Standardized ethograms and a device for assessing amphibian thermal responses in a warming world.

Most predictions of how populations and species of ectotherms will respond to global warming are based on estimates of the temperature at which organisms lose motor control (i.e., CTmax - the Critical Thermal Maximum). Here, we describe a non-lethal protocol and ethograms to estimate the relative tolerance of amphibians to increasing temperatures. These methods-suitable for field or laboratory conditions-are replicable, inexpensive and applicable to both post-metamorphic stages and organisms with direct development. We illustrate the use of this standardized protocol for four amphibians from a tropical cloud forest in Veracruz, Mexico with contrasting life histories: a lungless salamander (Aquiloeurycea cafetalera: Plethodontidae), a leaf-litter frog (Craugastor rhodopis: Craugastoridae), a semiaquatic frog (Lithobates berlandieri: Ranidae), and a tree frog (Rheohyla miotympanum: Hylidae). We identified four behavioral responses preceding CTmax for all amphibians included in this study: 1) Optimal Activity Range, 2) Supra-optimal Activity Range, 3) Heat Stress Range, and 4) Involuntary Movements Range. Additionally, we identified a fifth parameter associated with resilience to heat shock: 5) Recovery Stage after reaching CTmax. We conclude that the behavioral responses preceding the Critical Thermal Maximum are as informative as CTmax. Using behavioral responses to estimate thermal tolerance has the additional advantage of reducing the risk of injury or death of amphibians during physiological experiments.

Thermal biology mediates responses of amphibians and reptiles to habitat modification.

Human activities often replace native forests with warmer, modified habitats that represent novel thermal environments for biodiversity. Reducing biodiversity loss hinges upon identifying which species are most sensitive to the environmental conditions that result from habitat modification. Drawing on case studies and a meta-analysis, we examined whether observed and modelled thermal traits, including heat tolerances, variation in body temperatures, and evaporative water loss, explained variation in sensitivity of ectotherms to habitat modification. Low heat tolerances of lizards and amphibians and high evaporative water loss of amphibians were associated with increased sensitivity to habitat modification, often explaining more variation than non-thermal traits. Heat tolerances alone explained 24-66% (mean = 38%) of the variation in species responses, and these trends were largely consistent across geographic locations and spatial scales. As habitat modification alters local microclimates, the thermal biology of species will likely play a key role in the reassembly of terrestrial communities.

Averting biodiversity collapse in tropical forest protected areas.

The rapid disruption of tropical forests probably imperils global biodiversity more than any other contemporary phenomenon. With deforestation advancing quickly, protected areas are increasingly becoming final refuges for threatened species and natural ecosystem processes. However, many protected areas in the tropics are themselves vulnerable to human encroachment and other environmental stresses. As pressures mount, it is vital to know whether existing reserves can sustain their biodiversity. A critical constraint in addressing this question has been that data describing a broad array of biodiversity groups have been unavailable for a sufficiently large and representative sample of reserves. Here we present a uniquely comprehensive data set on changes over the past 20 to 30 years in 31 functional groups of species and 21 potential drivers of environmental change, for 60 protected areas stratified across the world's major tropical regions. Our analysis reveals great variation in reserve 'health': about half of all reserves have been effective or performed passably, but the rest are experiencing an erosion of biodiversity that is often alarmingly widespread taxonomically and functionally. Habitat disruption, hunting and forest-product exploitation were the strongest predictors of declining reserve health. Crucially, environmental changes immediately outside reserves seemed nearly as important as those inside in determining their ecological fate, with changes inside reserves strongly mirroring those occurring around them. These findings suggest that tropical protected areas are often intimately linked ecologically to their surrounding habitats, and that a failure to stem broad-scale loss and degradation of such habitats could sharply increase the likelihood of serious biodiversity declines.

Thermal quality influences habitat use of two anole species.

