Development and validation of a multiplex STR assay for three crane species: blue (Anthropoides paradiseus), wattled (Bugeranus carunculatus) and grey-crowned (Balearica regulorum) for monitoring legal trade

Worldwide, the illegal wildlife trade is growing and impacting on thousands of species. One aspect is the illegal trade of captive bred animals due to the perceived value of the species. Cranes are iconic species in Africa and are exploited by illegal trade. Although these species have been traded legally from captive facilities, there is a possibility that wild chicks are being laundered through this legal system. Crane species that have been affected by this trade
include blue cranes (Anthropoides paradiseus) and grey-crowned cranes (Balearica
regulorum) which are listed as Vulnerable (V) on the International Union for Conservation of Nature (IUCN) Red List and wattled cranes (Bugeranus carunculatus) which are listed as Endangered (EN). In order to monitor legal trade, DNA technologies are useful tools for the provision of evidence. The aim of this study was to develop and validate a set of STR markers for three crane species
(A. paradiseus, B. regulorum, B. carunculatus) occurring in South Africa to (1) monitor legal trade and (2) promote standardisation of genetic techniques to assist possible forensic investigations. A developed evidence based sampling protocol and the application of the STR assay in parentage confirmation of captive-bred cranes was described. An eighteen (18) STR loci multiplex assay was developed and optimised using samples from A. paradiseus (n=126), B. regulorum (n=104) and B. carunculatus (n=21) collected under following a chain of evidence protocol. The four multiplex assay yielded high discriminatory power for individual identification and parentage assignment of closely related individuals in all three species (A. paradiseus: PI = 1.6x10-24 PIsibs = 4.7x10-08
, B. carunculatus: PI = 1.4x10-19 PIsibs = 2.9x10-07 and B. regulorum: PI =1.3x10-12 PIsibs =5.0x10-05). The multiplex assay was further proven to be
reliable, reproducible and repeatable via a validation test and can successfully be applied in forensic investigations where possible illegal trade or capturing from the wild is suspected. In addition, the utility of the STR multiples assay to confirm parentage was emphasized through analysis of parent-offspring trios. The parentage of 26 offspring were verified with an average logarithm of odds (LOD) score of 18.01 under strict confidence levels (>95%). A management system was furthermore described to monitor legal trade of cranes including (1) the development of an evidence based sampling protocol, (2) the possible use of
morphological measurements for identification, (3) the establishment of a DNA database and (4) the verification of parentage and issuing of DNA passports for captive-bred compliance. The importance of complete genetic databases, maintaining chain-of-custody and integrity of samples was highlighted together with the need for effective communication between breeders, government agencies and laboratories to ensure admissibility of evidence and
successful convictions in future investigations of possible illegal trade. In this study, the first set of validated forensic genetic tools and management system to monitor legal trade and assist in the protection of wild populations of A. paradiseus, B. regulorum and B. carunculatus are presented.