Multi-method survey rediscovers critically endangered species and strengthens Madagascar's freshwater fish conservation.

Freshwater ecosystems are crucial for global biodiversity through supporting plant and animal species and providing essential resources. These ecosystems are under significant threat, particularly in island environments such as Madagascar. Our study focuses on the Amboaboa River basin, home to the rare and endemic fish species Rheocles derhami, last recorded in 2013. To assess the status of this and other threatened fish species including Ptychochromis insolitus and Paretroplus gymnopreopercularis, and to understand freshwater fish population dynamics in this biodiversity hotspot, we conducted a comprehensive survey using both environmental DNA (eDNA) and traditional fishing methods. While traditional methods effectively captured a diverse range of species, including several invasive aliens and the critically endangered endemic species that were the focus of this study, the eDNA approach detected only a fraction of these introduced species and struggled to identify some critically endangered endemics at the species level. This highlights the value of combining methods to enhance species detection. We also investigated the trade-offs associated with multi-primer assessments in eDNA analysis, focusing on three different primer combinations targeting the 12S mitochondrial gene: MiFish, Tele02, and Riaz. Additionally, we provided 12S reference barcodes for 10 species across 9 genera of fishes from the region to increase the coverage of the public reference databases. Overall, our study elucidates the current state of freshwater biodiversity in the Amboaboa River basin and underscores the value of employing multiple methods for effective conservation strategies.