Conservation genomics of an endangered montane amphibian reveals low population structure, low genomic diversity and selection pressure from disease.

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (Ne = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.

Captivity, Reintroductions, and the Rewilding of Amphibian-associated Bacterial Communities.

Many studies have noted differences in microbes associated with animals reared in captivity compared to their wild counterparts, but few studies have examined how microbes change when animals are reintroduced to the wild after captive rearing. As captive assurance populations and reintroduction programs increase, a better understanding of how microbial symbionts respond during animal translocations is critical. We examined changes in microbes associated with boreal toads (Anaxyrus boreas), a threatened amphibian, after reintroduction to the wild following captive rearing. Previous studies demonstrate that developmental life stage is an important factor in amphibian microbiomes. We collected 16S marker-gene sequencing datasets to investigate: (i) comparisons of the skin, mouth, and fecal bacteria of boreal toads across four developmental life stages in captivity and the wild, (ii) tadpole skin bacteria before and after reintroduction to the wild, and (iii) adult skin bacteria during reintroduction to the wild. We demonstrated that differences occur across skin, fecal, and mouth bacterial communities in captive versus wild boreal toads, and that the degree of difference depends on developmental stage. Skin bacterial communities from captive tadpoles were more similar to their wild counterparts than captive post-metamorphic individuals were to their wild counterparts. When captive-reared tadpoles were introduced to a wild site, their skin bacteria changed rapidly to resemble wild tadpoles. Similarly, the skin bacterial communities of reintroduced adult boreal toads also shifted to resemble those of wild toads. Our results indicate that a clear microbial signature of captivity in amphibians does not persist after release into natural habitat.

Effects of Herbicides and the Chytrid Fungus Batrachochytrium dendrobatidis on the growth, development and survival of Larval American Toads (Anaxyrus americanus).

Pesticides and pathogens adversely affect amphibian health, but their interactive effects are not well known. We assessed independent and combined effects of two agricultural herbicides and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on the growth, development and survival of larval American toads (Anaxyrus americanus). Wild-caught tadpoles were exposed to four concentrations of atrazine (0.18, 1.8, 18.0, 180 µg/L) or glyphosate (7, 70, 700, 7000 µg a.e./L), respectively contained in Aatrex® Liquid 480 (Syngenta) or Vision® Silviculture Herbicide (Monsanto) for 14 days, followed by two doses of Bd. At day 14, atrazine had not affected survival, but it non-monotonically affected growth. Exposure to the highest concentration of glyphosate caused 100% mortality within 4 days, while lower doses had an increasing monotonic effect on growth. At day 65, tadpole survival was unaffected by atrazine and the lower doses of glyphosate. Neither herbicide demonstrated an interaction effect with Bd on survival, but exposure to Bd increased survival among both herbicide-exposed and herbicide-control tadpoles. At day 60, tadpoles exposed to the highest concentration of atrazine remained smaller than controls, indicating longer-term effects of atrazine on growth, but effects of glyphosate on growth disappeared. Growth was unaffected by any herbicide-fungal interaction but was positively affected by exposure to Bd following exposure to atrazine. Atrazine exhibited a slowing and non-monotonic effect on Gosner developmental stage, while exposure to Bd tended to speed up development and act antagonistically toward the observed effect of atrazine. Overall, atrazine, glyphosate and Bd all showed a potential to modulate larval toad growth and development.

The structural discrepancy between the small and large gut microbiota of Asiatic toad (Bufo gargarizans) during hibernation.

Hibernating amphibians are suitable for the research on the adaptation of gut microbiota to long-term fasting and cold stresses. However, the previous studies mainly focus on the large or whole gut microbiota but not the small gut microbiota. To test the structural discrepancy between the small and large gut microbiota during hibernation, we performed two independent batches of 16S rRNA gene amplicon sequencing to profile the small and large gut microbiota of hibernating Asiatic toad (Bufo gargarizans) from two wild populations. Both batches of data revealed that Proteobacteria, Bacteroidetes, and Firmicutes were the three most dominant phyla in the small and large gut microbiota. Three core OTUs with 100% occurrence in all gut microbiotas were annotated as Pseudomonas. A significant structural discrepancy was detected between the small and large gut microbiota. For instance, Proteobacteria assembled in the small intestine with a higher proportion than it did in the large intestine, but Bacteroidetes and Firmicutes assembled in the large intestine with a higher proportion than they did in the small intestine. The large gut microbiota exhibited higher diversity than the small gut microbiota. Nevertheless, a severe batch effect existed in the structural analysis of the gut microbiotas. The large gut microbiota showed a better resistance to the batch effect than the small gut microbiota did. This study provides preliminary evidence that microbes assemble in the small and large intestines of amphibians with discrepant patterns during hibernation.

