DNA analysis and validation for species identification of seized helmeted hornbill (Rhinoplax vigil) casques

Helmeted hornbills (Rhinoplax vigil, J.R. Forster, 1781) are ‘Critically Endangered’ due to illegal hunting for their casques which are carved and traded for ornamental purposes. DNA species identification techniques can aid enforcement efforts, and validated wildlife forensic techniques for the species identification of R. vigil are needed. Here we tested multiple methods for sampling and extracting DNA from R. vigil casques and a validated a previously published assay using cytochrome B (cytB) primers to identity species and origin of traded casques. Phenol-chloroform: isoamyl alcohol extractions resulted in samples with higher quantity and quality of DNA than those extracted using the commercial Qiagen DNeasy Blood and Tissue kit. Samples collected from the caudal side of the casque yielded higher DNA quantity and quality than rostral and lateral sides, regardless of sampling method. We then assessed the repeatability, reproducibility, robustness, sensitivity, specificity, and phylogenetic resolution of a previously published species identification assay. We confirm the ability of this method to phylogenetically distinguish between R. vigil and closely related hornbills with high bootstrap support (99%). We also report the first genetic evidence of illegally traded R. vigil in Hong Kong using confiscated casques and provide more reference samples of R. vigil for future work. Overall, we provide multiple protocols for sampling and extracting DNA, and a validated species identification assay for amplifying DNA from R. vigil casques with potential to aid law enforcement in illegal wildlife crimes.