A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Environment rather than character displacement explains call evolution in glassfrogs.

The acoustic adaptation hypothesis (AAH) and ecological character displacement (ECD) are two potential mechanisms shaping call evolution that can predict opposite trends for the differentiation of signals. Under AAH, signals evolve to minimize environmental degradation and maximize detection against background noise, predicting call homogenization in similar habitats due to environmental constraints on signals. In contrast, ECD predicts greater differences in call traits of closely related taxa in sympatry because of selection against acoustic interference. We used comparative phylogenetic analyses to test the strength of these two selective mechanisms on the evolution of advertisement calls in glassfrogs, a highly diverse family of neotropical anurans. We found that, overall, acoustic adaptation to the environment may outweigh effects of species interactions. As expected under the AAH, temporal call parameters are correlated with vegetation density, but spectral call parameters had an unexpected inverse correlation with vegetation density, as well as an unexpected correlation with temperature. We detected call convergence among co-occurring species and also across multiple populations from the same species in different glassfrogs communities. Our results indicate that call convergence is common in glassfrogs, likely due to habitat filtering, while character displacement is relatively rare, suggesting that costs of signal similarity among related species may not drive divergent selection in all systems.

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.

The role of Central American barriers in shaping the evolutionary history of the northernmost glassfrog, Hyalinobatrachium fleischmanni (Anura: Centrolenidae).

The complex geological history of Central America has been useful for understanding the processes influencing the distribution and diversity of multiple groups of organisms. Anurans are an excellent choice for such studies because they typically exhibit site fidelity and reduced movement. The objective of this work was to identify the impact of recognized geographic barriers on the genetic structure, phylogeographic patterns and divergence times of a wide-ranging amphibian species, Hyalinobatrachium fleischmanni. We amplified three mitochondrial regions, two coding (COI and ND1) and one ribosomal (16S), in samples collected from the coasts of Veracruz and Guerrero in Mexico to the humid forests of Chocó in Ecuador. We examined the biogeographic history of the species through spatial clustering analyses (Geneland and sPCA), Bayesian and maximum likelihood reconstructions, and spatiotemporal diffusion analysis. Our data suggest a Central American origin of H. fleischmanni and two posterior independent dispersals towards North and South American regions. The first clade comprises individuals from Colombia, Ecuador, Panama and the sister species Hyalinobatrachium tatayoi; this clade shows little structure, despite the presence of the Andes mountain range and the long distances between sampling sites. The second clade consists of individuals from Costa Rica, Nicaragua, and eastern Honduras with no apparent structure. The third clade includes individuals from western Honduras, Guatemala, and Mexico and displays deep population structure. Herein, we synthesize the impact of known geographic areas that act as barriers to glassfrog dispersal and demonstrated their effect of differentiating H. fleischmanni into three markedly isolated clades. The observed genetic structure is associated with an initial dispersal event from Central America followed by vicariance that likely occurred during the Pliocene. The southern samples are characterized by a very recent population expansion, likely related to sea-level and climatic oscillations during the Pleistocene, whereas the structure of the northern clade has probably been driven by dispersal through the Isthmus of Tehuantepec and isolation by the Motagua-Polochic-Jocotán fault system and the Mexican highlands.

Local-scale Seasonality Shapes Anuran Community Abundance in a Cloud Forest of the Tropical Andes.

Analysing how seasonality shapes abundance patterns fosters understanding of the processes related to amphibian community assemblies. In this study we analyse the relationship between local seasonal patterns of abundance within the anuran community of the Monte Zerpa Cloud Forest, Mérida Mountain Range (Cordillera de Mérida), in the Venezuelan Andes. We hypothesized that variation in precipitation, temperature, and relative humidity affects the temporal abundance patterns of anurans. Data collection was performed through nocturnal biweekly inspections from 2002 to 2003. Air temperature, relative humidity, and monthly precipitation were considered as variables of climatic seasonality. Generalized linear models were used to evaluate the effect of climatic variables on anuran abundance across seasons. Overall, 542 individuals and four anuran species were recorded in stream tributaries only. The local anuran community was comprised of Hyalinobatrachium duranti, Hyloscirtus platydactylus, Hyloscirtus jahni, and Pristimantis vanadisae. The most abundant species were H. duranti (288 individuals) and H. platydactylus (145 individuals), representing 53% and 27% of the total anuran abundance, respectively. Differences in abundance between species were observed. Although the total abundance of anurans was higher during the low precipitation season, no significant differences between the two seasons were detected. The variation in anuran abundance was explained by relative humidity and temperature. Our results suggest that the highest abundance of anurans can be expected when temperatures reach favourable levels (15- 17°C), relative humidity increases, and precipitation remains constant.

