Rapid Evolution of Resistance and Tolerance Leads to Variable Host Recoveries following Disease-Induced Declines.

AbstractRecoveries of populations that have suffered severe disease-induced declines are being observed across disparate taxa. Yet we lack theoretical understanding of the drivers and dynamics of recovery in host populations and communities impacted by infectious disease. Motivated by disease-induced declines and nascent recoveries in amphibians, we developed a model to ask the following question: How does the rapid evolution of different host defense strategies affect the transient recovery trajectories of hosts following pathogen invasion and disease-induced declines? We found that while host life history is predictably a major driver of variability in population recovery trajectories (including declines and recoveries), populations that use different host defense strategies (i.e., tolerance, avoidance resistance, and intensity-reduction resistance) experience notably different recoveries. In single-species host populations, populations evolving tolerance recovered on average four times slower than populations evolving resistance. Moreover, while populations using avoidance resistance strategies had the fastest potential recovery rates, these populations could get trapped in long transient states at low abundance prior to recovery. In contrast, the recovery of populations evolving intensity-reduction resistance strategies were more consistent across ecological contexts. Overall, host defense strategies strongly affect the transient dynamics of population recovery and may affect the ultimate fate of real populations recovering from disease-induced declines.

Invasibility of a North American soil ecosystem to amphibian-killing fungal pathogens.

North American salamanders are threatened by intercontinental spread of chytridiomycosis, a deadly disease caused by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). To predict potential dispersal of Bsal spores to salamander habitats, we evaluated the capacity of soil microbial communities to resist invasion. We determined the degree of habitat invasibility using soils from five locations throughout the Great Smoky Mountains National Park, a region with a high abundance of susceptible hosts. Our experimental design consisted of replicate soil microcosms exposed to different propagule pressures of the non-native pathogen, Bsal, and an introduced but endemic pathogen, B. dendrobatidis (Bd). To compare growth and competitive interactions, we used quantitative PCR, live/dead cell viability assays, and full-length 16S rRNA sequencing. We found that soil microcosms with intact bacterial communities inhibited both Bsal and Bd growth, but inhibitory capacity diminished with increased propagule pressure. Bsal showed greater persistence than Bd. Linear discriminant analysis (LDA) identified the family Burkolderiaceae as increasing in relative abundance with the decline of both pathogens. Although our findings provide evidence of environmental filtering in soils, such barriers weakened in response to pathogen type and propagule pressure, showing that habitats vary their invasibility based on properties of their local microbial communities.

Chemical disinfection as a simple and reliable method to control the amphibian chytrid fungus at breeding points of endangered amphibians.

Chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is pushing amphibians towards extinction. Whilst mitigation methods were suggested a decade ago, we lack field trials testing their efficacy. We used the agrochemical fungicide, tebuconazole, to treat Bd infected breeding waterbodies of an endangered species that is highly susceptible to the fungus. Just two applications of tebuconazole led to a significant reduction in infection loads in the vast majority of sites, and at six sites the disinfection remained one/two-years post-application. Tebuconazole values drastically decreased in the waterbodies within a week after application, with no significant effects on their hydrochemical and hydrobiological characteristics. Although the use of chemicals in natural populations is undesirable, the growing existential threat to amphibians all over the world indicates that effective interventions in selected populations of endangered species are urgently needed.

An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence.

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

Field-based molecular detection of Batrachochytrium dendrobatidis in critically endangered Atelopus toads and aquatic habitats in Ecuador.

Batrachochytrium dendrobatidis (Bd) is a lethal fungal species that parasitizes vertebrates and is associated with the worldwide decline of amphibian populations. The development of sensitive, rapid detection methods, particularly DNA-based techniques, is critical for effective management strategies. This study evaluates the efficacy of DNA extraction and a portable PCR device in a mountable field laboratory setup for detecting Bd near the habitats of three critically endangered Atelopus toad species in Ecuador. We collected skin swabs from Atelopus balios, A. nanay, and A. bomolochos, and environmental DNA (eDNA) samples from streams in Andean and coastal regions of Ecuador. For eDNA, a comparison was made with duplicates of the samples that were processed in the field and in a standard university laboratory. Our findings revealed Bd detection in eDNA and swabs from 6 of 12 water samples and 10 of 12 amphibian swab samples. The eDNA results obtained in the field laboratory were concordant with those obtained under campus laboratory conditions. These findings highlight the potential of field DNA-based monitoring techniques for detecting Bd in amphibian populations and their aquatic habitats, particularly in remote areas. Furthermore, this research aligns with the National Action Plan for the Conservation of Ecuadorian Amphibians and contributes to the global effort to control this invasive and deadly fungus.

