Noninvasive Method for a Statewide Survey of Eastern Hellbenders Cryptobranchus alleganiensis Using Environmental DNA

Traditional survey methods of aquatic organisms may be difficult, lengthy, and destructive to the habitat. Some methods are invasive and can be harmful to the target species. The use of environmental DNA (eDNA) has proven to be effective at detecting low population density aquatic macroorganisms. This study refined the technique to support statewide surveys. Hellbender presence was identified by using hellbender specific primers (cytochrome b gene) to detect eDNA in water samples collected at rivers, streams and creeks in Ohio and Kentucky with historical accounts of the imperiled eastern hellbender (Cryptobranchus a. alleganiensis). Two sampling protocols are described; both significantly reduced the amount of water required for collection from the previously described 6？L collection. Two-liter samples were adequate to detect hellbender presence in natural waterways where hellbenders have been previously surveyed in both Ohio and Kentucky—1？L samples were not reliable. DNA extracted from 3？L of water collected onto multiple filters (1？L/filter) could be combined and concentrated through ethanol precipitation, supporting amplification of hellbender DNA and dramatically reducing the filtration time. This method improves the efficiency and welfare implications of sampling methods for reclusive aquatic species of low population density for statewide surveys that involve collecting from multiple watersheds. 1. Introduction Hellbenders (Cryptobranchus spp.) are North America’s largest amphibian species comprised of two subspecies. The Ozark hellbender (Cryptobranchus alleganiensis Bishop) is federally endangered, existing in fragment populations in southern Missouri and northern Arkansas, whereas the eastern hellbender, a species of special concern throughout its range, is found in Appalachian forests from southern New York to northern Alabama. Their native habitat consists of large slab rocks in clear and well aerated streams and rivers [1, 2]. In the last century, hellbender populations have declined due to anthropogenic factors [3, 4]. These include hunting and overcollecting [5], as well as degradation of habitat by pollution, dam construction, and sedimentation [6]. Collectively, these disturbances have had negative impacts on hellbender populations [1, 7, 8], particularly in the recruitment of juveniles [9]. Population trends of the hellbender have indicated an ~80% decrease in Missouri and Ohio when compared to data from the previous decades [4, 9]. Because of their sensitivity to environmental disturbances, hellbenders are considered important