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Amphibians are experiencing devastating population declines globally. A major driver is chytridiomycosis, an emerging infectious disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Bd was described in 1999 and has been linked with declines since the 1970s, while Bsal is a more recently discovered pathogen that was described in 2013. It is hypothesized that Bsal originated in Asia and spread via international trade to Europe, where it has been linked to salamander die-offs. Trade in live amphibians thus represents a significant threat to global biodiversity in amphibians. We review the current state of knowledge regarding Bsal and describe the risk of Bsal spread. We discuss regional responses to Bsal and barriers that impede a rapid, coordinated global effort. The discovery of a second deadly emerging chytrid fungal pathogen in amphibians poses an opportunity for scientists, conservationists, and governments to improve global biosecurity and further protect humans and wildlife from a growing number of emerging infectious diseases.

In North America, ranid frogs (Ranidae) have experienced larger declines than any other amphibian family, particularly species native to the southwestern USA and adjacent Mexico; however, our knowledge of their conservation status and threats is limited in Mexico. We assessed the status of the federally-threatened (USA) Chiricahua Leopard Frog (Lithobates chiricahuensis) in Sonora, Mexico, based on a search of museum specimens, published records, unpublished accounts, and surveys of 84 sites within the geographical and elevational range of the species from 2000-2016. We also provide information on occurrence of three other native ranid frog species encountered opportunistically during our surveys. The Chiricahua Leopard Frog is known in Sonora from only 20 historical (pre-2000) localities. Searches of three historical sites from 2000-2016 did not reveal any Chiricahua Leopard Frogs; however, we found it at three previously undocumented sites in 2016, all near Cananea. To our knowledge, these records are the first observations of Chiricahua Leopard Frogs in Sonora since 1998. Differences in conservation status between the USA and Sonora are likely due to differing magnitude and distribution of threats and a comparatively aggressive recovery program in the USA. For example, key non-native predators important in the decline of the Chiricahua Leopard Frog are much less widespread in Sonora compared to the USA Southwest, but there are fewer protections and recovery actions for the frog in Sonora than in the USA. Additional surveys for the Chiricahua Leopard Frog and other amphibians in Sonora should be a priority to fully assess threats and conservation status.

Accurate pathogen detection is essential for developing management strategies to address emerging infectious diseases, an increasingly prominent threat to wildlife. Sampling for free-living pathogens outside of their hosts has benefits for inference and study efficiency, but is still uncommon. We used a laboratory experiment to evaluate the influences of pathogen concentration, water type, and qPCR inhibitors on the detection and quantification of Batrachochytrium dendrobatidis (Bd) using water filtration. We compared results pre- and post-inhibitor removal, and assessed inferential differences when single versus multiple samples were collected across space or time. We found that qPCR inhibition influenced both Bd detection and quantification in natural water samples, resulting in biased inferences about Bd occurrence and abundance. Biases in occurrence could be mitigated by collecting multiple samples in space or time, but biases in Bd quantification were persistent. Differences in Bd concentration resulted in variation in detection probability, indicating that occupancy modeling could be used to explore factors influencing heterogeneity in Bd abundance among samples, sites, or over time. Our work will influence the design of studies involving amphibian disease dynamics and studies utilizing environmental DNA (eDNA) to understand species distributions.

rom 2004 to 2006, we used a variety of sampling techniques to survey the amphibians of Savannah National Wildlife Refuge (SNWR), a large protected area straddling the lower portions of the Savannah River on the border between South Carolina and Georgia, USA. We documented 22 amphibian species, 15 frogs and 7 salamanders, with a possible 23rd species present. Species richness was lower than what might be expected from amphibian field guides, likely due to a lack of specialized habitats, such as temporary ponds and upland pine forest, for many of the species inhabiting the adjacent Coastal Plain. Amphibians occupied a variety of habitats and appeared tolerant of the mildly acidic and low oxygen conditions of many of the wetlands. Although additional species may be found at SNWR, this initial survey provides a historic baseline for monitoring amphibian populations as areas adjacent to the refuge are disturbed, the climate changes, and as multi-use management objectives are implemented within refuge boundaries.