Host Identity Matters-Up to a Point: The Community Context of Batrachochytrium dendrobatidis Transmission.

AbstractThe level of detail on host communities needed to understand multihost parasite invasions is an unresolved issue in disease ecology. Coarse community metrics that ignore functional differences between hosts, such as host species richness, can be good predictors of invasion outcomes. Yet if host species vary in the extent to which they maintain and transmit infections, then explicitly accounting for those differences may be important. Through controlled mesocosm experiments and modeling, we show that interspecific differences between host species are important for community-wide infection dynamics of the multihost fungal parasite of amphibians (Batrachochytrium dendrobatidis [Bd]), but only up to a point. The most abundant host species in our system, fire salamander larvae (Salamandra salamandra), did not maintain or transmit infections. Rather, two less abundant "auxiliary" host species, Iberian tree frog (Hyla molleri) and spiny toad (Bufo spinosus) larvae, maintained and transmitted Bd. Frogs had the highest mean rates of Bd shedding, giving them the highest contributions to the basic reproduction number, R0. Toad contributions to R0 were substantial, however, and when examining community-level patterns of infection and transmission, the effects of frogs and toads were similar. Specifying more than just host species richness to distinguish salamanders from auxiliary host species was critical for predicting community-level Bd prevalence and transmission. Distinguishing frogs from toads, however, did not improve predictions. These findings demonstrate limitations to the importance of host species identities in multihost infection dynamics. Host species that exhibit different functional traits, such as susceptibility and infectiousness, may play similar epidemiological roles in the broader community.

Comparative Analysis of Skin Bacterial Diversity and Its Potential Antifungal Function Between Desert and Pine Forest Populations of Boreal Toads Anaxyrus boreas.

The skin microbiome in amphibians has gained a lot of attention as some of its members play a protective role against pathogens such as the fungus Batrachochytrium dendrobatidis (Bd). The composition of skin bacterial communities has been suggested as one of the factors explaining differences in susceptibility to Bd among amphibian species and populations. The boreal toad Anaxyrus boreas is known to be susceptible to Bd, and severe population declines in its southeastern range have been documented. However, throughout A. boreas distribution, populations present differences in susceptibility to Bd infections which may be associated with differences in skin microbial diversity. This study compared the skin bacterial diversity and Bd infection levels of A. boreas in one desert population and one pine forest population from Baja California, Mexico. We found that desert and pine forest toad populations exhibit differences in skin bacterial community structure but show similar Bd infection levels. Using a predictive method, we found that the abundance of bacteria with potential Bd-inhibitory properties differed between uninfected and infected individuals but not between populations. Our data suggest that several bacteria in the skin community may be offering protection from Bd infections in these A. boreas populations. This study provides foundational evidence for future studies seeking to understand the skin-microbial variation among boreal toads' populations and its relation with Bd susceptibility.

Identifying fungal-host associations in an amphibian host system.

Host-associated microbes can interact with macro-organisms in a number of ways that affect host health. Few studies of host-associated microbiomes, however, focus on fungi. In addition, it is difficult to discern whether a fungal organism found in or on an ectotherm host is associating with it in a durable, symbiotic interaction versus a transient one, and to what extent the habitat and host share microbes. We seek to identify these host-microbe interactions on an amphibian, the Colorado boreal toad (Anaxyrus boreas boreas). We sequenced the ITS1 region of the fungal community on the skin of wild toads (n = 124) from four sites in the Colorado Rocky Mountains, across its physiologically dynamic developmental life stages. We also sampled the common habitats used by boreal toads: water from their natal wetland and aquatic pond sediment. We then examined diversity patterns within different life stages, between host and habitat, and identified fungal taxa that could be putatively host-associated with toads by using an indicator species analysis on toad versus environmental samples. Host and habitat were strikingly similar, with the exception of toad eggs. Post-hatching toad life stages were distinct in their various fungal diversity measures. We identified eight fungal taxa that were significantly associated with eggs, but no other fungal taxa were associated with other toad life stages compared with their environmental habitat. This suggests that although pre- and post-metamorphic toad life stages differ from each other, the habitat and host fungal communities are so similar that identifying obligate host symbionts is difficult with the techniques used here. This approach does, however, leverage sequence data from host and habitat samples to predict which microbial taxa are host-associated versus transient microbes, thereby condensing a large set of sequence data into a smaller list of potential targets for further consideration.

