DOI: 10.5061/dryad.866t1g1r2
title: Data for: Reproductive colonization of land by frogs: Embryos and larvae excrete urea to avoid ammonia toxicity

description: 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.
description: 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 LC<sub>50</sub> 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.
description: 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.