Forensic application of DNA barcoding for identification of illegally traded African pangolin scales.

The escalating growth in illegal wildlife trade and anthropogenic habitat changes threaten the survival of pangolin species worldwide. All eight extant species have experienced drastic population size reductions globally with a high extinction risk in Asia. Consequently, forensic services have become critical for law enforcement, with a need for standardised and validated genetic methods for reliable identifications. The seizure of three tonnes of pangolin scales, believed to have originated from Africa, by Hong Kong Customs Authorities provided an opportunity for the application of DNA barcoding in identifying scales. Three mitochondrial DNA gene regions (COI, Cyt b, and D-loop) were amplified for a subsample of the confiscated material and compared with taxonomically verified references. All four African species were recovered as monophyletic with high interspecific uncorrected p-distance estimates (0.048-0.188) among genes. However, only three of four African species (Phataginus tricuspis, Phataginus tetradactyla, and Smutsia gigantea, originating from West and Central Africa) and one of four Asian species (Manis javanica from Southeast Asia) were identified among scales. Although the assignment of unknown scales to specific species was reliable, additional genetic tools and representative reference material are required to determine geographic origins of confiscated pangolin specimens.

Development and validation of a novel forensic STR multiplex assay for blue (Anthropoides paradiseus), wattled (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum).

The blue crane (Anthropoides paradiseus), wattled crane (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum) are species of concern as their populations are declining and they face several threats including habitat loss, disturbance and illegal trade. In South Africa, these species are bred in captivity for trade purposes which is permitted and regulated globally under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Legal sustainable trade through captive breeding of endangered wildlife species such as cranes has been promoted to counteract the illegal trade of individuals from the wild. Captive breeding independent of wild populations may reduce the harvest pressures on wild bird populations which in turn benefit the recovery of exploited species. This approach is considered to be controversial by some individuals. Although captive breeding of endangered species, for both population sustainability and commercial purposes, is promoted to aid in conserving species, concerns have been raised with regards to breeding facilities being used for laundering of animals. To monitor the legal trade of cranes in South Africa a short tandem repeat (STR) assay following recommendations of the International Society for Forensic Genetics (ISFG) was developed and validated. An STR assay comprising of four multiplexes that include 16 STR markers and two gender determination markers was proven to be highly informative with average polymorphic information content (PIC) values of 0.806, 0.646 and 0.725 for A. paradiseus, B. regulorum and B. carunculatus respectively. In addition, the assay showed sufficient discriminatory power for parentage assignment of closely related individuals in all three species (A. paradiseus: PI = 1.7×10-24, PIsibs = 4.7×10-08, and B. carunculatus: PI = 1.4×10-19, PIsibs = 2.9×10-07 and B. regulorum: PI = 1.7×10-12, PIsibs = 5.0×10-05). Analysis of 251 samples suggested that the validated multiplex assay ensures reliability, reproducibility, and repeatability for applications in forensic case work where illegal trade of offspring is suspected through verifying parentage of captive birds in breeding facilities.

Monitoring compliance of CITES lion bone exports from South Africa.

From 2008 to 2018, South Africa permitted the export of captive-bred African lion (Panthera leo) skeletons to Southeast Asia under CITES Appendix II. Legal exports rose from approximately 50 individuals in 2008 to a maximum of 1,771 skeletons in 2016, and has led to ongoing concerns over possible laundering of non-lion, multiple-source and wild-sourced bones. South Africa is required under its obligations to CITES to employ mechanisms for monitoring and reporting trade, and to limit the potential for illegal trade and laundering of lion and other large felid bones. Monitoring tools for legal trade are critical to compliance with CITES. Here we evaluate the CITES-compliance procedure implemented by South Africa for export of lion bones and identify six essential general points for consideration in the implementation of animal export quota compliance protocols. We provide specific insight into the South African lion bone export monitoring system through: i) outlining the protocols followed; ii) assessing the utility of cranial morphology to identify species; iii) evaluating skeleton consignment weight as a monitoring tool; and iv) presenting molecular (DNA) species assignment and pairwise-comparative sample matching of individuals. We describe irregularities and illicit behaviour detected in the 2017 and 2018 lion bone quotas. Notably, we report that the compliance procedure successfully identified and prevented the attempted laundering of a tiger (P. tigris) skeleton in 2018. We emphasise the utility of mixed-method protocols for the monitoring of compliance in CITES Appendix II export quota systems.