Spatio-temporal dynamics and drivers of highly pathogenic avian influenza H5N1 in Chile.

Introduction: Highly pathogenic avian influenza A H5N1 clade 2.3.4.4b (hereafter H5N1) is causing vast impacts on biodiversity and poultry around the globe. In Chile, lethal H5N1 cases have been reported in a wide range of wild bird species, marine mammals, backyard and industrial poultry, and humans. This study describes the spatio-temporal patterns of the current epizootic of H5N1 in Chile and test drivers that could be associated with outbreak occurrence. Methods: We used H5N1 cases reported by the Chilean National Animal Health Authority from 5 December 2022 to 5 April 2023. These included wild bird cases confirmed through an avian influenza-specific real-time reverse transcription PCR assay (RT-qPCR), obtained from passive and active surveillance. Data were analyzed to detect the presence of H5N1 clusters under space-time permutation probability modeling, the association of H5N1 with distance and days since the first outbreak through linear regression, and the correlation of H5N1 presence with a number of ecological and anthropogenic variables using general linear modeling. Results: From 445 H5N1 identified outbreaks involving 613 individual cases in wild birds, a consistent wave-like spread of H5N1 from north to south was identified, which may help predict hotspots of outbreak risk. For instance, seven statistically significant clusters were identified in central and northern Chile, where poultry production and wildlife mortality are concentrated. The presence of outbreaks was correlated with landscape-scale variables, notably temperature range, bird richness, and human footprint. Discussion: In less than a year, H5N1 has been associated with the unusual mortality of >100,000 individuals of wild animals in Chile, mainly coastal birds and marine mammals. It is urgent that scientists, the poultry sector, local communities, and national health authorities co-design and implement science-based measures from a One Health perspective to avoid further H5N1 spillover from wildlife to domestic animals and humans, including rapid removal and proper disposal of wild dead animals and the closure of public areas (e.g., beaches) reporting high wildlife mortalities.

Is Xenopus laevis introduction linked with Ranavirus incursion, persistence and spread in Chile?

Ranaviruses have been associated with amphibian, fish and reptile mortality events worldwide and with amphibian population declines in parts of Europe. Xenopus laevis is a widespread invasive amphibian species in Chile. Recently, Frog virus 3 (FV3), the type species of the Ranavirus genus, was detected in two wild populations of this frog near Santiago in Chile, however, the extent of ranavirus infection in this country remains unknown. To obtain more information about the origin of ranavirus in Chile, its distribution, species affected, and the role of invasive amphibians and freshwater fish in the epidemiology of ranavirus, a surveillance study comprising wild and farmed amphibians and wild fish over a large latitudinal gradient (2,500 km) was carried out in 2015-2017. In total, 1,752 amphibians and 496 fish were tested using a ranavirus-specific qPCR assay, and positive samples were analyzed for virus characterization through whole genome sequencing of viral DNA obtained from infected tissue. Ranavirus was detected at low viral loads in nine of 1,011 X. laevis from four populations in central Chile. No other amphibian or fish species tested were positive for ranavirus, suggesting ranavirus is not threatening native Chilean species yet. Phylogenetic analysis of partial ranavirus sequences showed 100% similarity with FV3. Our results show a restricted range of ranavirus infection in central Chile, coinciding with X. laevis presence, and suggest that FV3 may have entered the country through infected X. laevis, which appears to act as a competent reservoir host, and may contribute to the spread the virus locally as it invades new areas, and globally through the pet trade.

Interpopulation differences in male reproductive effort drive the population dynamics of a host exposed to an emerging fungal pathogen.

