Sample selection
We chose 10 samples from the Amphibian and Reptile collection at the Smithsonian Institution’s National Museum of Natural History for which replicated sampling of frozen allozyme supernatant (supernatant), frozen allozyme homogenate tissue pellet (pellet), frozen blood cells (blood), and formalin-fixed voucher specimens (formalin-fixed) were available. Frozen blood cells were used as the DNA source to develop a set of SNPs using the standard ddRADseq approach (see below). This sampling approach allowed us to compare the performance of allozyme supernatant and formalin-fixed tissues to the frozen tissue pellet. Although the pellet is composed of tissue that has been subjected to homogenization (grinding that might result in both mechanical and heat damage) it served as a point of reference for the other hDNA preservation types with the expectation that the pellet would produce consistently higher quality and more complete data using our target-capture method.
Specimens were collected by A. Wynn and Jeremy Jacobs between 1985 and 1990 from two sites in Virginia (VA) and Ohio (OH) for allozyme-based studies (Table S1). Salamanders were euthanized and prepared as voucher specimens 4 to 25 days after capture. Blood samples were collected from euthanized specimens with heparinized capillary tubes and centrifuged to separate blood cells from the serum (to be used for polyacrylamide gel electrophoresis) and were subsequently stored at -70° C. Further, mixed tissue samples (including heart, liver, intestine, spleen, and muscle from the body wall) were taken from each specimen and stored at -70° C. In the case of two of the three larval samples used in this study, the entire animal except the head and forearms were used for allozyme analysis. Tissues were homogenized in distilled water at a ratio of 1 g tissue weight to 2 mL water with a powered grinder using a conically tipped, frosted-glass grinding head and mating grinding tube, and centrifuged for 20 min at 10,000 rpm at 0 to -5° C. The supernatant was decanted from the pellet, and both supernatant and pellet were subsequently stored at -70° C. We note that many researchers did not retain the tissue pellet at this stage of their investigation and only archived the supernatants. Consequently, this particular collection provided an important opportunity to directly compare these sources of DNA.
After tissue removal, voucher specimens were fixed in 10% formalin. Fixation time in formalin was not recorded but ranged from at least one day to a month or more. Specimens were then soaked in at least one change of 60–70% non-denatured ethanol, and afterwards stored in 70% non-denatured ethanol. Larval samples were stored in buffered formalin from the time of processing (1985 or 1988) until September 1998 when they were transferred to 70% denatured ethanol. Because liver tissue was exhausted from most specimens, we harvested ~30 μg of muscle tissue (and also ~30 μg of liver from USNM 525133) and stored the samples in RNA-later for 2–12 hours before freezing the tissues at -80°C. We extracted DNA from ~100 ul of allozyme supernatant, ~15 μg of frozen tissue pellet, ~30 μg of formalin-fixed tissue, and for blood samples we digested all blood cells from a single ~3cm capillary tube.