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Fungal ecology /631/326/193/2539 1 Fungal evolution /631/326/193/2540 2 Fungal genomics /631/326/193/2541 3 Fungal pathogenesis /631/326/193/2542 4 Invasive species /631/158/2178 5 Conservation biology /631/158/672 6 Herpetology /631/601/2721 7

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Chytrid fungi and global amphibian declines 9 10 Matthew C. Fisher1* and Trenton W. J. Garner2 11 12 1 MRC Centre for Global Infectious Disease Analysis, School of Public Health, Imperial 13 College London, London, UK 14 15 2 Institute of Zoology, Zoological Society of London, London, UK 16 17 * corresponding author: matthew.fisher@imperial.ac.uk 18 19 20 ABSTRACT: 21 22 Discovering that chytrid fungi cause chytridiomycosis in amphibians represented a 23 paradigm shift in our understanding of how emerging infectious diseases contribute 24 to global patterns of biodiversity loss. In this Review, we describe how the use of 25 multidisciplinary biological approaches has been essential to pinpoint the origins of 26 amphibian-parasitising chytrid fungi, including Batrachochytrium dendrobatidis and 27 Batrachochytrium salamandrivorans, to time their emergence, to track their cycles of 28 expansion and to identify the core mechanisms that underpin their pathogenicity. We 29 discuss the development of experimental methods and bioinformatics toolkits that 30 provide a fuller understanding of batrachochytrid biology and inform policy and 31 control measures. 32 33

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[H1] Introduction 34 35 The reasons why modern-day amphibians are suffering rates of extinction that far 36 exceed those of any other class of vertebrates long mystified conservation biologists. 37 The discovery of the disease chytridiomycosis and its aetiological agents, chytrid fungi 38 in the genus Batrachochytrium, provided the link between emerging infections and 39 global amphibian declines. Historically underappreciated and infrequently studied, 40 these ancient, aquatic, flagellate fungi have earned notoriety as the leading infectious 41 disease threat to biodiversity. Following the concurrent detection of chytridiomycosis 42 in Central America and Australia in the late 1990’s 1 and identification of the cause 2, 43 Batrachochytrium dendrobatidis (Bd) has been found to infect species across all 44 continents where suitable hosts occur 3,4. Although Batrachochytrium was initially 45 thought to contain only one species the local extinction of fire salamanders in the 46 Netherlands by chytridiomycosis in 2010 led to the discovery of another pathogenic 47 species in the genus, Batrachochytrium salamandrivorans sp. nov. (Bsal 5). Both 48 pathogens (here called ‘batrachochytrids’ for brevity) infect the skin of amphibians. 49 This leads to ulceration due to infection of epidermal cells by Bsal whereas Bd infects 50 and develops in subcutaneous epidermal cells. Because amphibians need to 51 osmoregulate and respire through their water-permeable skin, skin disruption impairs 52 its essential homeostatic functions and leads to the death of heavily-infected 53 individuals. 54 55 Despite over 1,000 studies published since the discovery of Bd the original questions 56 regarding the extent of this panzootic [G] are still relevant today: where did these 57 pathogenic chytrids come from, when did they emerge, how do they cause disease in 58 amphibians and what can we do to prevent their impact? In this Review, we describe 59 how the adoption of new techniques and methods from across biology and 60 informatics has recently led to a radical change in our understanding of 61 batrachochytrids and chytridiomycosis. To achieve these advances, we explain how a 62 multidisciplinary scientific community built global networks for sharing data, 63 combined field research with modern biological techniques to dissect complex 64 biological systems, and improved the integration of resulting epidemiological data into 65 policy and law with the aim to limit the further spread of these pathogens. 66 67 [H1] Mapping the chytrid panzootic 68 69 By their very definition, panzootics are a global problem and cannot be tackled by 70 individual people or specialities. The realization that similar patterns of amphibian 71 declines occurred on several continents at the same time was a wake-up call and 72 highlights that an interdisciplinary scientific approach is needed to understand and 73 respond to novel conservation threats. In isolation, herpetologists had recorded rapid 74

