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.

The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Chytrid fungus in amphibians from the lowland Brazilian Amazon.

Infectious diseases are one of the main threats to biodiversity. The fungus Batrachochytrium dendrobatidis (Bd) is associated with several amphibian losses around the globe, and environmental conditions may dictate the success of pathogen spread. The Brazilian Amazon has been considered climatically unsuitable for chytrid fungus, but additional information on Bd dynamics in this ecoregion is still lacking. We sampled 462 amphibians (449 anurans, 4 caudatans and 9 caecilians), representing 57 species from the Brazilian Amazon, and quantified Bd infections using qPCR. We tested whether abiotic variables predicted the risk of Bd infections, and tested for relationships between biotic variables and Bd. Finally, we experimentally tested the effects of Bd strains CLFT 156 and CLFT 102 (from the southern and northern Atlantic Forest, respectively) on Atelopus manauensis. We detected higher Bd prevalence than those previously reported for the Brazilian Amazon, and positive individuals in all 3 orders of amphibians sampled. Both biotic and abiotic predictors were related to prevalence, and no variable explained infection load. Moreover, we detected higher Bd prevalence in forested than open areas, while the host's reproductive biology was not a factor. We detected higher mortality in the experimental group infected with CLFT 156, probably because this strain was isolated from a region characterized by discrepant climatic conditions (latitudinally more distant) when compared with the host's sampling site in Amazon. The lowland Brazilian Amazon is still underexplored and future studies targeting all amphibian orders are essential to better understand Bd infection dynamics in this region.

Amphibian chytrid infections along the highest elevational gradient of the Brazilian Atlantic Forest.

Environmental variation along elevational gradients shapes conditions for pathogen development, which influences disease outcomes. Chytridiomycosis is a non-vectored disease caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd) and is responsible for massive declines of amphibian populations all over the world. Several biotic and abiotic factors are known to influence Bd infection dynamics in amphibians, including temperature and host species richness. Here, we quantified Bd prevalence and load along an elevational gradient in the Caparaó National Park (CNP), Brazil, and tested for associations of Bd infections with elevation, temperature, and species richness. We hypothesized that Bd infections would increase as local species richness decreased with elevation. We detected Bd along the entire elevational gradient and found a negative association between infection load and elevation. We did not detect significant associations between infection prevalence and elevation. Our findings are consistent with other wide elevational gradient studies, but are contrary to 2 other studies performed in the Atlantic Forest. We did not find the minimum elevational range that should be sampled to detect the influence of elevation on Bd variation. Our study represents the widest elevational gradient that has been sampled in Brazil and contributes to a better understanding of Bd distribution and dynamics in natural systems.

Seasonal variation of Batrachochytrium dendrobatidis in a threatened anuran species from Uruguay.

Chytridiomycosis, an emergent infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), is considered one of the drivers of the current amphibian biodiversity loss. To inform endangered species conservation efforts, it is essential to improve our knowledge about the abiotic and biotic factors that influence Bd infection dynamics in the wild. Here, we analyzed variation of Bd infection in the redbelly toad Melanophryniscus montevidensis, a threatened bufonid from Uruguay. We tested the influence of temperature, precipitation, season, and host population size on Bd prevalence and intensity. Additionally, considering the sub-lethal effects of Bd, we tested if these variables, potentially through their effect on Bd, also explain the variation in host body condition. We determined a high Bd prevalence of 41% (100/241), and that population size influenced both Bd prevalence and infection intensity. We identified an effect of precipitation and season on Bd infection intensity and an effect of season on toad body condition. In addition, we found a negative effect of infection intensity on body condition; moreover, while some toads cleared the infection, their body condition did not improve, suggesting a long-term cost. This is the first report on host population size as an important factor in Bd infection dynamics in a threatened anuran species, and seasonal demographic changes appear to play an important role in the dynamics. Finally, we highlight the need for monitoring Bd in this and other endangered amphibian populations, especially those within the genus Melanophryniscus, which includes several Endangered and Data Deficient species in South America.

Forest cover influences chytrid infections in populations of Boana curupi, a threatened treefrog of south Brazil.

Complex interactions among hosts, pathogens, and the environment affect the vulnerability of amphibians to the emergence of infectious diseases such as chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Boana curupi is a forest-dwelling amphibian endemic to the southern Atlantic Forest of South America, a severely fragmented region. Here, we evaluated whether abiotic factors (including air and water temperature, relative air humidity, and landscape) are correlated with chytrid infection intensity and prevalence in B. curupi. We found individuals infected with Bd in all populations sampled. Prevalence ranged from 25-86%, and the infection burden ranged from 1 to over 130000 zoospore genomic equivalents (g.e.) (mean ± SD: 4913 ± 18081 g.e.). The infection load differed among populations and was influenced by forest cover at scales of 100, 500, and 1000 m, with the highest infection rates recorded in areas with a higher proportion of forest cover. Our results suggest that the fungus is widely distributed in the populations of B. curupi in southern Brazil. Population and disease monitoring are necessary to better understand the relationships between host, pathogen, and environment, especially when, as in the case of B. curupi, threatened species are involved.