The advertisement call of Centrolene notostictum (Anura, Centrolenidae) with a new record of geographic distribution in Tolima, Colombia.

Centrolene notostictum Ruiz-Carranza Lynch,1991 is a glassfrog known from the Cordillera Oriental of Colombia-in the Departments of Boyacá, Cundinamarca, Norte de Santander and Santander (Frost 2017)-and the Venezuelan versant of the Sierra de Perijá (Rojas et al. 2012) from 1600 to 2440 masl. Although males have been found calling on the upper surface of the leaves of ferns and Heliconeaceae on the margins of a small fast-flowing stream (Rojas et al. 2012), no quantitative description of the advertisement call of C. notostictum is currently available. During fieldwork in the Galilea Natural Forest (on the western slope of Cordillera Oriental in the municipality of Villarrica, department of Tolima, Colombia) in 2016 and 2017, we found three vocalizing males of C. notostictum. Herein, we report the first geographic record of this species for the department of Tolima and describe its previously unknown advertisement call.

A marvelous new glassfrog (Centrolenidae, Hyalinobatrachium) from Amazonian Ecuador.

Hyalinobatrachium is a behaviorally and morphologically conserved genus of Neotropical anurans, with several pending taxonomic problems. Using morphology, vocalizations, and DNA, a new species from the Amazonian lowlands of Ecuador is described and illustrated. The new species, Hyalinobatrachium yakusp. n., is differentiated from all other congenerics by having small, middorsal, dark green spots on the head and dorsum, a transparent pericardium, and a tonal call that lasts 0.27-0.4 s, with a dominant frequency of 5219.3-5329.6 Hz. Also, a mitochondrial phylogeny for the genus is presented that contains the new species, which is inferred as sister to H. pellucidum. Conservation threats to H. yakusp. n. include habitat destruction and/or pollution mainly because of oil and mining activities.

Resumen Hyalinobatrachium es un género de ranas Neotropicales con una morfología y comportamiento sumamente conservados, y con varios problemas taxonómicos no resueltos. Utilizando datos morfológicos, cantos y ADN, en el presente trabajo describimos una nueva especie de las tierras bajas de la Amazonía del Ecuador. La nueva especie, Hyalinobatrachium yakusp. n., se diferencia de todos sus congenéricos por tener una serie de puntos mediodorsales color verde oscuros en la cabeza y cuerpo, pericardio transparente, y un canto tonal con una duración de 0.27­0.4 s, con una frecuencia dominante 5219.3­5329.6 Hz. También presentamos una filogenia mitocondrial del género, la cual incluye la nueva especie y a su especie hermana, H. pellucidum. Las amenazas de conservación para H. yakusp. n. incluyen principalmente la destrucción y/o contaminación del hábitat debido a actividades mineras y petroleras.

Out of Amazonia: the unexpected trans-Andean distribution of Cochranella resplendens (Lynch and Duellman, 1978) (Anura: Centrolenidae).

The glassfrog genus Cochranella, with nine recognized species, is distributed in the lowlands and mid elevation of the Neotropical forests, from Nicaragua to Bolivia (Guayasamin et al. 2009; Twomey et al. 2014). Four species are trans-Andean-C. granulosa (Taylor 1949) occurs in the lowlands and mountains, at mid elevation, of Central America, C. litoralis (Ruiz-Carranza & Lynch 1996) and C. mache Guayasamin & Bonaccorso 2004 occur in the Pacific lowlands and the western cloud forests of Colombia and Ecuador, and C. euknemos (Savage & Starrett 1967) occurs both in Central America and South America (northwestern Colombia).-The other five species have cis-Andean distributions in the Amazonian slopes and lowlands, from Colombia to Bolivia: C. nola Harvey 1996, C. guayasamini Twomey, Delia & Castroviejo-Fisher 2014, C. resplendens (Lynch & Duellman 1973), C. erminea Torres-Gastello, Suárez-Segovia & Cisneros-Heredia 2007, and C. phryxa Aguayo-Vedia & Harvey 2006. In Colombia, C. resplendens is known from the foothills of the Amazon versant in Caquetá (Malambo et al. 2013) and Putumayo (Lynch & Duellman 1973; Ruiz-Carranza et al. 1996). The species is also known from Ecuador (Lynch & Duellman 1973) and Peru (Twomey et al. 2014). Here, we report two new records of Cochranella resplendens, extending the species distribution beyond the Amazonian lowlands into the northern Cordillera Central in Colombia.

