Response to thermal and hydric regimes point to differential inter- and intraspecific vulnerability of tropical amphibians to climate warming.

In Puerto Rico, an island threatened by climate warming, only one of two species of frogs that share part of their distribution has undergone a recent range contraction to higher elevations. We questioned if differences in their physiological response to temperature and dehydration might explain this distributional change. We studied a lowland and a highland population of Eleutherodactylus coqui, a widespread generalist, and E. portoricensis, an endangered species that is currently found only above 600 m. We compared various physiological aspects: operative temperature; temperature selection; critical temperatures; and their response to jumping performance tests at various thermal and hydric regimes. Results revealed that E. portoricensis had the highest CTmin and lowest CTmax and selected a cooler range of temperatures from the experimental gradient. Jumping performance increased with temperature for the three populations until attaining maximum performance. Afterwards, performance dropped drastically until reaching CTmax. Dehydration had a negative effect on performance for both species, particularly on maximum performance. This effect was greatest for E. portoricensis, followed by high-elevation E. coqui. The significantly greater thermo-hydric physiological limitations of E. portoricensis may explain its recent range contraction, potentially, as a response to climate warming. Low-elevation E. coqui had the lowest operative warming tolerance and was the only population to select temperatures like those encountered in their environment, indicating it may be narrowly adapted to local thermal conditions and thus, also vulnerable to climate change. Our results point towards plasticity in the response of E. coqui to varying climatic conditions, and present evidence of different physiological responses between closely related species at the same locality. This work highlights the importance of studying the combined effects of temperature and hydration to understand the response of ectotherms to warming environments and presents further evidence that desiccation may be a limiting factor determining which species may survive.

Genetic analysis of post-epizootic amphibian chytrid strains in Bolivia: Adding a piece to the puzzle.

The evolutionary history and dispersal pattern of Batrachochytrium dendrobatidis (Bd), an emergent fungal pathogen responsible for the decline and extinctions of many species of amphibians worldwide, is still not well understood. In South America, the tropical Andes are known as an important site for amphibian diversification, but also for being a place where hosts are at greater risk of chytridiomycosis. In an attempt to understand the history and the geographic pattern of Bd-associated amphibian declines in Bolivia, we isolated Bd from hosts at two locations that differ in their chronology of Bd prevalence and host survival outcome, the cloud forests of the Amazonian slopes of the Andes and Lake Titicaca in the altiplano. We genotyped Bd from both locations and sequenced the genome from the cloud forest isolate and then compared them to reference sequences of other Bd strains across the world. We found that the Bolivian chytrid isolates were nearly genotypically identical and that they belong to the global panzootic lineage (Bd-GPL). The Bolivian Bd strain grouped with other tropical New World strains but was closest to those from the Brazilian Atlantic Forest. Our results extend the presence of Bd-GPL to the central Andes in South America and report this hypervirulent strain at Lago Titicaca, where Bd has been detected since 1863, without evidence of amphibian declines. These findings suggest a more complex evolutionary history for this pathogen in Bolivia and may point to the existence of an old lineage of Bd that has since been extirpated following the arrival of the panzootic Bd-GPL or that the timing of Bd-GPL emergence is earlier than generally acknowledged.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

The Skin Microbiota of Eleutherodactylus Frogs: Effects of Host Ecology, Phylogeny, and Local Environment.

Amphibian skin microbiota has a potential protective role against diseases. However, the effects of environmental and host factors on symbiotic bacterial communities are not well understood. Caribbean frogs in the genus Eleutherodactylus represent a case of congeneric species that differ in ecological specialization by the process of adaptive radiation. For a small clade of Eleutherodactylus from Puerto Rico, we investigated the role of local environments, host species, and microhabitat in the composition of their skin microbiome. The potential congruence between microbial communities in hosts that are most closely related phylogenetically was also addressed. We hypothesized that the skin microbiota of Eleutherodactylus frogs would be mostly associated to microhabitat use, but also differ according to locality, and to a lesser extent to host species. To test this hypothesis, we swabbed the skin of a total of 98 adult individuals of seven Eleutherodactylus species distributed in two nearby localities in Puerto Rico, and sequenced the V4 region of the 16S rRNA gene. Results showed that locality had the greatest effect on determining skin bacterial communities of amphibian hosts, but this effect was stronger on the composition (based on presence/absence) than on its structure (based on sequence abundance). The most ecologically distinct host, E. cooki, and the generalist E. coqui presented, respectively, the most dissimilar and similar microbiota compared to other hosts. Host phylogeny showed a weak influence on skin microbiota. Results suggest that both local environment and ecological specialization are structuring the skin bacterial community in these Eleutherodactylus species, but that characteristics intrinsic to species may also render unique hosts the ability to maintain distinct microbiotas.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

A new species of Microkayla (Anura: Craugastoridae: Holoadeninae) from Department La Paz, Bolivia.