Compensatory recruitment is a key demographic mechanism that has allowed the coexistence of populations of susceptible amphibians with Batrachochytrium dendrobatidis (Bd), a fungus causing one of the most devastating emerging infectious disease ever recorded among vertebrates. However, the underlying processes (e.g. density-dependent increase in survival at early life stages, change in reproductive traits) as well as the level of interpopulation variation in this response are poorly known. We explore potential mechanisms of compensatory recruitment in response to Bd infection by taking advantage of an amphibian system where male reproductive traits are easy to quantify in free-living populations. The Southern Darwin's frog Rhinoderma darwinii is a vocal sac-brooding species that exhibits a high susceptibility to lethal Bd infection. Using a 7-year capture-recapture study at four populations with contrasting Bd infection status (one high prevalence, one low prevalence and two Bd-free populations), we evaluated whether Bd-positive populations exhibited a higher adult recruitment and a higher male reproductive effort than Bd-negative populations. We also estimated population growth rates to explore whether recruitment compensated for the negative impacts of Bd on the survival of adults. In addition, we evaluated a potential demographic signal of compensatory recruitment (i.e. positive relationship between the proportion of juveniles and Bd prevalence) in response to Bd infection using raw count data from 13 R. darwinii populations. The high Bd prevalence population exhibited the highest male reproductive effort and the highest recruitment among the four monitored populations. This led to a growing population during the study period despite high mortality of adult hosts. In contrast, males from the population with low Bd prevalence had a low reproductive effort and this population, which had the lowest adult recruitment, was declining during the study period despite adults having a higher survival in comparison to the high Bd prevalence population. We also found a demographic signal of compensatory recruitment in response to Bd infection in our broader analysis of 13 R. darwinii populations. Our study underlines the importance of interpopulation variation in life-history strategies on the fate of host populations after infectious disease emergence. Our results also suggest that an increase in reproductive effort can be one of the processes underlying compensatory recruitment in populations of Bd-susceptible amphibians.

Chytridiomycosis Outbreak in a Chilean Giant Frog (Calyptocephalella gayi) Captive Breeding Program: Genomic Characterization and Pathological Findings.

Emerging infectious diseases in wildlife are increasingly associated with animal mortality and species declines, but their source and genetic characterization often remains elusive. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions at the global scale. We used histology and whole-genome sequencing to describe the lesions caused by, and the genetic variability of, two Bd isolates obtained from a mass mortality event in a captive population of the threatened Chilean giant frog (Calyptocephalella gayi). This was the first time an association between Bd and high mortality had been detected in this charismatic and declining frog species. Pathological examinations revealed that 30 dead metamorphosed frogs presented agnathia or brachygnathia, a condition that is reported for the first time in association with chytridiomycosis. Phylogenomic analyses revealed that Bd isolates (PA1 and PA2) from captive C. gayi group with other Bd isolates (AVS2, AVS4, and AVS7) forming a single highly supported Chilean Bd clade within the global panzootic lineage of Bd (BdGPL). These findings are important to inform the strengthening of biosecurity measures to prevent the impacts of chytridiomycosis in captive breeding programs elsewhere.

Bioclimatic and anthropogenic variables shape the occurrence of Batrachochytrium dendrobatidis over a large latitudinal gradient.

Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused the greatest known loss of biodiversity due to an infectious disease. We used Bd infection data from quantitative real-time PCR (qPCR) assays of amphibian skin swabs collected across Chile during 2008-2018 to model Bd occurrence with the aim to determine bioclimatic and anthropogenic variables associated with Bd infection. Also, we used Bd presence/absence records to identify geographical Bd high-risk areas and compare Bd prevalence and infection loads between amphibian families, ecoregions, and host ecology. Data comprised 4155 Bd-specific qPCR assays from 162 locations across a latitudinal gradient of 3700 km (18º to 51ºS). Results showed a significant clustering of Bd associated with urban centres and anthropogenically highly disturbed ecosystems in central-south Chile. Both Bd prevalence and Bd infection loads were higher in aquatic than terrestrial amphibian species. Our model indicated positive associations of Bd prevalence with altitude, temperature, precipitation and human-modified landscapes. Also, we found that macroscale drivers, such as land use change and climate, shape the occurrence of Bd at the landscape level. Our study provides with new evidence that can improve the effectiveness of strategies to mitigate biodiversity loss due to amphibian chytridiomycosis.

Not just a pathogen: The importance of recognizing genetic variability to mitigate a wildlife pandemic.

