The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines, and the extinctions of stream-breeding amphibian species worldwide. While the factors that limit the distribution and abundance of B. dendrobatidis across large geographical regions are fairly well understood, little is known about the distribution of the fungus within localized areas such as individual catchments. The accurate identification of amphibian populations likely to be exposed to the fungus is urgently required for effective disease management. We conducted disease surveys of frogs representing five ecological guilds in south-east Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of stream-breeding amphibian populations, infection prevalence and intensity would be greatest in frogs breeding in permanent, flowing water. Overall, 30.3% of the 519 frogs we sampled were infected with B. dendrobatidis . However, infections were not evenly distributed across the ecological guilds, being almost completely restricted to frogs breeding at permanent waterbodies. Of these, stream breeders were significantly more likely to be infected than were pond breeders, though the intensity of frogs' infections did not differ significantly between the two guilds. Batrachochytrium dendrobatidis was detected on only one of the 117 frogs that were found at ephemeral ponds, ephemeral streams, or terrestrial sites. These findings provide strong support for the hypothesis that B. dendrobatidis was responsible for many of the unexplained disappearances of stream-breeding amphibian populations in recent decades, and will enable wildlife managers to more accurately focus conservation efforts on those species at highest risk of disease-related decline.     

Where are the survivors? Tracking relictual populations of endangered frogs in Costa Rica

Aim We used abiotic environmental variables and historical locality records to infer distributions of endangered anuran species of Costa Rica to promote efficient strategies for future amphibian surveys. Location Costa Rica. Methods We used a Maximum Entropy Algorithm (Maxent) to predict potential distribution maps for 17 species of endangered anurans and create a consensus map of species richness. We compared the environmental conditions from localities where relictual amphibian populations were recently rediscovered with the conditions across their historical range to evaluate the possibility that these relictual populations might occur in specific climatic conditions that could explain their persistence. We used a multicriteria analysis considering the following factors: the intersection zones between the consensus map, conservation areas, potential Batrachochytrium dendrobatidis (Bd) distribution, collecting effort and areas within the precipitation range at which reappearances had occurred to locate sites for future surveys. Results The resulting predictions suggest that suitable areas for the highest number of species occur between 1300 and 2500 m.a.s.l and are concentrated along the Pacific slopes of the Cordillera de Talamanca and Cordillera Volcánica Central. Around 45% of the high potential richness area is under protection. Relictual populations of declined species seem to persist mainly in highly humid localities (2500–3500 mm of mean annual precipitation). Around 240 km² has an ideal environment for the rediscovery of relictual populations. The multicriteria analysis showed that around 0.5% of the Costa Rican territory should be surveyed exhaustively for frogs. Main conclusions Many of the potential refugia we identified here have not been surveyed since 2000, the areas identified by the best model predictions correspond well with the localities of the relictual populations recently reported. We suggest future surveys of missing amphibian species should focus on these areas. The discovery of populations of endangered species can be used to propose conservation areas.     

Temperature alters reproductive life history patterns in Batrachochytrium dendrobatidis,a lethal pathogen associated with the global loss of amphibians

Understanding how pathogens respond to changing environmental conditions is a central challenge in disease ecology. The environmentally sensitive fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the amphibian disease chytridiomycosis, has spread globally causing amphibian extirpations in a wide variety of climatic regions. To gain an in‐depth understanding of Bd's responses to temperature, we used an integrative approach, combining empirical laboratory experiments with mathematical modeling. First, we selected a single Bd isolate and serially propagated two lineages of the isolate for multiple generations in two stable thermal conditions: 4°C (cold‐adapted lineage) and 23°C (warm‐adapted lineage). We quantified the production of infectious zoospores (fecundity), the timing of zoospore release, and zoospore activity in reciprocal temperature transplant experiments in which both Bd lineages were grown in either high or low temperature conditions. We then developed population growth models for the Bd lineages under each set of temperature conditions. We found that Bd had lower population growth rates, but longer periods of zoospore activity in the low temperature treatment (4°C) compared to the high temperature treatment (23°C). This effect was more pronounced in Bd lineages that were propagated in the low temperature treatment (4°C), suggesting a shift in Bd's response to low temperature conditions. Our results provide novel insights into the mechanisms by which Bd can thrive in a wide variety of temperature conditions, potentially altering the dynamics of chytridiomycosis and thus, the propensity for Bd to cause amphibian population collapse. We also suggest that the adaptive responses of Bd to thermal conditions warrant further investigation, especially in the face of global climate change.     

