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Environmental DNA (eDNA) methods for detecting and estimating abundance of aquatic species are emerging rapidly, but little is known about how processes such as secretion rate, environmental degradation, and time since colonization or extirpation from a given site affect eDNA measurements. Using stream-dwelling salamanders, we conducted three experiments to assess eDNA from qPCR analysis: (1) production rate, (2) persistence time under different temperature and light conditions, and (3) detectability and concentration through time following experimental introduction and removal of salamanders into previously unoccupied streams. We found that 44–50 g individuals held in aquaria produced 20.2 ng eDNA/L/hr for two hours, after which production either decreased considerably or equilibrated with degradation and settling rates. eDNA in both full-sun and shaded treatments degraded exponentially to <1% of the original concentration after 3 days. eDNA was no longer detectable in full-sun samples after 8 days, whereas eDNA was detected in 20% of shaded samples after 11 days and 100% of control samples after 18 days. When translocated into unoccupied streams, salamanders were detectable after 6 hours, but only when densities were relatively high (0.2481 individuals/m2) and when samples were collected within 5 m of the animals. Concentrations of eDNA detected were very low and increased steadily from 6–24 hours after introduction, reaching 0.0022 ng/L. Within 1 hour of removing salamanders from the stream, eDNA was no longer detectable. These results suggest that eDNA detectability and concentration depend on production rates of individuals, environmental conditions, density of animals, and their residence time.

Environmental DNA (eDNA) methods for detecting aquatic species are advancing rapidly, but with little evaluation of field protocols or precision of resulting estimates. We compared sampling results from traditional field methods with eDNA methods for two amphibians in 13 streams in central Idaho, USA. We also evaluated three water collection protocols and the influence of sampling location, time of day, and distance from animals on eDNA concentration in the water. We found no difference in detection or amount of eDNA among water collection protocols. eDNA methods had slightly higher detection rates than traditional field methods, particularly when species occurred at low densities. eDNA concentration was positively related to field-measured density, biomass, and proportion of transects occupied. Precision of eDNA-based abundance estimates increased with the amount of eDNA in the water and the number of replicate sub-samples collected, but eDNA concentration did not vary significantly with sample location in the stream, time of day, or distance downstream from animals. Our results further advance the implementation of eDNA methods for monitoring aquatic vertebrates in stream habitats.

Prevalence of the amphibian chytrid fungus on Buenos Aires National Wildlife Refuge is low. Very few bullfrogs (a carrier of Bd) on the refuge test positive and very few Chiricahua Leopard Frogs test positive (only at sites off of the refuge). Bullfrogs have been nearly eradicated from the refuge, but because they carry the disease, there is potential for bullfrogs to act as a vector, carrying the disease to the Chiricahua Leopard Frogs which are an endangered species if they move back onto the refuge.

Breeding is limited by energetic or environmental constraints and long-lived species sometimes skip breeding opportunities. Environmental conditions may vary considerably across the geographic and elevational range of a species and species that can respond through variation in life history strategies are likely to maintain populations at the extremes of their ranges. The decision to skip breeding enables animals to adjust life history to circumstances, and plasticity in behavior allows implementation of adjustments. Elevational patterns suggest that breeding may be limited physiologically at high elevations (e.g., greater probability of skipped breeding; resources and environmental conditions more variable) in contrast to low elevations (probability of skipping breeding lower; resources and environmental conditions more predictable). We estimated the probabilities of survival and skipped breeding in a high-elevation population of Common Toads and compared estimates to existing data for Common Toads at low elevations, and to another toad species inhabiting a similar high elevation environment. Female Common Toads at high elevations tend to have high probabilities of skipping breeding and survival relative to data for Common Toads at low elevations, and appear to use a similar strategy of skipping breeding in response to similar environmental constraints as other toads at high elevations. We provide evidence of variability in this aspect of life history for Common Toads. Understanding variation in life history within widely distributed species is critical. Knowing that certain life history strategies are employed on a continuum informs conservation efforts, especially as impacts of climate change are likely to be different depending on elevation.

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.

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 Candidate Species, breeds in shallow water associated with meadows. Our generalized linear model (GLM) model 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.

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 mg/kg to 235 mg/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.