Infection Status as the Basis for Habitat Choices in a Wild Amphibian.

AbstractAnimals challenged with disease may select specific habitat conditions that help prevent or reduce infection. Whereas preinfection avoidance of habitats with a high risk of disease exposure has been documented in both captive and free-ranging animals, evidence of switching habitats after infection to support the clearing of the infection is limited to laboratory experiments. The extent to which wild animals proximately modify habitat choices in response to infection status thus remains unclear. We investigated preinfection behavioral avoidance and postinfection habitat switching using wild, radio-tracked boreal toads (Anaxyrus boreas boreas) in a population challenged with Batrachochytrium dendrobatidis (Bd), a pathogenic fungus responsible for a catastrophic panzootic affecting hundreds of amphibian species worldwide. Boreal toads did not preemptively avoid microhabitats with conditions conducive to Bd growth. Infected individuals, however, selected warmer, more open habitats, which were associated with elevated body temperature and the subsequent clearing of infection. Our results suggest that disease can comprise an important selective pressure on animal habitat and space use. Habitat selection models, therefore, may be greatly improved by including variables that quantify infection risk and/or the infection status of individuals through time.

Molecular detection of the reptile-associated Borrelia group in Amblyomma dissimile, Mexico.

Recently, the first record of Borrelia associated with reptiles in Mexico was published; however, no studies have been done to assess the role of Mexican ticks as potential vectors of this Borrelia group. Amblyomma dissimile is a hard tick mainly associated with amphibians and reptiles in this country. The aim of this study was to evaluate the presence of Borrelia in A. dissimile from Mexico. We collected 60 A. dissimile individuals attached to 16 Rhinella horribilis. DNA was extracted and all specimens were screened individually for Borrelia by amplification of a fragment of the 16S rDNA and an additional fragment of the flagellin gene. Five ticks were positive for Borrelia, DNA sequences corresponded to Borrelia sp. and group with sequences of the reptile-associated Borrelia group. This is the first report of Borrelia in A. dissimile and the second report of the reptile-associated Borrelia group in Mexico. This study also highlights the importance of this tick species as potential vector of this group.

Comparison of the characteristics of intestinal microbiota response in Bufo gargarizans tadpoles: Exposure to the different environmental chemicals (Cu, Cr, Cd and NO3-N).

Environmental pollutants could change the intestinal microbiota communities, while data concerning the dynamics of the intestinal microbiota in response to different environmental chemicals in amphibian are lacking. We compared the effects of Cu, Cd, Cr and NO3-N on intestinal microbiota of B. gargarizans tadpoles by using high-throughput 16S rRNA sequencing technology. Our results revealed that responses of intestinal microbiota to three metals and NO3-N showed different characteristics. At the phylum level, the most 100 OTUs were predominantly colonized by Proteobacteria, and meanwhile, expansion of Proteobacteria was observed in Cu 64 µg/L, Cd (100 µg/L and 200 µg/L) and NO3-N100 mg/L treatment groups. In addition, the abundance of Bacteroidetes significantly increased in the gut administrate with Cu, Cd, Cr, NO3-N 20 mg/L exposures, while declined abundance of Fusobacteria was observed in Cu64 µg/L Cd100 µg/L Cd200µg/L-exposed groups. At the genus level, several genera exhibited increased prevalence of abundance such as Shewanella, Azospira and Flavobacterium. The functional prediction revealed that exposures of three metals and NO3-N increase the risks of metabolic disorders and diseases. Our research could be an important step toward an assessment of the ecological risks of different chemicals to aquatic organisms using intestinal microbiota.

Changes in the community structure of the symbiotic microbes of wild amphibians from the eastern edge of the Tibetan Plateau.

