The Avian DNA Barcoding Reference Library in South Africa: A Tool for Conservation and Combating Illegal Wildlife Trade

South Africa’s avian biodiversity faces challenges from both illegal activities and legal trade (e.g., breeding and parentage), requiring accurate species identification to inform conservation efforts, enforce regulations, and monitor wildlife trade. Morphological identification presents challenges due to variation within and similarities between species. DNA barcoding offers a reliable approach if supported by comprehensive reference libraries. This study established a DNA barcode reference library using Cytochrome c oxidase 1 (COI) sequences from 14 protected bird species targeted by illegal trade and at risk due to their conservation status, validated by strict Chain-of-Custody (CoC) protocols to ensure the reliability and admissibility of DNA barcoding in wildlife crime prosecutions. The study generated 69 COI sequences, covering 17.30% of South African bird species. Phylogenetic analysis distinguished 97.47% and 87.34% of species using Neighbour-Joining and Bayesian Inference trees, respectively, effectively differentiating protected species from each other and from species morphologically and phylogenetically similar. The limitations detected in this approach included high intraspecific variation, low interspecific distances, single barcode records, database gaps, and outdated taxonomic nomenclature. This study confirms DNA barcoding’s effectiveness and highlights the need for expanded reference databases for improved accuracy in forensic and research applications.