Characterization, optimization, and validation of environmental DNA (eDNA) markers to detect an endangered aquatic mammal

Environmental DNA (eDNA) represents a sensitive and efficient method for noninvasively sampling rare or otherwise hard to monitor taxa, potentially making it a powerful tool for conservation management. Still, this novel method can be affected by sampling protocols, abiotic characteristics of the microhabitat, the focal taxa itself, and primer design. Here we designed 12 species-specific primers for the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) inhabiting the middle to lower Yangtze River in China to enable complementary population survey tools for conservation efforts. Representing primer pairs that amplify a range of DNA fragment sizes, we test these primers for sensitivity at amplifying finless porpoise DNA using conventional PCR from serial diluted blood samples, and from eDNA in aquaria and in the wild, including a nature reserve and a negative control site. We further investigated the capacity for these primers to detect finless porpoise DNA signals from water samples over a 30-day period. Our study presents which primers were successful at amplifying finless porpoise DNA from aquaria and in the wild, and further demonstrates no significant amplicon size effects on primer sensitivity or longevity. We summarize the impact primer design may have on eDNA applications in general and suggest future considerations for conservation efforts with the Yangtze finless porpoise.