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Review this slideshow then answer the questions below.
Amphibian die off from Lumen Learning
- What phylum of fungi is responsible for chytridiomycosis?
- Why is this fungi successful at killing amphibians?
- Why be concerned about the world’s amphibian populations? What role do amphibians play in ecosystems?
- How might Bd be spreading through the environment?
- Propose a hypothesis that would explain why Bd is spreading like a new disease throughout the world.
- Some scientists are attempting to prevent the extinction of amphibians by capturing amphibians in the wild, breeding them in chytrid free labs, then releasing some of the amphibians into the wild. Discuss how such a strategy could save some species of amphibians.
- How else might we prevent the spread of Bd throughout the world?
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- Amphibian Die Off. Authored by: Lynette Hauser.
Frog-Killing Chytrid Fungus Far Deadlier than Scientists Realized
Mar 29, 2019
R esearchers have tallied the global death toll of the frog-killing chytrid fungus and its devastation is far worse than scientists thought, according to a study published yesterday (March 28) in Science.
The fungal pathogens that cause the disease chytridiomycosis ravage the skin of frogs, toads, and other amphibians, throwing off their balance of water and salt and eventually causing heart failure, Nature reports. One fungus implicated in causing chytridiomycosis was discovered in 1988 while another was found in 2013. Scientists realized the fungi caused vast declines in amphibian populations, they hadn’t examined its effects on a global scale until now.
A team of scientists from across the world banded together to search out the species and populations affected by the disease. They pored over literature reports and unpublished data and interviewed amphibian experts. Their survey found that chytrid fungi played a role in the decline of 501 species between 1965 and 2015. Of these, 90 went extinct.
“That’s fairly seismic,” biologist Wendy Palen of Simon Fraser University tells The New York Times. Palen coauthored a commentary that accompanies the report. “It now earns the moniker of the most deadly pathogen known to science.”
“It’s a staggering thing to consider,” Jonathan Kolby, a herpetologist at James Cook University in Australia, tells The Washington Post. “We’ve never before had a single disease that had the power to make multiple species extinct, on multiple continents, all at the same time.” The most declines were observed in populations of Central and South America and tropical Australia with far fewer declines in amphibians of Africa, North America, and Europe.
To protect frogs and prevent more species from going extinct, researchers are calling for biosecurity measures, including restrictions on the wildlife trade. “What concerns me is there’s going to be a next time. By not using this as a learning experience about what happens when we aren’t being careful, it almost undoubtedly ensures that the wildlife trade is moving other pathogens right now, be it for mammals, birds, fish. You name it,” Kolby tells the Post.
Scientists are concerned about possible future outbreaks in places that haven’t been touched by the fungi, particularly New Guinea, home of many amphibians found nowhere else, reports the Times. “It could be a meltdown of the ecosystems over there,” says Daniel Greenberg, also of Simon Fraser University and coauthor of the commentary, to the Times.
Frogs play in the environment: chewing up algae, as tadpoles, to help keep algal blooms at bay, devouring mosquitos that can spread disease, and providing food for birds and other animals. It’s yet unclear how declines of these species will change the ecosystems they are part of, the Post reports.
The survey’s results were bleak overall with one sliver of hope. Some 20 percent of species that declined because of chytrid had populations that showed evidence of recovering, indicating that maybe others could bounce back or possibly evolve to resist the disease, according to the Post.
'Carpets of Dead Frogs'
The fungus—called Bd for short—is so deadly because it targets amphibians' porous skin, which the animals use to breathe and drink water. Bd unspools the skin's proteins and feasts on the resulting spaghetti of amino acids. As it does, infected animals grow lethargic, shedding their skin in a death spiral that ends with heart failure in a matter of weeks. Some amphibians can tolerate or resist Bd, but the fungus can infect at least 695 species to varying degrees.
“This is pretty unusual for a disease, to see it affect so many different species,” says University of Maryland biologist Karen Lips, an amphibian-decline expert who wasn't involved with the new study.
In person, Bd infestations can look like biblical plagues. Each August, adult midwife toads in the French Pyrenees climb out of their birth lakes for the first time. The infected toads barely make it to shore. “They'll do one last hop, and then they'll expire in your hands,” says Fisher, one of the study's coauthors. “You can walk the lakes—it's just carpets of dead frogs.”
Similar die-offs started popping up in the 1970s, but researchers didn't realize these “enigmatic declines” were a global phenomenon until the 1990s. In 1997, researchers first described Bd, and within a decade, they had connected it to the slaughters. Meanwhile, Bd's killing spree continued. From 2004 to 2008, one site in Panama lost 41 percent of its amphibian species to the fungus.
Most of the once-mysterious slaughters are now attributed to the “Global Panzootic Lineage,” a lethal strain nicknamed BdGPL. But where did this killer come from? And when and how did it blaze a trail around the world?
Amphibian die-offs worsened malaria outbreaks in Central America
New research finds the global collapse of frogs and other amphibians exacerbated malaria outbreaks in Costa Rica and Panama during the 1990s and 2000s. Credit: David Mark from Pixabay.
The global collapse of frogs and other amphibians due to the amphibian chytrid fungus exacerbated malaria outbreaks in Costa Rica and Panama during the 1990s and 2000s, according to new research.
The findings provide the first evidence that amphibian population declines have directly affected human health and show how preserving biodiversity can benefit humans as well as local ecosystems.
"This is like a small building block showing that there could be unwanted human health consequences of amphibian collapses, and so we should really be trying to account for these impacts," said Joakim Weill, an environmental economist at the University of California Davis who will present the results Tuesday, 8 December at AGU's Fall Meeting 2020. "We really view this as an important first step leveraging this type of interdisciplinary work, trying to tease out causal relationship between environmental change and human health."