1. Conservation managers rely on accurate estimates of disease parameters, such as pathogen prevalence and infection intensity, to assess disease status of a host population. However, these disease metrics may be biased if low-level infection intensities are missed by sampling methods or laboratory diagnostic tests. These false negatives underestimate pathogen prevalence and overestimate mean infection intensity of infected individuals.

2. Our objectives were two-fold. First, we quantified false negative error rates of Batrachochytrium dendrobatidis on non-invasive skin swabs collected from an amphibian community in El Cop?, Panama. We swabbed amphibians twice in sequence, and we used a recently developed hierarchical Bayesian estimator to assess disease status of the population. Second, we developed a novel hierarchical Bayesian model to simultaneously account for imperfect pathogen detection from field sampling and laboratory diagnostic testing. We evaluated the performance of the model using simulations to quantify the magnitude of bias in estimates of pathogen prevalence and infection intensity.

3. We show that Bd detection probability from skin swabs was related to host infection intensity, where Bd infections < 10 zoospores have < 95 % probability of being detected. If imperfect Bd detection was not considered, then Bd prevalence was underestimated by as much as 16%. In the Bd-amphibian system, this indicates a need to correct for imperfect pathogen detection. More generally, our results have implications for study designs in other disease systems, particularly those with similar objectives, biology, and sampling decisions.

4. Uncertainty in pathogen detection is an inherent property of most sampling protocols and diagnostic tests, where the magnitude of bias depends on the study system, type of infection, and false negative error rates. Given that it may be difficult to know this information in advance, we advocate that the most cautious approach is to assume all errors are possible and to accommodate them by adjusting sampling designs. The modeling framework presented here improves the accuracy in estimating pathogen prevalence and infection intensity.

Abstract. There are many examples where the use of chemicals have had profound unanticipated consequences, such as fertilizers reducing crop yields (paradox of enrichment) and insecticides increasing insect pests (by reducing natural biocontrol). Recently, the application of agrochemicals, such as agricultural disinfectants and fungicides, has been explored as an approach to curb the pathogenic fungus, Batrachochytrium dendrobatidis (Bd), which is associated with worldwide amphibian declines. However, the long-term, net effects of early-life exposure to these chemicals on amphibian disease risk have not been thoroughly investigated. Using a combination of laboratory experiments and analysis of data from the literature, we explored the effects of fungicide exposure on Bd infections in two frog species. Extremely low concentrations of the fungicides azoxystrobin, chlorothalonil, and mancozeb were directly toxic to Bd in culture. However, estimated environmental concentrations of the fungicides did not reduce Bd on Cuban tree frog (Osteopilus septentrionalis) tadpoles exposed simultaneously to any of these fungicides and Bd, and fungicide exposure actually increased Bd-induced mortality. Additionally, exposure to any of these fungicides as tadpoles resulted in higher Bd abundance and greater Bd-induced mortality when challenged with Bd post-metamorphosis, an average of 71 days after their last fungicide exposure. Analysis of data from the literature revealed that previous exposure to the fungicide itraconazole, which is commonly used to clear Bd infections, made the critically endangered booroolong frog (Litoria booroolongensis) more susceptible to Bd. Finally, a field survey revealed that Bd prevalence was positively associated with concentrations of fungicides in ponds. Although fungicides show promise for controlling Bd, these results suggest that, if fungicides do not completely eradicate Bd or if Bd re-colonizes, exposure to fungicides has the potential to do more harm than good. To ensure that fungicide applications have the intended consequence of curbing amphibian declines, researchers must identify which fungicides do not compromise the pathogen resistance mechanisms of amphibians.

