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Infectious diseases are a major driver of the global amphibian decline. In addition, many factors, from genetics and stress to pollution and climate change, can influence the response to pathogens. Therefore, it is important to be able to evaluate amphibian immunity in the field and in the laboratory. The phytohemagglutinin (PHA) assay is an inexpensive and relatively non-invasive tool that has been used extensively to assess immunocompetence, especially in birds, and more recently in amphibians. However, there is substantial variation in experimental methodology among amphibian PHA studies in terms of species and life stages, PHA doses and injection sites, and use of experimental controls. Here, we compile and compare all known PHA studies in amphibians in order to identify knowledge gaps and develop best practices for future work. We found that research has only been conducted on a limited number of species, which may not reflect the diversity of amphibians as a whole. There is also a lack of validation studies in most species, so that doses and timing of PHA injection and subsequent swelling measurements may not effectively evaluate immunocompetence. Based on these and other findings, we put forward a set of recommendations to make future PHA studies more consistent and improve the ability to utilize this assay in wild populations, where immune surveillance is greatly needed.

Geographic distribution parish record for this snake species

Turtles are one of the most imperiled vertebrate groups in the world. With habitat destruction unabated in many places, urban and suburban greenspaces may serve as refugia for turtles, at least those species able to tolerate heavily altered landscapes. In south-central Louisiana, we have conducted a turtle capture-mark-recapture effort in two ponds in an urban greenspace for 13 years to understand species composition, survival, and individual growth rates. We had 574 total captures of 251 individuals of five species from 2009–2021, with Trachemys scripta elegans (Red-eared Sliders) and Sternotherus odoratus (Eastern Musk Turtles) being most common. Apparent annual survival for T. scripta (0.79) was similar to estimates reported in other studies in altered habitats, whereas apparent annual survival for S. odoratus (0.89) was slightly or much higher than other published studies. Growth rates of T. scripta were comparable to other studies and showed both sexes have similar rates of growth until maturity, which is earlier and at a smaller size in males. The two ponds showed marked differences in captures by size, with significantly more juvenile T. scripta captured in the pond with more vegetation, depth, and a softer bottom. Most T. scripta (78.5%) that were recaptured came from the same pond they were originally captured. The basic demographic data gained in this study can serve as a starting point for broader questions on urbanization effects and as a comparison to more natural populations.

National parks and other protected areas are important for preserving landscapes and biodiversity worldwide. An essential component of the mission of the United States (U.S.) National Park Service (NPS) requires understanding and maintaining accurate inventories of species on protected lands. We describe a new, national-scale synthesis of amphibian species occurrence in the NPS system. Many park units have a list of amphibian species observed within their borders compiled from various sources and available publicly through the NPSpecies platform. However, many of the observations in NPSpecies remain unverified and the lists are often outdated. We updated the amphibian dataset for each park unit by collating old and new park-level records and had them verified by regional experts. The new dataset contains occurrence records for 292 of the 424 NPS units and includes updated taxonomy, international and state conservation rankings, hyperlinks to a supporting reference for each record, specific notes, and related fields which can be used to better understand and manage amphibian biodiversity within a single park or group of parks.

Forest harvest is a primary landscape-scale management action affecting riparian forests. Although concerns about impacts of forest harvest on stream amphibians is generally limited to areas adjacent to harvest, there is a paucity of information regarding potential downstream effects of forest harvest on these species. We designed a before-after, control-impact (BACI) experiment to quantify potential impacts of clearcut logging that included 12-m buffers or smaller variable-width buffers on the distribution and abundance of headwater stream amphibians in adjacent and downstream areas. We sampled larval coastal tailed frogs (Ascaphus truei), coastal giant salamanders (Dicamptodon tenebrosus), and Columbia torrent salamanders (Rhyacotriton kezeri) across 3,915 sampling occasions that spanned 13 study reaches in 2008–2011 (pre-harvest) and 2013–2016 (post-harvest) as part of the Trask River Watershed Study in the Oregon Coast Range, U.S.A. We analyzed these data using occupancy models to estimate occupancy and (when possible) relative abundance, while accounting for various sources of imperfect detection. All species exhibited reduced occupancy adjacent to clearcuts with variable-width buffers (odds ratios [ORs] ranged = 0.24–0.48), and these negative impacts were not always diminished when increasing the buffer size to 12 m (ORs ranged = 0.20–3.56). Dicamptodon tenebrosus was the only species to have occupancy impacted in downstream areas, and this negative impact was related to clearcut logging with uniform 12-m buffers (OR = 0.60). This species was also the only species to have abundance negatively impacted by forest harvest in downstream areas (OR = https://0.41 with uniform 12-m buffers, OR = https://0.38 with variable-width buffers), albeit impacts to abundance were not evaluated for R. kezeri. Ascaphus truei abundance increased in areas downstream of clearcut logging with uniform 12-m buffers (OR = 2.92). Although we found the direction and magnitude of responses varied by species, our study confirms that clearcut logging can have negative impacts on amphibians that inhabit the adjacent stream areas. Perhaps more importantly, we also found that forest harvest can have negative effects on stream amphibians downstream of the harvested area and that increasing the buffer size to 12 m did not necessarily diminish these impacts in adjacent and downstream areas. Altogether, our study provides a nuanced picture of adjacent and downstream effects of forest harvest on three endemic headwater stream amphibians, and our findings demonstrate that forest management practices should consider downstream effects on aquatic taxa when assessing the impact of harvesting trees near headwater streams.

