Environmental DNA (eDNA) barcoding has emerged as an important non-invasive sampling technique for the detection of rare and endangered species that can be difficult to sample. Our objective was to develop a low-cost eDNA barcoding approach for the detection of an Endangered freshwater stingray species Fluvitrygon kittipongi in a tropical river system in Peninsular Malaysia. We designed a species-specific primer for a fragment of F. kittipongi cytochrome oxidase subunit I mtDNA (244 bp). The effectiveness of this primer to detect the stingray was evaluated using water samples taken from the upper and middle reaches of the Pahang River coupled with social media reports on sightings of F. kittipongi. Five of 14 water samples tested showed positive PCR amplification for the targeted species. These results represent the first successful application of eDNA to detect freshwater stingrays in Malaysia. Using a combination of freshly obtained carcass samples, social media reporting and target species eDNA detection, this study provides formal occurrence records of F. kittipongi in Malaysia in 3 major watersheds: the Perak, Pahang and Kelantan Rivers.

Alshari et al. (2023; Endang Species Res 50:311-313) were able to collect 3 Fluvitrygon kittipongi specimens from the Pahang River, which were not available to us at the time of our study (Lim & Then 2022; Endang Species Res 48:43-50). The cytochrome c oxidase I (COI) sequences (or haplotypes) of these 3 stingrays were identical to each other but differed from the haplotypes of our Perak River specimens. The result of 100% similarity between COI sequences of our Perak River rays and the eDNA water samples isolated from Pahang River in our study was suggested by Alshari et al. (2023) as DNA contamination in our study, which is plausible. However, further population genetics studies would be necessary to ascertain that the Perak River haplotype does not occur in stingrays of the Pahang River.