Regeneration of secondary forests on previously deforested or degraded land is one of the most dominant forms of land-use change in the tropics. However, the response of animal communities to forest regeneration is poorly understood. To evaluate support for thermal quality as a mechanism driving reptile species distributions during secondary forest succession, we measured operative temperatures and occupancy in three successional forest stages (pasture, secondary forest, and old growth forest) for two anole species common in the landscape (Norops humilis and Norops limifrons). We then measured thermal preference in laboratory experiments and used operative temperature and temperature preference measurements to determine how thermal quality of habitat changes over the course of secondary forest succession, and if occupancy varies as a function of thermal quality. We found that thermal quality was lowest in pasture habitat because of a large frequency of temperatures above the thermal preference range. However, in low thermal quality pasture sites, riparian habitats and remnant trees provided a thermal refuge for both lizard species. Our results support thermal quality as a mechanism for reptile species distributions in altered landscapes and highlight the importance of the maintenance of riparian corridors.

Tropical amphibians in shifting thermal landscapes under land-use and climate change.

Land-cover and climate change are both expected to alter species distributions and contribute to future biodiversity loss. However, the combined effects of land-cover and climate change on assemblages, especially at the landscape scale, remain understudied. Lowland tropical amphibians may be particularly susceptible to changes in land cover and climate warming because many species have narrow thermal safety margins resulting from air and body temperatures that are close to their critical thermal maxima (CTmax ). We examined how changing thermal landscapes may alter the area of thermally suitable habitat (TSH) for tropical amphibians. We measured microclimates in 6 land-cover types and CTmax of 16 frog species in lowland northeastern Costa Rica. We used a biophysical model to estimate core body temperatures of frogs exposed to habitat-specific microclimates while accounting for evaporative cooling and behavior. Thermally suitable habitat area was estimated as the portion of the landscape where species CTmax exceeded their habitat-specific maximum body temperatures. We projected changes in TSH area 80 years into the future as a function of land-cover change only, climate change only, and combinations of land-cover and climate-change scenarios representing low and moderate rates of change. Projected decreases in TSH area ranged from 16% under low emissions and reduced forest loss to 30% under moderate emissions and business-as-usual land-cover change. Under a moderate emissions scenario (A1B), climate change alone contributed to 1.7- to 4.5-fold greater losses in TSH area than land-cover change only, suggesting that future decreases in TSH from climate change may outpace structural habitat loss. Forest-restricted species had lower mean CTmax than species that occurred in altered habitats, indicating that thermal tolerances will likely shape assemblages in changing thermal landscapes. In the face of ongoing land-cover and climate change, it will be critical to consider changing thermal landscapes in strategies to conserve ectotherm species.

Infection risk decreases with increasing mismatch in host and pathogen environmental tolerances.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused the greatest known wildlife pandemic, infecting over 500 amphibian species. It remains unclear why some host species decline from disease-related mortality whereas others persist. We introduce a conceptual model that predicts that infection risk in ectotherms will decrease as the difference between host and pathogen environmental tolerances (i.e. tolerance mismatch) increases. We test this prediction using both local-scale data from Costa Rica and global analyses of over 11 000 Bd infection assays. We find that infection prevalence decreases with increasing thermal tolerance mismatch and with increasing host tolerance of habitat modification. The relationship between environmental tolerance mismatches and Bd infection prevalence is generalisable across multiple amphibian families and spatial scales, and the magnitude of the tolerance mismatch effect depends on environmental context. These findings may help explain patterns of amphibian declines driven by a global wildlife pandemic.

The importance of defining focal assemblages when evaluating amphibian and reptile responses to land use.

Habitat loss and degradation are primary threats to amphibians and reptiles, but the relative effects of common land uses on assemblages and the mechanisms that underlie faunal responses are poorly studied. We reviewed the effects of four prevalent types of habitat alteration (urbanization, agriculture, livestock grazing, and silviculture) on amphibian and reptile species richness and abundance by summarizing reported responses in the literature and by estimating effect sizes across studies for species richness in each land-use type. We then used a multinomial model to classify species as natural habitat specialists, generalists, and disturbed habitat specialists and examined variation in effect sizes for each land-use type according to habitat specialization categories. There were mixed conclusions from individual studies, some reporting negative, neutral, or positive effects of land use on species richness and total abundance. A large proportion of studies reported species-specific effects of individual species abundance. However, in our analysis of effect sizes, we found a general trend of negative effects of land use on species richness. We also demonstrate that habitat associations of common species and species turnover can explain variation in the effect of land use on herpetofauna. Our review highlights the pervasive negative effects of common land uses on amphibians and reptiles, the importance of identifying groups vulnerable to land-use change (e.g., forest-associated species) in conservation studies, and the potential influence of disturbance-associated species on whole assemblage analyses.

