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

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 <i>FOXL2</i> and <i>FGF9</i>. <i>FOXL2</i> is associated with ovarian development, and<i> FGF9</i> 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 <i>FOXL2</i> and <i>FGF9</i> across 3 developmental stages and 8 juvenile tissue types in male and female American alligators, <i>Alligator mississippiensis. </i>We report surprisingly high expression of <i>FOXL2</i> before the stage of embryonic development when sex is determined in response to temperature, and sustained and variable expression of <i>FGF9</i> 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.