The global spread of Batrachochytrium dendrobatidis, an extremely virulent fungal pathogen known as amphibian chytrid fungus, has been responsible for massive worldwide die-offs of amphibians since the 1980s. A 2019 study found the fungal disease has played a role in the decline of over 500 amphibian species over the past five decades and presumably caused extinctions of 90 species. The authors of that study referred to the die-offs as "the greatest recorded loss of biodiversity attributable to a disease."
Chytrid fungal disease traveled across Costa Rica and Panama from the early 1980s through the 2000s. Both countries experienced large increases in malaria cases following this rolling collapse of amphibian populations.
In the new study, researchers investigated whether these malaria outbreaks were connected to the amphibian declines because amphibians eat mosquitoes that transmit the disease. They compared the timing and spatial extent of amphibian die-offs with malaria cases in Costa Rica and Panama at the county level from 1976 to 2016.
The researchers found a significant increase in malaria cases in these countries that started immediately after the amphibian die-offs began and peaked 5 to 6 years after. In 1980, there were fewer than 1,000 cases of malaria in the two countries, but cases began to rise in 1990 and peaked at about 7,000 in Costa Rica in the mid-1990s and 5,000 in Panama in the mid-2000s.
Malaria cases went back down after this peak, and the researchers suspect this is due to local public health interventions like spraying of insecticides.
The results show some of the first evidence that species extinctions and biodiversity loss can directly affect human health, according to the researchers.
Other environmental factors like deforestation also played a role in exacerbating the outbreaks, but no other factor had as much of an impact on malaria cases as the amphibian declines, according to the study.
"We are able to find what really seems to be this striking causal relationship between amphibian declines and malaria," Weill said. "It's pretty incredible that we are finding anything in the first place, because these are events that occurred 40 years ago and the right people were in the right place to make observations about amphibian populations and human disease that we can use today to arrive at new insights."
Origin and emergence
Chytrids ‘out of Asia’
The debate on how chytridiomycosis emerged as a cause of amphibian declines has revolved around two competing arguments. The ‘novel pathogen hypothesis’ (NPH) stated that chytridiomycosis emerged locally after it had been seeded by intercontinental trade routes into naive ecosystems. The counterargument, known as the ‘endemic pathogen hypothesis’ (EPH), held that Bd was a widespread endemic commensal of amphibians that had become more virulent owing to global change forcing imbalanced infection dynamics 20 . Early molecular clues from multilocus sequence typing supported the NPH, as the isolates of Bd sampled at the time showed no signs of phylogeographic structure among the different regions with amphibian declines owing to chytridiomycosis 21,22 . This molecular evidence matched the observed patterns of chytridiomycosis observed in the Americas 1 , Australia 20 and the Caribbean islands 23 (Fig. 1). Later, sequencing of two complete genomes by the Joint Genome Institute (isolate JAM81 from Rana muscosa in California) and the Broad Institute (isolate JEL423 from Phyllomedusa lemur in Panama) in 2008 (ref. 24 ), along with the development of high-throughput shotgun sequencing, enabled a genome-scale genetic analysis of Bd. Early ABI SOLiD genome resequencing of 20 globally distributed isolates from sites experiencing chytridiomycosis uncovered striking patterns in comparison with sites without the disease. Resequencing identified three deeply diverged lineages: BdGPL (globally distributed), BdCAPE (named owing to its discovery in the Cape region of South Africa) and BdCH (a single, deeply branched isolate from Gamlikon in Switzerland 25 ). Only BdGPL was found across four continents and was associated with epizootics in North America, Central America, the Caribbean, Australia and Europe. The extraordinary global range, limited genomic diversity and relatively high virulence of this lineage, as well as its origin in the early 20th century, based on the phylogeny of BdGPL, supported the NPH over the EPH 25 . Heterozygous and triallelic single-nucleotide polymorphisms were threefold to fourfold more common than homozygous ones in BdGPL, which was held as evidence that the genesis of BdGPL was the result of a sexual ‘hybridization’ between two dissimilar parental genotypes 26 .
As of 2019, Batrachochytrium dendrobatidis (Bd) had invaded and caused chytridiomycosis in six regions globally: eastern Australia, the Mesoamerican peninsula, South America, the western United States, Africa and Europe. Five lineages of Bd, as well as recombinants, have been identified. In addition, another species, Batrachochytrium salamandrivorans (Bsal), was discovered in 2010. Batrachochytrids cause severe amphibian declines. The figure shows declines that match the Scheele et al. 4 criteria for category 3 or above: 3, extreme decline with >90% of individuals lost 4, presumed extinct in the wild (no known extant populations, and no individuals detected at known historical locations, but some reasonable doubt that the last individual has died) 5, confirmed extinct in the wild (as per International Union for Conservation of Nature [IUCN] listing). Adapted from ref. 65 , courtesy of P. Ghosh, Imperial College London, UK.
Subsequent analysis of a larger panel of isolates cast doubt on the findings of this earlier study 25 , suggesting both greater genetic diversity and an estimated origin of BdGPL 10,000–40,000 years before the present 27 . The authors interpreted these results as supporting the EPH rather than the NPH. Neither study could resolve the geographic origin of Bd, which was variously proposed to be African 28 , Japanese 29 , East Asian 30 , South American 31 or North American 32 .