Mechanistic insights into landscape genetic structure of two tropical amphibians using field-derived resistance surfaces.

Conversion of forests to agriculture often fragments distributions of forest species and can disrupt gene flow. We examined effects of prevalent land uses on genetic connectivity of two amphibian species in northeastern Costa Rica. We incorporated data from field surveys and experiments to develop resistance surfaces that represent local mechanisms hypothesized to modify dispersal success of amphibians, such as habitat-specific predation and desiccation risk. Because time lags can exist between forest conversion and genetic responses, we evaluated landscape effects using land-cover data from different time periods. Populations of both species were structured at similar spatial scales but exhibited differing responses to landscape features. Litter frog population differentiation was significantly related to landscape resistances estimated from abundance and experiment data. Model support was highest for experiment-derived surfaces that represented responses to microclimate variation. Litter frog genetic variation was best explained by contemporary landscape configuration, indicating rapid population response to land-use change. Poison frog genetic structure was strongly associated with geographic isolation, which explained up to 45% of genetic variation, and long-standing barriers, such as rivers and mountains. However, there was also partial support for abundance- and microclimate response-derived resistances. Differences in species responses to landscape features may be explained by overriding effects of population size on patterns of differentiation for poison frogs, but not litter frogs. In addition, pastures are likely semi-permeable to poison frog gene flow because the species is known to use pastures when remnant vegetation is present, but litter frogs do not. Ongoing reforestation efforts will probably increase connectivity in the region by increasing tree cover and reducing area of pastures.

Evaluating connectivity for tropical amphibians using empirically derived resistance surfaces.

Agricultural expansion continues to drive forest loss in species-rich tropical systems and often disrupts movement and distributions of organisms. The ability of species to occupy and move through altered habitats likely depends on the level of contrast between natural forest and surrounding land uses. Connectivity models, such as circuit theory models, are widely used in conservation biology, and their primary input consists of resistance surfaces representing movement costs associated with landscape features. Cost values are most frequently determined by expert opinion, which may not capture relevant levels of contrast among features. We developed resistance surfaces using experiments that represent different local mechanisms hypothesized to affect connectivity for two Neotropical amphibian species. Response ratios were calculated to translate experimental results to cost values used in connectivity modeling. We used relative abundance data in three land-cover types to generate resistance surfaces for evaluating independent support of models derived from experiments. Finally, we analyzed agreement among movement pathways predicted for each species and among three commonly used connectivity measures: Euclidean, least cost, and resistance distances. Experiments showed that extreme microclimates associated with altered habitats significantly increased desiccation and mortality risk for both species. Resistances estimated from microclimate experiments were concordant with those from survey data for both species. For one focal species, resistance estimates derived from predator encounter rates were also highly correlated with abundance-derived resistances. There was generally low agreement among the three alternative distance measures, which underscores the importance of choosing connectivity models that are most appropriate for the study objectives. Overall, similarity among linkages modeled for each species was high, but decreased with declining forest cover. Our results highlight the value of experiments for drawing inferences about processes in resistance modeling, as well as the need to consider model differences and species-specific responses when developing strategies to maintain connectivity.

Infection and co-infection by the amphibian chytrid fungus and ranavirus in wild Costa Rican frogs.

Amphibian populations are globally threatened by emerging infectious diseases, and 2 pathogens in particular are recognized as major threats: the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses. Here, we evaluated the prevalence of infection by Bd and ranavirus in an assemblage of frogs from a lowland wet forest in Costa Rica. We found an overall prevalence of 21.3% for Bd and 16.6% for ranavirus, and detected both pathogens widely among our 20 sampled species. We found a positive association between ranavirus and Bd infection in one of our 4 most commonly sampled species. We also found a positive but non-significant association between infection by ranavirus and infection by Bd among species overall. Our study is among the first detailed evaluations of ranavirus prevalence in the American tropics, and to our knowledge is the first to detect a positive association between Bd and ranavirus in any species. Considerable research attention has focused on the ecology of Bd in tropical regions, yet we argue that greater research focus is necessary to understand the ecology and conservation impact of ranaviruses on amphibian populations already decimated by the emergence of Bd.

