In species with temperature-dependent sex determination, the embryo commits to its sexual fate during a time window called the thermosensitive period (TSP). Although substantial research has focused on the effect of the temperature during this canonical TSP, the effect of temperatures experienced before this time (pre-TSP) on the onset and duration of the TSP is less understood. Here, we incubated eggs of the endangered Magdalena River turtle, Podocnemis lewyana, under 3 relatively constant temperatures and 6 shift-twice treatments. Constant treatments included two 100% masculinizing temperatures that fostered a relatively slower and faster embryo development and one 100% feminizing temperature. The shift treatments included a masculinizing temperature and later a feminizing temperature for a period of time at different incubation stages. Sex ratios were determined by a geometric-morphometric sexing approach developed for this species and validated by gonadal histology. This technique is a nonlethal sexing alternative; thus, it contributes to the conservation of this endangered species. The sex ratios obtained under constant temperatures were more feminized than the sex ratio of another population native to a warmer climate. Shift-twice experiments revealed that pre-TSP temperatures affected the duration of the TSP and as such influenced sex determination. Specifically, at 29°C pre-TSP, the TSP lasted 20 d (28% of the incubation period), whereas at 31°C pre-TSP, the TSP was extended for 30 d (52% of the incubation period). An approximation of developmental stages based on the congeneric Podocnemis expansa is provided. Conservation programs should monitor temperatures throughout the entire incubation period; otherwise important effects on sex ratios and other traits may be obscured.

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