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.