Widespread occurrence of the amphibian chytrid panzootic lineage in Uruguay is constrained by climate.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease among the main causes of amphibian declines worldwide. However, Bd studies on Neotropical amphibians from temperate areas are scarce. We present a comprehensive survey of Bd in Uruguay, in temperate central eastern South America, carried out between 2006 and 2014. Skin swabs of 535 specimens of 21 native and exotic frogs were tested by PCR. We used individual-level data to examine the relationship between infection, climatic variables, and their effects on body condition and the number of prey items found in stomach contents. Infection was widespread in free-ranging anurans with an overall prevalence of 41.9%, detected in 15 native species, wild American bullfrogs Aquarana catesbeiana, and captive specimens of Ceratophrys ornata and Xenopus laevis. Three haplotypes of the Bd ITS region were identified in native amphibians, all belonging to the global panzootic lineage (BdGPL), of which only one was present in exotic hosts. Despite high infection frequencies in different anurans, we found no evidence of morbidity or mortality attributable to chytridiomycosis, and we observed no discernible impact on body condition or consumed prey. Climatic conditions at the time of our surveys suggested that the chance of infection is associated with monthly mean temperature, mean humidity, and total precipitation. Temperatures below 21°C combined with moderate humidity and pronounced rainfall may increase the likelihood of infection. Multiple haplotypes of BdGPL combined with high frequencies of infection suggest an enzootic pattern in native species, underscoring the need for continued monitoring.

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.

Chiggers (Acariformes: Trombiculoidea) do not increase rates of infection by Batrachochytrium dendrobatidis fungus in the endemic Dwarf Mexican Treefrog Tlalocohyla smithii (Anura: Hylidae).

Amphibian populations are globally declining at an alarming rate, and infectious diseases are among the main causes of their decline. Two micro-parasites, the fungus Batrachochytrium dendrobatidis (Bd) and the virus Ranavirus (RV) have caused mass mortality of amphibians and population declines. Other, less understood epizootics are caused by macro-parasites, such as Trombiculoidea chiggers. Infection with chiggers can affect frog behavior and survival. Furthermore, synergistic effects of co-infection with both macro and micro-parasites may lead to higher morbidity. To better understand these potential synergies, we investigated the presence and co-infection by chiggers, Bd and RV in the endemic frog Tlalocohyla smithii (T. smithii). Co-infection of Bd, RV, and/or chiggers is expected in habitats that are suitable for their co-occurrence; and if infection with one parasite facilitates infection with the others. On the other hand, co-infection could decrease if these parasites were to differ in their micro-environmental requirements (i.e. niche apportionment). A total of 116 frogs of T. smithii were studied during 2014 and 2016 in three streams within the Chamela-Cuixmala Biosphere Reserve in Jalisco, Mexico. Our results show that 31% of the frogs were infected with Trombiculoidea chiggers (Hannemania sp. and Eutrombicula alfreddugesi); Hannemania prevalence increased with air temperature and decreased in sites with high canopies and with water pH values above 8.5 and below 6.7. Bd prevalence was 2.6%, RV prevalence was 0%, and none of the frogs infected with chiggers were co-infected with Bd. Together, this study suggests that chiggers do not facilitate infection with Bd, as these are apportioned in different micro-habitats. Nevertheless, the statistical power to assure this is low. We recommend further epidemiological monitoring of multiple parasites in different geographical locations in order to provide insight on the true hazards, risks and conservation options for amphibian populations.

Novel taxa in Cladochytriales (Chytridiomycota): Karlingiella (gen. nov.) and Nowakowskiella crenulata (sp. nov.).

Six Nowakowskiella species from Brazil were identified and purified on corn meal agar (CMA) plus glucose and Peptonized Milk-Tryptone-Glucose (PmTG) media and placed into a phylogenetic framework for the genus. New sequence data are presented for four species: N. elongata, N. multispora, and N. ramosa and the new species N. crenulata. Our maximum likelihood and Bayesian analyses of combined 18S, 5.8S, and 28S subunits of nuc rDNA showed that Nowakowskiella is not a monophyletic clade because of the position of Nowakowskiella elongata, which is more related to Cladochytrium. We reclassify N. elongata as the type of a new genus, Karlingiella.

