Supplementary Material for: Sex-Biased Expression of Sex-Differentiating Genes FOXL2 and FGF9 in American Alligators, Alligator mississippiensis

Across amniotes, sex-determining mechanisms exhibit great variation, yet the genes that govern sexual differentiation are largely conserved. Studies of evolution of sex-determining and sex-differentiating genes require an exhaustive characterization of functions of those genes such as FOXL2 and FGF9. FOXL2 is associated with ovarian development, and FGF9 is known to play a role in testicular organogenesis in mammals and other amniotes. As a step toward characterization of the evolutionary history of sexual development, we measured expression of FOXL2 and FGF9 across 3 developmental stages and 8 juvenile tissue types in male and female American alligators, Alligator mississippiensis. We report surprisingly high expression of FOXL2 before the stage of embryonic development when sex is determined in response to temperature, and sustained and variable expression of FGF9 in juvenile male, but not female tissue types. Novel characterization of gene expression in reptiles with temperature-dependent sex determination such as American alligators may inform the evolution of sex-determining and sex-differentiating gene networks, as they suggest alternative functions from which the genes may have been exapted. Future functional profiling of sex-differentiating genes should similarly follow other genes and other species to enable a broad comparison across sex-determining mechanisms.