O’Hanlon et al. 33 resolved much of the debate by publishing new sequence data for 177 Bd isolates collected using the RACE protocols 18 . The complete dataset of 234 isolates had been collected over nearly two decades and spanned the geographical distribution of Bd, numerous events of lethal chytridiomycosis and all three extant orders of Amphibia. This analysis redefined the evolutionary relationships among the lineages of Bd, aided by the first genome data from Asian isolates. Bd from the Korean peninsula comprised a new, fourth lineage, BdASIA-1, and this lineage showed signs of an ancestral relationship with the other lineages. Bayesian-based haplotype clustering revealed that the hyperdiverse BdASIA-1 lineage shared more diversity with the global population of Bd than did any other lineage and branched at the base of the Bsal-rooted Bd phylogeny. Tellingly, BdASIA-1 was the only lineage in mutation–drift equilibrium, a characteristic of endemism. All other lineages showed pronounced departures from equilibrium values of Tajima’s D statistic 34 , which are indicative of outbreak dynamics. Molecular screening of museum specimens of amphibians from Korea showed that infection has been present in the region for over 100 years 35 , and contemporary surveillance has demonstrated a widespread yet patchy and rare distribution of batrachochytrids throughout East Asia 12,36,37 , further suggesting endemism of Bd in this region. Multilocus genotyping confirmed the results of O’Hanlon et al. 33 and discovered a novel fifth lineage, BdASIA-3, also found in East Asia (the Philippines, Indonesia and China) 38 . This ‘chytrid-out-of-Asia’ hypothesis supporting the NPH was strengthened by the finding that, following discovery of chytridiomycosis caused by Bsal in Europe, this chytrid could only be detected elsewhere in Southeast Asia (Vietnam) 12 . The comprehensive lack of lethal outbreaks or population declines caused by chytridiomycosis in Asia, despite the widespread occurrence of Bd and Bsal 4,11 , is evidence for endemic host–pathogen interactions 39 . Batrachochytrids appear to have been infecting amphibians in the region for over 50 million years, leaving ample time for fungal speciation events and relatively stable host–pathogen dynamics to establish 11 . Accordingly, there is a need for more intensive pathogen discovery across Southeast Asia, as unmapped batrachochytrid diversity will likely yield further insights into the past emergence and present distribution of these pathogens.
Timing the panzootic
Final proof of the NPH required congruency between the timing of introductions of Bd and the onset of declines caused by chytridiomycosis. Chytridiomycosis declines peaked globally in the 1980s 4 , in keeping with the timing of regional wave-like dynamics suggesting epizootic spread from point sources 40,41 . To time introductions, O’Hanlon et al. 33 used two quasi-independent genomic regions to generate time-calibrated phylogenies, as well as a Bayesian framework to estimate the time to their most recent common ancestor (TMRCA, Box 1). These analyses estimated a substitution rate for Bd, one that was broadly similar to that estimated for other unicellular fungi. The updated TMRCA for the ancestor of BdGPL ranged between 120 and 50 years ago (1890s–1960s), which broadly agrees with the first inferred chytridiomycosis-related declines in regions that are currently dominated by BdGPL (Australia 20,42 , the Mesoamerican peninsula 13 and South America 14,40 ). Molecular dating also suggests that the widespread, and still largely unattributed, amphibian declines reported in Europe and North America in the 1950s and 1960s were driven by BdGPL, which has now achieved widespread endemicity across these regions 6,43 .
What has fuelled the global expansion of Bd? That all known lineages of Bd are circulating in globally traded amphibians proves that trade is disseminating amphibian vectors of batrachochytrids worldwide 44 today 33 (Fig. 2). For example, ‘African’ BdCAPE invaded the island of Mallorca through the reintroduction of captive-reared Mallorcan midwife toads infected in captivity by African endemic amphibians (Xenopus gilli) 45 . More widely, infection-tolerant species such as the African clawed frogs Xenopus laevis 28 and the North American bullfrogs Rana catesbeiana 44 are internationally traded in their millions and have been since the early 20th century. Other infection-tolerant species, such as the cane toad Rhinella marinus, have established feral populations from their origins in South and Central America. It is likely that these species had an important role in amplifying the worldwide emergence of Bd, and indeed, molecular methods have identified transcontinental links involving these species 46 . The evidence therefore suggests that the original out-of-Asia vectors of batrachochytrids were likely amphibians exported for food, research or collections, or perhaps were passively hiding in traded goods. However, identifying these original panzootic ‘sparks’ will likely prove a challenging task.
Intercontinental movements of Batrachochytrium dendrobatidis (Bd) have been inferred from the genome sequences of geographically separated isolates that form closely related phylogenetic clades, with high bootstrap support (≥90% data from ref. 33 ). Numbers show where isolates of Bd have been recovered from traded amphibians, with pictures of the species involved shown at the bottom of the figure. Also shown are the movements of traded, CITES-listed amphibians, showing the global connectivity of the amphibian trade, which involved over 15 million specimens during the period 2000–2010. Data are from TRAFFIC (a global network that monitors wildlife trade), CITES and refs 131,132 .