Environment has a potential effect on the animal symbiotic microbiome. Here, to study the potential relationship of the symbiotic microbiomes of wild amphibians with altitude, we collected the gut and skin samples from frogs (nine species) and the environmental samples (water and soil samples) from the Leshan Mountains (altitude: 360-410 m) and Gongga Mountains (altitude: 3340-3989 m) on the eastern edge of the Tibetan Plateau. Bufo gargarizans (Bg) samples were collected from both the Leshan and Gongga mountain regions (Bg was the only species sampled on both mountains). The DNA extracted from each sample was performed high-throughput sequencing (MiSeq) of bacterial 16S rRNA gene amplicons. High relative abundance of Caulobacteraceae and Sphingomonadaceae was found in skin samples from both Bg and the other high-altitude amphibians (nine species combined). High relative abundance of Coxiellaceae and Mycoplasmataceae was found in gut samples from both Bg and the other high-altitude amphibians. Furthermore, the alpha and beta diversities of skin and gut samples from Bg and the other amphibian species (nine species combined) were similar. In terms of the symbiotic microbial community, the low-altitude samples were less diverse and more similar to each other than the high-altitude samples were. We speculated that extreme high-altitude environments and host phylogeny may affect the amphibian microbiome. Despite the distinct microbial community differences between the skin and gut microbiomes, some functions were similar in the Bg and combined high-altitude samples. The Bg and high-altitude skin samples had higher oxidative stress tolerance and biofilm formation than the low-altitude skin samples. However, the opposite results were observed for the Bg and high-altitude gut samples. Further study is required to determine whether these characteristics favor high-altitude amphibian adaptation to extreme environments.

Shifts in temperature influence how Batrachochytrium dendrobatidis infects amphibian larvae.

Many climate change models predict increases in frequency and magnitude of temperature fluctuations that might impact how ectotherms are affected by disease. Shifts in temperature might especially affect amphibians, a group with populations that have been challenged by several pathogens. Because amphibian hosts invest more in immunity at warmer than cooler temperatures and parasites might acclimate to temperature shifts faster than hosts (creating lags in optimal host immunity), researchers have hypothesized that a temperature shift from cold-to-warm might result in increased amphibian sensitivity to pathogens, whereas a shift from warm-to-cold might result in decreased sensitivity. Support for components of this climate-variability based hypothesis have been provided by prior studies of the fungus Batrachochytrium dendrobatidis (Bd) that causes the disease chytridiomycosis in amphibians. We experimentally tested whether temperature shifts before exposure to Batrachochytrium dendrobatidis (Bd) alters susceptibility to the disease chytridiomycosis in the larval stage of two amphibian species-western toads (Anaxyrus boreas) and northern red legged frogs (Rana aurora). Both host species harbored elevated Bd infection intensities under constant cold (15° C) temperature in comparison to constant warm (20° C) temperature. Additionally, both species experienced an increase in Bd infection abundance after shifted from 15° C to 20° C, compared to a constant 20° C but they experienced a decrease in Bd after shifted from 20° C to 15° C, compared to a constant 15° C. These results are in contrast to prior studies of adult amphibians highlighting the potential for species and stage differences in the temperature-dependence of chytridiomycosis.

Chlamydia pneumoniae Polioencephalomyelitis and Ganglionitis in Captive Houston Toads (Anaxyrus houstonensis).

Chlamydia pneumoniae is a ubiquitous pathogen causing disease in humans, mammals, birds, reptiles, and amphibians. Since 2012, C. pneumoniae infection has caused neurologic disease and mortality in a breeding colony of endangered Houston toads (Anaxyrus houstonensis) at the Houston Zoo. The purpose of this report is to present the histopathologic and ultrastructural characteristics of C. pneumoniae infection in Houston toads. Fourteen cases were evaluated by histopathology and 1 case was evaluated by electron microscopy. The major histopathologic finding was necrotizing and histiocytic polioencephalomyelitis and ganglionitis. Bacteria formed intracytoplasmic inclusions within neurons but frequently extended into the surrounding tissue from necrotic cells. Ultrastructural evaluation showed the bacteria formed reticulate and elementary bodies characteristic of Chlamydia spp.