Advances in drilling techniques have facilitated a rapid increase in hydrocarbon extraction from energy shales, including the Williston Basin in central North America. This area overlaps with the Prairie Pothole Region, a region densely populated with wetlands that provide numerous ecosystem services. Historical (legacy) disposal practices often released saline co-produced waters (brines) with high chloride concentrations, affecting wetland water quality directly or persisting in sediments. Despite the potential threat of brine contamination to aquatic habitats, there has been little research into its ecological effects. We capitalized on a gradient of legacy brine-contaminated wetlands in northeast Montana to conduct laboratory experiments to assess variation in survival of larval Boreal Chorus Frogs (Pseudacris maculata) reared on sediments from 3 local wetlands and a control source. To help provide environmental context for the experiment, we also measured chloride concentrations in 6 brine-contaminated wetlands in our study area, including the 2 contaminated sites used for sediment exposures. Survival of frog larvae during 46- and 55-day experiments differed by up to 88% among sediment sources (Site Model) and was negatively correlated with potential chloride exposure (Chloride Model). Five of the 6 contaminated wetlands exceeded the U.S. EPA acute benchmark for chloride in freshwater (860 mg/L) and all exceeded the chronic benchmark (230 mg/L). However, the Wetland Site model explained more variation in survival than the Chloride Model, suggesting that chloride concentration alone does not fully reflect the threat of contamination to aquatic species. Because the profiles of brine-contaminated sediments are complex, further surveys and experiments are needed across a broad range of conditions, especially where restoration or remediation actions have reduced brine-contamination. Information provided by this study can help quantify potential ecological threats and help land managers prioritize conservation strategies as part of responsible and sustainable energy development.

Estimating population trends provides valuable information for resource managers, but monitoring programs face trade-offs between the quality and quantity of information gained and the number of sites surveyed. We compared the effectiveness of monitoring techniques for estimating population trends of Rana draytonii at Point Reyes National Seashore, California, USA, over a 13-year period. Our primary goals were 1) to estimate trends for a focal pond at Point Reyes National Seashore, and 2) to evaluate whether egg mass counts could reliably estimate an index of abundance relative to more intensive capture-mark-recapture methods. Capture-mark-recapture surveys of males indicated a stable population from 2005 through 2009, despite low annual apparent survival (26.3%). Egg mass counts from 2000 through 2012 indicated that despite some large fluctuations, the breeding female population was generally stable or increasing, with annual abundance varying between 26 and 130 individuals. Minor modifications to egg mass counts, such as marking egg masses, can allow estimation of egg mass detection probabilities necessary to convert counts to abundance estimates, even when closure of egg mass abundance cannot be assumed within a breeding season. High egg mass detection probabilities (mean per-survey detection probability = https://0.98 [0.89-0.99]) indicate that egg mass surveys can be an efficient and reliable method for monitoring population trends of federally threatened R. draytonii. Combining egg mass surveys to estimate trends at many sites with capture-mark-recapture methods to evaluate factors affecting adult survival at focal populations is likely a profitable path forward to enhance understanding and conservation of R. draytonii.

Habitat disturbance is an important cause of global amphibian declines, with especially strong effects in areas of high agricultural use. Determining the influence of site characteristics on amphibian presence and success is vital to developing effective conservation strategies. We used occupancy analysis to estimate presence of four anuran species at wetlands in northern Iowa as a function of eight environmental covariates hypothesized to affect occupancy: fish abundance, salamander abundance, invertebrate density, aquatic vegetative cover, wetland area, atrazine concentration in water, surrounding crop land use, and an overall wetland health score. We surveyed 27 wetlands in 2015 and 2016. Wetland site occupancy for all species in our study ranged from https://0.23 (Hyla spp. tadpoles) to https://0.95 (L. pipiens adults), indicating agricultural wetlands can provide refuge or habitat for amphibians. Detection probabilities ranged from https://0.13 (P. maculata adults) to https://0.61 (A. americanus adults). Fish abundance, percent of surrounding cropland within 500 m of the wetland, and salamander abundance were among the variables best supported in our model sets although their estimated effects were weak. The direction of predicted effects varied by species and life stage.Although we did not find strong evidence that the environmental factors we measured influenced amphibian populations in our study, we provide insight for managers and researchers on how amphibians use a landscape modified by agriculture.