Infection with Batrachochytrium dendrobatidis is common in tropical lowland habitats: Implications for amphibian conservation

Numerous species of amphibians declined in Central America during the 1980s and 1990s. These declines mostly affected highland stream amphibians and have been primarily linked to chytridiomycosis, a deadly disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Since then, the majority of field studies on Bd in the Tropics have been conducted in midland and highland environments (>800 m) mainly because the environmental conditions of mountain ranges match the range of ideal abiotic conditions for Bd in the laboratory. This unbalanced sampling has led researchers to largely overlook host–pathogen dynamics in lowlands, where other amphibian species declined during the same period. We conducted a survey testing for Bd in 47 species (n = 348) in four lowland sites in Costa Rica to identify local host–pathogen dynamics and to describe the abiotic environment of these sites. We detected Bd in three sampling sites and 70% of the surveyed species. We found evidence that lowland study sites exhibit enzootic dynamics with low infection intensity and moderate to high prevalence (55% overall prevalence). Additionally, we found evidence that every study site represents an independent climatic zone, where local climatic differences may explain variations in Bd disease dynamics. We recommend more detection surveys across lowlands and other sites that have been historically considered unsuitable for Bd occurrence. These data can be used to identify sites for potential disease outbreaks and amphibian rediscoveries.     

Predicting the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis in the New World1

One application of ecological niche modeling is predicting suitable areas for the establishment of invasive species. Herein, I model the fundamental niche of the chytrid fungus Batrachochytrium dendrobatidis, a pathogen linked to amphibian declines on several continents. Niche models were generated with the Genetic Algorithm of Rule‐Set Prediction using point distribution data of the pathogen and digital maps of environmental variables integrated in a GIS environment. The distribution of regions suitable for B. dendrobatidis in the New World is extensive and includes significant portions of: (1) Sierra Madre Occidental pine‐oak forest; (2) Sonoran and Sinaloan dry forest; (3) Veracruz moist forest; (4) Central America east from the Isthmus of Tehuantepec; (5) Caribbean Islands; (6) temperate forest in Chile and western Argentina south of latitude 30°S; (7) Andes above 1000 m of altitude in Venezuela, Colombia, and Ecuador; (8) eastern slopes of the Andes in Peru and Bolivia; (9) Brazilian Atlantic forest; (10) Uruguay, Paraguay, and northeastern Argentina; (11) southwestern and Madeira‐Tapajós Amazonian tropical rainforests. The regions with the highest suitability for B. dendrobatidis include habitats that contain the world's most diverse amphibian faunas. Models were built with New World localities, but also showed strong predictability for B. dendrobatidis localities in the Old World. Out of a total of 59 reported Old World localities for B. dendrobatidis, 56 occurred within regions with high predicted suitability. I also present analyses of the environmental envelope of B. dendrobatidis and discuss the implications of the results for the conservation of amphibians in the neotropics.     

Fluorescent probes as a tool for labelling and tracking the amphibian chytrid fungus Batrachochytrium dendrobatidis

