DNA based method for determining source country of the short beaked echidna (Tachyglossus aculeatus) in the illegal wildlife trade

•The short beaked echidna (Tachyglossus aculeatus) is a species targeted in the illegal wildlife trade.•Non-invasive sampling techniques were used to extract DNA from echidna quills, using four common extraction methods.•Phylogenetically informative markers for the D-loop were developed and validated. This allows echidnas from New Guinea and Australia to be distinguished, and can be implemented when an echidna is trafficked. The illegal trade in wild animals being sold as ‘captive bred’ is an emerging issue in the pet and zoo industry and has both animal welfare and conservation implications. DNA based methods can be a quick, inexpensive, and definitive way to determine the source of these animals, thereby assisting efforts to combat this trade. The short beaked echidna (Tachyglossus aculeatus) is currently one of the species suspected to be targeted in this trade. As this species is distributed throughout Australia and in New Guinea (currently comprising of five recognised sub-species), this project aimed to develop a DNA based method to definitively determine the source country of an echidna and explore the use of non-invasive sampling techniques. Here we use non-invasively sampled echidna quills and demonstrate the extraction of mitochondrial DNA and amplification of a region of the mitochondrial genome. Phylogenetically informative markers for analysis of a 322bp segment of the D-loop region were developed, and subsequently validated, using animals with known source locations allowing us to reliably distinguish between echidnas from New Guinea, and Australia. This research presents the first validated forensic protocols for short beaked echidnas and will be an integral tool in understanding the movement of animals in this emerging trade.