Emerging infectious diseases (EIDs) are a salient threat to many animal taxa, causing local and global extinctions, altering communities and ecosystem function. The EID chytridiomycosis is a prominent driver of amphibian declines, which is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). To guide conservation policy, we developed a predictive decision-analytic model that combines empirical knowledge of host-pathogen metapopulation dynamics with expert judgment regarding effects of management actions, to select from potential conservation strategies. We apply our approach to a boreal toad (Anaxyrus boreas boreas) and Bd system, identifying optimal strategies that balance tradeoffs in maximizing toad population persistence and landscape-level distribution, while considering costs. The most robust strategy is expected to reduce the decline of toad breeding sites from 53% to 21% over 50 years. Our findings are incorporated into management policy to guide conservation planning. Our online modeling application provides a template for managers of other systems challenged by EIDs.

The effects of contemporary logging practices on headwater stream amphibians have received considerable study but with conflicting or ambiguous results. We posit that focusing inference on specific life stages may help refine understanding, as aquatic and terrestrial impacts may differ considerably. Within a before-after timber harvest experiment, we used recaptures of individually-marked amphibians and a joint probability model of survival, movement, and capture probability, to measure relationships with stream reach, stream gradient, pre- and post-harvest periods, and the timber harvest intensity. Downstream biased movement occurred in both species but was greater for Coastal Tailed Frog (Ascaphus truei) larvae than aquatic Coastal Giant Salamanders Dicamptodon tenebrosus.. For D. tenebrosus, downstream biased movement occurred early in life, soon after an individual?s first summer. Increasing timber harvest intensity reduced downstream movement bias and reduced survival D. tenebrosus but neither of these effects were detected for A. truei larvae. The limited distribution of A. truei among study reaches diminished our ability to measure treatment effects compared to D. tenebrosus.

The reconstruction of US Highway 26/287 over Togwotee Pass, Wyoming, impacted or caused the loss of natural wetlands. To comply with U.S. Army Corps of Engineers permit conditions, the Wyoming Department of Transportation (WYDOT) recently completed construction or restoration of 38 mitigation wetlands along the Highway 26-287 corridor and at the aggregate pit site at the U.S. Forest Service Blackrock Ranger Station. This study provides WYDOT information on differences among wetlands created to mitigate for wetland loss (n=10), wetlands impacted but not destroyed (n=7), and natural wetlands (n=16) relative to various aspects of wildlife that use these habitats. We compare characteristics of amphibians, a pathogenic fungus, invertebrates, and birds. Created wetlands in this study area were significantly shallower than natural and impacted wetlands and had shorter hydroperiods; but impacted wetlands were similar in physical habitat characteristics to natural wetlands. Boreal toads (Anaxyrus boreas) rapidly colonized newly constructed wetlands and annual survival and recruitment rates were similar in created and natural wetlands. Boreal chorus frogs (Pseudacris maculata) were less than half as likely to occupy created wetlands as natural and impacted wetlands but population sizes were high in at least one created wetland. Barred tiger salamanders (Ambystoma mavortium) occurred in natural and impacted wetlands at similar levels, but we observed reproduction by Columbia spotted frogs (Rana luteiventris) at only one created wetland-they were common in natural and impacted wetlands. There was no difference in the prevalence of the pathogenic fungus between created and natural wetlands. Species richness of invertebrates was lower in constructed wetlands than in natural and impacted wetlands and the community composition of invertebrates differed among wetland types. Communities in created wetlands were more likely to be dominated by flying species compared to communities in natural wetlands that had more passive dispersers such as snails and clams. We recorded bird calls in two created and two natural wetlands; species richness was similar but some riparian specialists (e.g., willow flycatcher, Wilson's warbler) were not detected at either created wetland. Our results suggest that wetland creation can be an important tool for conserving wetland-dependent wildlife. Understanding how animals use created wetlands sites is a critical component to understanding the efficacy of mitigation efforts and determining alternative (e.g., earlier) endpoints. This report highlights characteristics in created sites that are advantageous to species that are perhaps non-focal, but important members of the natural community. The data presented here provide support for earlier endpoints for determining success in created wetlands and a baseline for continued monitoring of these or other created sites.