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and persistent amphibian declines as early as the 1970s; however, these declines were 75 only recognized as a global phenomenon at the landmark first World Congress of 76 Herpetology held in Canterbury in 1989 and quantified over a decade after the 77 Canterbury meeting 6. Many declines were initially classified as ‘enigmatic’, occurring 78 in pristine habitats largely untouched by habitat destruction. These observations 79 spurred a search for the underlying cause, which ultimately led to the discovery and 80 description of Bd and its life cycle through a multidisciplinary collaboration 1,2,7. 81 82 The development of a non-invasive, robust and probe-based quantitative molecular 83 diagnostic for Bd 8 enabled several regional surveillance efforts eventually compiled in 84 an online database, the Global Bd Mapping Project. This web-based system for 85 collating Bd incidence and associated metadata is an early example of a web-86 accessible database with application programming interfaces (APIs) for data storage, 87 data uploading, summary statistics, and visualisation of spatial data using Google 88 Maps. The Global Bd Mapping project is being integrated into the core AmphibiaWeb 89 site (https://amphibiaweb.org) where data compilation will continue in the 90 foreseeable future. Global mapping provided the first overview of the panzootic: as of 91 May 2019, Bd was found infecting 1,015 of 1,854 (54%) species and at 3,705 of 9,503 92 (39%) field sites (personal communication by D. Olson and K. Ronnenberg, US Forest 93 Service). In 2014, Bd infected 50% of tested frog species (order Anura), 55% of 94 salamander and newt species (clade Caudata) and 29% of caecilian species 95 (Gymnophiona) 9 testifying to an extraordinary and heretofore unmatched pathogen 96 host range. By comparison, the host range of Bsal is largely restricted to Caudata, with 97 only transient infection of anurans reported10. Global surveillance and molecular 98 diagnostics enabled rapid outbreak analysis of Bsal in the year of its discovery and in 99 doing so identified an Asian origin of the European Bsal outbreak 11,12. As with Bd, 100 surveillance of Bsal is being managed and coordinated using online databases and 101 informatic tools (http://www.salamanderfungus.org/about-bsal/ and 102 https://amphibiandisease.org). 103 104 Reconstructing the impact of the emergence of the batrachochytrids on amphibian 105 biodiversity has proven a complex task. This difficulty owes to declines occurring years 106 before the identification of Bd and frequently in remote areas where amphibian 107 surveillance efforts are haphazard at best. Nevertheless, a meta-analysis4 synthesised 108 data from multiple sources, including peer-reviewed studies, the International Union 109 for Conservation of Nature (IUCN) Red List of Threatened Species and consultations 110 with the scientists investigating the declines as they occurred (e.g. 13), and 111 retrospectively (e.g. 14,15). This meta-analysis revealed that chytridiomycosis has 112 contributed to the decline of at least 501 species (6.5% of all amphibian species), 113 leading to 90 presumed extinctions and decreases in abundance exceeding 90% in 114 another 124 species. At the time of writing, this represents the greatest documented 115

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loss of biodiversity attributable to a non-human species. Truly, Bd has earned its nom 116 de guerre as the ‘doomsday fungus’ 16. 117 118 Alongside the collation of epidemiological data, a worldwide effort to collect and 119 archive isolates of Bd for molecular and phenotypic analyses was initiated by the 120 European Union Project RACE (Risk Assessment of Chytridiomycosis to European 121 amphibian diversity). This project focussed on modifying the original protocols for 122 isolating Bd developed by Joyce Longcore2 and methods for cryopreservation17. RACE 123 developed a largely non-destructive procedure for isolating chytrids from amphibians 124 that, during a decade, was successfully used by a broad collective of researchers 125 working across 5 continents, 23 countries and 62 amphibian species. As a result, Bd 126 was isolated from all three orders of Amphibia and from all continents on which 127 infection occurs18. This project integrated online databases and digital mapping to 128 store project-related data in a way that enabled access from study sites using the GPS-129 smartphone enabled epidemiological software EpiCollect 19 130 (https://five.epicollect.net/project/bd-global-isolation-protocol). With these 131 webtools and smartphone-based technology, research groups working on the 132 batrachochytrids communicated and shared data on a scale that had never before 133 been used to track wildlife diseases, which has been essential for subsequent tracing 134 of the evolutionary history of these pathogens. 