We describe a new species of direct-developing frog of the genus Microkayla from the Cordillera Real of the Bolivian Andes, in the Department of La Paz. The new species, Microkayla huayna sp. nov., is closely related to M. teqta and can be distinguished from other species of the genus by its brown dorsal skin and the presence of a large dark brown vocal sac in males. This is the second species of Microkayla known from the Zongo Valley, and the ninth in the Cordillera Real, contributing to a total of 22 described species in Bolivia. Given its small distribution range, we recommend to considering it as Vulnerable according to IUCN criteria.

Skin bacterial microbiome of a generalist Puerto Rican frog varies along elevation and land use gradients.

Host-associated microbial communities are ubiquitous among animals, and serve important functions. For example, the bacterial skin microbiome of amphibians can play a role in preventing or reducing infection by the amphibian chytrid fungus, Batrachochytrium dendrobatidis. Evidence suggests that environmental bacteria likely serve as a source pool for at least some of the members of the amphibian skin bacterial community, underscoring the potential for local environmental changes to disrupt microbial community source pools that could be critical to the health of host organisms. However, few studies have assessed variation in the amphibian skin microbiome along clear environmental gradients, and so we know relatively little about how local environmental conditions influence microbiome diversity. We sampled the skin bacterial communities of Coqui frogs, Eleutherodactylus coqui (N = 77), along an elevational gradient in eastern Puerto Rico (0-875 m), with transects in two land use types: intact forest (N = 4 sites) and disturbed (N = 3 sites) forest. We found that alpha diversity (as assessed by Shannon, Simpson, and Phylogenetic Diversity indices) varied across sites, but this variation was not correlated with elevation or land use. Beta diversity (community structure), on the other hand, varied with site, elevation and land use, primarily due to changes in the relative abundance of certain bacterial OTUs (∼species) within these communities. Importantly, although microbiome diversity varied, E. coqui maintained a common core microbiota across all sites. Thus, our findings suggest that environmental conditions can influence the composition of the skin microbiome of terrestrial amphibians, but that some aspects of the microbiome remain consistent despite environmental variation.

Detection of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis in Museum Specimens of Andean Aquatic Birds: Implications for Pathogen Dispersal.

The occurrence of the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) in the feet of live waterfowl has been documented, but the potential role of birds as dispersers has not been studied. We report the presence of Bd in the feet of preserved aquatic birds in the Bolivian high Andes during the time of drastic amphibian declines in the country. We sampled 48 aquatic birds from the Bolivian Andes that were preserved in museum collections. Birds were sampled for the presence of Bd DNA by swabbing, taking small pieces of tissue from toe webbing, or both. We detected Bd by DNA using quantitative PCR in 42% of the birds sampled via toe tissue pieces. This method was significantly better than swabbing at detecting Bd from bird feet. We confirmed Bd presence by sequencing Bd -positive samples and found 91-98% homology with Bd sequences from GenBank. Our study confirms that aquatic birds can carry Bd and thus may serve as potential vectors of this pathogen across large distances and complex landscapes. In addition, we recommend using DNA from preserved birds as a novel source of data to test hypotheses on the spread of chytridiomycosis in amphibians.

Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research.

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co-evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen "hot spots," we need to identify pathogen "cold spots" so that we can better understand what limits the pathogen's distribution. Finally, we introduce the concept of "the Ghost of Epizootics Past" to discuss expected patterns in postepizootic host communities.

Genomic Correlates of Virulence Attenuation in the Deadly Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis.