Novel findings on the impact of chytridiomycosis on the cardiac function of anurans: sensitive vs. tolerant species.

BACKGROUND: Understanding of the physiological effects of chytridiomycosis is crucial to worldwide amphibian conservation. Therefore, we analyzed the cardiac function of two anuran species (Xenopus laevis and Physalaemus albonotatus) with different susceptibilities to infection by the causative agent of chytridiomycosis, Batrachochytrium dendrobatidis (hereafter Bd). METHODS: We analyzed the in situ heart rate (f H - bpm), relative ventricular mass (RVM -%), and Ca2+ handling in heart of Bd infected animals compared to uninfected controls of both study species. RESULTS: Bd infection resulted in a 78% decrease in contraction force values in P. albonotatus when compared to the less susceptible X. laevis. This negative effect was even more evident (82%) for the cardiac pumping capacity. The time to reach peak tension was 125% longer in P. albonotatus than in X. laevis, and cardiac relaxation was 57% longer. DISCUSSION: These results indicate a delay in the cardiac cycle of P. albonotatus on a beat-to-beat basis, which was corroborated by the bradycardia observed in situ. In summary, Bd-sensitive species present impaired cardiac function, which could be a factor in mortality risk. The more pronounced effects of Bd in P. albonotatus may not only result from electrolyte imbalance, as previously reported, but also could be an effect of toxins produced by Bd. For X. laevis, the ability to promote cardiac adjustments seems to be an important homeostatic feature that allows greater tolerance to chytridiomycosis. This study provides new physiological mechanisms underlying the tolerance or susceptibility of amphibian species to chytridiomycosis, which determine their adaptability to survive in the affected environments.

Eco-evolutionary rescue promotes host-pathogen coexistence.

Emerging infectious pathogens are responsible for some of the most severe host mass mortality events in wild populations. Yet, effective pathogen control strategies are notoriously difficult to identify, in part because quantifying and forecasting pathogen spread and disease dynamics is challenging. Following an outbreak, hosts must cope with the presence of the pathogen, leading to host-pathogen coexistence or extirpation. Despite decades of research, little is known about host-pathogen coexistence post-outbreak when low host abundances and cryptic species make these interactions difficult to study. Using a novel disease-structured N-mixture model, we evaluate empirical support for three host-pathogen coexistence hypotheses (source-sink, eco-evolutionary rescue, and spatial variation in pathogen transmission) in a Neotropical amphibian community decimated by Batrachochytrium dendrobatidis (Bd) in 2004. During 2010-2014, we surveyed amphibians in Parque Nacional G. D. Omar Torríjos Herrera, Coclé Province, El Copé, Panama. We found that the primary driver of host-pathogen coexistence was eco-evolutionary rescue, as evidenced by similar amphibian survival and recruitment rates between infected and uninfected hosts. Average apparent monthly survival rates of uninfected and infected hosts were both close to 96%, and the expected number of uninfected and infected hosts recruited (via immigration/reproduction) was less than one host per disease state per 20-m site. The secondary driver of host-pathogen coexistence was spatial variation in pathogen transmission as we found that transmission was highest in areas of low abundance but there was no support for the source-sink hypothesis. Our results indicate that changes in the host community (i.e., through genetic or species composition) can reduce the impacts of emerging infectious disease post-outbreak. Our disease-structured N-mixture model represents a valuable advancement for conservation managers trying to understand underlying host-pathogen interactions and provides new opportunities to study disease dynamics in remnant host populations decimated by virulent pathogens.

Differential patterns of Batrachochytrium dendrobatidis infection in relict amphibian populations following severe disease-associated declines.

Global amphibian biodiversity has declined dramatically in the past 4 decades, and many amphibian species have declined to near extinction as a result of emergence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). However, persistent or recovering populations of several amphibian species have recently been rediscovered, and such populations may illustrate how amphibian species that are highly susceptible to chytridiomycosis may survive in the presence of Bd. We conducted field surveys for Bd infection in 7 species of Costa Rican amphibians (all species that have declined to near extinction but for which isolated populations persist) to characterize infection profiles in highly Bd-susceptible amphibians post-decline. We found highly variable patterns in infection, with some species showing low prevalence (~10%) and low infection intensity and others showing high infection prevalence (>80%) and either low or high infection intensity. Across sites, infection rates were negatively associated with mean annual precipitation, and infection intensity across sites was negatively associated with mean average temperatures. Our results illustrate that even the most Bd-susceptible amphibians can persist in Bd-enzootic ecosystems, and that multiple ecological or evolutionary mechanisms likely exist for host-pathogen co-existence between Bd and the most Bd-susceptible amphibian species. Continued monitoring of these populations is necessary to evaluate population trends (continuing decline, stability, or population growth). These results should inform efforts to mitigate impacts of Bd on amphibians in the field.

