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).