Amphibian supercooling capacity is not limited to sub-zero thermal environments

Freeze-tolerant amphibians initiate controlled freezing using ice nucleators and survive internal ice formation by accumulating cryoprotectants. In contrast, freeze-avoidant (supercooling) species rely on the inhibition of ice nucleators to prevent freezing altogether. All confirmed supercooling species are native to the Northern Hemisphere and regularly endure negative temperatures. The occurrence, ecological role, and underlying mechanisms of supercooling in amphibians remain poorly understood. Here, we demonstrate for the first time that amphibian supercooling capacity may be present even if not expressed (i.e., latent) and not limited to freezing thermal environments. Exploratory metagenomic data allow us to evaluate whether skin-associated bacteria could contribute to freeze avoidance. In addition, using field experiments, we assess cold and dehydration tolerance limits in two syntopic amphibian species from a high tepui summit (Roraima-tepui in Venezuela) and explore the potential role of cryoprotective dehydration in facilitating supercooling. Despite being syntopic, these species showed striking differences in thermal and dehydration tolerance. Physiological freeze avoidance in tropical montane amphibians is shown to be associated with low critical thermal minima, high dehydration tolerance and possibly antifreeze-producing skin microbiota, although the latter needs further investigation. These traits may determine species persistence under shifting climatic regimes, particularly in thermally variable montane systems.