Quantifying climate sensitivity and climate-driven change in North American amphibian communities.

Changing climate will impact species' ranges only when environmental variability directly impacts the demography of local populations. However, measurement of demographic responses to climate change has largely been limited to single species and locations. Here we show that amphibian communities are responsive to climatic variability, using >500,000 time-series observations for 81 species across 86 North American study areas. The effect of climate on local colonization and persistence probabilities varies among eco-regions and depends on local climate, species life-histories, and taxonomic classification. We found that local species richness is most sensitive to changes in water availability during breeding and changes in winter conditions. Based on the relationships we measure, recent changes in climate cannot explain why local species richness of North American amphibians has rapidly declined. However, changing climate does explain why some populations are declining faster than others. Our results provide important insights into how amphibians respond to climate and a general framework for measuring climate impacts on species richness.

Large-scale recovery of an endangered amphibian despite ongoing exposure to multiple stressors.

Amphibians are one of the most threatened animal groups, with 32% of species at risk for extinction. Given this imperiled status, is the disappearance of a large fraction of the Earth's amphibians inevitable, or are some declining species more resilient than is generally assumed? We address this question in a species that is emblematic of many declining amphibians, the endangered Sierra Nevada yellow-legged frog (Rana sierrae). Based on >7,000 frog surveys conducted across Yosemite National Park over a 20-y period, we show that, after decades of decline and despite ongoing exposure to multiple stressors, including introduced fish, the recently emerged disease chytridiomycosis, and pesticides, R. sierrae abundance increased sevenfold during the study and at a rate of 11% per year. These increases occurred in hundreds of populations throughout Yosemite, providing a rare example of amphibian recovery at an ecologically relevant spatial scale. Results from a laboratory experiment indicate that these increases may be in part because of reduced frog susceptibility to chytridiomycosis. The disappearance of nonnative fish from numerous water bodies after cessation of stocking also contributed to the recovery. The large-scale increases in R. sierrae abundance that we document suggest that, when habitats are relatively intact and stressors are reduced in their importance by active management or species' adaptive responses, declines of some amphibians may be partially reversible, at least at a regional scale. Other studies conducted over similarly large temporal and spatial scales are critically needed to provide insight and generality about the reversibility of amphibian declines at a global scale.

Quantitative evidence for the effects of multiple drivers on continental-scale amphibian declines.

Since amphibian declines were first proposed as a global phenomenon over a quarter century ago, the conservation community has made little progress in halting or reversing these trends. The early search for a "smoking gun" was replaced with the expectation that declines are caused by multiple drivers. While field observations and experiments have identified factors leading to increased local extinction risk, evidence for effects of these drivers is lacking at large spatial scales. Here, we use observations of 389 time-series of 83 species and complexes from 61 study areas across North America to test the effects of 4 of the major hypothesized drivers of declines. While we find that local amphibian populations are being lost from metapopulations at an average rate of 3.79% per year, these declines are not related to any particular threat at the continental scale; likewise the effect of each stressor is variable at regional scales. This result - that exposure to threats varies spatially, and populations vary in their response - provides little generality in the development of conservation strategies. Greater emphasis on local solutions to this globally shared phenomenon is needed.

In situ effects of pesticides on amphibians in the Sierra Nevada.

For more than 20 years, conservationists have agreed that amphibian populations around the world are declining. Results obtained through laboratory or mesocosm studies and measurement of contaminant concentrations in areas experiencing declines have supported a role of contaminants in these declines. The current study examines the effects of contaminant exposure to amphibians in situ in areas actually experiencing declines. Early larval Pseudacris regilla were translocated among Lassen Volcanic, Yosemite and Sequoia National Parks, California, USA and caged in wetlands in 2001 and 2002 until metamorphosis. Twenty contaminants were identified in tadpoles with an average of 1.3-5.9 (maximum = 10) contaminants per animal. Sequoia National Park, which had the greatest variety and concentrations of contaminants in 2001, also had tadpoles that experienced the greatest mortality, slowest developmental rates and lowest cholinesterase activities. Yosemite and Sequoia tadpoles and metamorphs had greater genotoxicity than those in Lassen during 2001, as determined by flow cytometry. In 2001 tadpoles at Yosemite had a significantly higher rate of malformations, characterized as hemimelia (shortened femurs), than those at the other two parks but no significant differences were observed in 2002. Fewer differences in contaminant types and concentrations existed among parks during 2002 compared to 2001. In 2002 Sequoia tadpoles had higher mortality and slower developmental rates but there was no difference among parks in cholinesterase activities. Although concentrations of most contaminants were below known lethal concentrations, simultaneous exposure to multiple chemicals and other stressors may have resulted in lethal and sublethal effects.