A new lungless caecilian (Amphibia: Gymnophiona) from Guyana.

We report the discovery of a single specimen of a small, terrestrial, lungless caecilian, the second known taxon of lungless caecilians. It differs from all other caecilians in lacking open external nares, and from the large aquatic lungless species described by Nussbaum & Wilkinson (Nussbaum, R. A. & Wilkinson, M. 1995 Proc. R. Soc. Lond. B 261, 331-335) in having no significant skull modifications. All modifications are of 'soft morphology' (covered external nares and choanae, lung and pulmonary vessel loss, etc.). A new genus and species are described to accommodate this form. Aspects of its skull and visceral morphology are described and considered in terms of the possible life history and evolution of the species, and compared with those of other lungless amphibians.

Sex-related differences in alkaloid chemical defenses of the dendrobatid frog Oophaga pumilio from Cayo Nancy, Bocas del Toro, Panama.

Poison frogs contain an alkaloid-based chemical defense that is sequestered directly from a diet of alkaloid-containing arthropods. Geographic and temporal variation in alkaloid defense is common in poison frogs and is generally attributed to differences in the availability of alkaloid-containing arthropods. Variable chemical defense in poison frogs may have important consequences for predator-prey interactions, requiring a full understanding of the factors involved in explaining such variation. In the present study, we examine alkaloid variation in the dendrobatid poison frog Oophaga pumilio between males and females on Cayo Nancy (Isla Solarte), located in the Bocas del Toro archipelago of Panama. On average, females contained a significantly larger number and quantity of alkaloids when compared to males. Alkaloid composition varied significantly between males and females, illustrating that chemical defense in this population of O. pumilio is sex-dependent. The variation in alkaloids between sexes is attributed to differences in feeding and behavior between males and females. The majority of alkaloids present in the skin of O. pumilio appear to be of oribatid mite origin, supporting the importance of these dietary arthropods in the chemical defense of poison frogs.

Spatial patterns of the frog Oophaga pumilio in a plantation system are consistent with conspecific attraction.

The conspecific attraction hypothesis predicts that individuals are attracted to conspecifics because conspecifics may be cues to quality habitat and/or colonists may benefit from living in aggregations. Poison frogs (Dendrobatidae) are aposematic, territorial, and visually oriented-three characteristics which make dendrobatids an appropriate model to test for conspecific attraction. In this study, we tested this hypothesis using an extensive mark-recapture dataset of the strawberry poison frog (Oophaga pumilio) from La Selva Biological Station, Costa Rica. Data were collected from replicate populations in a relatively homogenous Theobroma cacao plantation, which provided a unique opportunity to test how conspecifics influence the spatial ecology of migrants in a controlled habitat with homogenous structure. We predicted that (1) individuals entering a population would aggregate with resident adults, (2) migrants would share sites with residents at a greater frequency than expected by chance, and (3) migrant home ranges would have shorter nearest-neighbor distances (NND) to residents than expected by chance. The results were consistent with these three predictions: Relative to random simulations, we observed significant aggregation, home-range overlap, and NND distribution functions in four, five, and six, respectively, of the six migrant-resident groups analyzed. Conspecific attraction may benefit migrant O. pumilio by providing cues to suitable home sites and/or increasing the potential for social interactions with conspecifics; if true, these benefits should outweigh the negative effects of other factors associated with aggregation. The observed aggregation between migrant and resident O. pumilio is consistent with conspecific attraction in dendrobatid frogs, and our study provides rare support from a field setting that conspecific attraction may be a relevant mechanism for models of anuran spatial ecology.

Spatial and temporal patterns of alkaloid variation in the poison frog Oophaga pumilio in Costa Rica and Panama over 30 years.

