Holistic multi-method survey to enhance Madagascar's freshwater fish conservation

Freshwater ecosystems are crucial for global biodiversity, supporting plant and animal species and providing essential resources. However, these ecosystems, particularly in island environments such as Madagascar, are under significant threat. 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. Our findings reveal updated records of Rheocles derhami and an expanded species list within these freshwater environments. We also explore the implications of combining these two monitoring approaches and investigate the trade-offs associated with multi-primer assessments in eDNA analysis, focusing on three different primers targeting the 12S mitochondrial gene: MiFish, Tele02, and Riaz. Additionally, we provide 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 enlightens the current state of freshwater biodiversity in Amboaboa River basin and underscores the value of employing multiple methods for effective conservation strategies.