Eye Darkening Under Dehydration Stress in the Neotropical Frog <i>Boana punctata</i> (Schneider, 1799)

Amphibians face significant dehydration challenges due to their permeable skin and weak water retention mechanisms, especially under ongoing global warming that predicts increasing land dryness. This makes the identification of physiological stress indicators crucial to assessing hydric risk in amphibian populations. This study examines iris darkening as a response to dehydration in the neotropical tree frog (Boana punctata) under controlled conditions. Twenty-five adult frogs were collected in the province of Chaco, Argentina, and subjected to sequential dehydration treatments (10, 20, and 30 min) using two methods (constant airflow of 0.2 m/s or desiccant rice), followed by 2-h rehydration. Linear mixed‑effects models revealed a strong negative relationship between hydration level and iris darkening, increasing about 1.5% in dark area for each 1% loss in hydration level, consistent across dehydration methods and with similar sensitivity across body sizes and individuals, although larger frogs showed lower baseline darkening. Iris darkening showed partial reversal post-rehydration. This validates iris darkening as a robust, non-invasive biomarker for hydric stress in wild amphibian populations facing climate-driven aridity.