Emerging infectious diseases (EIDs) are increasingly recognized as a threat to both biodiversity and human health (Scheele et al., 2019; Wells et al., 2020). But pathogens cannot been seen as unique entities; their intraspecific genetic variability represented in variants, strains, antigenic types or genetic lineages may cause different impacts at the population level (Nelson and Holmes, 2007; Greenspan et al., 2018). The global spread of pathogens has been largely facilitated by globalization of transport, which particularly intensified during the last century (O'Hanlon et al., 2018). As seen with SARS-CoV-2, air travel can rapidly spread a pathogen globally (Wells et al., 2020). Furthermore, after initial introduction subsequent translocations of a pathogen may cause the contact of different variants facilitating the rise of novel genotypes that may have higher pathogenicity or transmissibility (Nelson and Holmes, 2007; Greenspan et al., 2018). Chytridiomycosis is an EID caused by the fungus Batrachochytrium dendrobatidis (Bd), that infects amphibian skin causing population declines to extinction in susceptible species. Now a wildlife pandemic, Bd has been recognized as the single pathogen causing the greatest loss of biodiversity on Earth (Scheele et al., 2019). Recent advances in genetics have made novel tools for pathogen detection and characterization more accessible and reliable (Boyle et al., 2004; Byrne et al., 2019). In this issue of Molecular Ecology Resources, Ghosh et al. (2021) report the development of a new genotyping qPCR assay targeting mitochondrial DNA (mtDNA) of Bd, and based on noninvasive swab samples (Figure 1), discriminate between the two most globally widespread and pathogenic genetic lineages of Bd. Having a better understanding of how the genetic diversity of a pathogen is distributed is crucial to understand their spread patterns and develop timely mitigation strategies.

Persistence, bioaccumulation and vertical transfer of pollutants in long-finned pilot whales stranded in Chilean Patagonia.

Long-finned pilot whales (LFPW) are cetaceans with strong social groups often involved in mass strandings worldwide. However, these beachings occur for reasons that are not fully understood. In 2016, 124 LFPW were stranded on the Chilean Patagonian islands, offering a unique opportunity to obtain crucial information on the ecology, biology, and genetics of this population. In addition, we examined whether persistent organic pollutants (POPs) and trace elements (TEs) were responsible for this mass mortality. Stable isotopes (δ13C & δ15N) and genetic analyses were used to reconstruct the trophic ecology, social structure, and kinship of LFPW and compared to POPs and TEs levels found in LFPW. Mitochondrial DNA analyses on 71 individuals identified four maternal lineages within the stranded LFPW. Of these animals, 32 individuals were analyzed for a suite of POPs, TEs, and lipid content in blubber. The highest levels were found for ΣDDXs (6 isomers) (542.46 ± 433.46 ng/g, lw) and for total Hg (2.79 ± 1.91 mg/kg, dw). However, concentrations found in these LFPW were lower than toxicity thresholds and those reported for LFPW stranded in other regions. Evidence was found of ΣDDX, Σ7PCBs, and Cd bioaccumulation and maternal transfer of POPs in mother/offspring groups. Nevertheless, no clear relationship between contaminant concentrations and LFPW mortality was established. Further research is still needed to assess LFPW populations including conservations status and exposure to chemicals in remote areas such as Patagonia.

In memoriam: a eulogy for Daniel González-Acuña, 1963-2020

Dr. Daniel González-Acuña was born on February 8, 1963, in San Fernando, central Chile, and became a veterinarian in 1988, at the University of Concepción. Keen on science and wildlife, he did his PhD studies in Germany, at the University of Veterinary Medicine in Hannover (Tierärztliche Hochschule Hannover), receiving his degree in 1997. Professor Eberhard Mey, an expert on lice, was his mentor. His doctoral dissertation was on the ecology and taxonomy of ectoparasites and endoparasites of Chilean birds. When he returned to Chile, he won a faculty position at the University of Concepción as a zoology professor in the School of Veterinary Sciences in Chillán. He spent the rest of his career at the same University, where he became a full professor(AU)

Author Correction: 50 Years of Cetacean Strandings Reveal a Concerning Rise in Chilean Patagonia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