A network extension of species occupancy models in a patchy environment applied to the Yosemite Toad (Anaxyrus canorus).

A central challenge of conservation biology is using limited data to predict rare species occurrence and identify conservation areas that play a disproportionate role in regional persistence. Where species occupy discrete patches in a landscape, such predictions require data about environmental quality of individual patches and the connectivity among high quality patches. We present a novel extension to species occupancy modeling that blends traditional predictions of individual patch environmental quality with network analysis to estimate connectivity characteristics using limited survey data. We demonstrate this approach using environmental and geospatial attributes to predict observed occupancy patterns of the Yosemite toad (Anaxyrus (= Bufo) canorus) across >2,500 meadows in Yosemite National Park (USA). A. canorus, a Federal Proposed Species, breeds in shallow water associated with meadows. Our generalized linear model (GLM) accurately predicted ~84% of true presence-absence data on a subset of data withheld for testing. The predicted environmental quality of each meadow was iteratively 'boosted' by the quality of neighbors within dispersal distance. We used this park-wide meadow connectivity network to estimate the relative influence of an individual Meadow's 'environmental quality' versus its 'network quality' to predict: a) clusters of high quality breeding meadows potentially linked by dispersal, b) breeding meadows with high environmental quality that are isolated from other such meadows, c) breeding meadows with lower environmental quality where long-term persistence may critically depend on the network neighborhood, and d) breeding meadows with the biggest impact on park-wide breeding patterns. Combined with targeted data on dispersal, genetics, disease, and other potential stressors, these results can guide designation of core conservation areas for A. canorus in Yosemite National Park.

Accumulation of pesticides in Pacific chorus frogs (Pseudacris regilla) from California's Sierra Nevada Mountains, USA.

Pesticides are receiving increasing attention as potential causes of amphibian declines, acting singly or in combination with other stressors, but limited information is available on the accumulation of current-use pesticides in tissue. The authors examined potential exposure and accumulation of currently used pesticides in pond-breeding frogs (Pseudacris regilla) collected from 7 high elevations sites in northern California. All sites sampled are located downwind of California's highly agricultural Central Valley and receive inputs of pesticides through precipitation and/or dry deposition. Whole frog tissue, water, and sediment were analyzed for more than 90 current-use pesticides and pesticide degradates using gas chromatography-mass spectrometry. Two fungicides, pyraclostrobin and tebuconazole, and one herbicide, simazine, were the most frequently detected pesticides in tissue samples. Median pesticide concentration ranged from 13 µg/kg to 235 µg/kg wet weight. Tebuconazole and pyraclostrobin were the only 2 compounds observed frequently in frog tissue and sediment. Significant spatial differences in tissue concentration were observed, which corresponded to pesticide use in the upwind counties. Data generated indicated that amphibians residing in remote locations are exposed to and capable of accumulating current-use pesticides. A comparison of P. regilla tissue concentrations with water and sediment data indicated that the frogs are accumulating pesticides and are potentially a more reliable indicator of exposure to this group of pesticides than either water or sediment.

Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species.

Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

Trends in amphibian occupancy in the United States.

Though a third of amphibian species worldwide are thought to be imperiled, existing assessments simply categorize extinction risk, providing little information on the rate of population losses. We conducted the first analysis of the rate of change in the probability that amphibians occupy ponds and other comparable habitat features across the United States. We found that overall occupancy by amphibians declined 3.7% annually from 2002 to 2011. Species that are Red-listed by the International Union for Conservation of Nature (IUCN) declined an average of 11.6% annually. All subsets of data examined had a declining trend including species in the IUCN Least Concern category. This analysis suggests that amphibian declines may be more widespread and severe than previously realized.

Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan.

Butachlor is the most commonly used herbicide on paddy fields in Taiwan and throughout Southeast Asia. Since paddy fields provide habitat for pond breeding amphibians, we examined growth, development, time to metamorphosis, and survival of alpine cricket frog tadpoles (Fejervarya limnocharis) exposed to environmentally realistic concentrations of butachlor. We documented negative impacts of butachlor on survival, development, and time to metamorphosis, but not on tadpole growth. The 96 h LC(50) for tadpoles was 0.87 mg/l, much lower than the 4.8 mg/l recommended dosage for application to paddy fields. Even given the rapid breakdown of butachlor, tadpoles would be exposed to concentrations in excess of their 96 h LC(50) for an estimated 126 h. We also documented DNA damage (genotoxicity) in tadpoles exposed to butachlor at concentrations an order of magnitude less than the 4.8 mg/l recommended application rate. We did not find that butachlor depressed cholinesterase activity of tadpoles, unlike most organophosphorus insecticides. We conclude that butachlor is likely to have widespread negative impacts on amphibians occupying paddy fields with traditional herbicide application.

Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses.

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.

Toxicity of two insecticides to California, USA, anurans and its relevance to declining amphibian populations.

Contaminants have been associated with population declines of several amphibian species in California (USA). Pesticides from the Central Valley of California are transported by winds into the Sierra Nevada Mountains and precipitate into wet meadows where amphibians breed. The present study examined the chronic toxicity of two of the insecticides most commonly used in the Central Valley and found in the mountains, chlorpyrifos and endosulfan, to larval Pacific treefrogs (Pseudacris regilla) and foothill yellow-legged frogs (Rana boylii) and discusses the implications of this toxicity to declining amphibian populations. Larvae were exposed to the pesticides from Gosner stages 25 to 26 through metamorphosis. The estimated median lethal concentration (LC50) for chlorpyrifos was 365 microg/L in P. regilla and 66.5 microg/L for R. boylii. Time to metamorphosis increased with concentration of chlorpyrifos in both species, and cholinesterase activity declined with exposure concentration in metamorphs of both species at Gosner stages 42 to 46. For endosulfan, the estimated LC50 was 15.6 microg/L for P. regilla and 0.55 microg/L for R. boylii. All R. boylii exposed to concentrations of greater than 0.8 microg/L died before they entered metamorphosis. Pseudacris regilla remains relatively abundant and is broadly distributed throughout California. In contrast, R. boylii is among the species experiencing severe population declines. The present study adds to the increasing evidence that pesticides are very harmful to amphibians living in areas that are miles from sources of pesticide application.

Pesticides in mountain yellow-legged frogs (Rana muscosa) from the Sierra Nevada Mountains of California, USA.

In 1997, pesticide concentrations were measured in mountain yellow-legged frogs (Rana muscosa) from two areas in the Sierra Nevada Mountains of California, USA. One area (Sixty Lakes Basin, Kings Canyon National Park) had large, apparently healthy populations of frogs. A second area (Tablelands, Sequoia National Park) once had large populations, but the species had been extirpated from this area by the early 1980s. The Tablelands is exposed directly to prevailing winds from agricultural regions to the west. When an experimental reintroduction of R. muscosa in 1994 to 1995 was deemed unsuccessful in 1997, the last 20 (reintroduced) frogs that could be found were collected from the Tablelands, and pesticide concentrations in both frog tissue and the water were measured at both the Tablelands and at reference sites at Sixty Lakes. In frog tissues, dichlorodiphenyldichloroethylene (DDE) concentration was one to two orders of magnitude higher than the other organochlorines (46+/-20 ng/g wet wt at Tablelands and 17+/-8 Sixty Lakes). Both gamma-chlordane and trans-nonachlor were found in significantly greater concentrations in Tablelands frog tissues compared with Sixty Lakes. Organophosphate insecticides, chlorpyrifos, and diazinon were observed primarily in surface water with higher concentrations at the Tablelands sites. No contaminants were significantly higher in our Sixty Lakes samples.

Polychlorinated biphenyls and toxaphene in Pacific tree frog tadpoles (Hyla regilla) from the California Sierra Nevada, USA.

Pacific tree frog (Hyla regilla) tadpoles were collected throughout the Sierra Nevada mountain range, California, USA, in 1996 and 1997 and analyzed for the presence of polychlorinated biphenyls (PCBs) and toxaphene. Whole-tadpole sigma PCB levels ranged from 244 ng/g (wet wt) at lower elevations on the western slope to 1.6 ng/g high on the eastern slope, whereas sigma toxaphene levels ranged from 15.6 to 1.5 ng/g. Linear regression of PCB and toxaphene residue levels versus elevation indicated a significant relationship, with an r2 value of 0.33 for PCB and 0.45 for toxaphene indicating a significant elevation effect on PCB and toxaphene bioaccumulation in Sierra Nevada H. regilla. Tadpole samples from sites in east-facing versus west-facing drainage basins showed significant differences in PCB and toxaphene residue levels, suggesting the possibility of a rain-shadow effect in the long-range atmospheric transport of these contaminants to the Sierra Nevada Mountains.