The decline of amphibian populations is one of the starkest examples of the biodiversity crisis. For stream-breeding amphibians, alteration of natural flow regimes by dams, water diversions, and climate change have been implicated in declines and extirpations. Identifying drivers of amphibian declines requires long time series of abundance data because amphibian populations can exhibit high natural variability. Multiple population viability analysis (MPVA) models integrate abundance data and share information from different populations to estimate how environmental factors influence population growth. Flow alteration has been linked to declines and extirpations in the Foothill Yellow-Legged Frog (Rana boylii), a stream-breeding amphibian native to California and Oregon. To date, no study has jointly analyzed abundance data from populations throughout the range of R. boylii in an MPVA model. We compiled time series of egg mass counts (an index of adult female abundance) from R. boylii populations in 36 focal streams and fit an MPVA model to quantify how streamflow metrics, stream temperature, and surrounding land cover affect population growth. We found population growth was positively related to stream temperature and was higher in the years following a wet year with high total annual streamflow. Density-dependence was weakest (i.e., carrying capacity was highest) for streams with high seasonality of streamflow and intermediate rates of change in streamflow during the spring. Our results highlight how altered streamflow can further increase the risk of decline for R. boylii populations. Managing stream conditions to better match natural flow and thermal regimes would benefit the conservation of R. boylii populations.

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats, including contaminants. While the bi-phasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used non-lethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017–2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ? 0.67) whereas total Hg in sediment was not (R2 ? 0.04). Our study, the largest broadscale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for non-lethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify potential threats of MeHg to amphibians.

Non-invasive methods are important to conservation physiology to reduce negative effects on the species being studied. Glucocorticoid (GC) hormones are often used to assess health of individuals, but collection methods can be invasive. Many amphibians are imperiled worldwide, and saliva is a non- or semi-invasive matrix to measure GCs that has been partially validated for only four amphibian species. Validation ensures that assays are reliable and can detect changes in saliva corticosterone (sCORT) following exposure to stressors, but it is also necessary to ensure sCORT concentrations are correlated with plasma concentrations. To help validate the use of saliva in assessing CORT responses in amphibians, we captured uniquely marked Columbia Spotted Frogs (Rana luteiventris) on sequential days and collected baseline and stress-induced (after handling) samples. For a subset of individuals, we collected and quantified CORT in both saliva and blood samples, which have not been compared for amphibians. We tested several aspects of CORT responses and, by collecting across separate days, measured repeatability of CORT responses across days. We also evaluated whether methods common to amphibian conservation, such as handling alone or handling, clipping a toe, and tagging elevated sCORT. Similar to previous studies, we show that sCORT is reliable concerning parallelism, recovery, precision, and sensitivity. Saliva CORT was weakly correlated with plasma CORT (R2 = 0.21), and we detected elevations in sCORT after handling, demonstrating biological validation. Toe-clipping and tagging did not increase sCORT over handling alone, but repeated handling elevated sCORT for ~72 hours. However, sCORT responses were highly variable and repeatability was low within individuals and among capture sessions, contrary to previous studies with urinary and waterborne CORT. Saliva CORT is a semi-invasive and rapid technique that could be useful to assess effects of anthropogenic change, and conservation efforts, but will require careful study design and future validation.

Interventions of host-pathogen dynamics provide strong tests of relationships, yet they are still rarely applied across multiple populations. After American Bullfrogs (Rana catesbeiana) invaded a wildlife refuge where federally threatened Chiricahua Leopard Frogs (R. chiricahuensis) were reintroduced 12 years prior, managers launched a landscape-scale eradication effort to help ensure continued recovery of the native species. We used a before-after-control-impact (BACI) design and environmental DNA sampling of 19 eradication sites and 18 control sites between fall 2016 and winter 2020–2021 to measure community-level responses to bullfrog eradication, including for 2 pathogens. Dynamic occupancy models revealed successful eradication from 94% of treatment sites. Native amphibians did not respond to bullfrog eradication, but the pathogens amphibian chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses were co-extirpated with bullfrogs. Our spatially replicated experimental approach provides strong evidence that management of invasive species can simultaneously reduce predation and disease risk for imperiled species.