Since the early 1990s, > 5,000 ha of historic wetlands (and adjacent prairie)
have been restored on the row-crop agricultural landscape of Winnebago County, Iowa, USA.
From 2008?2011, we surveyed 22 of these sites for probabilities of occupancy and colonization
by Boreal Chorus Frogs (BCF; Pseudacris maculata), Northern Leopard Frogs (NLF; Lithobates
pipiens), and American Toads (AT; Anaxyrus americanus). We used radio telemetry to measure
patterns of movement and habitat use by 22 NLF and 54 AT, and deployed biophysical models
in available habitats to estimate their physiological costs. BCF occupied 100% of restored
wetlands; NLF and AT occupied 59?91% and 71?89%, respectively, varying according to annual
weather conditions. BCF colonized new sites within a year; NLF and AT required 3 and 2 yr,
respectively.

Drought has fueled an increased frequency and severity of large wildfires in many ecosystems. Despite an increase in research on wildfire effects on vertebrates, the vast majority of it has focused on short-term (<5 yrs) effects and there is still little information on the time scale of population recovery for species that decline in abundance after fire. In 2003, a large wildfire in Montana (USA) burned the watersheds of four of eight streams that we sampled for larval Rocky Mountain tailed frogs (Ascaphus montanus) in 2001. Surveys during 2004?2005 revealed reduced abundance of larvae in burned streams relative to unburned streams, with greater declines associated with increased fire extent. Rocky Mountain tailed frogs have low vagility and have several unusual life-history traits that could slow population recovery, including an extended larval period (4 yrs), delayed sexual maturity (6?8 yrs), and low fecundity (<50 eggs/yr). To determine if abundance remained depressed since the 2003 wildfire, we repeated surveys during 2014?2015 and found relative abundance of larvae in burned and unburned streams had nearly converged to pre-fire conditions within two generations. The negative effects of burn extent on larval abundance weakened >58% within 12 yrs after the fire. We also found moderate synchrony among populations in unburned streams and negative spatial autocorrelation among populations in burned streams. We suspect negative spatial autocorrelation among spatially-clustered burned streams reflected increased post-fire patchiness in resources and different rates of local recovery. Our results add to a growing body of work that suggests populations in intact ecosystems tend to be resilient to habitat changes caused by wildfire. Our results also provide important insights into recovery times of populations that have been negatively affected by severe wildfire.

Emerging infectious diseases such as chytridiomycosis and ranavirus infections are considered important contributors to the worldwide decline of amphibian populations. We reviewed data on 247 anuran mortality events in 43 states of the United States from 1999 ? 2015. Our findings suggest that a severe infectious disease of tadpoles caused by a protist belonging to the phylum Perkinsea might represent the third most common infectious disease of anurans after ranavirus infections and chytridiomycosis. Severe Perkinsea infections (SPI) were systemic and led to multiorganic failure and death. The SPI mortality events affected numerous anuran species and occurred over a broad geographic area, from boreal to subtropical habitats. Livers from all PCR-tested SPI-tadpoles were positive for the Novel Alveolate Group 01 (NAG01) of Perkinsea, while only 2.5% of apparently normal tadpole livers tested positive, suggesting that subclinical infections are uncommon. Phylogenetic analysis demonstrated that SPI is associated with a genetically distinct clade of NAG01 Perkinsea. These data suggest that this virulent Perkinsea clade is an important pathogen of frogs in the United States. Given its association with mortality events and tendency to be overlooked, the potential role of this emerging pathogen in amphibian declines on a broad geographic scale warrants further investigation

The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class II&#61538; chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under significant positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a broad range of Ranid taxa.

Over 2 years of surveys of 3 stream networks, stream salamander occupancy was better described by covariates related to the position of the site within the network than microhabitat features. Via simulation, we found that changes in occupancy can be detected by preferentially sampling headwaters, but causes of variation in extinction rates will require more intensive effort.