Assessing amphibian disease risk across tropical streams while accounting for imperfect pathogen detection.

Ecologists studying emerging wildlife diseases need to confront the realism of imperfect pathogen detection across heterogeneous habitats to aid in conservation decisions. For example, spatial risk assessments of amphibian disease caused by Batrachochytrium dendrobatidis (Bd) has largely ignored imperfect pathogen detection across sampling sites. Because changes in pathogenicity and host susceptibility could trigger recurrent population declines, it is imperative to understand how pathogen prevalence and occupancy vary across environmental gradients. Here, we assessed how Bd occurrence, prevalence, and infection intensity in a diverse Neotropical landscape vary across streams in relation to abiotic and biotic predictors using a hierarchical Bayesian model that accounts for imperfect Bd detection caused by qPCR error. Our model indicated that the number of streams harboring Bd-infected frogs is higher than observed, with Bd likely being present at ~ 43% more streams than it was detected. We found that terrestrial-breeders captured along streams had higher Bd prevalence, but lower infection intensity, than aquatic-breeding species. We found a positive relationship between Bd occupancy probability and stream density, and a negative relationship between Bd occupancy probability and amphibian local richness. Forest cover was a weak predictor of Bd occurrence and infection intensity. Finally, we provide estimates for the minimum number of amphibian captures needed to determine the presence of Bd at a given site where Bd occurs, thus, providing guidence for cost-effective disease risk monitoring programs.

Seasonal prevalence of the amphibian chytrid in a tropical pond-dwelling tadpole species.

Infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians and has caused catastrophic global declines of amphibian populations. Some studies have detected a seasonal pattern of infection associated with the local climate, and although most of them have focused on investigating the seasonality of Bd in relation to its impacts on amphibians, fewer have aimed to understand the chytrid persistence in the amphibian assemblage over seasons by investigating reservoir hosts. Since tadpoles are generally tolerant to Bd infection, they often play a relevant role in local disease dynamics. Thus, we hypothesized that tadpoles of Boana faber, a species that can be found in permanent ponds throughout the seasons, would function as Bd reservoirs. We therefore investigated Bd infection prevalence in tadpoles of this species over 2 yr in a nature reserve. As expected, we detected a seasonal variation of Bd infection, with a higher prevalence of Bd during the coldest months (winter) when compared to the warmer months (summer). Interestingly, our seasonal-trend decomposition analysis showed that Bd prevalence is increasing annually in the area, which could represent either a natural fluctuation of this pathogen, or an imminent threat to that anuran assemblage. With this study, we highlight the tadpole of B. faber as a potential reservoir for Bd, and we suggest that monitoring Bd in such hosts could be a powerful tool for identifying priority areas for amphibian conservation.

Low-load pathogen spillover predicts shifts in skin microbiome and survival of a terrestrial-breeding amphibian.

Wildlife disease dynamics are strongly influenced by the structure of host communities and their symbiotic microbiota. Conspicuous amphibian declines associated with the waterborne fungal pathogen Batrachochytrium dendrobatidis (Bd) have been observed in aquatic-breeding frogs globally. However, less attention has been given to cryptic terrestrial-breeding amphibians that have also been declining in tropical regions. By experimentally manipulating multiple tropical amphibian assemblages harbouring natural microbial communities, we tested whether Bd spillover from naturally infected aquatic-breeding frogs could lead to Bd amplification and mortality in our focal terrestrial-breeding host: the pumpkin toadlet Brachycephalus pitanga. We also tested whether the strength of spillover could vary depending on skin bacterial transmission within host assemblages. Terrestrial-breeding toadlets acquired lethal spillover infections from neighbouring aquatic hosts and experienced dramatic but generally non-protective shifts in skin bacterial composition primarily attributable to their Bd infections. By contrast, aquatic-breeding amphibians maintained mild Bd infections and higher survival, with shifts in bacterial microbiomes that were unrelated to Bd infections. Our results indicate that Bd spillover from even mildly infected aquatic-breeding hosts may lead to dysbiosis and mortality in terrestrial-breeding species, underscoring the need to further investigate recent population declines of terrestrial-breeding amphibians in the tropics.

Arthropod-bacteria interactions influence assembly of aquatic host microbiome and pathogen defense.

