Determination of tortoises origin based on SNPSTR markers: a case study of melanistic Testudo hermanni

Abstract

Nine out of the ten most commonly seized CITES-listed reptiles are tortoises and freshwater turtles. It is often necessary to prove the geographic origin of these animals, which may be challenging when using only morphological characters. Molecular tools are needed to detect and prove illegal wildlife trade. Therefore, in this study, we developed a set of DNA markers for one species of Testudines, Testudo hermanni. The set includes two short tandem repeat (STR) markers and ten STR markers that contain at least one flanking single nucleotide polymorphism (SNP), so-called SNPSTR markers. We identified 72 SNPs and 201 STR alleles by analysing 97 samples. The set has an average observed heterozygosity rate of 0.607 ± 0.047 and an expected heterozygosity rate of 0.868 ± 0.017. The probability of exclusion values based on the 12 markers is greater than 99.95%, while the probability of identity values exceeds the number of individuals worldwide, allowing the set to be used for both individual identification and relationship testing. By analysing samples from a broad spectrum of the T. hermanni distribution range, we have further shown that SNPSTR markers can be used to identify the subspecies and the geographical origin with a high degree of certainty. In addition, given the sampled range, we were able to match a fully melanistic T. hermanni to the Serbian population, and the origin of four partially melanistic individuals to France/Spain. Overall, this study provides a method to combat the illegal trade in T. hermanni and will serve as a great asset against illegal overexploitation.