How to spot a black-footed cat? Successful application of cross-species markers to identify captive-bred individuals from non-invasive genetic sampling

The black-footed cat (Felis nigripes) is the smallest felid of Southern Africa, endemic to the arid steppe and savannah habitats. However, though threatened and characterized by decreasing sizes of its populations, a number of ecological, demographic, sanitary, and genetic aspects, essential for the long-term conservation of the species, still remain poorly known. Non-invasive genetic sampling may represent an appropriate and cost-effective tool to fill this lack of information. Thus, for the first time so far, we developed a protocol for species and individual identification of black-footed cats, starting from markers originally designed for the domestic cat and from 23 non-invasively collected samples of captive-bred individuals. We then tested its genotyping efficiency and reliability for future applications in non-invasive genetic monitoring programs of the wild populations. Most of the samples (65%), corresponding to 15 individuals, were successfully genotyped at 316 bp of the mtDNA ND5 and at nine autosomal microsatellites. We detected two black-footed cat mtDNA ND5 haplotypes that were clearly distinguishable from all the other wild and domestic felids. All microsatellites were polymorphic and showed low error rates, probabilities of identity < 0.001 and a mean observed heterozygosity H O = 0.68. Subsequent approximate Bayesian computation simulations confirmed that black-footed cats and African and European wildcats likely experienced sequential population splittings that started during the Late Pliocene and continued through the Early Pleistocene. Our study provided the first reliable and cost-effective molecular multilocus characterization of non-invasively collected samples of black-footed cats. Though solely tested on captive-bred individuals, our method could be applied to design and implement effective long-term monitoring and conservation plans of poorly investigated black-footed cat wild populations.