Risk analysis protocols for prioritizing the management of non-native species are numerous, yet few incorporate risk and management in the same analysis or accommodate a broad diversity of taxa outside of a specific geographic area. We adapted a protocol that accounts for these factors to address non-native animal species in the Southern California/Northern Baja California Coast Ecoregion near the international border in San Diego County, an area with high indigenous biodiversity and high numbers of species of conservation concern. This stepwise, semi-quantitative protocol is applicable to any animal group in any predefined geographic area, relies on consensus-building among taxonomic experts, and has been vetted through previous use and in peer-reviewed literature. Our results show that the final prioritization was driven mainly by management feasibility, with top-ranked species having multitrophic effects that favor other non-native invaders over native residents. Conditions within the assessment area required some modification to the protocol as it was originally designed, namely a shift in emphasis from eradication to control, given that eradication is implausible for most non-native species in the assessment area. We call attention to taxon-specific issues that surfaced during the analysis, identify areas for improvement in this first-ever risk assessment for invasive animal species in the Natural Communities Conservation Plan/Habitat Conservation Plan (NCCP/HCP) reserve system of San Diego County, and provide suggestions for further refinement of the protocol. This study builds on the effort to standardize risk analysis for invasive species globally, given that many of the same invaders present threats to indigenous biodiversity worldwide.

Introduction: Designs for safe and effective road crossing structures for small animals are typically under-road microtunnels and culverts which have varying levels of effectiveness reported in the scientific literature. Many species, particularly migratory amphibians, may have limited ability to find and use passages if they are too far apart, resulting in substantial barrier effects.

Methods: We designed a novel open elevated passage (elevated road segment: ERS), similar to a low terrestrial bridge, that could theoretically be built to any length based upon species needs and movement characteristics. A 30 m length prototype ERS was installed along a forest road with a history of amphibian road mortality in Sierra National Forest, Fresno County, CA, USA. From 2018 to 2021, we monitored small animal activity under the ERS in relation to surrounding roadside and forest habitats using active infrared cameras.

Results: We documented a total of 8,815 unique use events, using species specific independence criteria, across 22 species of amphibians (3), reptiles (4), and small mammals (15). Poisson regression modeling of taxonomic group activity under the ERS, roadside and forest, showed that amphibian activity was highest in the forest habitat, no differences were observed for reptiles, and small mammal activity was highest under the ERS. However, mean activity estimates under the ERS were equal to or greater than the open roadside habitat for all 22 species, suggesting that adding cover objects, such as downed logs and vegetation may further enhance passage use.

Discussion: Overall, results showed that the design of the ERS crossing has potential to provide high connectivity for a wide range of amphibian, reptile, and small mammal species while reducing road mortality. ERS systems can also be used in areas with challenging terrain and other hydrological and environmental constraints. Incorporating current road ecology science, we provide supplemental ERS concept designs for secondary roads, primary roads and highways to help increase the options available for road mitigation planning for small animals.

1. Surveillance programs are essential for detecting emerging pathogens and often rely on molecular methods to make inference about the presence of a target disease agent. However, molecular methods rarely detect target DNA perfectly. For example, molecular pathogen detection methods can result in misclassification (i.e., false positives and false negatives) or partial detection errors (i.e., detections with ‘ambiguous’, ‘uncertain’, or ‘equivocal’ results). Then, when data are to be analyzed, these?partial observations?are?either?discarded?or censored;?this, however, disregards information that could be used to make inference about the true state of the system. There is a critical need for more direction and guidance related to how many samples is enough to declare a unit of interest ‘pathogen-free’.
2. Here, we develop a Bayesian hierarchal framework that accommodates false negative, false positive, and uncertain detections to improve inference related to the occupancy of a pathogen. We apply our modeling framework to a case study of the fungal pathogen Pseudogymnoascus destructans (Pd) identified in Texas bats at the invasion front of white-nose syndrome. To improve future surveillance programs, we provide guidance on sample sizes required to be 95% certain a target organism is absent from a site.
3. We found that the presence of uncertain detections increased the variability of resulting posterior probability distributions of pathogen occurrence, and that our estimates of required sample size were very sensitive to prior information about pathogen occupancy, pathogen prevalence, and diagnostic test specificity. In the Pd case study, we found that the posterior probability of occupancy was very low in 2018, but occupancy probability approached 1 in 2020, reflecting increasing prior probabilities of occupancy and prevalence elicited from the site manager.
4. Our modeling framework provides the user a posterior probability distribution of pathogen occurrence, which allows for subjective interpretation by the decision-maker. To help readers apply and use the methods we developed, we provide an interactive?RShiny?app?that generates target species?occupancy estimation and sample size estimates to make these methods more accessible?to the scientific community (https://rmummah.shinyapps.io/ambigDetect_sampleSize).?This modeling framework and sample size guide may be useful for improving inferences from molecular surveillance data about emerging pathogens, non-native invasive species, and endangered species where misclassifications and ambiguous detections occur.