50 Years of Cetacean Strandings Reveal a Concerning Rise in Chilean Patagonia.

Cetacean strandings (CS) have been reported in increasing numbers in coastal areas worldwide. Although the causes of these strandings are unknown, a number of anthropogenic and environmental factors have been suggested. This paper aims to characterize CS patterns and describe their fine-scale spatiotemporal dynamics. We analysed spatial and spatiotemporal CS patterns in Chile from January 1968 to January 2020. We identified a total of 389 CS events affecting eight cetacean families, 21 genera, and 35 species, which represent more than 85% of the reported species richness for the country. Most CS events (94.1%) were single (i.e., ≤two individuals). There were also 18 mass stranding (three to 24 individuals, 4.1%) and nine unusually large mass stranding events (>25 individuals, 2%). Purely spatial tests showed CS events appearing in random occurrence along the Chilean coast. Local tests for spatio-temporal clusters, however, identified a greater number of hotspots reported in the southernmost part of the country, namely, Chilean Patagonia. Specifically, significant spatio-temporal clusters were identified and defined as containing three or more individuals within a two-month period as a focal coastal event (<1 km radius). It is a cause of concern that CS events in Chile have been increasing consistently over the last decades, and although we were not able to identify their causes, we are able to highlight the importance of changes in climate conditions and of an increase in monitoring activities as primary drivers for such patterns, particularly important in Chilean Patagonia.

Assessing the Present and Future Habitat Suitability of Caligus rogercresseyi (Boxshall and Bravo, 2000) for Salmon Farming in Southern Chile.

The sea louse (Caligus rogercresseyi) is the most relevant parasite for the farmed salmon industry in Chile, the second largest producer worldwide. Although spatial patterns of C. rogercresseyi have been addressed from data obtained from established monitoring and surveillance programs, studies on its spatial ecology are limited. A wide geographic distribution of C. rogercresseyi is presumed in Chile; however, how this species could potentially be distributed in space is unknown. Our study presents an analysis of the habitat suitability for C. rogercresseyi in the entire area occupied by marine sites of salmon farms in Chile. Habitat suitability modeling was used to explore the likelihood of species spatial occurrence based on environmental characteristics. Due to the expanding salmon industry in southern Chile, we studied C. rogercresseyi habitat suitability models for present (average of 2005-2010) and two future projections (2050 and 2100) under different climate change scenarios. Models were constructed with the maxent algorithm using a large database of spatial C. rogercresseyi occurrences from the Chilean fisheries health authority and included 23 environmental variables obtained from the Ocean Rasters for Analysis of Climate and Environment (Bio-ORACLE). Habitat suitability models indicated that water temperature, water salinity, and current velocity of waters were the most important characteristics limiting C. rogercresseyi distribution in southern Chile. Habitat suitability models for current climate indicated a heterogeneous pattern with C. rogercresseyi being present in waters with temperature range 12.12-7.08°C (sd = 0.65), salinity range 33.7-25.5 pss (sd = 1.73), and current water velocity range 0.23-0.01 m-1 (sd = 0.02). Predictions for future projections in year 2050 and year 2100 suggest new clumped dispersion of the environmental conditions for C. rogercresseyi establishment. Our results suggest complexity and a wide dispersion of the biogeographic distribution of the C. rogercresseyi habitat suitability with potential implications for control strategies and environmental issues for salmon farming in Chile. Further investigations are required into C. rogercresseyi distribution in southern Chile considering the possible effect of climate change.

High prevalence of chigger mite infection in a forest-specialist frog with evidence of parasite-related granulomatous myositis.

Amphibians are hosts for a wide variety of micro- and macro-parasites. Chigger mites from the Hannemania genus are known to infect a wide variety of amphibian species across the Americas. In Chile, three species (H. pattoni, H. gonzaleacunae and H. ortizi) have been described infecting native anurans; however, neither impacts nor the microscopic lesions associated with these parasites have been described. Here, we document 70% prevalence of chigger mite infection in Eupsophus roseus and absence of infection in Rhinoderma darwinii in the Nahuelbuta Range, Chile. Additionally, we describe the macroscopic and microscopic lesions produced by H. ortizi in one of these species, documenting previously undescribed lesions (granulomatous myositis) within the host's musculature. These findings highlight that further research to better understand the impacts of chigger mite infection on amphibians is urgently required in Chile and elsewhere.

Cryptic disease-induced mortality may cause host extinction in an apparently stable host-parasite system.

