The bog turtle (Glyptemys muhlenbergii) is listed as critically endangered with over 80% of their natural habitat lost. Multi-state efforts are underway to better characterize extant populations. Traditional sampling methods for bog turtles can be ineffective due to the turtle’s 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 collected from bog turtle habitat are often rich in organics and substances that frequently inhibit both DNA extraction and qPCR amplification. Our primary goal was to determine if methodological improvements in primer design, sample processing, and qPCR chemistry could overcome previous hurdles in developing eDNA methods for this important species. We tested 61 turtle blood DNA extracts with newly designed assays to determine assay sensitivity and specificity. To simultaneously determine DNA extraction efficiency and detect possible PCR inhibition we included a novel internal control using a genetically modified strain of Caenorhabditis elegans.
On standard curves composed of serially diluted gBlocksTM the amplification efficiency of the new assay, BT3, was 98-100%. Blood samples were collected from 22 different sites in Pennsylvania, New York, Connecticut, and New Jersey. After testing 50 bog turtle, 5 wood turtle (Glyptemys insculpta), 4 spotted turtle (Clemmys guttata) and 2 box turtle (Terrapene sp.) blood DNA extracts, all bog turtle extracts had 485-3,533 gene copies/ng blood DNA, while all other species tested did not amplify indicating 100% sensitivity and specificity of the assay. Initial laboratory simulations with the C. elegans internal control indicated that DNA extraction efficiency from environmental samples was significantly inhibited presumably due to the complex nature of the sampled substrate. We show that internal controls such as these not only allow more accurate estimations of eDNA concentrations in the environment, but also may be needed for valid site to site comparisons. Such information is necessary to prioritize habitat protection and restoration efforts. Further testing of blood samples from additional sites is needed to confirm high sensitivity and specificity over a wide geographic range. Likewise, further testing of environmental samples and optimization of DNA extraction methods is needed to demonstrate assay utility in a practical setting.