DNA contamination and taxonomic diversity hinder eDNA research on freshwater stingray species: Comment on Lim & Then (2022)

Recently, Lim & Then (2022; Endang Species Res 48:43-50) used an environmental DNA (eDNA) PCR-based method for detecting the presence of an endangered species of freshwater stingray, Fluvitrygon kittipongi (Dasyatidae), in the Pahang River basin, east Peninsular Malaysia. For that, they designed a species-specific pair of primers to amplify a 196 base pair (bp) fragment of the cytochrome c oxidase I (COI) gene. Lim & Then (2022) reported positive detection of this species along an upstream stretch of the Pahang River during the inter-monsoon and the southwest monsoon seasons, with the PCR-amplified eDNA sequences identical to the reference COI sequences of F. kittipongi collected from the Perak River basin, west Peninsular Malaysia. Here, we argue that such positive results are likely the consequence of a DNA contamination because the 196 bp COI fragments of F. kittipongi from the Pahang River and the Perak River differ from each other by 4 nucleotide substitutions. The source of contamination in Lim & Then (2022) could have been the Perak samples the authors handled to develop the primers. We also briefly discuss the potential impact of the presumed co-occurrence of freshwater stingrays of the genus Fluvitrygon in the Pahang River on the specificity of the F. kittipongi-specific primers designed by Lim & Then (2022).