A sex ratio by incubation temperature reaction norm for the American alligator ( Alligator mississippiensis ) based on fluctuating thermal regimes

Environmental conditions during embryonic development wield critical influence on organismal traits. For example, in species with temperature-dependent sex determination (TSD), the incubation temperatures an embryo experiences determines sex. Reaction norms quantifying this relationship are essential tools for predicting how climatic variability will shift population sex ratios. However, existing TSD reaction norms are derived from constant temperature laboratory conditions, leaving open the question of their applicability to natural conditions where incubation temperatures fluctuate. In this study, we developed a TSD reaction norm for the American alligator (Alligator mississippiensis) using fluctuating temperature treatments. We incubated eggs under five fluctuating temperature regimes, determined offspring sex using gonadal gene expression, and used the resulting sex ratios to fit reaction norms based on mean temperature and constant temperature equivalents (CTE). The reaction norms followed the canonical Female-Male-Female pattern of crocodilians. At lower incubation temperatures, our fluctuating-temperature reaction norms closely aligned with that derived from constant temperatures, suggesting that mean temperature and CTE statistics perform reasonably well in this range. However, at higher incubation temperatures, the range most relevant to climate warming projections, our reaction norms diverged substantially from constant temperature reaction norms with upper pivotal temperature estimates differing by up to 1.36°C. Because small thermal shifts at this critical transition can dramatically alter predicted sex ratios, these discrepancies carry real consequences for conservation forecasting. Our results underscore the importance of incorporating ecologically realistic thermal variation into TSD reaction norms and highlight the upper male-to-female transition as a priority for mechanistic study.