Nor climate nor human impact factors: Chytrid infection shapes the skin bacterial communities of an endemic amphibian in a biodiversity hotspot.

The bacterial communities of the amphibian skin (i.e., the bacteriome) are critical to the host's innate immune system. However, it is unclear how different drivers can alter this function by modulating the bacteriome's structure. Our aim was to assess the extent to which different host attributes and extrinsic factors influence the structure of the bacterial communities of the skin. Skin bacterial diversity was examined in 148 individuals of the four-eyed frog (Pleurodema thaul) from 16 localities spanning almost 1800 km in latitude. The richness and beta diversity of bacterial families and the richness and abundance of Bd-inhibitory bacterial genera were used to describe their structure. Predictors associated with the host (developmental stage, genetic lineage, individual Batrachochytrium dendrobatidis [Bd] infection status) and the landscape (current climate, degree of anthropogenic disturbance) were used in the statistical modeling in an information theoretical approach. Bd infection and host developmental stage were the only predictors affecting bacteriome richness, with Bd+ individuals and postmetamorphic stages (adults and juveniles) having higher richness than Bd- ones and tadpoles. High diversity in Bd+ individuals is not driven by bacterial genera with known anti-Bd properties. Beta diversity was not affected by Bd infection and was mainly a consequence of bacterial family turnover rather than nestedness. Finally, for those bacterial genera known to have inhibitory effects on chytrid, Bd+ individuals had a slightly higher diversity than Bd- ones. Our study confirms an association between Bd infection and the host developmental stage with the bacterial communities of the skin of P. thaul. Unexpectedly, macroclimate and human impact factors do not seem to play a role in shaping the amphibian skin microbiome. Our study exemplifies that focusing on a single host-parasite system over a large geographic scale can provide essential insights into the factors driving host-parasite-bacteriome interactions.

Ongoing declines for the world's amphibians in the face of emerging threats.

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

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.

Testing a global standard for quantifying species recovery and assessing conservation impact.

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard.

RESUMEN: Reconociendo que era imperativo evaluar la recuperación de especies y el impacto de la conservación, la Unión Internacional para la Conservación de la Naturaleza (UICN) convocó en 2012 al desarrollo de una "Lista Verde de Especies" (ahora el Estatus Verde de las Especies de la UICN). Un marco de referencia preliminar de una Lista Verde de Especies para evaluar el progreso de las especies hacia la recuperación, publicado en 2018, proponía 2 componentes separados pero interconectados: un método estandarizado (i.e., medición en relación con puntos de referencia de la viabilidad de especies, funcionalidad y distribución antes del impacto) para determinar el estatus de recuperación actual (puntuación de recuperación de la especie) y la aplicación de ese método para estimar impactos en el pasado y potenciales de conservación basados en 4 medidas (legado de conservación, dependencia de conservación, ganancia de conservación y potencial de recuperación). Probamos el marco de referencia con 181 especies representantes de diversos taxa, historias de vida, biomas, y categorías (riesgo de extinción) en la Lista Roja de la IUCN. Con base en la distribución observada de la puntuación de recuperación de las especies, proponemos las siguientes categorías de recuperación de la especie: totalmente recuperada, ligeramente mermada, moderadamente mermada, mayormente mermada, gravemente mermada, extinta en estado silvestre, e inderterminada. Cincuenta y nueve por ciento de las especies se consideraron mayormente o gravemente mermada. Aunque hubo una relación negativa entre el riesgo de extinción y la puntuación de recuperación de la especie, la variación fue considerable. Algunas especies en las categorías de riesgo bajas fueron evaluadas como más lejos de recuperarse que aquellas con alto riesgo. Esto enfatiza que la recuperación de especies es diferente conceptualmente al riesgo de extinción y refuerza la utilidad del Estado Verde de las Especies de la UICN para comprender integralmente el estatus de conservación de especies. Aunque el riesgo de extinción no predijo el legado de conservación, la dependencia de conservación o la ganancia de conservación, se correlacionó positivamente con la potencial de recuperación. Solo 1.7% de las especies probadas fue categorizado como cero en los 4 indicadores de impacto de la conservación, lo que indica que la conservación ha jugado, o jugará, un papel en la mejoría o mantenimiento del estatus de la especie la gran mayoría de ellas. Con base en nuestros resultados, diseñamos una versión actualizada del marco de referencia para la evaluación que introduce la opción de utilizar una línea de base dinámica para evaluar los impactos futuros de la conservación en el corto plazo y redefine corto plazo como 10 años.

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.

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.

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.

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.

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.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

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.