135 136 [H1] Origin and emergence 137 138 [H2] Chytrids ‘out-of-Asia’ 139 140 Debate on how chytridiomycosis emerged as a cause of amphibian declines revolved 141 around two competing arguments. The ‘novel pathogen hypothesis’ (NPH) stated that 142 chytridiomycosis was emerging locally after it has been seeded by intercontinental 143 trade routes into naïve ecosystems. The counterargument, known as the ‘endemic 144 pathogen hypothesis’ (EPH), held that Bd was a widespread endemic commensal of 145 amphibians that had become more virulent owing to global change forcing 146 imbalanced infection dynamics20. Early molecular clues from multilocus sequence 147 typing [G] supported the NPH, as the isolates of Bd sampled at the time showed no 148 signs of phylogeographic structure from the different regions with amphibian declines 149 due chytridiomycosis 21,22. This molecular evidence matched the observed patterns of 150 chytridiomycosis observed in the Americas1, Australia20 and the Caribbean islands23 151 (Figure 1). Later, sequencing of two complete genomes by the Joint Genome Institute 152 (isolate JAM81 from Rana muscosa in California) and the Broad Institute (isolate 153 JEL423 from Phyllomedusa lemur in Panama) in 2008 24 and the development of high-154 throughput shotgun-sequencing enabled genome-scale genetic analysis of Bd. Early 155 ABI SOLiD genome resequencing of 20 globally distributed isolates from sites 156

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experiencing chytridiomycosis uncovered striking patterns in comparison to sites 157 without disease. Resequencing identified three deeply diverged lineages: BdGPL 158 (globally distributed), BdCAPE (named owing to its discovery in the Cape region of 159 South Africa) and BdCH (a single deeply branched isolate from Gamlikon in Switzerland 160 25). Only BdGPL was found across four continents and associated with epizootics [G] 161 in North America, Central America, the Caribbean, Australia, and Europe. The 162 extraordinary global range, limited genomic diversity, relatively high virulence, and 163 origin in the early 20th Century based on the phylogeny of BdGPL supported the NPH 164 over the EPH 25. Heterozygous and triallelic single nucleotide polymorphisms were 3-165 4 fold more common than homozygous ones in BdGPL, which was held as evidence 166 that the genesis of BdGPL was the result of a sexual ‘hybridization’ between two 167 dissimilar parental genotypes 26. 168 169 Subsequent analysis of a larger panel of isolates cast doubt upon the findings of this 170 earlier study 25, suggesting both greater genetic diversity and an estimated origin of 171 BdGPL 10,000–40,000 years before present 27. The authors interpreted these results 172 to support the EPH rather than the NPH. Neither study could resolve the geographic 173 origins of Bd, variously proposed to be African28, Japanese29, East Asian30, South 174 American31, or North American32. 175 176 O’Hanlon et al 33 resolved much of the debate, publishing new sequence data for 177 177 Bd isolates collected using the RACE protocols 18. The complete dataset of 234 isolates 178 were collected over nearly two decades and spanned the geographical distribution of 179 Bd, events of lethal chytridiomycosis and all three extant orders of the Amphibia. This 180 analysis redefined the evolutionary relationships amongst lineages of Bd, aided by the 181 first genome data from Asian isolates. Bd from the Korean peninsula comprised a new 182 4th lineage, BdASIA-1, and this lineage showed signs of an ancestral relationship with 183 the other lineages. Bayesian-based haplotype clustering [G] revealed that the 184 hyperdiverse BdASIA-1 lineage shared more diversity with the global population of Bd 185 than any other lineage and branched at the base of the Bsal-rooted Bd phylogeny. 186 Tellingly, BdASIA-1 was the only lineage in mutation-drift equilibrium [G], a 187 characteristic of endemism. All other lineages showed pronounced departures from 188 equilibrium values of Tajima’s D statistic [G] 34, which are indicative of outbreak 189 dynamics. Molecular screening of museum specimens of amphibians from Korea 190 showed infection has been present in the region for over 100 years35 and 191 contemporary surveillance has demonstrated a widespread yet patchy and rare 192 distribution of batrachochytrids throughout East Asia12,36,37, further suggesting 193 endemism of Bd in this region. Multilocus genotyping confirmed the results of 194 O’Hanlon et al. 33 and discovered a novel 5th lineage, BdASIA-3, also found in East Asia 195 (the Philippines, Indonesia and China) 38. This ‘chytrid-out-of-Asia’ hypothesis 196 supporting the NPH was strengthened by the finding that, following discovery of 197