A total of 232 alkaloids, representing 21 structural classes were detected in skin extracts from the dendrobatid poison frog Oophaga pumilio, collected from 53 different populations from over 30 years of research. The highly toxic pumiliotoxins and allopumiliotoxins, along with 5,8-disubstitiuted and 5,6,8-trisubstituted indolizidines, all of which are proposed to be of dietary mite origin, were common constituents in most extracts. One decahydroquinoline (DHQ), previously shown be of ant origin, occurred in many extracts often as a major alkaloid, while other DHQs occurred rather infrequently. Histrionicotoxins, thought to be of ant origin, did not appear to possess a specific pattern of occurrence among the populations, but when present, were usually found as major components. Certain 3,5-disubstituted pyrrolizidines and indolizidines, known to be of ant origin, did occur in extracts, but infrequently. Alkaloid composition differed with regard to geographic location of frog populations, and for populations that were sampled two or more times during the 30-year period significant changes in alkaloid profiles sometimes occurred. The results of this study indicate that chemical defense in a dendrobatid poison frog is dependent on geographic location and habitat type, which presumably controls the abundance and nature of alkaloid-containing arthropods.

Additive effects of mean temperature, temperature variability, and chlorothalonil to red-eyed treefrog (Agalychnis callidryas) larvae.

Amphibian populations are declining globally, and multiple anthropogenic stressors, including contamination by pesticides and shifting climates, are driving these declines. Climate change may increase average temperatures or increase temperature variability, either of which may affect the susceptibility of nontarget organisms to contaminants. Eight-day ecotoxicological assays were conducted with red-eyed treefrog (Agalychnis callidryas) larvae to test for additive and interactive effects of exposure to the fungicide chlorothalonil, average temperature, and temperature variability on tadpole growth and survival. Egg masses were collected from seasonal ponds at La Selva Biological Station in Costa Rica, and tadpoles were exposed to a series of chlorothalonil concentrations across a range of ecologically relevant mean temperatures (23.4-27.3 °C) and daily temperature fluctuations (1.1-9.9 °C). Survival was measured each day, and tadpole growth was measured at the end of each trial. Concentrations of chlorothalonil ≥60 µg/L reduced survival, although survival was not affected by mean temperature or daily temperature range, and there were no synergistic interactions between chlorothalonil and temperature regime on survival. Chlorothalonil suppressed tadpole growth at relatively low concentrations (∼15 µg/L). There were impacts of both average temperature and daily temperature range on tadpole growth, although there were no synergistic interactions between temperature regimes and chlorothalonil. The results should inform efforts to manage ecosystems impacted by multiple large-scale anthropogenic stressors as well as methods for the design of ecologically appropriate toxicology trials. Environ Toxicol Chem 2016;35:2998-3004. © 2016 SETAC.

Variation of total mercury concentrations in pig frogs (Rana grylio) across the Florida Everglades, USA.

The Pig Frog (Rana grylio) is an aquatic frog that is an abundant component of the Everglades ecosystem. South Floridians recreationally and commercially hunt pig frogs in marshes throughout Water Conservation Areas (WCA) and Big Cypress National Preserve (BCNP) in South Florida. Most of these areas are under fish consumption advisories because of high levels of methylmercury present in game fish tissues. It is important to understand how mercury is distributed throughout Pig Frog populations because their consumption from certain areas may present a risk to human health. We sampled 88 pig frogs along a north-south transect through the Florida Everglades. There were substantial differences in total mercury (THg) concentrations from leg muscle tissue among sites. Total mercury in frog leg tissue was highest from areas protected from harvest in Everglades National Park (ENP), with a maximum concentration of 2051 ng/g wet mass. The THg levels in R. grylio leg tissue from most harvested areas are below Federal advisory limits. However, many pig frogs collected near Frog City, and one from WCA 3B and 3AN, harvested sites, had THg levels above the USEPA 0.3 mg/kg Fish Tissue Residue Criterion. Spatial patterns in the mercury found among pig frogs were similar to those of other wildlife species from the Everglades. We found frogs to have high THg levels in areas where alligators and mosquito fish also have high THg. THg in ENP frogs had an exponential relationship to SVL, we found no other relationship in frogs from other sites. Our data suggests that pig frogs should not be harvested or consumed from sites that exceed federal limits.

