Experimental design and data collection:
In June and July of 2021, 7-8 clutches of alligator eggs were collected
from each of four, geographically distinct populations, including Par
Pond on the United States Department of Energy’s Savannah River Site in
Aiken, South Carolina (SR), Tom Yawkey Wildlife Center in Georgetown,
South Carolina (YK), Lake Woodruff National Wildlife Refuge in De Leon
Springs, Florida (WO), and Lake Apopka in Apopka, Florida (AP; Figure
1A). After locating nests by helicopter or airboat, all eggs were
removed from a nest cavity shortly after oviposition. Eggs were placed
in plastic bus pans with natal nesting material for transport back to
the University of Georgia’s Savannah River Ecology Laboratory in Aiken,
SC, where they were individually weighed and 1-2 eggs from each clutch
were staged according to Ferguson (1985). The remaining eggs were
transferred into new bus pans with dampened sphagnum moss and kept in
commercial incubators (model I36NLC, Percival Scientific, Perry, IA,
USA) at 32°C, an intermediate temperature that produces mixed sex ratios
(Lang and Andrews, 1994). During this period, eggs were misted twice
daily, and bins were rotated once daily within each incubator to limit
the effect of intra-incubator temperature variation. Incubator
temperatures were also monitored with HOBO TidbiT® v2 Temp Loggers
(Onset, Bourne, MA, USA) placed within bus pans and incubator set
temperatures were adjusted daily to maintain a constant 32°C.
At embryonic stage 15, just prior to the opening of the thermosensitive
period of sex determination (McCoy et al., 2015), eggs from each
population were randomly assigned in a split-clutch design to one of two
temperature treatments: a constant MPT (33.5°C) or a constant FPT
(29.5°C). Since full clutches were collected for multiple studies, a
random subset of 3-10 eggs/clutch/temperature/site were chosen at this
time to raise until hatch for this experiment. Throughout the entire
incubation period, eggs were continually monitored and incubator
temperature set points were adjusted to maintain consistent
temperatures.
Once hatchlings pierced the eggshell (“pipped”), the date was
recorded, and eggs were placed in glass Mason jars (one egg/jar) with
damp sphagnum moss. Embryos were given 48 hours to hatch from the egg
before being assisted if they did not hatch on their own. Once fully
hatched, neonates were weighed using a digital balance (± 0.01g) and SVL
and tail girth at the cloaca (TG) were measured using a flexible ruler
(±0.1 mm), and head length (HL) and head width (HW) were measured using
calipers (± 1 mm). Hatchlings were then individually marked using
unique, numbered toe tags and transported to large, indoor, fiberglass
holding tanks where they were maintained under common-garden conditions
and natural light cycles for 10-days. During this period, hatching
alligators relied on maternal yolk reserves and were not fed. Water was
changed daily, and hatchlings were monitored twice daily for overall
health and survival. At 10-days post-hatch (10-DPH), hatchlings were
remeasured, euthanized via cervical severance and pithing, and dissected
to obtain residual yolk mass and fat body mass. Phenotypes analyzed
included morphological traits of mass, BMI, SVL, TG, HL, and HW at
hatch, and metabolic traits including incubation duration (measured in
days from stage 15 to pip), change in morphological traits between
10-DPH and hatch (∆ mass ∆ BMI, ∆ SVL, ∆ TG), residual yolk mass, and
fat body mass. All experiments were approved by the University of
Georgia Animal Care and Use Committee (A2021 05-007-Y3-A0) and
collections were carried out under permits from the South Carolina
Department of Natural Resources (SC-08-2021) and Florida Fish and
Wildlife Conservation Commission (SPGS-18-33).