Evolutionary history

Zimbabwe, and South-Africa, as well as captive populations across North America. The only exception to this trend is the isolated population of the Angolan Giant sable (H. n.

variani), considered as “Critically Endangered” by the IUCN Red List of Threatened Species (IUCN SSC Antelope Specialist Group 2008). During Angolan Civil War (1975-2002), this population suffered a severe decline due to hunting pressure, decreasing from an estimated 2,000 to 3,000 individuals before 1970, to around 300 in 2007 (IUCN SSC Antelope Specialist Group 2008). The population was feared extinct until its rediscovery in 2006 (Pitra et al. 2006), which led to the development of an in situ Conservation Plan to ensure suitable habitat and management of surviving individuals, from both an ecological and genetic perspective (Vaz Pinto 2009; Estes 2013; Vaz Pinto et al. 2016). Currently, 100 Giant sable individuals are estimated to live within remaining areas of Cangandala National Park and Luando Reserve (IUCN SSC Antelope Specialist Group 2008). In recent years, increasing numbers of sable antelopes occurs in private land in Zambia, Namibia, Zimbabwe, and South-Africa, as well as captive populations across North America, mainly because it has become one of the most highly prized trophy hunting species in Africa (Bothma and van Royen 2005; Lindsey et al. 2013; Piltz, Sorensen and Ferrie 2016).

Inbreeding and outbreeding depression risks are well documented within endangered species (Edmands 2006). Interspecific hybridization between the roan and sable antelope can occur under exceptional circumstances, as the one reported for the Angolan Giant sable (Vaz Pinto et al. 2016). However, intraspecific hybridization may be of more generalized concern for these species, owing to animal translocation across different subspecies ranges, as documented recently for roan antelopes (van Wyk et al.

2019). Contemporary conservation studies rely on defining units of management based on both genetic and ecological distinctiveness of a species, subspecies, or population, inferred by phylogenetic history. They are defined as evolutionary significant units (ESUs) or management units (MUs), either representing a slower or faster isolation response, respectively (Moritz 1994, 1999). Genomic data allows for the inference of conservation units (CUs), integrating adaptive genetic variation (Funk et al. 2012). From a conservation and management perspective, it is of extreme importance to define such units and prevent further depuration of genetic diversity.

subspecific validity. Observed differentiation patterns can be associated with specific evolutionary histories, shaped by both intrinsic and extrinsic factors.

The first attempt to study the roan antelope using molecular data was by Matthee and Robinson (1999), analysing a fragment of the mitochondrial DNA (mtDNA) control region.

They included 13 individuals covering the range of all, but two recognized subspecies, the north-central charicus and north-eastern bakeri. Phylogenetic analysis retrieved four mtDNA lineages, supporting the division between northwest koba (Benin), eastern langheldi (north Tanzania and Kenya), south-central cottoni (south Tanzania, Malawi, and Zambia) and southern equinus (Namibia and South-Africa) subspecies. These associations were related to vicariance or the effects of isolation by distance, and a clear division between individuals in East and Southern Africa. However, the low number of individuals and the use of a single mtDNA fragment is limiting for evolutionary inferences.

In a subsequent study, Alpers and co-workers (2004) extended the analysis of the same mtDNA fragment plus eight microsatellite loci, to more than one hundred individuals, covering the same four subspecies but with a more comprehensive sampling, and adding one sample from the subspecies charicus from Cameroon. Based on phylogeny and individual clustering, these authors proposed the presence of two ESUs corresponding to the northwest koba, and remaining distribution range, respectively. The sample representing the subspecies charicus from Cameroon was not strongly associated with any of the analysed subspecies and its phylogenetic position was left unresolved.

Additionally, these authors proposed two refugial areas for the species during the Pleistocene climatic cycles, located in the West and East of Africa, respectively. The West refuge, associated with H. e. koba, was proposed to be specifically located around Ghana, while the East refuge comprised the remaining subspecies, with the most likely location of the refugial population around the northeast. Both studies added relevant information on roan’s genetic diversity and evolutionary history, but the poor geographic sampling, particularly across north-central and northeast distribution of the species, left uncertainty on the number of genetic clusters across the species distributional rangeand have not investigated the time of their divergence (Figure 1.12). Considering previous studies on the roan antelope, Lorenzen et al. (2012) associated the highest levels of gene flow for the eastern samples to a possible recent admix and a mosaic of several other possible refugia in the area.

Matthee and Robinson (1999) also analysed the mtDNA control region for 15 sable individuals, covering the four described subspecies. Phylogenetic analysis retrieved two mtDNA lineages, dividing specimens from west Tanzania from the remaining ones. This differentiation was related to a geographic barrier, including vegetational differences and rifting, that disrupted gene flow between both lineages. Soon after, Pitra and co-workers

Figure 1. 12 The roan antelope intraspecific analysis. On the left, subspecies geographical distribution, according to Ansell (1972) and current species distribution (IUCN SSC Antelope Specialist Group 2017a). On the right, a schematic cladogram showing intraspecific phylogenetic relationships using mitochondrial data, adapted from Alpers et al. (2004);

clade identification following the ones from the authors. Each colour corresponds to a subspecies, according to the legend.

(2002) supported this observation by combining mtDNA control region and cytochrome b sequences of 95 individuals from several localities, across East and Southern Africa.

They identified three mtDNA lineages, one in west Tanzania, another in the East, and a third one in the Southern Africa, but also including some specimens from west Tanzania.