Despite the high profile of amphibian declines and the increasing threat of drought and frag- mentation to aquatic ecosystems, few studies have examined long-term rates of change for a single species across a large geographic area. We analyzed growth in annual egg-mass counts of the Columbia spotted frog (<i>Rana luteiventris</i>) across the northwestern United States, an area encompassing 3 genetic clades. On the basis of data collected by multiple partners from 98 water bodies between 1991 and 2011, we used state-space and linear-regression models to measure effects of patch characteristics, frequency of summer drought, and wetland restoration on population growth. Abundance increased in the 2 clades with greatest decline history, but declined where populations are consideredmost secure. Population growthwas negatively associated with temporary hydroperiods and landscape modification (measured by the human footprint index), but was similar in modified and natural water bodies. The effect of drought was mediated by the size of the water body: populations in large water bodies maintained positive growth despite drought, whereas drought magnified declines in small water bodies. Rapid growth in restored wetlands in areas of historical population declines provided strong evidence of successful management. Our results highlight the importance of maintaining large areas of habitat and underscore the greater vulnerability of small areas of habitat to environmental stochasticity. Similar long-term growth rates inmodified and natural water bodies and rapid, positive responses to restoration suggest pond construction and other forms of management can effectively increase population growth. These tools are likely to become increasingly important to mitigate effects of increased drought expected from global climate change.
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Amphibians in California are facing serious population declines. Contaminants, especially pesticides, have been linked to these declines. This study reports on a survey of central and northern California wetlands sampled along four transects associated with Lassen National Park, Lake Tahoe, Yosemite National Park and Sequoia National Park; each transect was sampled from the coast to the Sierra Nevada mountains. Pacific treefrogs (Pseudacris regilla), water, and sediment were collected from 49 wetlands in 2001 and frogs and sediments were collected from 55 wetlands in 2002. Twenty-three pesticides were found in frog, water, or sediment samples. Eleven contaminants including trifluralin, endosulfan I, chlordanes, trans-nonachlor and polybrominated diphenyl ethers (PBDEs) were found in the tissues of adult P. regilla. Seventeen contaminants were found in sediments including endosulfan sulfate, chlordanes, DDE, and chlorpyrifos. The average (SD) number of chemicals detected per pond in sediments was 2.4 (2.5). In water 17 chemicals were detected with endosulfan II being present in almost all samples. Trifluralin, chlordanes, and chlorpyrifos were the next most common. The mean number of chemicals in water per pond was 7.8 (2.9). With the possible exception of chlorpyrifos oxon in sediments and total endosulfans, none of the contaminants by themselves exceeded known lethal or sublethal concentrations. Principal Components Analysis showed that the concentrations of endosulfans, chlorpyrifos, and trifluralin were associated with historic and present day population status of amphibians.

Based on 2431 captures of 757 individual frogs over a 9-yr period, we found that the population of Rana sierrae in one meadow–stream complex in Yosemite National Park ranged from an estimated 45 to 115 adult frogs. Rana sierrae at our relatively low elevation site (2200 m) grew at a fast rate (K¼0.73–0.78), had high overwintering survival rates (44.6–95%), lived a long time (up to 16 yr), and tended to be fairly sedentary during the summer (100% minimum convex polygon annual home ranges of 139 m2) but had low year-to-year site fidelity. Even though the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) has been present in the population for at least 13 yr, there was no clear downward trend as might be expected from reports of R. sierrae population declines associated with Bd or from reports of widespread population decline of R. sierrae throughout its range.

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. sierra 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.

1. Worldwide declines in amphibian populations are often attributed to loss of habitat and exploitation; additionally, climate change may play an important role in future declines. Despite protection of habitat, amphibians relying on seasonal habitats, such as vernal pools, may need active management to maintain their populations under forecasts of warming temperatures and more variable precipitation. However, few studies have examined the factors influencing where these species choose to breed (breeding occurrence) and the conditional likelihood of successful metamorphosis.<br />
2. We developed an occupancy model and estimated parameters within a Bayesian framework to investigate the factors influencing probabilities of wood frog (Lithobates sylvatica) breeding and successful metamorphosis at Patuxent Research Refuge, Maryland, USA. Our objectives were to obtain estimates of breeding occurrence and metamorph occupancy and evaluate the success of current management actions.<br />
3. The probabilities of wood frog breeding and successful metamorphosis varied by year and were positively related to the pond’s typical hydroperiod length and annual precipitation. Contrary to our predictions, previous occupancy states had little effect on breeding and metamorph occupancy probabilities, which is likely due to high correlation of occupancy with hydroperiod. Though sample sizes were small, management actions resulted in an increase in both breeding and metamorph occupancy probabilities.<br />
4. Synthesis and applications. We demonstrate that management actions targeting short-hydroperiod pools favorably influence both components of breeding success. Still, continued monitoring is needed to determine whether managed pools remain suitable for wood frogs. With predicted changes in climate and a positive relationship between <br />
breeding occupancy and winter precipitation, a proactive focus on active management of vernal pools may provide a means to maintain wood frog populations into the future.

