Early onset of urea synthesis and ammonia detoxification pathways in three terrestrially developing frogs.

Frogs evolved terrestrial development multiple times, necessitating mechanisms to avoid ammonia toxicity at early stages. Urea synthesis from ammonia is a key adaptation that reduces water dependence after metamorphosis. We tested for early expression and plasticity of enzymatic mechanisms of ammonia detoxification in three terrestrial-breeding frogs: foam-nest-dwelling larvae of Leptodactylus fragilis (Lf) and arboreal embryos of Hyalinobatrachium fleischmanni (Hf) and Agalychnis callidryas (Ac). Activity of two ornithine-urea cycle (OUC) enzymes, arginase and CPSase, and levels of their products urea and CP in tissues were high in Lf regardless of nest hydration, but reduced in experimental low- vs. high-ammonia environments. High OUC activity in wet and dry nests, comparable to that under experimental high ammonia, suggests terrestrial Lf larvae maintain high capacity for urea excretion regardless of their immediate risk of ammonia toxicity. This may aid survival through unpredictably long waiting periods before rain enables their transition to water. Moderate levels of urea and CP were present in Hf and Ac tissues and enzymatic activities were lower than in Lf. In both species, embryos in drying clutches can hatch and enter the water early, behaviorally avoiding ammonia toxicity. Moreover, glutamine synthetase was active in early stages of all three species, condensing ammonia and glutamate to glutamine as another mechanism of detoxification. Enzyme activity appeared highest in Lf, although substrate and product levels were higher in Ac and Lf. Our results reveal that multiple biochemical mechanisms of ammonia detoxification occur in early life stages of anuran lineages that evolved terrestrial development.

Reproductive colonization of land by frogs: Embryos and larvae excrete urea to avoid ammonia toxicity.

Vertebrate colonization of land has 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 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 suggest 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.

La colonización del medio terrestre ha ocurrido en múltiples ocasiones, incluyendo más de 50 orígenes de oviposición terrestre en ranas. Algunos factores ambientales y respuestas fenotípicas que facilitaron estas transiciones se conocen, pero las respuestas a la restricción al agua y el riesgo de toxicidad por el amonio durante el desarrollo temprano no son bien entendidas. Examinamos si la acumulación de amonio y el riesgo de deshidratación inducen el cambio de excreción de amonio a urea en estadios tempranos del desarrollo de cuatro especies de anuros, en tres orígenes de desarrollo en tierra. Cuantificamos la concentración de amonio y urea durante el desarrollo temprano en tierra en condiciones de hidratación adecuada y de desecación. Cuando encontramos excreción de urea, evaluamos su incremento plástico en condiciones de desecación y de acumulación de amonio en los ambientes de desarrollo. Evaluamos el papel adaptativo de la excreción de urea comparando la tolerancia al amonio medida en pruebas LC50 por 96h con los niveles de amonio acumulados en los ambientes de desarrollo. El amonio se acumuló en los nidos de espuma y el fluido perivitelino, incrementando durante el desarrollo y alcanzando la concentración más alta en condiciones de desecación. Las cuatro especies presentan una elevada tolerancia al amonio al compararlas con peces y ranas con reproducción acuática. Las larvas nidícolas de Leptodactylus fragilis y los embriones avanzados de Hyalinobatrachium fleischmanni excretaron urea, mostrando un incremento plástico en condiciones de desecación. Estas dos especies pueden tener los periodos más extensos de desarrollo en tierra y la excreción de urea parece adaptativa, al prevenir su exposición a niveles de amonio potencialmente letales. Ni los embriones avanzados de Agalychnis callidryas ni las larvas nidícolas de Engystomops pustulosus experimentan niveles tóxicos de amonio en condiciones de desecación, y tampoco excretaron urea. Nuestros resultados sugieren que un inicio temprano en la excreción de urea, su incremento en condiciones de desecación y una elevada tolerancia al amonio pueden ayudar a prevenir la toxicidad del amonio durante el desarrollo terrestre. El amonio elevado es un riesgo común para el desarrollo que puede ser exacerbado a medida que el cambio climático incrementa el riesgo de desecación en ranas con reproducción terrestre. También puede ser una señal que lleva a respuestas fisiológicas adaptativas durante el desarrollo temprano.

DNA Barcoding Survey of Anurans across the Eastern Cordillera of Colombia and the Impact of the Andes on Cryptic Diversity.

Colombia hosts the second highest amphibian species diversity on Earth, yet its fauna remains poorly studied, especially using molecular genetic techniques. We present the results of the first wide-scale DNA barcoding survey of anurans of Colombia, focusing on a transect across the Eastern Cordillera. We surveyed 10 sites between the Magdalena Valley to the west and the eastern foothills of the Eastern Cordillera, sequencing portions of the mitochondrial 16S ribosomal RNA and cytochrome oxidase subunit 1 (CO1) genes for 235 individuals from 52 nominal species. We applied two barcode algorithms, Automatic Barcode Gap Discovery and Refined Single Linkage Analysis, to estimate the number of clusters or "unconfirmed candidate species" supported by DNA barcode data. Our survey included ~7% of the anuran species known from Colombia. While barcoding algorithms differed slightly in the number of clusters identified, between three and ten nominal species may be obscuring candidate species (in some cases, more than one cryptic species per nominal species). Our data suggest that the high elevations of the Eastern Cordillera and the low elevations of the Chicamocha canyon acted as geographic barriers in at least seven nominal species, promoting strong genetic divergences between populations associated with the Eastern Cordillera.