The decline of wildlife populations due to emerging infectious disease often shows a common pattern: the parasite invades a naive host population, producing epidemic disease and a population decline, sometimes with extirpation. Some susceptible host populations can survive the epidemic phase and persist with endemic parasitic infection. Understanding host-parasite dynamics leading to persistence of the system is imperative to adequately inform conservation practice. Here we combine field data, statistical and mathematical modelling to explore the dynamics of the apparently stable Rhinoderma darwinii-Batrachochytrium dendrobatidis (Bd) system. Our results indicate that Bd-induced population extirpation may occur even in the absence of epidemics and where parasite prevalence is relatively low. These empirical findings are consistent with previous theoretical predictions showing that highly pathogenic parasites are able to regulate host populations even at extremely low prevalence, highlighting that disease threats should be investigated as a cause of population declines even in the absence of an overt increase in mortality.

Dispersal and extrapolation on the accuracy of temporal predictions from distribution models for the Darwin's frog.

Climate change is a major threat to biodiversity; the development of models that reliably predict its effects on species distributions is a priority for conservation biogeography. Two of the main issues for accurate temporal predictions from Species Distribution Models (SDM) are model extrapolation and unrealistic dispersal scenarios. We assessed the consequences of these issues on the accuracy of climate-driven SDM predictions for the dispersal-limited Darwin's frog Rhinoderma darwinii in South America. We calibrated models using historical data (1950-1975) and projected them across 40 yr to predict distribution under current climatic conditions, assessing predictive accuracy through the area under the ROC curve (AUC) and True Skill Statistics (TSS), contrasting binary model predictions against temporal-independent validation data set (i.e., current presences/absences). To assess the effects of incorporating dispersal processes we compared the predictive accuracy of dispersal constrained models with no dispersal limited SDMs; and to assess the effects of model extrapolation on the predictive accuracy of SDMs, we compared this between extrapolated and no extrapolated areas. The incorporation of dispersal processes enhanced predictive accuracy, mainly due to a decrease in the false presence rate of model predictions, which is consistent with discrimination of suitable but inaccessible habitat. This also had consequences on range size changes over time, which is the most used proxy for extinction risk from climate change. The area of current climatic conditions that was absent in the baseline conditions (i.e., extrapolated areas) represents 39% of the study area, leading to a significant decrease in predictive accuracy of model predictions for those areas. Our results highlight (1) incorporating dispersal processes can improve predictive accuracy of temporal transference of SDMs and reduce uncertainties of extinction risk assessments from global change; (2) as geographical areas subjected to novel climates are expected to arise, they must be reported as they show less accurate predictions under future climate scenarios. Consequently, environmental extrapolation and dispersal processes should be explicitly incorporated to report and reduce uncertainties in temporal predictions of SDMs, respectively. Doing so, we expect to improve the reliability of the information we provide for conservation decision makers under future climate change scenarios.

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.

Xenopus laevis and Emerging Amphibian Pathogens in Chile.

Amphibians face an extinction crisis with no precedence. Two emerging infectious diseases, ranaviral disease caused by viruses within the genus Ranavirus and chytridiomycosis due to Batrachochytrium dendrobatidis (Bd), have been linked with amphibian mass mortalities and population declines in many regions of the globe. The African clawed frog (Xenopus laevis) has been indicated as a vector for the spread of these pathogens. Since the 1970s, this species has been invasive in central Chile. We collected X. laevis and dead native amphibians in Chile between 2011 and 2013. We conducted post-mortem examinations and molecular tests for Ranavirus and Bd. Eight of 187 individuals (4.3 %) tested positive for Ranavirus: seven X. laevis and a giant Chilean frog (Calyptocephallela gayi). All positive cases were from the original area of X. laevis invasion. Bd was found to be more prevalent (14.4 %) and widespread than Ranavirus, and all X. laevis Bd-positive animals presented low to moderate levels of infection. Sequencing of a partial Ranavirus gene revealed 100 % sequence identity with Frog Virus 3. This is the first report of Ranavirus in Chile, and these preliminary results are consistent with a role for X. laevis as an infection reservoir for both Ranavirus and Bd.

Geographic body size variation in ectotherms: effects of seasonality on an anuran from the southern temperate forest.

