Environmental DNA and Next-Generation Sequencing to Detect Rare Freshwater Fish Species in Southern Ontario

Finding novel techniques to sample species that are uncommon, small, or secretive are essential to collect genetic diversity and distribution information with minimal impact on habitat or harm to the species (e.g. stress). Environmental DNA (eDNA) was extracted from water samples from two major river systems in Southwest Ontario, Canada: Grand River and Sydenham River. Using PCR and massively parallel (“NextGen”) sequencing we mapped habitat use and overlap for three species at risk (Northern madtoms (Noturus stigmosus), Silver shiners (Notropis photogenis), and Eastern sand darters (Ammocrypta pellucida)) and the invasive Round goby (Neogobius melanostomus) in the two river systems. Each eDNA sample was PCR amplified using COI, 16S, and Cytbprimers designed to target all 4 species at once (cross species primers), as well as species-specific primers. The resulting PCR products were NextGen sequence characterized with a target of 5000 reads per sample and all sequences were verified for match with the target species. Positive eDNA detections are used for habitat-use analysis by mapping the occurrence of the species on river habitat maps. This project will propose a new method of utilizing eDNA analysis as a non-invasive sampling tool to study both endangered and invasive aquatic species.