Identifying Cryptic Mammals With Non-Invasive Methods: An Effective Molecular Species Identification Tool to Survey Southern African Terrestrial Carnivores

Carnivores play a vital role in ecosystem health and are thus an important focus for conservation management. Non-invasive methods have gained traction for carnivore monitoring as carnivores are often elusive and wide-ranging, making visual counts particularly difficult. Faecal mini-barcoding combines field collection of scats with genetic analysis for species identification. Here, we assessed the applicability of a mini-barcode based on the mitochondrial ATP6 gene in southern Africa. We predicted amplification success based on in silico evaluation of reference sequences from 34 of the 42 terrestrial carnivore species existing in southern Africa, including the Congo clawless otter (Aonyx congicus) for which we contributed a mitochondrial assembly. We further tested amplification success on available reference samples of 23 species. We expanded the existing ATP6 mini-barcode reference database by contributing additional sequences for 22 species, including the Cape genet (Genetta tigrina) and the side-striped jackal (Lupulella adusta) for which no complete mini-barcode sequences were available on GenBank, and compiled a representative reference dataset of 61 unique sequences as a tool for species identification. As a proof of principle, we applied the ATP6 mini-barcode to a small scat-based carnivore survey conducted in Namibia 13 years prior, which showed a 95% identification success and detected six species among 157 samples collected. With southern Africa's mammalian carnivores facing escalating threats, this robust mini-barcode offers a vital tool for accurate species identification from non-invasive samples, enabling crucial monitoring and conservation efforts.