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chytridiomycosis caused by Bsal in Europe, this chytrid could only be detected 198 elsewhere in south-east Asia (Vietnam)12. The comprehensive lack of lethal outbreaks 199 or population declines caused by chytridiomycosis in Asia, despite the widespread 200 occurrence of Bd and Bsal 4,11, are evidence for endemic host-pathogen interactions39. 201 Batrachochytrids appear to have been infecting amphibians in the region for over 50 202 million years, leaving ample time for fungal speciation events and relatively stable 203 host-pathogen dynamics to establish11. Accordingly, there is a need for more intensive 204 pathogen discovery across south-east Asia as unmapped batrachochytrid diversity will 205 likely yield further insights into the past emergence and present distribution of these 206 pathogens. 207 208 [H2] Timing the panzootic 209 210 Final proof of the NPH required congruency between the timing of introductions of Bd 211 and the onset of declines caused by chytridiomycosis. Chytridiomycosis-declines 212 peaked globally in the 1980’s 4, in keeping with the timing of regional wave-like 213 dynamics suggesting epizootic spread from point sources 40,41. To time introductions, 214 O’Hanlon et al. 33 used two quasi-independent genomic regions to generate time-215 calibrated phylogenies and a Bayesian framework to estimate their time to most 216 recent common ancestor (TMRCA, Box 1). These analyses estimated a substitution 217 rate for Bd, one that was broadly similar to that estimated for other unicellular fungi. 218 The updated TMRCA for the ancestor of BdGPL ranged between 120 and 50 years ago 219 (1890’s–1960’s), which broadly agrees with the first inferred chytridiomycosis-related 220 declines in regions that are currently dominated by BdGPL (Australia20,42, the 221 Mesoamerican peninsula13 and South America14,40). Molecular dating also suggests 222 that the widespread, and still largely unattributed, amphibian declines reported in 223 Europe and North America in the 1950’s and 1960’s were driven by BdGPL, which has 224 now achieved widespread endemicity across these regions6,43. 225 226 What has fuelled the global expansion of Bd? That all known lineages of Bd are 227 circulating in globally-traded amphibians proves that trade is disseminating amphibian 228 vectors of batrachochytrids worldwide 44 today 33 (Figure 2). For example, ‘African’ 229 BdCAPE invaded the island of Mallorca through the reintroduction of captive reared 230 Mallorcan midwive toads infected in captivity by African endemic amphibians 231 (Xenopus gilli) 45. More widely, infection-tolerant species such as the African clawed 232 frogs Xenopus laevis 28 and north American bullfrogs Lithobates catesbeiana 44 are 233 internationally traded in their millions and have been since early the 20th century. 234 Other infection-tolerant species, such as the cane toad Rhinella marinus, have 235 established feral populations from their origins in South and Central America. It is 236 likely that these species had an important role in amplifying the worldwide emergence 237 of Bd and indeed, molecular methods have identified transcontinental links involving 238

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these species 46. The evidence therefore suggests that the original out-of-Asia vectors 239 of batrachochytrids were likely amphibians exported either for food, research or 240 collections, or perhaps passively hiding in traded goods. However, identifying these 241 original panzootic ‘sparks’ will likely prove a challenging task. 242 243 [H2] Cycles and circles of expansion 244 245 Occurrence of the divergent BdCAPE in Africa, Central America and Europe 33,38, 246 BdASIA-2/BRAZIL in the Brazilian Atlantic forests 31 and Korea 33, and the ASIA-1-like 247 BdCH in Switzerland show that the evolutionary history of Bd is complex and 248 characterized by at least three out-of-Asia emergences of lineages other than BdGPL. 