Genome-wide DNA methylation patterns harbor signatures of hatchling sex and past incubation temperature in a species with environmental sex determination

Conservation of thermally sensitive species depends on monitoring
organismal and population-level responses to environmental change in real
time. Epigenetic processes are increasingly recognized as key integrators
of environmental conditions into developmentally plastic responses, and
attendant epigenomic datasets hold potential for revealing cryptic
phenotypes relevant to conservation efforts. Here, we demonstrate the
utility of genome-wide DNA methylation (DNAm) patterns in the face of
climate change for a group of especially vulnerable species, those with
temperature-dependent sex determination (TSD). Due to their reliance on
thermal cues during development to determine sexual fate, contemporary
shifts in temperature are predicted to skew offspring sex ratios and
ultimately destabilize sensitive populations. Using reduced-representation
bisulfite sequencing, we profiled the DNA methylome in blood cells of
hatchling American alligator (Alligator mississippiensis), a TSD species
lacking reliable markers of sexual dimorphism in early life-stages. We
identified 120 sex-associated differentially methylated cytosines (DMCs;
FDR < 0.1) in hatchlings incubated under a range of temperatures,
as well as 707 unique temperature-associated DMCs. We further developed
DNAm-based models capable of predicting hatchling sex with 100% accuracy
(in 20 training samples and 4 test samples) and past incubation
temperature with a mean absolute error of 1.2˚C (in 4 test samples) based
on the methylation status of 20 and 24 loci, respectively. Though largely
independent of epigenomic patterning occurring in the embryonic gonad
during TSD, DNAm patterns in blood cells may serve as non-lethal markers
of hatchling sex and past incubation conditions in conservation
applications. These findings also raise intriguing questions regarding
tissue-specific epigenomic patterning in the context of developmental
plasticity.