Patterns of parental care in Neotropical glassfrogs: fieldwork alters hypotheses of sex-role evolution.

Many animals provide parental care to offspring. Parental sex-roles vary extensively across taxa, and such patterns are considered well documented. However, information on amphibians is lacking relative to other vertebrate groups. We combine natural history observations with functional and historical analyses to examine the evolution of egg care in glassfrogs (Centrolenidae). Parental care was considered rare and predominately provided by males. Our field observations of 40 species revealed that care occurs throughout the family, and the caregiving sex changes across lineages. We discovered that a brief period of maternal care is widespread and occurs in species previously thought to lack care. Using a combination of female-removal experiments, prey-choice tests with egg-eating katydids, and parental disturbance-tolerance assays, we confirm the adaptive benefits of short-term maternal care in wild Cochranella granulosa and Teratohyla pulverata. To examine historical transitions between caregiving sexes, we assembled a molecular phylogeny and estimated ancestral care states using our data and the literature. We assessed patterns indicative of sex-specific constraints by testing whether transitions between the sexes are associated with changes in care levels. Our analyses support that male-only care evolved 2-3 times from female-only care, and this change is associated with substantial increases in care levels - a pattern supporting the hypothesis that male-only care evolved via constraints on maternal expenditure. Many groups of amphibians remain poorly studied, with emerging evidence indicating that care patterns are more diverse than currently appreciated. Natural history remains fundamental to uncovering this diversity and generating testable hypotheses of sex-role evolution.

Explaining Andean megadiversity: the evolutionary and ecological causes of glassfrog elevational richness patterns.

The Tropical Andes are an important global biodiversity hotspot, harbouring extraordinarily high richness and endemism. Although elevational richness and speciation have been studied independently in some Andean groups, the evolutionary and ecological processes that explain elevational richness patterns in the Andes have not been analysed together. Herein, we elucidate the processes underlying Andean richness patterns using glassfrogs (Centrolenidae) as a model system. Glassfrogs show the widespread mid-elevation diversity peak for both local and regional richness. Remarkably, these patterns are explained by greater time (montane museum) rather than faster speciation at mid-elevations (montane species pump), despite the recency of the major Andean uplift. We also show for the first time that rates of climatic-niche evolution and elevational change are related, supporting the hypothesis that climatic-niche conservatism decelerates species' shifts in elevational distributions and underlies the mid-elevation richness peak. These results may be relevant to other Andean clades and montane systems globally.

Cyanosis by methemoglobinemia in tadpoles of Cochranella granulosa (Anura: Centrolenidae)

Tadpoles inhabit generally well oxygenated rivers and streams, nevertheless they were found in areas with limited oxygen availability inside the rivers. To assess this feature, I examined factors that influence centrolenid tadpole behaviour using Cochranella granulosa. The tadpoles were reared in well-oxygenated and hypoxic environments and their development, survivorship and growth were compared. The tadpoles in oxygenated water acquired a pale color, while tadpoles in hypoxic water grew faster and were bright red and more active. In the oxygenated water, the ammonium, which had its origin in the tadpoles’ urine and feces, was oxidized to nitrate. In contrast, in the hypoxic treatment, the nitrogen compounds remained mainly as ammonium. Presumably, the nitrate in oxygenated water was secondarily reduced to nitrite inside the long intestine coils, because all symptoms in the tadpoles point to methemoglobinemia, which can occur when the nitrite passes through the intestine wall into the bloodstream, transforming the hemoglobin into methemoglobin. This could be checked by a blood test where the percentage of methemoglobin was 2.3% in the blood of tadpoles reared in hypoxic condition, while there was a 19.3% level of methemoglobin in the blood of tadpoles reared in oxygenated water. Together with the elevated content of methemoglobin, the growth of the tadpoles was delayed in oxygenated water, which had high nitrate content. The study about quantitative food-uptake showed that the tadpoles benefit more from the food in hypoxic water, although they spent there more energy moving around than the tadpoles living in oxygenated but nitrate-charged water. Rev. Biol. Trop. 58 (4): 1467-1478. Epub 2010 December 01.