Box 1 Dating the emergence of Batrachochytrium dendrobatidis
Sequence data is increasingly being used to time epidemiological events, ranging across different infections (for example, the emergence of HIV-1 (ref. 124 ), the spread and diversification of plague 125 and the emergence of Cryptococcus gattii in North America 126 ). For microbial species with rapidly evolving genomes or short generation times, genetic lineages may measurably diverge over observable time spans, allowing substitution rates to be calculated directly rather than assumed 127 . Calculation is based on known dates of isolation in order to determine the rate of evolution. For example, the amount of sequence change that has occurred between cultures of Batrachochytrium dendrobatidis (Bd) isolated from Xenopus and Litoria frogs, together with these groups’ dates of isolation (T(Xenopus) and T(Litoria) in the figure, part a), can be used to estimate an evolutionary rate and thus the time at which the pathogen lineages in the two frogs most recently shared a common ancestor (TMRCA). This method is known as tip dating 128 , and several computational packages exist to carry out such analyses (reviewed in 129 ). Measurable molecular evolution has occurred between T(Xenopus) and T(Litoria), and these data, together with data from other isolates (figure, part b), can be used to estimate the rate of evolution. A core assumption of tip dating is that sequences are not recombining, as this introduces additional divergence that is not linearly related to TMRCA. To avoid this bias, genome sequences can be statistically ‘cleaned’ of recombining sites, using programs such as Gubbins 130 , or analysis can focus on recombination-free genomic regions, such the mitochondrial (mt) genome. Attempts to date the emergence of Bd either have assumed a rate of molecular evolution extrapolated from other eukaryotic species 27 or have used tip dating on nuclear genomes in which major recombination breakpoints had been taken into account 25 . The former method dated the origin of Bd to approximately 26,400 years ago, whereas the latter method estimated a more recent origin of 35–257 years ago. At 299,707 bp, Bd has the largest mitochondrial genome of any fungus 33 and contains substantial diversity. Tip dating based on the mitochondrial DNA of Bd has estimated a TMRCA for the emergence of BdGPL of 1962 (1859–1988), substantiating earlier estimates based on nuclear DNA and matching the onset of global amphibian declines 4 (figure part c the arrow indicates when Bd was discovered, and the severity of declines is shown as the cumulative number of lost individuals). Part c is adapted with permission from ref. 4 , AAAS.
Cycles and circles of expansion
The occurrence of the divergent BdCAPE variant in Africa, Central America and Europe 33,38 BdASIA-2/BRAZIL in the Brazilian Atlantic forests 31 and Korea 33 and the ASIA-1-like BdCH in Switzerland show that the evolutionary history of Bd is complex and has been characterized by at least three out-of-Asia emergences of lineages other than BdGPL. With too few isolates to allow for confidently deriving measurable evolutionary rates, the TMRCAs for these lineages have thus far not been estimated. Notwithstanding this fact, levels of diversity exceed those seen in BdGPL, suggesting that their out-of-Asia dispersal predates that of BdGPL 33 . The detection of molecular signatures of Bd in Brazilian museum collections of amphibians indicates that Brazil was invaded by Bd as far back as 1894 (ref. 31 ). While molecular confirmation is needed, it appears that the early invasion was by BdASIA-2/BRAZIL, followed by a secondary introduction of BdGPL into Brazil in the 1970s. The result was a peak of declines in the 1970s, owing to the higher virulence of this lineage 14 and the founding of a region of contact between the two lineages in the Brazilian Atlantic forest 47,48,49 . To complicate matters further, BdASIA-2/BRAZIL is itself found in Korean populations of introduced North American bullfrogs, suggesting that these widely traded frogs have been vectors for this lineage, re-establishing it in its ancestral Asian homeland 33 .
Surveillance across Africa shows that this continent also has a complex history of Bd introductions 50 . The pathogen is widely present, occurring in Cameroon from at least 1933, Kenya in 1934, Uganda in 1935, South Africa in 1938, the Democratic Republic of Congo in 1950 and the island Bioko in 1966 (refs 28,51,52,53,54 ). The infection status of the amphibians of Madagascar remains unclear 18,55,56 . The extent to which Africa has suffered amphibian declines as a consequence of chytridiomycosis is largely undetermined. However, at least one extinction in the wild has occurred (the Tanzanian Kihansi spray toad, Nectophrynoides asperginis 15 ), and the presence of Bd has been correlated with declines of amphibian species in Cameroon 57 and South Africa 58 . Genome sequencing 33 and multilocus genotyping 38 have shown the widespread occurrence of both BdCAPE and BdGPL, the former widely distributed in Cameroon, including in caecilians 59 , and the latter occurring in both Ethiopia and Uganda. Both lineages occur in Southern Africa, where, as in Brazil, different lineages are in spatial contact. The patchy distribution of BdCAPE in Central America and Europe suggests that secondary waves of expansion for this lineage have occurred.
Recombinants, not hybrids
Genotyping has identified recombinants of BdASIA-2/BRAZIL and BdGPL in the Brazilian Atlantic forest 48 , as well as genetic mosaics of BdCAPE and BdGPL in South Africa 33 . Within lineages, alleles segregate 47,60 , intrachromosomal recombination breakpoints have been detected 25 , and when single-nucleotide polymorphisms have been phased, crossovers have been observed in all lineages tested 26 . Clearly the extreme genetic bottlenecks that characterize the out-of-Asia evolutionary history of Bd have not impaired the ability of this species to recombine. Whereas chytrids such as Allomyces and Rhizophydium undergo meiosis, recombinant mating structures have not been described for Bd or Bsal, nor have canonical fungal mating-type alleles been identified, suggesting that recombination in batrachochytrids may not be meiotic. In support of this idea, some ‘meiotic toolbox’ genes defined in yeast are missing in the genome of Bd, and signatures of sex-associated, repeat-induced point mutations in transposable elements are also absent 61 . Furthermore, widespread chromosomal copy number variation 26 is also evidence that recombination may not be due to meiosis. Accordingly, it has been proposed 25,62 that non-meiotic recombination (called ‘parasexual’ recombination) may be generating the polyploid heterozygous mosaics that characterize Bd. However, the cell biology that underpins the widespread recombination in Bd, either meiotic or non-meiotic, remains wholly unexplored.
The description of the global Bd population as stemming from a genetically diverse Asian population in mutation–drift equilibrium and recombining when the opportunity arises shows that the global Bd population is currently behaving as a cohesive biological species. Prior to the discovery of this Asian origin, interlineage recombination events were termed ‘hybridizations’, and BdGPL was suggested to result from a hybridization event among two related chytrid species 25 . However, the simplest description of the global population genetic structure of Bd is that each lineage represents a separate genealogical ‘draw’ from a recombining parental population that is most likely Asian. As multiple founding events do not appear to have appreciably blunted the ability of Bd to shuffle its genome if given the opportunity, it is premature to give these lineages species status and to name recombinants ‘hybrids’. Accordingly, the most biologically accurate description of the genomic mosaics that are increasingly being described is ‘recombinants’.
