Assessing the utility of DNA barcoding in wildlife forensic cases involving South African antelope

Poaching of South African wildlife is considered a threat to biodiversity. In the absence of diagnostic
morphometric traits, DNA barcoding is considered as a method of choice for species identification. Here, we
report on forensic case work involving the illegal hunting of antelope species. Three forensic cases which
included confiscated material were submitted between 2018 and 2019 and for species identification. Laboratory
procedures including DNA extraction and sequencing of cytochrome c oxidase 1 (COI) and cytochrome b (cytb)
were conducted following forensic procedures to determine species identification. Generated sequences matched
to reference sequences on the National Centre for Biotechnology Information (NCBI) and the Barcode of Life Data
Systems (BOLD) to impala (Aepyceros melampus, 99.4–99.7 % homology), eland (Tragelaphus oryx, 99.8–100 %
homology) and kudu (T. strepsiceros, 99.6–99.7 % homology). Phylogenetic analysis and intra- and interspecies
distance further confirmed species identification with high bootstrap support (96–100 %). Average intraspecies
sequence divergence was 0–1.15% and pairwise comparisons between taxa satisfied the 10-fold genetic distance.
Thus both COI and cytb barcoding genes are suitable methodologies for forensic identification of species in the
cases presented here. However, analysis of the reference samples identified species where barcoding may
potentially fail. These include taxa that have undergone recent, rapid radiations resulting in high intraspecies
distance or species that can hybridize. We thus recommend in these cases a reference database that includes
geographically widespread samples is required and analysis with additional mitochondrial and/or nuclear
markers.