Los renacuajos por lo general viven en ríos y arroyos bien oxigenados, sin embargo, como han sido encontrados en áreas con disponibilidad de oxígeno limitada en los ríos, se estudió como influye este factor en su comportamiento. Renacuajos de Cochranella granulosa fueron criados en ambientes bien oxigenados y de hipoxia para comparar su desarrollo, supervivencia y crecimiento. En el agua que no fue cambiada durante al menos un mes, los renacuajos mostraron diferencias en su desarrollo cuando vivían en agua hipóxica u oxigenada. Los renacuajos en el agua aireada tenían un color pálido, mientras que en la hipóxica fueron más activos y de un color rojo brillante. En el agua hipóxica, el nitrógeno que se originó de la orina y las heces de los renacuajos se mantuvo principalmente en forma de amonio; en cambio, el amonio fue oxidado a nitrato en el agua aireada. Presumiblemente, el nitrato en el agua oxigenada se redujo secundariamente a nitrito dentro del intestino, ya que todos los síntomas en los renacuajos que vivían en esta agua apuntaron a una metahemoglobinemia, que se produce cuando el nitrito pasa a través de la pared del intestino a la corriente sanguínea transformando la hemoglobina en metahemoglobina. Esto pudo comprobarse mediante un análisis sanguíneo en donde el porcentaje de metahemoglobina fue del 2.3% en la sangre de los renacuajos criados en condición hipóxica y de un 19.3% de metahemoglobina en aquellos criados en agua aireada. En la misma forma en que la metahemoglobina aumenta en la sangre de los renacuajos que viven en agua oxigenada, su crecimiento disminuye en agua con alto contenido de nitrato. El estudio cuantitativo de la ingestión de nutrientes mostró que el crecimiento de los renacuajos se beneficia más de los alimentos en agua hipóxica, a pesar de que los renacuajos son más activos en sus movimientos que los que viven en agua oxigenada pero cargada de nitratos.

Fine scale gene flow and individual movements among subpopulations of Centrolene prosoblepon (Anura: Centrolenidae)

Dispersal capabilities determine and maintain local gene flow, and this has implications for population persistence and/or recolonization following environmental perturbations (natural or anthropogenic), disease outbreaks, or other demographic collapses. To predict recolonization and understand dispersal capacity in a stream-breeding frog, we examined individual movement patterns and gene flow among four subpopulations of the Neotropical glassfrog, Centrolene prosoblepon, at a mid-elevation cloud forest site at El Copé, Panama. We measured male movement directly during a two year mark-recapture study, and indirectly with gene flow estimates from mitochondrial DNA sequences (mtDNA). Individuals of this species showed strong site fidelity: over two years, male frogs in all four headwater streams moved very little (mean = 2.33 m; mode = 0 m). Nine individuals changed streams within one or two years, moving 675-1,108 m. For those males moving more than 10 m, movement was biased upstream (p < 0.001). Using mtDNA ND1 gene sequences, we quantified gene flow within and among headwater streams at two spatial scales: among headwater streams within two adjacent watersheds (2.5 km2) and among streams within a longitudinal gradient covering 5.0 km2. We found high gene flow among headwater streams (phi(ST) = 0.007, p = 0.325) but gene flow was more limited across greater distances (phi(CT) = 0.322, p = 0.065), even within the same drainage network. Lowland populations of C. prosoblepon potentially act as an important source of colonists for upland populations in this watershed.

La capacidad de dispersión determina y mantiene el flujo genético local, y esto tiene implicaciones para la persistencia poblacional y/o la recolonización que sigue a perturbaciones ambientales. Examinamos patrones individuales de movimiento y flujo genético entre subpoblaciones de Centrolene prosoblepon (Anura: Centrolenidae) en un sitio de elevación media en El Copé, Panamá. Medimos directamente el movimiento de los machos durante un estudio de marcado-recaptura, e indirectamente con estimaciones de flujo genético a partir de secuencias de ADN mitocondrial (mtDNA). Los individuos mostraron fuerte fidelidad a su lugar: por más de dos años, las ranas macho de los cuatro arroyos al inicio del río se movieron muy poco (promedio = 2.33 m; moda = 0 m). Nueve individuos cambiaron de corriente de agua en uno o dos años, moviéndose 675-1 108 m. Usando la secuencia genética ND1 del ADN mitocondrial, medimos el flujo genético en dos escalas espaciales: entre arroyos que originan el río (2.5 km2) y entre arroyos con un gradiente longitudinal en 5.0 km2. Encontramos un flujo genético alto entre los arroyos al inicio del río (f = 0.007, p = 0.325 y otro más limitado en distancias mayores (f = 0.322, p = 0.065).