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Subterranean habitats represent focal habitats in many conservation strategies; however, these environments are some of the most difficult to sample. New sampling methods, such as environmental DNA ( eDNA ), show promise to improve stygobiont detection, but sources of sampling bias are poorly understood. Therefore, we determined the factors affecting detection probability using traditional visual surveys and  eDNA  surveys for both cavefishes and cave crayfishes and demonstrated how detection affects survey efforts for these taxa. We sampled 40 sites (179 visual and 183  eDNA  surveys) across the Ozark Highlands ecoregion. We estimated the detection probability of cave crayfishes and cavefishes using both survey methods under varying environmental conditions. The effectiveness of  eDNA  or visual surveys varied by environmental conditions (i.e., water volume, prevailing substrate, and water velocity) and the target taxa. When sampling in areas with average water velocity, no flow, and coarse substrate,  eDNA  surveys had a higher detection probability (0.49) than visual surveys (0.35) for cavefishes and visual surveys (0.67) had a higher detection probability than  eDNA  surveys (0.40) for cave crayfishes. Under the same sampling conditions, 5 visual surveys compared to 10  eDNA  surveys would be needed to confidently detect cave crayfishes and 9 visual surveys compared to 4  eDNA  surveys for cavefishes. Environmental DNA is a complementary tool to traditional visual surveys; however, the limitations we identified indicate  eDNA  currently cannot replace visual surveys in subterranean environments. Although sampling designs that account for imperfect sampling are particularly useful, they may not be practical; thus, increasing sampling efforts to offset known detection bias would benefit conservation strategies.

Refining sampling protocols for cavefishes and cave crayfishes to account for environmental variation