The Class Amphibia is one of the most severely impacted taxa in an on-going global biodiversity crisis. Because amphibian reproduction is tightly associated with the presence of water, climatic changes that affect water availability pose a particularly menacing threat to both aquatic and terrestrial-breeding amphibians. We explore the impacts that one facet of climate change – that of extreme variation in precipitation - may have on amphibians. This variation is manifested principally as increases in the incidence and severity of both drought and major storm events. We stress the need to consider not only total precipitation amounts but also the pattern and timing of rainfall events. Such rainfall “pulses” are likely to become increasingly more influential on amphibians, especially in relation to seasonal reproduction. Changes in reproductive phenology can strongly influence the outcome of competitive and predatory interactions, thus potentially altering community dynamics in assemblages of co-existing species. We present conceptual models to illustrate possible landscape and metapopulation consequences of alternative climate change scenarios for pond-breeding amphibians, using the Mole Salamander, Ambystoma talpoideum, as an example. Although amphibians have evolved a variety of life history strategies that enable them to cope with environmental uncertainty, it is unclear whether adaptations can keep pace with the escalating rate of climate change. Climate change, especially in combination with other stressors, is a daunting challenge for the persistence of amphibians and, thus, the conservation of global biodiversity.

The Toiyable subpopulation of the Columbia spotted frog (Rana luteiventris) is located in central Nevada and is part of the Great Basin Distinct Population Segment (DPS). Columbia spotted frogs are of special concern as range-wide population declines have been documented and the species is a candidate for listing under the Endangered Species Act. Multiple state and federal agencies have cooperatively monitored this subpopulation over the last seven years, and will continue to do so in the near future. We restructured the database and estimated population parmeters using a Huggins Colosed Captures Robust Design Model. Derived estimates of population size did not show evidence of decline over the study years.

Landscapes of interwoven wetlands and uplands offer a rich set of ecosystem goods and services. Managing lands to maximize ecosystem services requires information that distinguishes change caused by local actions from broader-scale shifts in climate, land use, and other forms of global change. Satellite and airborne sensors collect valuable data for this purpose, especially when the data are analyzed along with data collected from ground-based sensors. The U.S. Geological Survey (USGS) is using remote sensing technology in this way as part of the Terrestrial Wetland Global Change Research Network to assess effects of climate change interacting with land-use change and other potential stressors along environmental gradients of wetland-upland landscapes in the United States and Canada.

Amphibians in freshwater coastal wetlands periodically experience acute exposure to salinity from hurricane-related overwash events, as well as chronic exposure associated with rising sea levels. In a comparative experimental approach, we examined whether seven species of anuran amphibians vary in their tolerance to changes in salinity. In a laboratory study, we exposed larval Hyla cinerea (Green Treefrog), H. squirella (Squirrel Treefrog), Lithobates catesbeianus (American Bullfrog), L. sphenocephalus (Southern Leopard Frog), Anaxyrus terrestris (Southern Toad), and Gastrophryne carolinensis (Eastern Narrow-mouthed Toad) from an inland population in north central Florida, USA, and Osteopilus septentrionalis (Cuban Treefrog) tadpoles from an inland population in southwest Florida, to acute salinity for 72 h. For each species, we replicated trials in which tadpoles were exposed to salinities of 0.2 (control), 5, 10, 12, 14, and 16 ppt. For all species, tadpoles reared in the control and 5 ppt treatments had 96.7 – 100% survival. No individuals of G. carolinensis survived at salinities exceeding 5 ppt and no individuals of any species survived in the 14 or 16 ppt treatments. For all other native species, survival at 10 ppt ranged from 46.7 to 80%, but declined to 0% at 12 ppt (except for H. cinerea, of which only 3.3% survived at 12 ppt). In contrast, all individuals of the invasive, non-native O. septentrionalis{/I] survived exposure to a salinity of 10 ppt, and survival in this species remained relatively high at 12 ppt. Our results illustrate that the non-native O. septentrionalis has a higher salinity tolerance than the native species tested, which may contribute to its invasion potential. Moreover, species commonly associated with coastal freshwater wetlands differ in their salinity tolerances, suggesting that salt water intrusion due to storm surges and sea level rise may affect the species composition of these ecosystems.