Countless species of animals—big game, birds, bats, insects, amphibians, reptiles, and fish—migrate to reach suitable habitats to feed, reproduce, and raise their young. Animal migrations developed over millennia commonly follow migration corridors—unique routes for each species—to move among seasonal habitats. Changes along those corridors, whether from human development (buildings, roads, dams) or from natural disturbances (for example, climate change, drought, fire, flooding, or invasive species), can make them harder to navigate. The U.S. Geological Survey’s Ecosystems Mission Area provides science that assists land managers in mapping, enhancing, protecting, and reconnecting migration corridors critical for diverse fish and wildlife populations that migrate, such as Odocoileus hemionus (mule deer) and Antilocapra americana (pronghorn), trout and salmon, salamanders, tortoises, bats, and Danaus plexippus (monarch butterflies).

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats including contaminants. While the biphasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used nonlethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017–2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether the concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ? 0.67), whereas total Hg in sediment was not (R2 ? 0.04). Our study, the largest broad-scale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for nonlethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify the potential threats of MeHg to amphibians.

Protected areas like national parks are essential elements of conservation because they limit human influence on the landscape, which protects biodiversity and ecosystem function. The role of national parks in conservation, however, often goes far beyond limiting human influence. The U.S. National Park Service and its system of land units contribute substantively to conservation by providing protected lands where researchers can document trends in species distributions and abundances, examine characteristics important for generating these trends, and identify and implement conservation strategies to preserve biodiversity. We reviewed the contribution of U.S. national parks to amphibian research and conservation and highlight important challenges and findings in several key areas. First, U.S. national parks were instrumental in providing strong support that amphibian declines were real and unlikely to be simply a consequence of habitat loss. Second, research in U.S. national parks provided evidence against certain hypothesized causes of decline, like UV-B radiation, and evidence for others, such as introduced species and disease. However, describing declines and identifying causes contributes to conservation only if it leads to management; importantly, U.S. national parks have implemented many conservation strategies and evaluated their effectiveness in recovering robust amphibian populations. Among these, removal of invasive species, especially fishes; conservation translocations; and habitat creation and enhancement stand out as examples of successful conservation strategies with broad applicability. Successful management for amphibians is additionally complicated by competing mandates and stakeholder interests; for example, past emphasis on increasing visitor enjoyment by introducing fish to formerly fishless lakes had devastating consequences for many amphibians. Other potential conflicts with amphibian conservation include increasing development, increased risk of introductions of disease and exotic species with increased visitation, and road mortality. Decision science and leveraging partnerships have proven to be key components of effective conservation under conflicting mandates in national parks. As resource managers grapple with large-scale drivers that are outside local control, public-private partnerships and adaptive strategies are increasing in importance. U.S. national parks have played an important role in many aspects of identifying and ameliorating the amphibian decline crisis and will continue to be essential for the conservation of amphibians in the future.

Amphibians are among the most sensitive taxa to climate change, and species inhabiting arid and semiarid landscapes at the extremes of their range are especially vulnerable to drought. The Jack Creek, Oregon, USA, population of Oregon spotted frogs (Rana pretiosa) faces unique challenges because it occupies the highest elevation site in the species' extant range and one that has been transformed by loss of American beavers (Castor canadensis[/]), which historically maintained open water. We evaluated the effects of drought mitigation (addition of excavated ponds) on relationships between local and regional water availability, inactive legacy beaver dams, and Oregon spotted frog population dynamics in the Jack Creek system. We conducted egg mass surveys and capture-mark-recapture sampling at a treatment reach with excavated ponds and 3 reference reaches over 13 years; surveys spanned a period before and after pond excavation at the treatment and 1 primary comparison reference reach. We analyzed data using a combination of robust design capture-mark-recapture estimators and generalized linear mixed models to characterize population dynamics. Adult Oregon spotted frog survival was approximately 19.5% higher at the treatment reach than the primary reference reach during the study period. Annual survival was most strongly associated with late summer vegetation greenness, a proxy for water availability, and males had higher survival than females. Among the 4 study reaches, the treatment reach consistently had higher late summer vegetation greenness, and the hydrology functioned more independently of regional precipitation patterns relative to the reference reaches; however, these dynamics were not linked to pond excavation. Breeding was concentrated in 2 legacy beaver ponds that were deepened by excavation during the study compared to an unexcavated beaver pond, 2 excavated ponds without legacy beaver dams, and 9 reference ponds. These results point to the benefit of enhancing existing beaver structures and indicate that management actions aimed at maintaining surface water for breeding in spring and saturated soils and ponded water for adults in late summer would benefit this unique population of Oregon spotted frogs in the face of drought.