Vertebrate colonization of land occurred multiple times, including over 50 origins of terrestrial eggs in frogs. Some environmental factors and phenotypic responses that facilitated these transitions are known, but responses to water constraints and risk of ammonia toxicity during early development are poorly understood. We tested if ammonia accumulation and dehydration risk induce a shift from ammonia to urea excretion during in early stages of four anurans, from three origins of terrestrial development. We quantified ammonia and urea concentrations during early development on land, under well-hydrated and dry conditions. Where we found urea excretion, we tested for a plastic increase under dry conditions and with ammonia accumulation in developmental environments. We assessed the potential adaptive role of urea excretion by comparing ammonia tolerance measured in 96h-LC₅₀ tests with ammonia levels in developmental environments. Ammonia accumulated in foam nests and perivitelline fluid, increasing over development and reaching higher concentrations under dry conditions. All four species showed high ammonia tolerance, compared to fishes and aquatic-breeding frogs. Both nest-dwelling larvae of Leptodactylus fragilis and late embryos of Hyalinobatrachium fleischmanni excreted urea, showing a plastic increase under dry conditions. These two species can develop the longest on land and urea excretion appears adaptive, preventing their exposure to potentially lethal levels of ammonia. Neither late embryos of Agalychnis callidryas nor nest-dwelling larvae of Engystomops pustulosus experienced toxic ammonia levels under dry conditions, and neither excreted urea. Our results suggests that an early onset of urea excretion, its increase under dry conditions, and elevated ammonia tolerance, can all help prevent ammonia toxicity during terrestrial development. High ammonia represents a general risk for development which may be exacerbated as climate change increases dehydration risk for terrestrial-breeding frogs. It may also be a cue that elicits adaptive physiological responses during early development.

We conducted field work in Gamboa and at the Soberanía National Park, in Panama. We worked with foam nests and gelatinous clutches from four frog species species, during 2016–2019 breeding seasons. We collected data with permission from the Panamanian Ministry of the Environment (details provided in the manuscript). We conducted experiments in an open-air laboratory, at ambient temperature and humidity, at the Smithsonian Tropical Research Institute (STRI) in Gamboa. We performed laboratory analysis at STRI facilities in Gamboa and Panama City. We exposed egg clutches to ecologically relevant, species-specific control (well-hydrated) and dry conditions in the laboratory, based on their natural history. We quantified ammonia and urea concentration in developmental environments at multiple times to assess their accumulation over development and the effect of dry conditions on the amount of urea excretion at the latest sampling age. We quantified nitrogen excretion using a colorimetric approach with a commercial enzymatic kit (Boehringer Mannheim Cat. No. 10542946035). We also conducted standard 96-h LC₅₀ tests for ammonia in all the study species to test ammonia tolerance during early development in terrestrial developmental environments. Aditional details are provided in the main manuscript and its repsective supporting information.

See README.txt file for description of all files included in this data publication. All statistical analysis can be performed using R v 3.6.1 (R Core Team 2019), using csv files generated from excel tabs for each species.