Land cover and forest connectivity alter the interactions among host, pathogen and skin microbiome.

Deforestation has detrimental consequences on biodiversity, affecting species interactions at multiple scales. The associations among vertebrates, pathogens and their commensal/symbiotic microbial communities (i.e. microbiomes) have important downstream effects for biodiversity conservation, yet we know little about how deforestation contributes to changes in host microbial diversity and pathogen abundance. Here, we tested the effects of landcover, forest connectivity and infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) on amphibian skin bacterial diversity along deforestation gradients in Brazilian landscapes. If disturbance to natural habitat alters skin microbiomes as it does in vertebrate host communities, then we would expect higher host bacterial diversity in natural forest habitats. Bd infection loads are also often higher in these closed-canopy forests, which may in turn impact skin-associated bacterial communities. We found that forest corridors shaped composition of host skin microbiomes; high forest connectivity predicted greater similarity of skin bacterial communities among host populations. In addition, we found that host skin bacterial diversity and Bd loads increased towards natural vegetation. Because symbiotic bacteria can potentially buffer hosts from Bd infection, we also evaluated the bi-directional microbiome-Bd link but failed to find a significant effect of skin bacterial diversity reducing Bd infections. Although weak, we found support for Bd increasing bacterial diversity and/or for core bacteria dominance reducing Bd loads. Our research incorporates a critical element in the study of host microbiomes by linking environmental heterogeneity of landscapes to the host-pathogen-microbiome triangle.

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.

Variation in Metabolite Profiles of Amphibian Skin Bacterial Communities Across Elevations in the Neotropics.

Both the structure and function of host-associated microbial communities are potentially impacted by environmental conditions, just as the outcomes of many free-living species interactions are context-dependent. Many amphibian populations have declined around the globe due to the fungal skin pathogen, Batrachochytrium dendrobatidis (Bd), but enivronmental conditions may influence disease dynamics. For instance, in Panamá, the most severe Bd outbreaks have occurred at high elevation sites. Some amphibian species harbor bacterial skin communities that can inhibit the growth of Bd, and therefore, there is interest in understanding whether environmental context could also alter these host-associated microbial communities in a way that might ultimately impact Bd dynamics. In a field survey in Panamá, we assessed skin bacterial communities (16S rRNA amplicon sequencing) and metabolite profiles (HPLC-UV/Vis) of Silverstoneia flotator from three high- and three low-elevation populations representing a range of environmental conditions. Across elevations, frogs had similar skin bacterial communities, although one lowland site appeared to differ. Interestingly, we found that bacterial richness decreased from west to east, coinciding with the direction of Bd spread through Panamá. Moreover, metabolite profiles suggested potential functional variation among frog populations and between elevations. While the frogs have similar bacterial community structure, the local environment might shape the metabolite profiles. Ultimately, host-associated community structure and function could be dependent on environmental conditions, which could ultimately influence host disease susceptibility across sites.

Effects of amphibian phylogeny, climate and human impact on the occurrence of the amphibian-killing chytrid fungus.