Newly identified diversity of Dinomycetaceae (Rhizophydiales, Chytridiomycota), a family of fungal parasites of marine dinoflagellates.

We identified two new parasite species of Chytridiomycota isolated during blooms of the dinoflagellate Alexandrium minutum in the coastal Mediterranean Sea. Light and electron microscopy together with molecular characterization of the nuclear 18S, ITS, and 28S rDNA regions led to their identification as two new species, Dinomyces gilberthii and Paradinomyces evelyniae, both belonging to the family Dinomycetaceae, order Rhizophydiales. Dinomyces gilberthii differs from the previously described D. arenysensis by the presence of discharge papillae and the development of a drop-shaped sporangium. Paradinomyces evelyniae differs from the previously described P. triforaminorum by the prominent lipid globule present in early sporangia and by the pointed end producing a rhizoid. The two chytrids differed in their geographical distribution. Dinomyces gilberthii was detected in several Mediterranean habitats, including harbours and beaches, and was particularly prevalent during summer dinoflagellate blooms. Its widespread occurrence in coastal ecosystems suggested a high level of adaptability to this environment. Paradinomyces evelyniae had a more restricted distribution in the coastal-marine environment, occurring in harbour sediments and only occasionally in the water column during winter and early spring. Paradinomyces evelyniae has previously been detected in the Baltic Sea, suggesting that its distribution encompasses contrasting coastal environments, although its presence is rare.

Coevolution of a generalist pathogen with many hosts: the case of the amphibian chytrid Batrachochytrium dendrobatidis.

Generalist pathogens maintain infectivity in numerous hosts; how this broad ecological niche impacts host-pathogen coevolution remains to be widely explored. Batrachochytrium dendrobatidis (Bd) is a highly generalist pathogenic fungus that has caused devastating declines in hundreds of amphibian species worldwide. This review examines amphibian chytridiomycosis host-pathogen interactions and available evidence for coevolution between Bd and its numerous hosts. We summarize recent evidence showing that Bd genotypes vary in geographic distribution and virulence, and that amphibian species also vary in Bd susceptibility according to their geographic distribution. How much variation can be explained by phenotypic plasticity or genetic differences remains uncertain. Recent research suggests that Bd genotypes display preferences for specific hosts and that some hosts are undergoing evolution as populations rebound from Bd outbreaks. Taken together, these findings suggest the potential for coevolution to occur and illuminate a path for addressing open questions through integrating historical and contemporary genetic data.

A target enrichment approach for enhanced recovery of Synchytrium endobioticum nuclear genome sequences.

Potato wart disease is caused by the obligate fungal pathogen Synchytrium endobioticum. DNA extraction from compost, purified spores and crude wart tissue derived from tuber galls of infected potatoes often results in low S. endobioticum DNA concentration or highly contaminated with DNA coming from other microorganisms and the potato host. Therefore, Illumina sequencing of these samples generally results in suboptimal recovery of the nuclear genome sequences of S. endobioticum. A hybridization-based target enrichment protocol was developed to strongly enhance the recovery of S. endobioticum DNA while off-target organisms DNA remains uncaptured. The design strategy involved creating a set of 180,000 molecular baits targeting both gene and non-gene regions of S. endobioticum. The baits were applied to whole genome amplified DNA samples of various S. endobioticum pathotypes (races) in compost, from purified spores and crude wart tissue samples. This was followed by Illumina sequencing and bioinformatic analyses. Compared to non-enriched samples, target enriched samples: 1) showed a significant increase in the proportion of sequenced bases mapped to the S. endobioticum nuclear genome, especially for crude wart tissue samples; 2) yielded sequencing data with higher and better nuclear genome coverage; 3) biased genome assembly towards S. endobioticum sequences, yielding smaller assembly sizes but higher representation of putative S. endobioticum contigs; 4) showed an increase in the number of S. endobioticum genes detected in the genome assemblies. Our hybridization-based target enrichment protocol offers a valuable tool for enhancing genome sequencing and NGS-based molecular detection of S. endobioticum, especially in difficult samples.