The dissemination of the virulent pathogen Batrachochytrium dendrobatidis (Bd) has contributed to the decline and extinction of many amphibian species worldwide. Several different strains have been identified, some of which are sympatric. Interactions between co-infecting strains of a pathogen can have significant influences on disease epidemiology and evolution; therefore the dynamics of multi-strain infections is an important area of research. We stained Bd cells with 2 fluorescent BODIPY fatty acid probes to determine whether these can potentially be used to distinguish and track Bd cell lines in multi-strain experiments. Bd cells in broth culture were stained with 5 concentrations of green-fluorescent BODIPY FL and red-fluorescent BODIPY 558/568 and visualised under an epifluorescent microscope for up to 16 d post-dye. Dyed strains were also assessed for growth inhibition. The most effective concentration for both dyes was 10 pM. This concentration of dye produced strong fluorescence for 12 to 16 d in Bd cultures held at 23 degrees C (3 to 4 generations), and did not inhibit Bd growth. Cells dyed with BODIPY FL and BODIPY 558/568 can be distinguished from each other on the basis of their fluorescence characteristics. Therefore, it is likely that this technique will be useful for research into multi-strain dynamics of Bd infections.     

Unlocking the story in the swab: A new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole‐genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field‐collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole‐genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.     

Experimental evolution alters the rate and temporal pattern of population growth in Batrachochytrium dendrobatidis,a lethal fungal pathogen of amphibians

Virulence of infectious pathogens can be unstable and evolve rapidly depending on the evolutionary dynamics of the organism. Experimental evolution can be used to characterize pathogen evolution, often with the underlying objective of understanding evolution of virulence. We used experimental evolution techniques (serial transfer experiments) to investigate differential growth and virulence of Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes amphibian chytridiomycosis. We tested two lineages of Bd that were derived from a single cryo‐archived isolate; one lineage (P10) was passaged 10 times, whereas the second lineage (P50) was passaged 50 times. We quantified time to zoospore release, maximum zoospore densities, and timing of zoospore activity and then modeled population growth rates. We also conducted exposure experiments with a susceptible amphibian species, the common green tree frog (Litoria caerulea) to test the differential pathogenicity. We found that the P50 lineage had shorter time to zoospore production (T_min), faster rate of sporangia death (d_s), and an overall greater intrinsic population growth rate (λ). These patterns of population growth in vitro corresponded with higher prevalence and intensities of infection in exposed Litoria caerulea, although the differences were not significant. Our results corroborate studies that suggest that Bd may be able to evolve relatively rapidly. Our findings also challenge the general assumption that pathogens will always attenuate in culture because shifts in Bd virulence may depend on laboratory culturing practices. These findings have practical implications for the laboratory maintenance of Bd isolates and underscore the importance of understanding the evolution of virulence in amphibian chytridiomycosis.     

Estimating soil carbon fluxes following land-cover change: a test of some critical assumptions for a region in Costa Rica

Changes in soil carbon storage that accompany land‐cover change may have significant effects on the global carbon cycle. The objective of this work was to examine how assumptions about preconversion soil C storage and the effects of land‐cover change influence estimates of regional soil C storage. We applied three models of land‐cover change effects to two maps of preconversion soil C in a 140 000 ha area of northeastern Costa Rica. One preconversion soil C map was generated using values assigned to tropical wet forest from the literature, the second used values obtained from extensive field sampling. The first model of land‐cover change effects used values that are typically applied in global assessments, the second and third models used field data but differed in how the data were aggregated (one was based on land‐cover transitions and one was based on terrain attributes). Changes in regional soil C storage were estimated for each combination of model and preconversion soil C for three time periods defined by geo‐referenced land‐cover maps. The estimated regional soil C under forest vegetation (to 0.3 m) was higher in the map based on field data (10.03 Tg C) than in the map based on literature data (8.90 Tg C), although the range of values derived from propagating estimation errors was large (7.67–12.40 Tg C). Regional soil C storage declined through time due to forest clearing for pasture and crops. Estimated CO₂ fluxes depended more on the model of land‐cover change effects than on preconversion soil C. Cumulative soil C losses (1950–1996) under the literature model of land‐cover effects exceeded estimates based on field data by factors of 3.8–8.0. In order to better constrain regional and global‐scale assessments of carbon fluxes from soils in the tropics, future research should focus on methods for extrapolating regional‐scale constraints on soil C dynamics to larger spatial and temporal scales.     