The finding that Bd is a recombining species is not only academically interesting the process of recombination through secondary contact is likely important in an epidemiological context. Outcrossing can purge deleterious alleles and generate variation that may facilitate host exploitation, exacerbating epizootics. Theory and experimentation have shown that interactions between diverse genotypes can lead to competitive interactions that result in increased transmission and may exacerbate infection dynamics 63,64 . Co-infections of Bd lineages have been observed in South Africa where BdGPL and BdCAPE co-occur 65 , and in the absence of a defined environmental developmental stage, co-infection is when recombination events will occur. That recombination can affect the virulence of Bd was demonstrated in a study 49 that showed that BdGPL and BdASIA-2/BRAZIL recombinant genotypes were more aggressive than either of the parent genotypes in two amphibian species. This result suggests that outcrossing in Bd results in genetic dominance and enhanced fitness. Whereas these hybrids were inferred to be F1, an F2 backcross in Brazil has been observed, suggesting that recombinants can survive beyond their immediate F1 genesis 48 .
2. Impacts of chytrid on amphibian populations
(a) Chytrids as invasive pathogens
Some of the earliest amphibian die-offs and unexplained declines were detected across many protected sites in tropical rainforest streams during the 1980s . At least six species of frogs have not been seen for one to three decades and are presumed extinct, whereas six other species have declined so greatly that the probability of extinction is high . Berger et al.  identified a skin infection associated with dead frogs and population declines from three regions that they would attribute to Bd, which had just been described . In the decades to follow, extensive research on many of the surviving species has confirmed the role of Bd in contributing to population declines across Australia , and has shown how species-specific differences in ecology and behaviour contribute to variation in disease dynamics [14,28]. Despite continued declines in many species , others have developed ways to persist with infection. Retallick et al.  described a population of Taudactylus eungellensis persisting with stable infections that had experienced an epidemic die-off 10 years earlier. A 7-year capture–mark–recapture study of the endangered Mixophyes fleayi showed increased abundance after an initial population decline that was attributed to Bd . Others have shown how warmer and drier microclimates might provide a refuge from Bd for some species . Recent evidence suggests that some Australian amphibians may be adapting to Bd in both laboratory  and field studies , populations of Litoria verreauxii alpina that had been previously exposed to Bd survived longer than those that had not.
(ii) Central America
Central America has played a key role in the Bd story for many reasons. First, it provided one of the early examples of enigmatic declines in the form of Monteverde's golden toad (Incilius periglenes) . Second, this was one of the first sites where Bd was found infecting wild animals at sites of population decline and mass mortality, and served as definitive proof that a pathogen could cause mortality and declines in multiple species [30,34,35]. Third, the spatio-temporal spread of Bd through Central America provided definitive proof of an invasive pathogen moving in an epidemic wave [8,36]. Subsequent genetic studies have revealed that these die-offs were attributable to a single lineage, identified as the invasive Bd-GPL .
The Neotropics have been especially hard hit by these declines , with hundreds of species threatened with extinction. The high rates of loss are associated with the exceptionally high diversity and endemism in this region, with over 50% of all described amphibian species found in the Neotropics . In the Neotropics amphibian endemism is concentrated in the montane areas, where the cool moist climate supports chytrid growth. For some tropical amphibians, an entire species could be eliminated by chytridiomycosis because of the small population size, small range and rapid spread of disease. The impact of Bd on Central American amphibians has been devastating [34,35,39]. In species-rich cloud forests of Lower Central America , mass die-offs were observed, followed by numerous population declines of many species, including extirpations of dozens of species in a matter of months . To the north in Mexico, Guatemala and Honduras, die-offs were not seen, but unexplained disappearances of dozens of species were noted throughout the region in the 1990s and 2000s [39,40]. Decades later, scientists were able to revisit historic collecting sites and search for Bd [39,40] or to test museum specimens collected from these sites . They found that the first appearance of Bd in the museum record was followed by the rapid loss of wild populations of both anurans  and salamanders , further extending the epidemic wave from Mexico through Panama.
In Central America, chytridiomycosis has reshaped the patterns of amphibian biodiversity, obscuring historical biogeographic patterns such that distance between sites is no longer correlated with community similarity. Community composition in the region was severely disrupted by epidemics at multiple sites and many of the unique species of these communities were eliminated, with disproportionate effects on endemic species. This resulted in a homogenization of the regional fauna and ecological homogenization of reproductive mode and habitat . As a result, the impacts of chytridiomycosis have altered community structure and our ability to detect biogeographic patterns.
Among Neotropical amphibians, the harlequin frogs (genus Atelopus) are an iconic species in both research and conservation. As a group, the harlequin frogs (genus Atelopus) are one of the most threatened groups of amphibians in the world, having experienced severe population declines and extinctions from Bd throughout their range from Costa Rica and Panama to Colombia, Ecuador, Venezuela and Peru . The similarity of response among such a large number of closely related species is the best example of taxonomy predicting response to disease. At least 40 of 97 described species have disappeared in the past 20 years, with three species listed as extinct and 82 species listed as endangered or critically endangered  only 10 species are not threatened [36,43]. Declines have been so widespread and so obvious that Lips et al.  used the reports of losses to map the spatio-temporal pattern of population declines in Atelopus to represent the hypothesized spread of Bd through South American Andes. Atelopus species are also important because they may play a disproportionate role in disease dynamics. DiRenzo et al.  demonstrated that Atelopus zeteki is an acute supershedder, producing exceptionally high numbers of zoospores over several weeks prior to death. This led to the hypothesis that Atelopus species might contribute to increasing the pool of zoospores in the environment and the prediction that the presence of Atelopus species in a community might amplify disease and cause populations of other species to decline. This will be a key concern as conservation programmes consider reintroduction of Atelopus and other susceptible species into communities where they have been extirpated and where surviving species have achieved coexistence with Bd. While some species that had been missing for many years have been ‘rediscovered’, the low numbers, continued mortality and lack of large-scale recovery indicate that disease must still be causing unobserved mortality or lack of recruitment in amphibian populations at these sites. To effectively manage Atelopus or other species where Bd is present, we need to know how disease has altered demographic rates of species such as survivorship, recruitment and transitions between life stages. Quantitative data from four species of Atelopus  can provide the baseline data for reintroductions and help understand stages limiting to natural population recovery. As in Australia, there is some evidence that warmer regions where environmental conditions could mitigate the impacts of disease  might be the best option for reintroductions.