Advances in Managing Chytridiomycosis for Australian Frogs: Gradarius Firmus Victoria.

Extensive knowledge gains from research worldwide over the 25 years since the discovery of chytridiomycosis can be used for improved management. Strategies that have saved populations in the short term and/or enabled recovery include captive breeding, translocation into disease refugia, translocation from resistant populations, disease-free exclosures, and preservation of disease refuges with connectivity to previous habitat, while antifungal treatments have reduced mortality rates in the wild. Increasing host resistance is the goal of many strategies under development, including vaccination and targeted genetic interventions. Pathogen-directed strategies may be more challenging but would have broad applicability. While the search for the silver bullet solution continues, we should value targeted local interventions that stop extinction and buy time for evolution of resistance or development of novel solutions. As for most invasive species and infectious diseases, we need to accept that ongoing management is necessary. For species continuing to decline, proactive deployment and assessment of promising interventions are more valid than a hands-off, do-no-harm approach that will likely allow further extinctions.

Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

FIRST RECORD OF BATRACHOCHYTRIUM DENDROBATIDIS IN THE NORTHERN NETHERLANDS.

Batrachochytrium dendrobatidis (Bd) infects amphibians and has been linked to the decline of hundreds of anuran amphibians all over the world. In the province of Groningen in the Netherlands, this fungal pathogen was not detected before this study. To determine whether Groningen was Bd-free, we surveyed 12 locations in this province in 2020 and 2021. Samples were then used to quantify the presence of Bd with a qPCR assay. In total, 2 out of 110 (∼0.02%) collected in 2020 and 11 out of 249 samples collected in 2021 tested positive for Bd. Infected amphibians were found in 4 out of the 12 sites, and the prevalence of Bd was estimated at 4% for both years combined. Our study provides the first record of Bd in Groningen, and we hypothesize that Bd is present throughout the Netherlands in regions currently considered "Bd-free." Furthermore, we warn scientists and policymakers to be apprehensive when calling a site free from Bd when sampling is limited or not recent.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. RESULTS: Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. CONCLUSIONS: These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents' detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Skin microbiome disturbance linked to drought-associated amphibian disease.

The onset of global climate change has led to abnormal rainfall patterns, disrupting associations between wildlife and their symbiotic microorganisms. We monitored a population of pumpkin toadlets and their skin bacteria in the Brazilian Atlantic Forest during a drought. Given the recognized ability of some amphibian skin bacteria to inhibit the widespread fungal pathogen Batrachochytrium dendrobatidis (Bd), we investigated links between skin microbiome health, susceptibility to Bd and host mortality during a die-off event. We found that rainfall deficit was an indirect predictor of Bd loads through microbiome disruption, while its direct effect on Bd was weak. The microbiome was characterized by fewer putative Bd-inhibitory bacteria following the drought, which points to a one-month lagged effect of drought on the microbiome that may have increased toadlet susceptibility to Bd. Our study underscores the capacity of rainfall variability to disturb complex host-microbiome interactions and alter wildlife disease dynamics.

Evolutionary analysis of major histocompatibility complex variants in chytrid-resistant and susceptible amphibians.

