DNA extraction
DNA extraction for pellet, supernatant, and blood samples followed a standard salt-extraction protocol (Sambrook & Russell 2001). DNA samples were quantified using a QUBIT® 2.0 Fluorometer HS (Life Technologies, Grand Island, NY, USA) and representative extractions of each were checked on a 1% Agarose gel using 1–5 μl of DNA.
Our formalin-fixed extraction protocol followed Bell et al. (2017) with some modifications. Extractions were conducted in a dedicated PCR-free lab within a UV irradiated fume hood; all surfaces were pre-wiped with bleach and materials (plastics, pipettes etc.) were UV irradiated before use. We soaked ~30 μg of tissue (muscle for all specimens plus liver for USNM 525133) in GTE Buffer for 24 hours, and repeated this for three washes (3 days total). We incubated for ~4 days at 55°C in a 1.5 ml tube containing 500 μl Cell Lysis Buffer, 100 μl Proteinase K, and 20 μl of 1 mM DTT, adding 20 μl of Proteinase K per day as needed. We placed the samples on ice for 5 min, added 200 μl of Protein Precipitate Solution, inverted 50 times, and spun samples on a centrifuge at 14,000 g for 3 min. We poured the supernatant into a new 1.5 ml tube containing 600 ul of cold 100% ETOH and 3 μl glycogen solution. We again inverted the samples 50 times, incubated samples for 48 hours at -20°C, then spun samples at 14000g for 30 min. After discarding the supernatant, we added 200 ul of 70% ETOH, inverted 50 times, spun at 14000 g for 3 min, then drained and dried the samples for ~3–6 hours. We resuspended samples in 40 μl Tris solution. If DNA yield was expected to be low, we conducted multiple extractions of the same sample and pooled the final product. One of the larval specimens (USNM 525132) yielded only 32 ng of total DNA (based on Qubit quantification) and we did not attempt to capture this sample. The full extraction protocol is available in Appendix I. Fragment distribution of three replicates of the supernatant and formalin-fixed extractions were quantified on a TapeStation (Agilent, Santa Clara, CA; Appendix II). Specifically, we compared the liver and muscle extractions of USNM 525133 to visualize the distribution of DNA fragments extracted from both tissue types.