BACKGROUND: Body size variation has played a central role in biogeographical research, however, most studies have aimed to describe trends rather than search for underlying mechanisms. In order to provide a more comprehensive understanding of the causes of intra-specific body size variation in ectotherms, we evaluated eight hypotheses proposed in the literature to account for geographical body size variation using the Darwin's frog (Rhinoderma darwinii), an anuran species widely distributed in the temperate forests of South America. Each of the evaluated hypotheses predicted a specific relationship between body size and environmental variables. The level of support for each of these hypotheses was assessed using an information-theoretic approach and based on data from 1015 adult frogs obtained from 14 sites across the entire distributional range of the species. RESULTS: There was strong evidence favouring a single model comprising temperature seasonality as the predictor variable. Larger body sizes were found in areas of greater seasonality, giving support to the "starvation resistance" hypothesis. Considering the known role of temperature on ectothermic metabolism, however, we formulated a new, non-exclusive hypothesis, termed "hibernation hypothesis": greater seasonality is expected to drive larger body size, since metabolic rate is reduced further and longer during colder, longer winters, leading to decreased energy depletion during hibernation, improved survival and increased longevity (and hence growth). Supporting this, a higher post-hibernation body condition in animals from areas of greater seasonality was found. CONCLUSIONS: Despite largely recognized effects of temperature on metabolic rate in ectotherms, its importance in determining body size in a gradient of seasonality has been largely overlooked so far. Based on our results, we present and discuss an alternative mechanism, the "hibernation hypothesis", underlying geographical body size variation, which can be helpful to improve our understanding of biogeographical patterns in ectotherms.

Is chytridiomycosis driving Darwin's frogs to extinction?

Darwin's frogs (Rhinoderma darwinii and R. rufum) are two species of mouth brooding frogs from Chile and Argentina that have experienced marked population declines. Rhinoderma rufum has not been found in the wild since 1980. We investigated historical and current evidence of Batrachochytrium dendrobatidis (Bd) infection in Rhinoderma spp. to determine whether chytridiomycosis is implicated in the population declines of these species. Archived and live specimens of Rhinoderma spp., sympatric amphibians and amphibians at sites where Rhinoderma sp. had recently gone extinct were examined for Bd infection using quantitative real-time PCR. Six (0.9%) of 662 archived anurans tested positive for Bd (4/289 R. darwinii; 1/266 R. rufum and 1/107 other anurans), all of which had been collected between 1970 and 1978. An overall Bd-infection prevalence of 12.5% was obtained from 797 swabs taken from 369 extant individuals of R. darwinii and 428 individuals representing 18 other species of anurans found at sites with current and recent presence of the two Rhinoderma species. In extant R. darwinii, Bd-infection prevalence (1.9%) was significantly lower than that found in other anurans (7.3%). The prevalence of infection (30%) in other amphibian species was significantly higher in sites where either Rhinoderma spp. had become extinct or was experiencing severe population declines than in sites where there had been no apparent decline (3.0%; x(2) = 106.407, P<0.001). This is the first report of widespread Bd presence in Chile and our results are consistent with Rhinoderma spp. declines being due to Bd infection, although additional field and laboratory investigations are required to investigate this further.

The population decline and extinction of Darwin's frogs.

Darwin's frogs (Rhinoderma darwinii and R. rufum) are two species of mouth-brooding frogs from Chile and Argentina. Here, we present evidence on the extent of declines, current distribution and conservation status of Rhinoderma spp.; including information on abundance, habitat and threats to extant Darwin's frog populations. All known archived Rhinoderma specimens were examined in museums in North America, Europe and South America. Extensive surveys were carried out throughout the historical ranges of R. rufum and R. darwinii from 2008 to 2012. Literature review and location data of 2,244 archived specimens were used to develop historical distribution maps for Rhinoderma spp. Based on records of sightings, optimal linear estimation was used to estimate whether R. rufum can be considered extinct. No extant R. rufum was found and our modelling inferred that this species became extinct in 1982 (95% CI, 1980-2000). Rhinoderma darwinii was found in 36 sites. All populations were within native forest and abundance was highest in Chiloé Island, when compared with Coast, Andes and South populations. Estimated population size and density (five populations) averaged 33.2 frogs/population (range, 10.2-56.3) and 14.9 frogs/100 m(2) (range, 5.3-74.1), respectively. Our results provide further evidence that R. rufum is extinct and indicate that R. darwinii has declined to a much greater degree than previously recognised. Although this species can still be found across a large part of its historical range, remaining populations are small and severely fragmented. Conservation efforts for R. darwinii should be stepped up and the species re-classified as Endangered.