An amphibian emerging infectious disease (EID), chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), originated in Asia but primarily led to declines and extinctions in amphibian populations outside of Asia. Host major histocompatibility complex (MHC) molecules exhibit high polymorphism, and the evolution of MHC can be influenced by recombination and pathogens. Previous studies have indicated that host MHC class II is associated with Bd resistance. In this study, I conducted recombination and selection tests on functional MHC IIß1 alleles from an Asian Bd-resistant anuran species (Bufo gargarizans) and an Australasian Bd-susceptible species (Litoria caerulea). Recombination at the same site was identified in both species, supporting the hypothesis that recombination contributes to MHC IIß1 diversity in amphibians. Positive selection was observed in MHC IIß1 alleles in both species. In L. caerulea, at least four amino acid sites were identified under significant positive selection in the MHC IIß1, whereas these sites were either negatively selected or conserved in B. gargarizans. This suggests these sites might be selected for Bd resistance. Hydrophobicity was detected in certain amino acid sites relating to Bd resistance, suggesting this physicochemical property may be a factor selected to counteract Bd infection. These findings of this study provide an evolutionary basis for understanding how amphibian MHC IIß1 may undergo selection in response to chytrid infection.

Widely used herbicide metolachlor can promote harmful bloom formation by stimulating cyanobacterial growth and driving detrimental effects on their chytrid parasites.

Metolachlor (MET) is a widely used herbicide that can adversely affect phytoplanktonic non-target organisms, such as cyanobacteria. Chytrids are zoosporic fungi ubiquitous in aquatic environments that parasitize cyanobacteria and can keep their proliferation in check. However, the influence of organic pollutants on the interaction between species, including parasitism, and the associated ecological processes remain poorly understood. Using the host-parasite system consisting of the toxigenic cyanobacterium Planktothrix agardhii and its chytrid parasite Rhizophydium megarrhizum, we investigated the effects of environmentally relevant concentrations of MET on host-parasite interactions under i) continuous exposure of chytrids and cyanobacteria, and ii) pre-exposure of chytrids. During a continuous exposure, the infection prevalence and intensity were not affected, but chytrid reproductive structures were smaller at the highest tested MET concentration. In the parasite's absence, MET promoted cyanobacteria growth possibly due to a hormesis effect. In the pre-exposure assay, MET caused multi- and transgenerational detrimental effects on parasite fitness. Chytrids pre-exposed to MET showed reduced infectivity, intensity, and prevalence of the infection, and their sporangia size was reduced. Thus, pre-exposure of the parasite to MET resulted in a delayed decline of the cyanobacterial cultures upon infection. After several parasite generations without MET exposure, the parasite recovered its initial fitness, indicating that detrimental effects are transient. This study demonstrates that widely used herbicides, such as MET, could favor cyanobacterial bloom formation both directly, by promoting cyanobacteria growth, and indirectly, by inhibiting their chytrid parasites, which are known to play a key role as top-down regulators of cyanobacteria. In addition, we evidence the relevance of addressing multi-organism systems, such as host-parasite interactions, in toxicity assays. This approach offers a more comprehensive understanding of the effects of pollutants on aquatic ecosystems.

Batrachochytrium dendrobatidis strain affects transcriptomic response in liver but not skin in latitudinal populations of the common toad (Bufo bufo).

Batrachochytrium dendrobatidis (Bd) is a fungal pathogen that has decimated amphibian populations worldwide for several decades. We examined the changes in gene expression in response to Bd infection in two populations of the common toad, Bufo bufo, in a laboratory experiment. We collected B. bufo eggs in southern and northern Sweden, and infected the laboratory-raised metamorphs with two strains of the global panzoonotic lineage Bd-GPL. Differential expression analysis showed significant differences between infected and control individuals in both liver and skin. The skin samples showed no discernible differences in gene expression between the two strains used, while liver samples were differentiated by strain, with one of the strains eliciting no immune response from infected toads. Immune system genes were overexpressed in skin samples from surviving infected individuals, while in liver samples the pattern was more diffuse. Splitting samples by population revealed a stronger immune response in northern individuals. Differences in transcriptional regulation between populations are particularly relevant to study in Swedish amphibians, which may have experienced varying exposure to Bd. Earlier exposure to this pathogen and subsequent adaptation or selection pressure may contribute to the survival of some populations over others, while standing genetic diversity in different populations may also affect the infection outcome.

Dietary carotenoid supplementation has long-term and community-wide effects on the amphibian skin microbiome.

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter-individual variation in the availability of micro-nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint-species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life-history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre-metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies.

Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.