California has been important because it is the site of some of the earliest enigmatic declines and die-offs of amphibians from remote protected areas. One of the earliest, enigmatic population declines in the USA was noted in the Wyoming toad (Anaxyrus hemiophrys baxteri ) in the mid-1970s it became extinct in the wild within 10 years , but no definitive cause has been attributed. The Yosemite toad (A. canorus) experienced population declines in 1976 . Some of those carcasses were later found to be infected with Bd , and it is presumed that Bd caused those declines. A mass die-off of mountain yellow-legged frogs (Lithobates muscosa) in the Sierra Nevada of California was seen in 1979, followed by population declines and extirpation by 1983 .
Understanding the mechanisms that drive a chytridiomycosis epidemic came from long-term studies of mountain yellow-legged frogs in alpine lakes across the California Sierra Nevada [9,54]. These researchers documented the invasion of Bd-GPL into naive populations of amphibians where it caused die-offs, population declines and extirpations. They identified intensity of infection as the key predictor of mortality, with higher intensities associated with individual mortality and the average intensity of the population determining whether a population persists or is extirpated . For mountain yellow-legged frogs in the Sierra Nevada, individuals die at 10 000 zoospores, and populations are extirpated when average infection is 10 000 zoospores . However, these values cannot be applied to all systems, as the mortality threshold varies among species [44,55]. Higher frog density was identified as the mechanism causing more infections, greater numbers of zoospores and a larger environmental pool of zoospores that caused higher intensities and mortality . Similar data do not exist for any other species or system, although other studies have shown that higher intensity of disease is generally associated with greater mortality or morbidity. The large number of ponds spread out across a large area has allowed repeated observations of epidemics, to document the epidemic spread of Bd across the landscape. These results are consistent with the epidemic wave hypothesis, and provide an example independent of those from Central America, Andean South America and Australia.
The single-species system of the California Sierra Nevada also offers a contrast to the multiple-species, highly complex tropical cloud forest amphibian fauna of Central America. For two systems that are so different in terms of climate, habitat and biological composition, it is remarkable that the patterns of disease spread and epizootics are so similar. The next step is to determine whether disease dynamics of the epidemics described in Panama  match those reported for the Sierra Nevada system, and whether frog density and disease load are the key predictors of tropical epizootics and species response to infection.
(iv) Puerto Rico
In Puerto Rico, three endemic species of amphibians became extinct in the 1970s . No die-offs were ever observed, but museum specimens showed that Bd has been present since 1976. Like species that disappeared from Panama and Costa Rica, these species were habitat specialists, and at least one was associated with streams. Using capture–mark–recapture and disease surveillance, researchers were able to document ongoing population declines associated with disease despite the lack of detectable mortality. Today, surviving species are at lower abundance than historically, and populations fluctuate with climate patterns . Burrowes et al.  proposed a synergism between disease and climatic conditions to explain fluctuations. They proposed that during the dry season when these frogs aggregate in retreat sites, the localized high density promotes the spread of Bd, and highly infected individuals die in the retreat site. It is not known how this Bd-GPL lineage got to the island however, severe outbreaks of chytridiomycosis on other Caribbean islands such as the extirpation of mountain chicken frogs (Leptodactylus fallax) on Dominica and Montserrat  have been caused by Bd-GPL, showing that this lineage is actively island-hopping across this region.
(v) Salamander chytrid
Batrachochytrium salamandrivorans (Bsal) is a recently discovered species of salamander-specific chytrid  that has been introduced into Europe, where it is causing mass die-offs and population declines in several species [58,59]. Field and museum sampling has shown that Bsal has been present in Asia for over 150 years and is present in the wild in at least three countries , although the impact on Asian populations is not known. Bsal has not been found in North America [46,61], although several species are highly susceptible in the laboratory. Because North America is a global hotspot for salamanders, with 10 families and 675 species , the US Fish and Wildlife Service has taken preventative measures and imposed a moratorium on imports of 201 species belonging to genera known to be carriers of Bsal. Because the USA lacks a formal policy requiring inspection or testing of live imports of most wildlife species for diseases or pathogens, native species remain at risk of invasion by these and other pathogens. Switzerland has taken similar steps and passed a law banning the import of all salamanders into that country.