The observed sympatry of the two highly divergent mtDNA lineages in west Tanzania was explained by an ancient unidirectional outbreeding event. Sable antelope’s evolutionary history was explained taking into account several historical events, including allopatric fragmentation between west Tanzania and the remaining populations, consistent with the discontinuous distribution of the miombo habitats around the mountain circles in East Africa, as well as alternated periods of short to long-distance dispersal between individuals in west Tanzania and those in eastern and southern ranges, respectively. Later, by including samples of the Angolan Giant sable in their previous dataset, Pitra et al. (2006) observed a close relationship between Angolan haplotypes and those observed in a few individuals from western Tanzania. Once again, these patterns were associated with long-distance dispersal between both regions, from a common source population. With this result, the legitimacy of the Giant sable as a separate clade was left unresolved. The validity of the variani subspecies was further investigated by Jansen van Vuuren and co-workers (2010), specifically its genetic distinction from geographically closer and phenotypically similar, particularly with respect to facial markings, individuals living across western Zambia. The authors concluded that

despite their morphological resemblance, significant genetic differences underpin these two evolutionary lineages.

Using whole-genome mitochondrial sequencing for more than 200 contemporary and historic samples, across the sable antelope’s entire distributional range, Rocha (2014) unveiled the species’ maternal evolutionary history. Four main lineages were identified, including the divergent lineage identified in previous studies in west Tanzania – which was assumed to be more closely related to kirkii subspecies, according to Ansell (1972), and later named relict by Vaz Pinto (2018); an eastern lineage present in Kenya and north Mozambique; southern lineage comprising individuals from south Mozambique, Zambia, Zimbabwe, Botswana, Namibia, and South-Africa; and a central lineage present in Angola, west Tanzania, and Malawi. High divergence levels (~3%) found between the relict and the remaining lineages, and associated vicariant event estimated around 1.4 Mya, led to the hypothesizes that the relic lineage was a ghost lineage of an extinct population that survived, as a result of an ancient introgression event. The origin of this lineage, dated to the early Pleistocene period (~1 to 2.5 Mya), was related to the increased tectonic activity of the EARS, probably contributing to the isolation of a population in the Tanzanian plateau, from the remaining individuals in the East. The second vicariant event, dated to 350 kya, subsequently separated the eastern lineage, delimited by the EARS and the EAM, across central Tanzania. Central and southern lineages split later, around 200 kya. The presence of the central lineage across individuals in west Tanzania, Malawi, and Angola was explained by a dispersal corridor through Congo. Individuals found today in west Tanzania carry both the relict and central lineage, which clarifies the proximity found in previous studies between variani, niger and west Tanzanian samples (Pitra et al. 2002, 2006; Jansen van Vuuren et al. 2010).

Further divisions observed within eastern and southern lineages were dated around 150 kya and coincide with the Pleistocene dry cold period MIS 6. These divergences were explained by vegetation change in both regions, caused by climatic transition, with subsequent reconnection by range expansion, once conditions improved. The split within haplogroups of the southern lineage occurred after 100 kya and was associated with the hydrological changes the Zambezi River experienced, separating individuals in the northern from those in the southern bank of the river.

The first study on the sable antelope’s evolutionary history using nuclear data was by Vaz Pinto (2018), genotyping 400 individuals for 57 polymorphic microsatellites previously developed (Vaz Pinto et al. 2015). Using clustering analysis, five geographically differentiated groups were obtained and named as follows: west Tanzanian, in the Tanzania plateau; eastern, distributed from Kenya to central Mozambique, Malawi, and eastern Zambia; southern, located south of the Zambezi

River; Zambian, across Zambia to the east and north of the Zambezi, including south DRC; and Angolan, across Cangandala National Park and Luando Reserve in Angola.

Angolan group registered the lowest levels of diversity and highest levels of differentiation, in agreement with its restricted location and recent population bottleneck.

These five groups are consistent with previous study based on mitogenomes (Rocha 2014) and their geographical delimitation supports vicariant events resulting from the same geographical features, such as the EARS, EAM and the Zambezi River, as well as Pleistocene climatic fluctuations (Figure 1.13).

Figure 1. 13 The sable antelope intraspecific analysis. On the left, subspecies geographical distribution, according to Ansell (1972) and current species distribution, adapted from IUCN SSC Antelope Specialist Group (2008, 2017b). On the right, a schematic cladogram showing intraspecific phylogenetic relationships using whole-mitochondrial data, adapted from Rocha (2014), as well as clustering analysis showing the assignment proportions of each individual using microsatellite loci, adapted from Vaz Pinto (2018); mtDNA lineages and nuclear groups identification following the ones from the authors. Each colour corresponds to a subspecies, according to the legend.

Based on the studies of Matthee and Robinson (1999) and Pitra et al. (2002, 2006), Lorenzen et al. (2012) proposed three main refugial areas for the sable antelope during the Pleistocene climatic cycles, located in the East, South, and Southwest Africa, respectively. The East refuge was associated with the highest divergence levels calculated for the population in west Tanzania, and therefore, supposed to be the oldest population. The South refuge comprised remaining sable antelopes’ distributional range, except for the isolated H. n. variani, which was associated with a Southwest refugial area, probably deriving from the South refuge, with subsequent independent evolution.

Based on later studies from Rocha (2014) and Vaz Pinto (2018), the reasons for the East and Southwest refugia were not validated.