Ancestral chytrid pathogen remains hypervirulent following its long coevolution with amphibian hosts.

Many amphibian species around the world, except in Asia, suffer morbidity and mortality when infected by the emerging infectious pathogen Batrachochytrium dendrobatidis (Bd). A lineage of the amphibian chytrid fungus isolated from South Korean amphibians (BdAsia-1) is evolutionarily basal to recombinant global pandemic lineages (BdGPL) associated with worldwide amphibian population declines. In Asia, the Bd pathogen and its amphibian hosts have coevolved over 100 years or more. Thus, resilience of Asian amphibian populations to infection might result from attenuated virulence of endemic Bd lineages, evolved immunity to the pathogen or both. We compared susceptibilities of an Australasian amphibian, Litoria caerulea, known to lack resistance to BdGPL, with those of three Korean species, Bufo gargarizans, Bombina orientalis and Hyla japonica, after inoculation with BdAsia-1, BdGPL or a blank solution. Subjects became infected in all experimental treatments but Korean species rapidly cleared themselves of infection, regardless of Bd lineage. They survived with no apparent secondary effects. By contrast, L. caerulea, after infection by either BdAsia-1 or BdGPL, suffered deteriorating body condition and carried progressively higher Bd loads over time. Subsequently, most subjects died. Comparing their effects on L. caerulea, BdAsia-1 induced more rapid disease progression than BdGPL. The results suggest that genomic recombination with other lineages was not necessary for the ancestral Bd lineage to evolve hypervirulence over its long period of coevolution with amphibian hosts. The pathogen's virulence may have driven strong selection for immune responses in endemic Asian amphibian host species.

Exposure to cadmium induced gut histopathological damages and microbiota alterations of Chinese toad (Bufo gargarizans) larvae.

Cadmium (Cd) is highly hazardous to both terrestrial and aquatic life and it also has multiple negative impacts on amphibian tadpoles and frogs. However, its effects on gut health of amphibian tadpoles are still poorly understood. We used Chinese toad (Bufo gargarizans) tadpoles to examine the effects of chronic cadmium exposure on gut histology and intestinal microbiota by using regular histology analysis and high-throughput sequencing techniques. Tadpoles were exposed to cadmium concentrations at 0, 5, 100 and 200 µg L-1 from Gosner stage 26 to 38. Our results showed 100 and 200 µg L-1 cadmium exposure caused severe gut histopathological alterations while 5 µg L-1 cadmium exposure induced subtle intestine damage. Moreover, species diversity, taxonomic composition and community structure of gut microbiota were influenced by cadmium exposure. Species diversity and richness decreased gradually with the increase of cadmium concentration. Microbial communities of tadpoles in 100 and 200 µg L-1 cadmium exposure groups were remarkably different from those in control group. Furthermore, the relative abundances of prevalent phyla such as Proteobacteria, Bacteroidetes and Firmicutes and dominant genera such as Klebsiella and Aeromonas were also affected by cadmium exposure. We concluded that cadmium could be harmful to tadpole health by inducing intestinal damages and gut microbiota changes.

An interaction between climate change and infectious disease drove widespread amphibian declines.

Climate change might drive species declines by altering species interactions, such as host-parasite interactions. However, few studies have combined experiments, field data, and historical climate records to provide evidence that an interaction between climate change and disease caused any host declines. A recently proposed hypothesis, the thermal mismatch hypothesis, could identify host species that are vulnerable to disease under climate change because it predicts that cool- and warm-adapted hosts should be vulnerable to disease at unusually warm and cool temperatures, respectively. Here, we conduct experiments on Atelopus zeteki, a critically endangered, captively bred frog that prefers relatively cool temperatures, and show that frogs have high pathogen loads and high mortality rates only when exposed to a combination of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) and high temperatures, as predicted by the thermal mismatch hypothesis. Further, we tested various hypotheses to explain recent declines experienced by species in the amphibian genus Atelopus that are thought to be associated with B. dendrobatidis and reveal that these declines are best explained by the thermal mismatch hypothesis. As in our experiments, only the combination of rapid increases in temperature and infectious disease could account for the patterns of declines, especially in species adapted to relatively cool environments. After combining experiments on declining hosts with spatiotemporal patterns in the field, our findings are consistent with the hypothesis that widespread species declines, including possible extinctions, have been driven by an interaction between increasing temperatures and infectious disease. Moreover, our findings suggest that hosts adapted to relatively cool conditions will be most vulnerable to the combination of increases in mean temperature and emerging infectious diseases.

