An application of PCR-RFLP species identification assay for environmental DNA detection

Recent advancement of eDNA methods for surveying species in aquatic ecosystems has been used for various organisms and contributed to monitoring and conservation of species and environments. Amphibians are one of the promising taxa which could be monitored efficiently by using eDNA. However, the cost of the eDNA detection can be quite high and requires instruments that are not usually installed in ecology laboratories. For aiding researchers foraying into eDNA studies of amphibians, especially those not specialized in molecular biology, we developed a cost efficient protocol using PCR-RFLP method. We attempted to detect eDNA of three Japanese Rana species ( R. japonica, R. ornativentris, and R. tagoi tagoi ) in various spatial scales including an area close to the Fukushima Nuclear Power Plant (FNPP) where the environment is recovering after the disaster in 2011. Our PCR-RFLP protocol was successful in detecting Rana species in static water in both laboratory and field; however, it could not detect Rana species in non-static water samples from the field. Even a more sensitive detection method (standard qPCR) was unable to detect frogs in all non-static water samples. We speculate that our new protocol is effective for frogs living in lentic habitats, but not for lotic habitats which may still require the gold standard of field observation for detection approach.