(b) Chytrid present, but impacts on amphibians varied or unknown
At least two lineages of Bd are present in Africa, Bd-Cape, an endemic lineage from South Africa, and the invasive Bd-GPL . Neither the geographical nor taxonomic distribution of either lineage has been described for Africa, and no demographic studies have assessed impacts of either lineage on wild populations. Bd has been present in South Africa since at least 1938 and is commonly found in Ghana, Kenya, South Africa and Western Africa . Bd is widespread in many of the highland regions , whereas tropical West African areas regions are reported to be clear of Bd . Recent reports from Cameroon  found Bd present in several species, several of which had shown recent declines and they suggested Bd as a likely cause. One epidemic of chytridiomycosis has been reported from Africa, that of the Kihansi Gorge in Tanzania. In 2003, a mass mortality event was followed by population declines of many species that was attributed to Bd . It is not known which lineage of Bd caused this mortality event, although patterns suggest the invasive Bd-GPL. It was hypothesized that infected frogs arrived at the site in construction material, although how the site remained Bd-free after a century of Bd presence in surrounding areas is curious. Luckily, a captive population of the endemic Kihansi spray toad (Nectophrynoides asperginis) had been established in captivity, and more than 2000 animals were reintroduced to the site in 2013, despite the fact that chytrid has never been eliminated from any site where it occurs. Many kinds of laboratory and field studies are needed to explain amphibian𠄼hytrid history at Kihansi Gorge, and across Africa.
Studies in Asia are likely to be very enlightening in understanding the history of the amphibian𠄼hytrid system. Bd is widely distributed throughout Asia  and has been present in the region since at least 1911 , but no reports exist of disease-driven mortality or population declines . Many distinct genotypes are being described from the regions, including Japan , Korea  and India  as well as Bsal . The growing diversity of chytrid pathogens of amphibians from Asia suggests a likely site of origin for these pathogens. Unfortunately, Asia has one of the poorest records of amphibian demographic research both in general and in relation to chytrid infection, and this greatly limits conclusions on the evolutionary history of the system and hampers effective conservation and management.
In their meta-analysis of published studies, Houlahan et al.  identified rapid and widespread population declines in many amphibians in the UK and western Europe starting in the late 1950s and lasting into the late 1960s. Further research is needed to assess causes and patterns of these historic declines, but disease is one of several possible causes. Europe is home to at least three species of chytrid: an endemic Swiss lineage, the invasive Bd-GPL lineage and invasive Bsal. Despite extensive research across the region, evidence for epidemics is limited to several recent die-offs and population declines in montane regions of Spain and the French Pyrenees. The earliest confirmed case of mass mortality and subsequent population declines from Bd was during 1997  from Central Spain. By 2007, populations of Salamandra salamandra also began to decline in this park and mass mortalities of Bufo bufo were observed [74,75]. Garner et al.  surveyed field and museum samples collected across Europe between 1994 and 2004, and found evidence for infections in Spain, Portugal, Italy, Switzerland and Great Britain from as early as 1998. They concluded that chytrid is widespread, even though epidemics and mass mortalities have rarely been reported. Walker et al.  modelled the geographical distribution of Bd in Iberia and looked for evidence of both spread and environmental forcing of disease emergence. They found multiple distinct genotypes consistent with either a history of multiple introductions or of a single ancient introduction of Bd into Iberia. The impact of chytrids on amphibians in other parts of Europe is less clear. Switzerland has its own unique, ancient lineage of Bd (Bd-CH ) that may be hypovirulent as there are no confirmed chytrid-related die-offs or population declines from that region . Many Swiss populations of amphibians are missing from many historical localities, although causes are complex and none have definitively been attributed to disease. The situation is similar to that in Italy, where Bd has been present for several decades and populations of some species are declining, but no causative link has been established [79,80]. Spitzen-Van Der Sluijs et al.  showed that in the Netherlands Bd was present in many species but at low prevalence and low intensity. They concluded that in Europe host responses are geographically and taxonomically inconsistent and are influenced by environmental factors or strain-dependent variation in virulence. For example, they found that Alytes obstetricans in the Netherlands is infected but not declining, whereas populations of that species in upland areas of Spain are highly susceptible. As Bsal continues to spread in the region , researchers will have opportunity to establish capture–mark–recapture studies to quantify the response of native amphibians to co-infection by multiple chytrid lineages.
Brazil presents one of the most complex amphibian𠄼hytrid stories and has contributed to our understanding of the amphibian𠄼hytrid system in several unique ways. Brazil was among the first sites to show evidence of mass die-offs of a diverse community of amphibians. Heyer et al.  observed a mass die-off of an entire community of amphibians in a protected site in the Atlantic Coastal Forest of Brazil in 1979. They attributed the loss to a particularly bad winter, but Bd has since been found throughout the Atlantic coastal forest , although no definitive link has been made between losses at this site and chytridiomycosis. This is the general situation for most of the country, where reports of mass mortality are lacking  and definitive impacts of chytrids on amphibian populations are inconsistent or lacking. Like Europe, Brazil hosts multiple lineages of chytrid but is the first to show evidence of hybridization between lineages [83,85,86]. Brazil is unique in having amphibians infected by both the endemic lineage (Bd-Brazil) and the invasive Bd-GPL for over 100 years. Importantly, the long history of Bd-GPL in Brazil suggests additional vectors of pathogen introduction beyond the trade in amphibians. The lack of clear evidence of population declines, the long occurrence of Bd in Brazil and the low but steady prevalence of infection in Brazil suggests that these amphibians have evolved ways of coexisting with chytrid  and may produce insights for long-term planning in other regions of the world.
(v) Eastern United States
Bd is widespread in North America  and generally occurs at low prevalence and low intensity in the eastern US . Bd has been present in the USA for at least 140 years, with the oldest records from southern leopard frogs (Lithobates sphenocephala) collected in Illinois in 1888 . In this region, no die-offs have been reported, no species have been lost, and no recent dramatic population declines have been identified. Disease prevalence in Illinois today is some of the highest reported, and infection intensity is sufficient to cause death in California ranids. It is not known which lineage of Bd is present in Illinois, and whether it is native or introduced. Such a long coexistence between host and pathogen in Illinois suggests that amphibians and chytrids may have a long coevolutionary history there or that the USA is home to an endemic lineage .