Effects of host species and environmental factors on the prevalence of Batrachochytrium dendrobatidis in northern Europe.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a major threat to amphibian populations. To assist efforts to address such threats, we examined differences in Bd host infection prevalence among amphibian species and its relations to both local environmental factors in breeding habitats and landscape variables measured at three scales (500, 2000 and 5000 m radii) around breeding sites in southernmost Sweden. We sampled 947 anurans of six species in 31 ponds and assessed their infection status. We then examined correlations of infection prevalence with canopy cover, pond perimeter and pH (treated as local-scale pond characteristics), and the number of ponds, area of arable land, area of mature forest, number of resident people and presence of sea within the three radii (treated as landscape variables). The Bd infection prevalence was very low, 0.5-1.0%, in two of the six anuran species (Bufo bufo and Rana temporaria), and substantially higher (13-64%) in the other four (Bombina bombina, Bufotes variabilis, Epidalea calamita, Rana arvalis). In the latter four species Bd infection prevalence was positively associated with ponds' pH (site range: 5.3-8.1), and negatively associated with areas of mature forest and/or wetlands in the surroundings. Our results show that the infection dynamics of Bd are complex and associated with host species, local pond characteristics and several landscape variables at larger spatial scales. Knowledge of environmental factors associated with Bd infections and differences in species' susceptibility may help to counter further spread of the disease and guide conservation action plans, especially for the most threatened species.

Changes in intestinal microbiota of Bufo gargarizans and its association with body weight during metamorphosis.

The assembly of intestinal microbial communities can play major roles in animal development. We hypothesized that intestinal microbial communities could mirror the developmental programs of amphibian metamorphosis. Here, we surveyed the morphological parameters of the body and intestine of Bufo gargarizans at varying developmental stages and inventoried the intestinal microbial communities of B. gargarizans at four key developmental stages via 16S rDNA gene sequencing. Firstly, our survey showed that during metamorphosis, body weight and intestinal weight were reduced by 56.8 and 91.8%, respectively. Secondly, the gut bacterial diversity of B. gargarizans decreased with metamorphosis and the composition of the tadpoles' intestinal microbiota varied across metamorphosis. Compared to aquatic larvae, terrestrial juveniles showed major shifts in microbial composition, including reduction in Proteobacteria and Actinobacteria, increases in Bacteroidetes and Fusobacteria, and the appearance of Verrucomicrobia. Firmicutes in four developmental stages showed similar abundance at the phylum level, but in each stage was driven by distinct genera. Enterobacter, Aeromonas, Mucinivorans and Bacteroides also changed in abundance and were found to be significantly correlated with loss of body or intestinal tissue during metamorphosis. These results indicate a shift in intestinal microbial community composition throughout amphibian metamorphosis.

Host-pathogen metapopulation dynamics suggest high elevation refugia for boreal toads.

Emerging infectious diseases are an increasingly common threat to wildlife. Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease that has been linked to amphibian declines around the world. Few studies exist that explore amphibian-Bd dynamics at the landscape scale, limiting our ability to identify which factors are associated with variation in population susceptibility and to develop effective in situ disease management. Declines of boreal toads (Anaxyrus boreas boreas) in the southern Rocky Mountains are largely attributed to chytridiomycosis but variation exists in local extinction of boreal toads across this metapopulation. Using a large-scale historic data set, we explored several potential factors influencing disease dynamics in the boreal toad-Bd system: geographic isolation of populations, amphibian community richness, elevational differences, and habitat permanence. We found evidence that boreal toad extinction risk was lowest at high elevations where temperatures may be suboptimal for Bd growth and where small boreal toad populations may be below the threshold needed for efficient pathogen transmission. In addition, boreal toads were more likely to recolonize high elevation sites after local extinction, again suggesting that high elevations may provide refuge from disease for boreal toads. We illustrate a modeling framework that will be useful to natural resource managers striving to make decisions in amphibian-Bd systems. Our data suggest that in the southern Rocky Mountains high elevation sites should be prioritized for conservation initiatives like reintroductions.