An efficient environmental DNA detection method for rare species: a case study of a small salamander (Hynobius boulengeri).

Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species.