That is not to say populations have not experienced declines or are at risk of future declines. An older, slower, silent wave of population declines occurred in the USA with little note, and is continuing today. Houlahan et al.  described widespread declines in amphibians in North America and Europe in the 1950s. Most of these populations did not recover, but experienced a second round of declines in the 1970s and 1980s. To date, no cause of those declines has been put forward. A pattern of slow, unnoted declines has been shown for amphibians across the USA . These authors synthesized amphibian monitoring data and detected a 3.7% annual decline in occupancy, with southern species of amphibians (especially salamanders) showing the greatest declines. They concluded that declines may be more widespread and severe here than previously recognized, but did not assign any specific cause to any of these declines. Grant et al.  compared the spatial patterns and intensities of four threats—including chytridiomycosis—to declines in species occupancy for those US Geological Survey data and concluded that no single threat was consistent in explaining observed trends and that amphibian response to the threats varied spatially.
One of the largest and most widespread reports of salamander declines comes from the Appalachian Mountains. Declines in populations in terrestrial forest salamanders (Family Plethodontidae: Plethodon) occurred throughout the eastern US in the late 1970s to early 1980s , although die-offs were not noted and no definitive cause has been identified. Caruso & Lips  resurveyed many of Highton's historic collecting sites and found that occupancy and detection were lower for many species of Plethodon in the Great Smoky Mountain National Park. They resurveyed more species at Highton's historic sites and found that many populations of multiple genera have declined in both occupancy and detection (Caruso et al. 2012, unpublished data). Extensive testing for disease in living animals and in preserved museum specimens from these sites produced very few animals infected with Bd. The lack of mortality, species extinction or disease epidemics all suggest either that the eastern US lineage of Bd has been present for a long time, and native species have evolved to avoid infection, that it is hypovirulent like Bd-CH, or that undiscovered lineages of chytrid fungi are present but not detected with current molecular assays.
(c) Chytrid-free areas
Places where Bd has not yet arrived are rare, but generally are oceanic islands such as Papua New Guinea, Fiji, the Solomon Islands [67,92] and the Seychelles . The Seychelles support an amphibian fauna that is 92% endemic , which makes it impossible to predict responses based on phylogeny. However, in the case of Papua New Guinea, the fauna has strong connections to that of Australia, which was severely affected by Bd in the 1980s , suggesting that impacts of introduced chytrids there are likely to be devastating. Understanding how are these areas have not become infected can inform efforts to prevent its establishment. Human movements and commercial trade have been linked to international movement of chytrid fungi across large scales [16,94]. The recent detection of Bd on Madagascar —which is 1800 km from the Seychelles—is especially worrisome. Conservationists were prepared for a devastating effect when Bd was first detected on Madagascar, but so far, these have not materialized . Unfortunately, the Madagascar chytrid has not been sequenced, so we do not know which lineage it is, or how it got there. Also lacking are experimental data on the response of Madagascar amphibians to infections. It will be important to institute disease surveillance and amphibian monitoring in these uninfected places to provide information on the arrival and spread of disease. Testing species in the laboratory for susceptibility will be critical to predict the response in the field and design conservation measures.
Historical and contemporary implications of panzootic chytridiomycosis
Our results point to endemism of B. dendrobatidis in Asia, out of which multiple panzootic lineages have emerged. These emergent diasporas include the virulent and highly transmissible BdGPL, which spread during the early 20th century via a yet unknown route to infect close to 700 amphibian species out of
1300 thus far tested (34). With more than 7800 amphibian species currently described, the number of affected species is likely to rise. The international trade in amphibians has undoubtedly contributed directly to vectoring this pathogen worldwide (Fig. 4) (35, 36), and within our phylogeny we identified many highly supported (≥90% bootstrap support) clades on short branches that linked isolates collected from wild amphibian populations across different continents (Fig. 4 and figs. S10 to S14). However, the role of globalized trade in passively contributing to the spread of this disease cannot be ruled out. It is likely no coincidence that our estimated dates for the emergence of BdGPL span the globalization “big bang”—the rapid proliferation in intercontinental trade, capital, and technology that started in the 1820s (37). The recent invasion of Madagascar by Asian common toads hidden within mining equipment (38) demonstrates the capacity for amphibians to escape detection at borders and exemplifies how the unintended anthropogenic dispersal of amphibians has also likely contributed to the worldwide spread of pathogenic chytrids.
The hyperdiverse hotspot identified in Korea likely represents a fraction of the Batrachochytrium genetic diversity in Asia, and further sampling across this region is urgently needed because the substantial global trade in Asian amphibians (39) presents a risk of seeding future outbreak lineages. Unique ribosomal DNA haplotypes of B. dendrobatidis have been detected in native amphibian species in India (40, 41), Japan (16), and China (42). Although caution should be observed when drawing conclusions about lineages based on short sequence alignments (fig. S3), other endemic lineages probably remain undetected within Asia. It is noteworthy that the northern European countryside is witnessing the emergence of B. salamandrivorans, which also has its origin in Asia. The emergence of B. salamandrivorans is linked to the amphibian pet trade (43), and the broad expansion of virulence factors that are found in the genomes of these two pathogens is testament to the evolutionary innovation that has occurred in these Asian Batrachochytrium fungi (23).
Our findings show that the global trade in amphibians continues to be associated with the translocation of chytrid lineages with panzootic potential. Ultimately, our work confirms that panzootics of emerging fungal diseases in amphibians are caused by ancient patterns of pathogen phylogeography being redrawn as largely unrestricted global trade moves pathogens into new regions, infecting new hosts and igniting disease outbreaks. Within this context, the continued strengthening of transcontinental biosecurity is critical to the survival of amphibian species in the wild (44).
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