Optimisation of an Environmental DNA‐Based Approach for Assessing Freshwater Fish Biodiversity in Southernmost South America

ABSTRACT In the Southernmost region of South America (52°‐55° S), the native freshwater fish biodiversity is extremely low, and invasive species pose a constant threat. Environmental DNA (eDNA) has emerged as a powerful tool for studying fish biodiversity and biomonitoring remote locations. This study evaluated the effectiveness of targeted eDNA and eDNA metabarcoding for assessing fish species composition from four basins of Tierra del Fuego, Patagonia. For targeted eDNA, we designed, validated, and implemented nine species‐specific primers in real‐time PCR assays. For eDNA metabarcoding, we performed high‐throughput sequencing of two barcodes on the mitochondrial 12S rRNA gene and constructed a local reference database. Also, eDNA‐based results were compared with those of electrofishing to verify consistency. The taxon‐specific approach proved the most effective for detecting native fish species. Meanwhile, no significant differences were observed between methods for documenting alien species. The targeted eDNA method successfully validated the presence of Aplochiton, a genus of high conservation value endemic to Patagonia and the Falkland/Malvinas Islands. Our findings have significant implications for documenting and monitoring native fish species in remote areas where field surveys can be particularly challenging. Also, this study underscores the importance of eDNA‐based methods to complement traditional methods, particularly in conservation efforts aimed at rare species in regions facing ecological threats from salmonids.