Abstract
Bog turtles (Glyptemys muhlenbergii) are listed endangered species in the United States. Multi-state efforts are underway to better characterize extant populations of the species and prioritize restoration efforts. Traditional sampling methods for bog turtles can be ineffective due to their wetland habitat, small size, and burrowing nature. New molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, such as wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Our first goal was to develop a qPCR assay that could correctly identify blood collected from seven species of turtle over a wide geographic range. The eDNA detection method was primarily validated using contrived positive samples from the environment. Furthermore, methods employing a genetically modified strain of C. elegans as a full-process internal control which was used in method optimization and to determine DNA recovery from field sample.