Effects of reproductive resource supplementation on space-use patterns in Dendrobates pumilio.

I increased the availability of tadpole-rearing sites (bromeliads) and oviposition sites (leaf litter) and examined the effects on space-use patterns in adult Dendrobates pumilio at the La Selva Biological Reserve in northeastern Costa Rica. I made 4158 observations of 986 marked adults during the seven-month study period. The addition of leaf litter did not result in increased density, but the addition of bromeliads did. The addition of leaf litter resulted in increased home-range size, but the effect depended on bromelial addition and sex. Home-range size was greater for females than males, and may be related to parental care and/or mate selection. Home-range size was not correlated with density. Spatio-temporal overlap was not affected by resource supplementation but the proportion of females captured in sites shared with males varied temporally. Dispersion was no affected by bromeliad supplementation but males on litter-addition plots were less clumped than males on other plots. These results indicate that the density increase observed on bromeliad-addition plots was accommodated by frogs utilizing previously unoccupied space. Frogs were captured more frequently at bromeliads on bromeliad-supplemented plots than on litter-addition and control plots. The size of the area defended by territorial males is smaller than home-range size indicating the absence of home-range defense.

Patterns of reproduction and habitat use in an assemblage of Neotropical hylid frogs.

We censused pond-breeding hylid frogs in northeastern Costa Rica weekly for 15 months to deseribe patterns of reproduction, habitat use, and to establish baseline data on relative abundance for members of the assemblage. Reproduction in the Costa Rican assemblage was seasonal and occurred only during wet months. Some species called, but none reproduced, during the dry season. Three species (Agalychnis callidryas, Hyla ebraccata, and Scinax elaeochroa) accounted for more than 75% of the observations made during the study. The species overlapped broadly in time and space, but differed in substrate use and phenology. Two species of leaf-breeding frogs (A. callidryas and A. saltator) used perches that were significantly higher than those used by the other species. Some phenological differences were associated with different mating strategies. Explosive breeders (Scinax elaeochroa and Smilisca baudinii) were most common early in the wet seasons. Prolonged breeders (A. callidryas and H. ebraccata) were the most persistent members of this assemblage. Predation affects early and late life history stages of these hylids. Predation on arboreal egg masses by two snake species was observed. Ctenid spiders preyed on recently metamorphosed frogs and small adults. Our weekly samples were pooled into 21-day periods so that we could compare our results with those obtained for two communities of breeding anurans from South America. The patterns observed in the Costa Rican assemblage differed from those reported for South American pond-breeding frogs, but in all three assemblages reproduction was associated with wet periods.

Acute toxicity tests and meta-analysis identify gaps in tropical ecotoxicology for amphibians.

Amphibian populations are declining worldwide, particularly in tropical regions where amphibian diversity is highest. Pollutants, including agricultural pesticides, have been identified as a potential contributor to decline, yet toxicological studies of tropical amphibians are very rare. The present study assesses toxic effects on amphibians of 10 commonly used commercial pesticides in tropical agriculture using 2 approaches. First, the authors conducted 8-d toxicity assays with formulations of each pesticide using individually reared red-eyed tree frog (Agalychnis callidryas) tadpoles. Second, they conducted a review of available data for the lethal concentration to kill 50% of test animals from the US Environmental Protection Agency's ECOTOX database to allow comparison with their findings. Lethal concentration estimates from the assays ranged over several orders of magnitude. The nematicides terbufos and ethoprophos and the fungicide chlorothalonil were very highly toxic, with evident effects within an order of magnitude of environmental concentrations. Acute toxicity assays and meta-analysis show that nematicides and fungicides are generally more toxic than herbicides yet receive far less research attention than less toxic herbicides. Given that the tropics have a high diversity of amphibians, the findings emphasize the need for research into the effects of commonly used pesticides in tropical countries and should help guide future ecotoxicological research in tropical regions.