Emerging infectious diseasespose a significant threat to global health, but predicting disease outcomes for particular species can be complicated when pathogen virulence varies across space, time, or hosts. The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in frog populations. Not only do Bd isolates from wild populations vary in virulence, but virulence shifts can occur over short timescales when Bd is maintained in the laboratory. We leveraged changes in Bd virulence over multiple generations of passage to better understand mechanisms of pathogen virulence. We conducted whole-genome resequencing of two samples of the same Bd isolate, differing only in passage history, to identify genomic processes associated with virulence attenuation. The isolate with shorter passage history (and greater virulence) had greater chromosome copy numbers than the isolate maintained in culture for longer, suggesting that virulence attenuation may be associated with loss of chromosome copies. Our results suggest that genomic processes proposed as mechanisms for rapid evolution in Bd are correlated with virulence attenuation in laboratory culture within a single lineage of Bd. Moreover, these genomic processes can occur over extremely short timescales. On a practical level, our results underscore the importance of immediately cryo-archiving new Bd isolates and using fresh isolates, rather than samples cultured in the laboratory for long periods, for laboratory infection experiments. Finally, when attempting to predict disease outcomes for this ecologically important pathogen, it is critical to consider existing variation in virulence among isolates and the potential for shifts in virulence over short timescales.

Coqui frogs persist with the deadly chytrid fungus despite a lack of defensive antimicrobial peptides.

The amphibian skin fungus Batrachochytrium dendrobatidis (Bd) occurs widely in Puerto Rico and is thought to be responsible for the apparent extinction of 3 species of endemic frogs in the genus Eleutherodactylus, known as coquis. To examine immune defenses which may protect surviving species, we induced secretion of skin peptides from adult common coqui frogs E. coqui collected from upland forests at El Yunque. By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we were unable to detect peptide signals suggestive of antimicrobial peptides, and enriched peptides showed no capacity to inhibit growth of Bd. Thus, it appears that E. coqui depend on other skin defenses to survive in the presence of this deadly fungus.

A new species of Psychrophrynella (Anura: Craugastoridae) from the Cordillera Real, Department La Paz, Bolivia.

We describe a new species of Psychrophrynella from a single locality in the Cordillera Real, in the Andes of Department of La Paz, Bolivia. Psychrophrynella teqta sp. nov. is highly variable in color pattern and differs from the species geographically closer mostly by presenting yellow or red blotches, usually absent in other species. In addition, the new species differs from all species in the genus because the males have a pulsed call. Males of this species show parental care, and the high number of eggs per nest suggest that perhaps more than one clutch might be attended by a single male. As other species in the genus, the new species is infected by Batrachochytrium dendrobatidis, and thus, a conservation threat is already present upon its discovery.

Genomic studies of disease-outcome in host--pathogen dynamics.

Pathogens act as agents of evolutionary change in host populations, altering the host's allele frequencies and phenotypes through selection. The mechanisms underlying these adaptive changes depend on which defense strategy the host adopts upon infection. With increased anthropogenic change and loss of biodiversity, ecological impacts on adaptive processes may reduce the ability of hosts to evolve resistance, or to persist within their tolerance limits, thus increasing the capacity of pathogens to cause disease and mortality. In this review, we use amphibians and a pathogenic chytrid fungus (Batrachochytrium dendrobatidis, Bd) to illustrate how integrating genomic approaches into current research, both for hosts and pathogens, will improve our understanding of factors promoting the outcome of disease. As new emerging pathogens continue threatening amphibian populations worldwide, we recommend that researchers focus on individuals that survive after natural epizootics or experimental challenges. These survivors represent an underutilized and underexploited genetic resource for characterizing adaptive traits involved in the clearance of pathogens or in their tolerance. We highlight two target areas that will benefit from focused research: (1) Identification of the genetic basis of the hosts' defense strategies (resistance and tolerance) and of Bd's pathogenicity traits and (2) genomic characterization of shifts in fitness that drive seasonal and/or temporal patterns in host-pathogen interactions. To provide insights into hosts' survival, we review recent literature--including experimental Bd challenges and longitudinal studies--that underscore the complexity of Bd infections as determined by a combination of genetic and environmental factors. Given the heterogeneity of disease-outcomes and broad diversity of host species, amphibians provide a unique opportunity to identify novel genetic determinants of resistance to a recently emerged fungal pathogen. Developing additional genetic resources (e.g., genomic profiles, resistance mapping, and dual RNA-seq) will advance our understanding of the components of the innate and adaptive immune system acting on infected hosts in varying environments. These ecoimmunomic applications, which link host-pathogen eco-evolutionary processes with applied conservation efforts, will specifically benefit threatened amphibians that remain safeguarded in captive colonies.

Susceptibility to the amphibian chytrid fungus varies with ontogeny in the direct-developing frog, Eleutherodactylus coqui.