The host-associated microbiome is vital to host immunity and pathogen defense. In aquatic ecosystems, organisms may interact with environmental bacteria to influence the pool of potential symbionts, but the effects of these interactions on host microbiome assembly and pathogen resistance are unresolved. We used replicated bromeliad microecosystems to test for indirect effects of arthropod-bacteria interactions on host microbiome assembly and pathogen burden, using tadpoles and the fungal amphibian pathogen Batrachochytrium dendrobatidis as a model host-pathogen system. Arthropods influenced host microbiome assembly by altering the pool of environmental bacteria, with arthropod-bacteria interactions specifically reducing host colonization by transient bacteria and promoting antimicrobial components of aquatic bacterial communities. Arthropods also reduced fungal zoospores in the environment, but fungal infection burdens in tadpoles corresponded most closely with arthropod-mediated patterns in microbiome assembly. This result indicates that the cascading effects of arthropods on the maintenance of a protective host microbiome may be more strongly linked to host health than negative effects of arthropods on pools of pathogenic zoospores. Our work reveals tight links between healthy ecosystem dynamics and the functioning of host microbiomes, suggesting that ecosystem disturbances such as loss of arthropods may have downstream effects on host-associated microbial pathogen defenses and host fitness.

Diverse genotypes of the amphibian-killing fungus produce distinct phenotypes through plastic responses to temperature.

Phenotypes are the target of selection and affect the ability of organisms to persist in variable environments. Phenotypes can be influenced directly by genes and/or by phenotypic plasticity. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has a global distribution, unusually broad host range, and high genetic diversity. Phenotypic plasticity may be an important process that allows this pathogen to infect hundreds of species in diverse environments. We quantified phenotypic variation of nine Bd genotypes from two Bd lineages (Global Pandemic Lineage [GPL] and Brazil) and a hybrid (GPL-Brazil) grown at three temperatures (12, 18 and 24°C). We measured five functional traits including two morphological traits (zoospore and zoosporangium sizes) and three life history traits (carrying capacity, time to fastest growth and exponential growth rate) in a phylogenetic framework. Temperature caused highly plastic responses within each genotype, with all Bd genotypes showing phenotypic plasticity in at least three traits. Among genotypes, Bd generally showed the same direction of plastic response to temperature: larger zoosporangia, higher carrying capacity, longer time to fastest growth and slower exponential growth at lower temperatures. The exception was zoospore size, which was highly variable. Our findings indicate that Bd genotypes have evolved novel phenotypes through plastic responses to temperature over very short timescales. High phenotypic variability likely extends to other traits and may facilitate the large host range and rapid spread of Bd.

Amphibian chytrid infection is influenced by rainfall seasonality and water availability.

Amphibians suffer from a number of factors that make them the most threatened group of vertebrates. One threat is the fungal disease chytridiomycosis caused by the emerging pathogen Batrachochytrium dendrobatidis (Bd), which has rapidly spread and caused the loss of massive amphibian biodiversity worldwide. Recently, Bd was associated with a few amphibian population declines and extinctions in some areas of the Brazilian Atlantic Forest. However, the mechanisms underlying such declines are not fully understood. Therefore, it is essential to improve our knowledge of abiotic factors that can possibly influence Bd prevalence and chytridiomycosis disease severity. Herein we tested the hypothesis that water availability (such as in perennial streams, where Bd is frequently present in larvae) and rainfall would increase the prevalence of Bd. To test this, we sampled frogs from 6 transects with different numbers of perennial waterbodies, and we report that the more water available in the area, the higher the probability of Bd infection on anurans. Seasonality also influenced both the Bd prevalence in the area and the intensity of infection in infected frogs. However, Bd prevalence was higher during the rainy months whereas the infection burden was lower. We suggest that Bd is likely spread during the summer, when most anuran species gather near the water for spawning and when rainfall overfills ephemeral wetlands. On the other hand, during the drier months, a higher infection burden may be explained by increased disease susceptibility.

Historical amphibian declines and extinctions in Brazil linked to chytridiomycosis.