Genetic evidence for a high diversity and wide distribution of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphibians

Population declines and extinctions of amphibians have been attributed to the chytrid fungus Batrachochytrium dendrobatidis (Bd), especially one globally emerging recombinant lineage (‘Bd‐GPL’). We used PCR assays that target the ribosomal internal transcribed spacer region (ITS) of Bd to determine the prevalence and genetic diversity of Bd in South Korea, where Bd is widely distributed but is not known to cause morbidity or mortality in wild populations. We isolated Korean Bd strains from native amphibians with low infection loads and compared them to known worldwide Bd strains using 19 polymorphic SNP and microsatellite loci. Bd prevalence ranged between 12.5 and 48.0%, in 11 of 17 native Korean species, and 24.7% in the introduced bullfrog Lithobates catesbeianus. Based on ITS sequence variation, 47 of the 50 identified Korean haplotypes formed a group closely associated with a native Brazilian Bd lineage, separated from the Bd‐GPL lineage. However, multilocus genotyping of three Korean Bd isolates revealed strong divergence from both Bd‐GPL and the native Brazilian Bd lineages. Thus, the ITS region resolves genotypes that diverge from Bd‐GPL but otherwise generates ambiguous phylogenies. Our results point to the presence of highly diversified endemic strains of Bd across Asian amphibian species. The rarity of Bd‐GPL‐associated haplotypes suggests that either this lineage was introduced into Korea only recently or Bd‐GPL has been outcompeted by native Bd strains. Our results highlight the need to consider possible complex interactions among native Bd lineages, Bd‐GPL and their associated amphibian hosts when assessing the spread and impact of Bd‐GPL on worldwide amphibian populations.     

Ants on the Move: Resource Limitation of a Litter‐nesting Ant Community in Costa Rica1

The leaf litter of tropical wet forests is replete with itinerant ant nests. Nest movement may help ants evade the constraints of stress and disturbance and increase access to resources. I studied how nest relocation and environmental factors may explain the density, size, and growth of leaf litter ant nests. I decoupled the relationships among litter depth, food abundance, and nest availability in a 4‐mo manipulation of food and leaf litter in a community of litter‐nesting ants in a lowland wet forest in Costa Rica. Over 4 mo, 290 1 m² treatment and control plots were sampled without replacement. Nest densities doubled in response to food supplementation, but did not decrease in response to litter removal or stress (from litter trampling). The supplementation of food increased the utilization of less favored nesting materials. In response to food supplementation and litter trampling, arboreal ants established nests in the litter, and growth rates of the most common ants (Pheidole spp.) increased. Colony growth was independent of colony size and growth rates of the most abundant ants. In general, I conclude that litter‐nesting ant density is driven primarily by food limitation, that nest relocation behavior significantly affects access to resource and the demographic structure of this community, and that nest fission may be a method to break the growth–reproduction trade‐off.     

Assessing the value of secondary forest for amphibians: <Emphasis Type="Italic">Eleutherodactylus</Emphasis> frogs in a gradient of forest alteration

Secondary forests constitute a growing portion of forested areas worldwide. They might have a substantial role for the conservation of biodiversity in tropical areas, but there is little information on their potential to support forest species and the recovery of faunal communities. We studied two forest frogs (Eleutherodactylus diastema and E. fitzingeri) in an area of Costa Rica composed of a mosaic of primary forest, young secondary forest and pasture, and we compared the density of calling males in areas with different forest alteration. Autoregressive models were used to compensate for potentially undesired effects of spatial autocorrelation and pseudoreplication. Both species were most abundant in riparian, primary forest. However, E. fitzingeri was also abundant in riparian secondary forests, and its density far from the river was similar in primary and secondary forest, suggesting that river proximity can influence the recovery of secondary forest for amphibians. Conversely, the density of E. diastema was similar in secondary forest and pasture, stressing interspecific differences for recovery rate. These frogs have a keystone role in nutrient cycling and food webs, and their prompt recovery might represent an important step for the functional recovery of forests. Nevertheless, the strong interspecific differences stress the complexity of these processes.