Functional genetic diversity of Toll-like receptor (TLR) genes for bontebok, blesbok and their hybrids

The bontebok (Damaliscus pygargus pygargus) and blesbok (Damaliscus pygargus phillipsi) originated from the endemic ancestral South African antelope (Damaliscus pygargus) which belonged to the Alcelaphini tribe. Several studies have classified the bontebok and blesbok as separated sub-species (Essop et al., 1991; Van der Walt et al., 2001; Van der Walt, 2002) and the two subspecies have different ecosystems preferences with their natural distribution ranges being largely non-overlapping. Bontebok is endemic within the Cape Floristic Region of the Western Cape in South Africa. In the early 1800s, during European settlement, the population experienced a severe bottleneck because of overhunting (van Rensburg, 1975). Due to efforts of farmers in the Bredasdorp region, the subspecies was brought back from the brink of extinction in the 1830s and populations today all originate from this historically protected population (n = 27) (Bigalke, 1955). The first Bontebok National Park (BNP) was established from the historically protected population (n = 17) in 1931 in an area near Bredasdorp (Bigalke, 1955). However, the vegetation of the park was not ideal and animals suffered from a copper deficiency as well as a massive worm infestation. In 1960, a total of 61 animals were successfully translocated to a newly established Bontebok National Park in Swellendam (Barnard & Van der Walt, 1961). Today, the largest bontebok sub-populations are located in the Bontebok National Park and the De Hoop Nature Reserve. The total number of bontebok in South Africa is estimated between 6,500 and 7,000 animals with less than 1,000 individuals occurring within its former distribution range (Unpublished data, CapeNature, 2014). The blesbok were distributed in the grassland biomes in Gauteng, Eastern Cape, Mpumalanga and the Free State (Skinner & Smithers, 1990) and were hunted for their hides by the settlers. In 1899, the Anglo Boer War brought the hunting and trade in their skins to an end and the remaining blesbok populations could only be found on farms by the end of the war. In 1962, the population was projected to be around 46,000 animals (Kettlitz, 1967) and the current population size is estimated between 235,000 and 240,000 (East, 1999).The International Union for Conservation of Nature (IUCN) listed the bontebok as near-threatened species on the Red List of Threatened Species (Radloff et al., 2017) whereas the blesbok is listed as least concern (Dalton et al., 2017).

Bontebok is threatened by low habitat availability, hybridisation with blesbok and low genetic diversity. Bontebok are almost exclusively grazers (Beukes 1984), with a preference for short grass and recently burnt veld (Beukes 1987; Novellie 1987; Kraaij & Novellie 2010). Within their natural distribution range, suitable habitat is predominantly limited to the remaining renosterveld patches in the Overberg region. Thus, the amount of optimal habitat is both limited and fragmented. In addition, the subspecies' continued survival is currently being jeopardized by the existence of hybrid species due to translocation of bontebok and blesbok outside their natural distribution range (Birss et al., 2013). The hybrid offspring are known to be fertile, which may lead to the extinction of bontebok due to introgression (Goodman et al., 1999; Allendorf et al., 2001). Genetic diversity plays a role in adaptation of the population to environmental changes such as climate change, parasites, food availability, predation, competition, pollution and infectious diseases (Frankham et al., 2010). A previous molecular investigation using neutral markers by van Wyk et al. (2013) determined that genetic diversity in pure South African bontebok was significantly lower than pure blesbok. The authors attributed the loss of genetic diversity in bontebok due to the genetic bottleneck described in the 1800s (van Wyk et al., 2013). Inbreeding and population bottlenecks can result in animals having lower resistance to disease. Thus, functional diversity in combination with neutral diversity needs to be considered for future management plans in bontebok.

Toll-like receptors (TLRs) are a protein family that is responsible for the detection of distinctive distinct pathogen-associated molecular patterns (PAMPs) of invading microorganisms including bacteria, viruses, fungi, and parasites (Janeway, 1989; Medzhitov, 2001). TLRs form part of the pathway which activate the innate and acquired active immune responses, and to control and eradicate pathogen and parasite challenges. Mammals have 13 TLRs which are organised into six major groups based on phylogenetic analysis, namely; the TLR2 group (TLRs 1, 2, 6, 10 and 14), known to bind di- and triacylated lipoproteins anchored in the cell wall of bacteria, fungi and parasites; the TLR3 group which sense double-stranded RNA; the TLR4 group, which target the lipopolisaccharides embedded in the cell wall of gram-negative bacteria; the TLR5 group, which detects flagellated bacteria through the recognition of flagellins; the TLR 7/8/9 group which detects single-stranded RNA and the TLR11 group (TLRs 11, 12, 13, 21, 22 and 23) which detect microbial DNA (Roach et al., 2005). TLRs have steadily become a significant tool to investigate specific loci relevant for immune system function (Alcaide and Edwards, 2011) as many of the TLR genetic mutations observed in humans are associated with susceptibility or resistance to infectious diseases (Schröder and Schumann, 2005; Barreiro et al., 2009; Werling et al., 2009).

In this study, we apply molecular techniques to examine the level of genetic diversity at adaptive and neutral sites in bontebok, blesbok and hybrid individuals. We further aimed to investigate the level of genetic diversity within and among bontebok populations from National parks and private game farms. Lastly, we discuss how variation at adaptive loci may be important for the long term viability of the bontebok. We hypothesize that TLR gene diversity will reflect microsatellite heterozygosity with lower diversity being detected in the bontebok in comparison to the more widespread blesbok due to the purifying/negative selection acting on the TLRs. In addition, we suspect that hybrid individuals will have intermediate diversity due to mixing of bontebok and blesbok genomes. Currently, little is known with regards to the relationship between diversity of immunity associated genes and genetic diversity at neutral loci in bontebok and blesbok species. To our knowledge, this is the first study to investigate TLR genetic diversity in pure and hybrid mammalian individuals.