assessing introgressive hybridization in roan antelope (Hippotragus equinus): Lessons from South Africa

Biological diversity is being lost at unprecedented rates, with genetic admixture and introgression presenting major threats to biodiversity. Our ability to accurately identify introgression is critical to manage species, obtain insights into evolutionary processes, and ultimately contribute to the Aichi Targets developed under the Convention on Biological Diversity. The current study concerns roan antelope, the second largest antelope in Africa. Despite their large size, these antelope are sensitive to habitat disturbance and interspecific competition, leading to the species being listed as Least Concern but with decreasing population trends, and as extinct over parts of its range. Molecular research identified the presence of two evolutionary significant units across their sub-Saharan range, corresponding to a West African lineage and a second larger group which includes animals from East, Central and Southern Africa. Within South Africa, one of the remaining bastions with increasing population sizes, there are a number of West African roan antelope populations on private farms, and concerns are that these animals hybridize with roan that naturally occur in the southern African region. We used a suite of 27 microsatellite markers to conduct admixture analysis. Our results indicate evidence of hybridization, with our developed tests using a simulated dataset being able to accurately identify F1, F2 and non-admixed individuals at threshold values of qi > 0.80 and qi > 0.85. However, further backcrosses were not always detectable with backcrossed-Western roan individuals (46.7–60%), backcrossed-East, Central and Southern African roan individuals (28.3–45%) and double backcrossed (83.3–98.3%) being incorrectly classified as non-admixed. Our study is the first to confirm ongoing hybridization in this within this iconic African antelope, and we provide recommendations for the future conservation and management of this species.


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The increased rate of human-driven global change is a major threat to biodiversity [1]. Factors such as climate 38 change, habitat fragmentation, and environmental degradation are influencing the distribution and abundance of 39 species, often in ways that are impossible to predict [2]. Thus, a central theme in conservation biology is how 40 best to manage for species persistence under rapidly changing and often unpredictable conditions. When faced 41 with environmental change, species may persist by moving (or being moved) to track suitable environments.

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Although there is sufficient evidence to suggest that species notably alter their ranges [3], facilitation of such 43 movement for larger vertebrate species (through the creation of habitat corridors, transfrontier parks or 44 translocations) often place insurmountable burdens on conservation agencies that are ultimately responsible for 45 the management of these populations. Notwithstanding, signatory countries to the Convention on Biological

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Diversity have an obligation to manage and protect biodiversity, as also set out more recently in the Aichi

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Admixture and introgression are major threats to species conservation (these threats are dealt with specifically 50 under Aichi Target 13; see https://www.cbd.int/sp/targets/). The ability to accurately identify introgression is 51 critical to the management of species [4][5][6][7][8][9], and may provide unprecedented insights into evolutionary 52 processes. Although admixture, or even genetic rescue, may have beneficial outcomes through the introduction 53 of new alleles into small or isolated populations, it can lead to outbreeding depression essentially disrupting 54 locally adapted gene-complexes [10][11][12][13]. Because of the movement of animals (either natural or human-55 facilitated), admixture and the effects thereof become increasingly more important to understand and manage.

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Roan antelope (Hippotragus equinus) is one of Africa's most iconic large antelope species. It has a sub-Saharan 58 range, is a water-dependant species, and prefers savanna woodlands and grasslands. [14]  on morphological analyses. However, subsequent genetic studies by [15] and [16] provided less support for 61 these subspecies designation. Although the [15] study included relatively few specimens (only 13 animals were 62 available at the time), [16]

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Our aim here is to expand on the limited and non-specific suite of microsatellite markers employed by [16] to 92 specifically test the validity of these anecdotal reports of trade in West African roan. Also, we assessed the ability 93 of these markers to discriminate between non-admixed animals and hybrid offspring (F2, F3, and F4). Our results

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will not only confirm whether suggestions of hybridization are true, but will also provide a valuable tool to ensure 95 genetic integrity in the conservation of roan antelope in Southern Africa.  (Fig 1A).

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The two distinct genetic clusters (K = 2) was supported by the Bayesian assignment analysis (Fig 1B, S1 Fig).

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Genetic diversity for each population is summarized in Table 5. Overall, the genetic diversity in the Western roan

Discussion
An increasing number of species experience dramatic declining population numbers globally, with ample evidence suggesting that we are entering a mass extinction event. Although the drivers of these population declines are numerous and varied, the underlying root cause inevitably stems from anthropogenic pressures. Not surprisingly, hybridization and admixture of groups with distinct evolutionary trajectories are increasing, raising concerns about the integrity of a large number of species, especially those that experience disproportionately large human interest. For roan antelope, one of Africa's most spectacular large antelope species, this is certainly the case. Although roan antelope numbers are increasing in South Africa (largely because of protection under private ownership), real concerns exist about their genetic integrity given admixture with West African roan antelope, also for export to neighbouring countries. We discuss our results here, and provide some suggestions for roan antelope conservation in South Africa.