The recent increase in emerging fungal diseases is causing unprecedented threats to biodiversity. The origin of spread of the frog-killing fungus Batrachochytrium dendrobatidis (Bd) is a matter of continued debate. To date, the historical amphibian declines in Brazil could not be attributed to chytridiomycosis; the high diversity of hosts coupled with the presence of several Bd lineages predating the reported declines raised the hypothesis that a hypervirulent Bd genotype spread from Brazil to other continents causing the recent global amphibian crisis. We tested for a spatio-temporal overlap between Bd and areas of historical amphibian population declines and extinctions in Brazil. A spatio-temporal convergence between Bd and declines would support the hypothesis that Brazilian amphibians were not adapted to Bd prior to the reported declines, thus weakening the hypothesis that Brazil was the global origin of Bd emergence. Alternatively, a lack of spatio-temporal association between Bd and frog declines would indicate an evolution of host resistance in Brazilian frogs predating Bd's global emergence, further supporting Brazil as the potential origin of the Bd panzootic. Here, we Bd-screened over 30 000 museum-preserved tadpoles collected in Brazil between 1930 and 2015 and overlaid spatio-temporal Bd data with areas of historical amphibian declines. We detected an increase in the proportion of Bd-infected tadpoles during the peak of amphibian declines (1979-1987). We also found that clusters of Bd-positive samples spatio-temporally overlapped with most records of amphibian declines in Brazil's Atlantic Forest. Our findings indicate that Brazil is post epizootic for chytridiomycosis and provide another piece to the puzzle to explain the origin of Bd globally.

Spatial distribution of Batrachochytrium dendrobatidis in South American caecilians.

The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) is linked to population declines in anurans and salamanders globally. To date, however, few studies have attempted to screen Bd in live caecilians; Bd-positive caecilians have only been reported in Africa and French Guiana. Here, we performed a retrospective survey of museum preserved specimens to (1) describe spatial patterns of Bd infection in Gymnophiona across South America and (2) test whether areas of low climatic suitability for Bd in anurans predict Bd spatial epidemiology in caecilians. We used quantitative PCR to detect Bd in preserved caecilians collected over a 109 yr period, and performed autologistic regressions to test the effect of bioclimatic metrics of temperature and precipitation, vegetation density, and elevation on the likelihood of Bd occurrence. We detected an overall Bd prevalence of 12.4%, with positive samples spanning the Uruguayan savanna, Brazil's Atlantic Forest, and the Amazon basin. Our autologistic models detected a strong effect of macroclimate, a weaker effect of vegetation density, and no effect of elevation on the likelihood of Bd occurrence. Although most of our Bd-positive records overlapped with reported areas of high climatic suitability for the fungus in the Neotropics, many of our new Bd-positive samples extend far into areas of poor suitability for Bd in anurans. Our results highlight an important gap in the study of amphibian chytridiomycosis: the potential negative impact of Bd on Neotropical caecilians and the hypothetical role of caecilians as Bd reservoirs.

Jumping into a trap: high prevalence of chytrid fungus in the preferred microhabitats of a bromeliad-specialist frog.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been identified as a major threat to several amphibian populations in tropical forests. Amphibians that inhabit the phytotelmata (water tanks) of bromeliads may be especially at risk of Bd infection since the humid, environmentally buffered microhabitat that they prefer might also be favorable for Bd persistence on the host. To test this hypothesis, we sampled adults and tadpoles of the bromeligenous anuran Phyllodytes edelmoi (endemic to the northern Brazilian Atlantic Forest) from the bromeliad Portea leptantha for Bd, using qPCR. We also analyzed 8 bromeliad characteristics: water tank temperature and pH, canopy closure, tank diameter, number of leaves, bromeliad maximum column depth to store water, bromeliad relative volume, and season. Adult frogs preferentially selected bromeliads with a smaller diameter, more leaves and a relatively higher volume of water. We found that Bd was more prevalent in frogs inhabiting bromeliad phytotelmata with smaller diameters, suggesting that the behavioral preferences of P. edelmoi may be driving Bd infection patterns. Therefore, species such as P. edelmoi will be trapped by their own natural history traits.

Physiological responses of Brazilian amphibians to an enzootic infection of the chytrid fungus Batrachochytrium dendrobatidis.

Pathophysiological effects of clinical chytridiomycosis in amphibians include disorders of cutaneous osmoregulation and disruption of the ability to rehydrate, which can lead to decreased host fitness or mortality. Less attention has been given to physiological responses of hosts where enzootic infections of Batrachochytrium dendrobatidis (Bd) do not cause apparent population declines in the wild. Here, we experimentally tested whether an enzootic strain of Bd causes significant mortality and alters host water balance (evaporative water loss, EWL; skin resistance, R(s); and water uptake, WU) in individuals of 3 Brazilian amphibian species (Dendropsophus minutus, n = 19; Ischnocnema parva, n = 17; Brachycephalus pitanga, n = 15). Infections with enzootic Bd caused no significant mortality, but we found an increase in R(s) in 1 host species concomitant with a reduction in EWL. These results suggest that enzootic Bd infections can indeed cause sub-lethal effects that could lead to reduction of host fitness in Brazilian frogs and that these effects vary among species. Thus, our findings underscore the need for further assessment of physiological responses to Bd infections in different host species, even in cases of sub-clinical chytridiomycosis and long-term enzootic infections in natural populations.

Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data.

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.