Genetic diversity in captive and free-ranging black-footed cats (Felis nigripes) from South Africa

The black-footed cat is a small and solitary felid species that is endemic to the arid to semi-arid subregions of southern Africa but mainly occurs in South Africa. The species is currently listed in the “vulnerable” category on the IUCN Red List with fewer than 10,000 adult individuals estimated to remain in the wild. Despite this status and dwindling population sizes, studies on the species are limited, particularly concerning their genetics. To date, only one study has focused solely on black-footed cat genetics in which captive black-footed cats were typed using a panel of nine autosomal microsatellite markers and partial ND5 gene sequencing. The ND5 analyses recovered two haplotypes, differing at four nucleotide sites across a 316 bp region, suggesting the likely occurrence of two discrete black-footed cat lineages. One lineage is represented by a publicly available complete mitochondrial genome that was generated through Illumina Sequencing technology. The other lineage is limited to partial ND5, 16S rRNA and COI gene data, highlighting the need to generate a complete mitochondrial genome reference for the unrepresented lineage. However, a pertinent issue in mitochondrial studies on various felid species is the occurrence of nuclear-mitochondrial (numt) paralogs. Due to the similarity of numts and mitochondrial DNA, primers often bind to numts in conjunction with or in preference to mitochondrial DNA, leading to numt pollution in datasets. To avoid this, the present study aimed to design mitochondrial-specific DNA markers. Therefore, this study aimed to generate a complete mitochondrial genome for an individual cat with an ND5 haplotype that was confirmed to correspond to the unrepresented lineage. This was achieved by designing mitochondrion-specific primers that target six overlapping regions that span the complete mitochondrial genome using a primer-walking approach. In this manner, a near-complete mitochondrial genome (16,000 nt in length) was generated with only a stretch of the D-loop gene region (623 nt) being omitted. Using these established primers, additional genetic data was generated for four commonly sequenced mitochondrial markers encoding the COI, ND5, ND6 and cyt b genes for 21 black-footed cats sourced from one captive (n=7) and three free-ranging (n=14) populations. Whilst the sample size is small, the value is that it includes wild-caught animals of known provenance from three localities within South Africa, to which captive cats have not been reintroduced. Analysis of phylogeographical structure using concatenated datasets that included all four gene regions recovered two genetically distinct but geographically indistinct lineages. One possible explanation for the lack of geographical structure is that a historical isolating event led to the accumulation of mutations over time, followed by range expansion, resulting in the two lineages seen today. Whereas this study made use of high-quality genomic DNA, many genetic studies are limited to generating partial gene sequences due to cost and time constraints, in addition to limitations associated with poor DNA quality when working with non-invasive samples; a requirement for a species with this conservation status. Thus, the comparative phylogenetic utility of the four gene regions was assessed based on genetic diversity, mutational distribution and levels of phylogenetic resolution to determine the genetic marker/s best suited to conservation genetics efforts. Haplotype network and sliding window analyses determined that ND5 and cyt b had high nucleotide diversity and mutational hotspots that make both suitable for targeted amplification of short but informative fragments (<500 bp) and that ND6 and COI should be avoided. Overall, the present study successfully expanded on available genetic data on black-footed cats by generating a near-complete mitochondrial genome for an unrepresented lineage and by establishing mitochondrion-specific markers that can be used in future studies on this vulnerable species.