Chytridiomycosis, due to the fungus Batrachochytrium dendrobatidis (Bd), has been associated with the alarming decline and extinction crisis of amphibians worldwide. Because conservation programs are implemented locally, it is essential to understand how the complex interactions among host species, climate and human activities contribute to Bd occurrence at regional scales. Using weighted phylogenetic regressions and model selection, we investigated geographic patterns of Bd occurrence along a latitudinal gradient of 1500 km within a biodiversity hot spot in Chile (1845 individuals sampled from 253 sites and representing 24 species), and its association with climatic, socio-demographic and economic variables. Analyses show that Bd prevalence decreases with latitude although it has increased by almost 10% between 2008 and 2013, possibly reflecting an ongoing spread of Bd following the introduction of Xenopus laevis. Occurrence of Bd was higher in regions with high gross domestic product (particularly near developed centers) and with a high variability in rainfall regimes, whereas models including other bioclimatic or geographic variables, including temperature, exhibited substantially lower fit and virtually no support based on Akaike weights. In addition, Bd prevalence exhibited a strong phylogenetic signal, with five species having high numbers of infected individuals and higher prevalence than the average of 13.3% across all species. Taken together, our results highlight that Bd in Chile might still be spreading south, facilitated by a subset of species that seem to play an important epidemiological role maintaining this pathogen in the communities, in combination with climatic and human factors affecting the availability and quality of amphibian breeding sites. This information may be employed to design conservation strategies and mitigate the impacts of Bd in the biodiversity hot spot of southern Chile, and similar studies may prove useful to disentangle the role of different factors contributing to the emergence and spread of this catastrophic disease.

A new species of Psychrophrynella (Amphibia, Anura, Craugastoridae) from the humid montane forests of Cusco, eastern slopes of the Peruvian Andes.

We describe a new species of Psychrophrynella from the humid montane forest of the Department Cusco in Peru. Specimens were collected at 2,670-3,165 m elevation in the Área de Conservación Privada Ukumari Llakta, Japumayo valley, near Comunidad Campesina de Japu, in the province of Paucartambo. The new species is readily distinguished from all other species of Psychrophrynella but P. bagrecito and P. usurpator by possessing a tubercle on the inner edge of the tarsus, and from these two species by its yellow ventral coloration on abdomen and limbs. Furthermore, the new species is like P. bagrecito and P. usurpator in having an advertisement call composed of multiple notes, whereas other species of Psychrophrynella whose calls are known have a pulsed call (P. teqta) or a short, tonal call composed of a single note. The new species has a snout-vent length of 16.1-24.1 mm in males and 23.3-27.7 mm in females. Like other recently described species in the genus, this new Psychrophrynella inhabits high-elevation forests in the tropical Andes and likely has a restricted geographic distribution.

Long-term endemism of two highly divergent lineages of the amphibian-killing fungus in the Atlantic Forest of Brazil.

The recent global spread of the amphibian-killing fungus [Batrachochytrium dendrobatidis (Bd)] has been closely tied to anthropogenic activities; however, regional patterns of spread are not completely understood. Using historical samples, we can test whether Bd was a spreading or endemic pathogen in a region within a particular time frame, because those two disease states provide different predictions for the regional demographic dynamics and population genetics of Bd. Testing historical patterns of pathogen prevalence and population genetics under these predictions is key to understanding the evolution and origin of Bd. Focusing on the Atlantic Forest (AF) of Brazil, we used qPCR assays to determine the presence or absence of Bd on 2799 preserved postmetamorphic anurans collected between 1894 and 2010 and used semi-nested PCRs to determine the frequency of rRNA ITS1 haplotypes from 52 samples. Our earliest date of detection was 1894. A mean prevalence of 23.9% over time and spatiotemporal patterns of Bd clusters indicate that Bd has been enzootic in the Brazilian AF with no evidence of regional spread within the last 116 years. ITS1 haplotypes confirm the long-term presence of two divergent strains of Bd (BdGPL and Bd-Brazil) and three spatiotemporally broad genetic demes within BdGPL, indicating that Bd was not introduced into southeast Brazil by the bullfrog trade. Our data show that the evolutionary history and pathogen dynamics of Bd in Brazil is better explained by the endemic pathogen hypothesis.

Three new genera in Chytridiales from aquatic habitats in Argentina.

Sampling for chytrids in a variety of habitats has resulted in pure cultures that when analyzed have yielded hypotheses of relationships based on molecular and zoospore ultrastructural markers. To extend our understanding of diversity of Chytridiales in eastern Argentina and USA, we isolated and examined the morphology, ultrastructure and 28S and ITS1-5.8S-ITS2 rDNA sequences of numerous chytrids from aquatic habitats from these two regions. Three family-level lineages (Chytridiaceae, Chytriomycetaceae, family incertae sedis) are represented in our molecular phylogeny, and three new genera (Avachytrium, Odontochytrium in Chytriomycetaceae, Delfinachytrium in family incertae sedis) are described. These findings of new genera and species emphasize the potential for discovery of additional diversity.