Evidence of hybridization
Using a suite of variable and informative microsatellite markers, we provide unequivocal evidence of hybridization and introgression between roan antelope naturally occurring in South Africa (East, Central and Southern African origin), and those of West African decent (a separate evolutionary significant unit; see [16]).
More problematic, the identification of first and second generation backcrosses with q-values close to threshold values strongly suggest that hybrid individuals are viable and fertile; as also suggested from anecdotal evidence from some game farms. Although genetic diversity estimates were moderately higher in the known and putative hybrid individuals, it has previously been reported that F2 hybrids can display reduced fitness as a result of disruption of sets of co-adapted gene complexes by recombination [52,53], thereby weakening the entire gene pool of naturally occurring individuals. Our marker set was able to accurately identify F1 and F2 hybrids, as well as non-admixed individuals at thresholds of q = 0.20 and q = 0.15. However, the accurate classification of further backcrosses was less accurate at these thresholds (40% to 83%) with backcrossed individuals being incorrectly classified as non-admixed. The use of higher thresholds (qi = 0.10 and qi = 0.05) did increase the number of individuals correctly classified as backcrosses, however, this also resulted in an increase in the number of nonadmixed individuals being incorrectly classified as hybrids. Thus in certain instances, backcrossed and double backcrossed individuals extend beyond the detection power of the current microsatellite marker panel.
The minimum number of markers required to accurately and consistently identify backcrosses is currently being debated. Simulation analysis in the grey wolf (Canis lupus) that hybridizes with domestic dogs (C. lupus familiaris) indicated that simply increasing the number of microsatellite markers used does not equate to an increase in the number of correctly identified admixed individuals [54]. It may be important to evaluate single nucleotide polymorphisms (SNPs) with high discriminating power to increase the ability to detect backcrossed and double backcrossed individuals, but in all likelihood thousands of SNPs may be required. Notwithstanding, the marker set described here represents the first step in assessing hybridization in roan antelope, and in the identification of hybrid individuals.

Conservation management
As signatories to the Convention on Biological Diversity, South Africa has an obligation to conserve the genetic integrity of its biological diversity. Furthermore, admixture between distinct wildlife subspecies is prohibited under national and provincial legislation. Within South Africa, wildlife can be privately owned. There has been some debate about the legal rights of an owner to act in a certain manner with its property, and whether farming with wildlife should be managed and regulated any differently than, for example, agricultural stock such as cattle.
Notwithstanding, current international, national and provincial legislation is clear in prohibiting admixture, irrespective of ownership.
The private ownership of biological diversity has been advantages for a large number of species, and the high commercial value attached to many of these species has undoubtedly aided in their conservation and protection; to the point where a number of species are doing better under private ownership compared with in protected areas or national parks [55]. Roan antelope is a prime example, but others include sable antelope, white and black rhinoceros, and bontebok to name but a few. Unfortunately, many of these species are intensively managed, with selection for specific desired traits. These management practises have unintended consequences, notably a loss of genetic diversity. In our study, a number of loci showed deviations from HWE and linkage disequilibrium; all which can be ascribed to small numbers of founding individuals and genetic drift on farms [56] which may, in the long term, compromise local adaptation [57]. To fully understand the impact that farming practises, notably intensive management and selection, have on wildlife populations, comparisons need to be done with naturally occurring populations on nature reserves.
Currently, the full extent of hybridization in South Africa between roan antelope belonging to the two distinct ESUs is unknown. Laboratory screening for permitting purposes (to either sell, or translocate animals) suggest that the occurrence of widespread introgression is low, and largely confined to specific game farms.
Animals of West African decent are no longer maladapted to South African conditions and have, over the span of 20 years, adapted to local conditions. The question that needs consideration is whether South Africa should safeguard the genetic integrity and genetic variability of both roan ESUs. If historic occurrence is considered, then all West African animals should be removed from South African populations. However, the South African situation has spawned several ex situ breeding programmes and agreements and/or animals that could be allowed to be backcrossed to obtain some form of purity, over four or five generations. This might improve genetic variation within the national population, but may not be desirable given that the impact of hybridization on the South African roan full genome is not known. Thus, we recommend the implementation and continuation of strict genetic monitoring for hybridization in roan antelope in South Africa. With the microsatellite marker set described here, and using a threshold of qi = 0.15, it is possible to detect F1 and F2 hybrids prior to translocation, thereby reducing and ultimately eliminating Western roan antelope alleles in the indigenous roan gene pools. In addition, management of roan in South Africa would benefit from a national meta-population conservation plan to inform translocations and reintroductions and to effectively monitor genetic diversity and further hybridization events.