Age-related differences in susceptibility to infectious disease are known from a wide variety of plant and animal taxonomic groups. For example, the immature immune systems of young vertebrates, along with limited prior exposure to pathogens and behavioral factors, can place juveniles at greater risk of acquiring and succumbing to a pathogen. We studied the ontogenetic susceptibility of terrestrial direct-developing frogs (Eleutherodactylus coqui) to the fungal pathogen, Batrachochytrium dendrobatidis (Bd), which is responsible for the decline of amphibian species worldwide. By exposing juvenile and adult frogs to the same dose and strain of Bd, we uncovered ontogenetic differences in susceptibility. Froglets exposed to the pathogen had significantly lower survival rates compared with control froglets, while adult frogs largely cleared infection and had survival rates indistinguishable from control frogs, even when exposed to a much higher dose of Bd. The high disease-induced mortality rate of juveniles may explain ongoing population declines in eastern Puerto Rico, where Bd is endemic and juveniles experience higher prevalence and infection intensity compared to adults. Our results have important implications for understanding and modeling the decline, possibly to extinction, of amphibian populations and species.

A fungal pathogen of amphibians, Batrachochytrium dendrobatidis, attenuates in pathogenicity with in vitro passages.

Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen's role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.

ITS1 copy number varies among Batrachochytrium dendrobatidis strains: implications for qPCR estimates of infection intensity from field-collected amphibian skin swabs.

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.

Lability in host defenses: terrestrial frogs die from chytridiomycosis under enzootic conditions.

Chytridiomycosis-induced mortalities often occur upon the emergence of Batrachochytrium dendrobatidis (Bd) in naïve amphibian populations. We report chytridiomycosis-associated mortalities in the wild of the coqui (Eleutherodactylus coqui), a declining direct-developing frog with persistent Bd infections. These findings provide additional evidence of decreased host defenses during cool-dry seasons in Puerto Rico.

Teaching biology through statistics: application of statistical methods in genetics and zoology courses.

Incorporation of mathematics into biology curricula is critical to underscore for undergraduate students the relevance of mathematics to most fields of biology and the usefulness of developing quantitative process skills demanded in modern biology. At our institution, we have made significant changes to better integrate mathematics into the undergraduate biology curriculum. The curricular revision included changes in the suggested course sequence, addition of statistics and precalculus as prerequisites to core science courses, and incorporating interdisciplinary (math-biology) learning activities in genetics and zoology courses. In this article, we describe the activities developed for these two courses and the assessment tools used to measure the learning that took place with respect to biology and statistics. We distinguished the effectiveness of these learning opportunities in helping students improve their understanding of the math and statistical concepts addressed and, more importantly, their ability to apply them to solve a biological problem. We also identified areas that need emphasis in both biology and mathematics courses. In light of our observations, we recommend best practices that biology and mathematics academic departments can implement to train undergraduates for the demands of modern biology.

Persistence with Chytridiomycosis does not assure survival of direct-developing frogs.

The chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been linked to extinction and decline of numerous amphibians. We studied the population-level effects of Bd in two post-decline anuran species, Eleutherodactylus coqui and E. portoricensis, at El Yunque National Forest, Puerto Rico. Data on amphibian abundance was updated to report long-term population trends. Mark-recapture data was used to monitor Bd-infection status and estimate survival probabilities of infected versus uninfected adults. Prevalence of Bd (number of infected/total sampled) and individual infection level (number of zoospores) were compared among age classes at Palo Colorado Forest (661 m) and Elfin Forest (850 m). Results revealed that both species continued to decrease in Palo Colorado Forest, while in the Elfin Forest, E. portoricensis recuperated from drastic declines. Age class, season, and locality significantly predicted zoospore load. Age was also significantly associated with high zoospores loads among Bd-positive frogs, and the prevalence of Bd was higher in juveniles than adults in all populations studied. We suggest that early age represents a critical life stage in the survival of direct-developing frogs infected by this fungus. Survival probability was always higher for uninfected frogs, but recapture rates of infected versus uninfected adults were significantly different only in Palo Colorado, alerting that the negative effect of Bd infection under enzootic conditions is greater at mid-elevations. This work contributes to our understanding of how direct-developing amphibians persist with Bd, pointing to critical life stages and synergistic interactions that may induce fluctuations and/or declines in the wild.

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence.

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.