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 pre
viously 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.