249 With too few isolates to confidently derive measurable evolutionary rates, the TMRCA 250 for these lineages have thus far not been estimated. Notwithstanding, levels of 251 diversity exceed those seen in BdGPL suggesting that their out-of-Asia dispersal 252 predates that of BdGPL 33. The detection of molecular signatures of Bd in Brazilian 253 museum collections of amphibians indicates that Brazil was invaded by Bd as far back 254 as 1894 31. While awaiting molecular confirmation, it appears that the early invasion 255 was by BdASIA-2/BRAZIL, followed by a secondary introduction of BdGPL into Brazil in 256 the 1970s. The result was a peak of declines in the 1970s owing to the higher virulence 257 of this lineage 14 and the founding of a region of contact between the two lineages in 258 the Brazilian Atlantic forest 47-49. To complicate matters further, BdASIA-2/BRAZIL is 259 itself found in Korean populations of introduced North American bullfrogs, suggesting 260 that these widely-traded frogs have been vectors for this lineage, re-establishing it in 261 its ancestral Asian homeland 33. 262 263 Surveillance across Africa shows that this continent also has a complex history of Bd 264 introductions50. The pathogen is widely present, occurring in Cameroon from at least 265 1933, Kenya in 1934, Uganda in 1935, South Africa in 1938, the Democratic Republic 266 of Congo in 1950 and Bioko island in 1966 28,51-54. The infection status of the 267 amphibians of Madagascar remains unclear 18,55,56. The extent that Africa has suffered 268 amphibian declines as a consequence of chytridiomycosis is largely undetermined. 269 However, at least one extinction in the wild has occurred (the Tanzanian Kihansi Spray 270 Toad, Nectophrynoides asperginis 15) and the presence of Bd has been correlated with 271 declines of amphibian species in Cameroon 57 and South Africa 58. Genome sequencing 272 33 and multilocus genotyping 38 has shown the widespread occurrence of both BdCAPE 273 and BdGPL, the former widely distributed in Cameroon, including in caecilians 59, and 274 the latter occurring in both Ethiopia and Uganda. Both lineages occur in Southern 275 Africa where, similar as in Brazil, lineages are in spatial contact. The patchy distribution 276 of BdCAPE in central America and Europe suggests that secondary waves of expansion 277 for this lineage have occurred. 278 279

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[H2] Recombinants, not hybrids 280 281 Genotyping has identified recombinants of BdASIA-2/BRAZIL and BdGPL in the 282 Brazilian Atlantic forest 48, and genetic mosaics of BdCAPE and BdGPL in South Africa 283 33. Within lineages, alleles segregate 47,60, intrachromosomal recombination 284 breakpoints have been detected 25 and when single nucleotide polymorphisms are 285 phased [G] , crossovers [G] are observed in all lineages that have been tested 26. 286 Clearly, the extreme genetic bottlenecks that characterise the out-of-Asia 287 evolutionary history of Bd have not impaired the ability of this species to recombine. 288 Whereas chytrids such as Allomyces and Rhizophydium undergo meiosis [G], 289 recombinant mating structures have not been described for Bd or Bsal nor canonical 290 fungal mating-type alleles [G] identified, suggesting that recombination in 291 batrachochytrids may not be meiotic. In support of this, some ‘meiotic toolbox’ genes 292 defined in yeast are missing in the genome of Bd and signatures of sex-associated, 293 repeat-induced point mutations in transposable elements are also absent 61. Further, 294 widespread chromosomal copy number variation [G] 26 is also evidence that 295 recombination may not owe to meiosis. Accordingly, it has been proposed 25,62 that 296 non-meiotic recombination (called ‘parasexual’ recombination) may be generating the 297 polyploid heterozygous mosaics that characterise Bd. However, the cell biology that 298 underpins the widespread recombination, either meiotic or non-meiotic, in Bd 299 remains wholly unexplored. 300 301 That the global population of Bd stems from a genetically diverse Asian population in 302 mutation or drift equilibria and recombines when the opportunity arises, shows that 303 the global Bd population is currently behaving as a cohesive biological species. Prior 304 to the discovery of its Asian origin, inter-lineage recombination events were termed 305 ‘hybridisations’, and the origin of BdGPL was suggested to result from a hybridisation 306 event amongst two related chytrid species 25. However, the simplest description of 307 the global population genetic structure of Bd is that each lineage represents separate 308 genealogical ‘draws’ from a recombining parental population that is most likely Asian. 309 As multiple founding events do not appear to have appreciably blunted the ability of 310 Bd to shuffle its genome if given the opportunity, it is premature to give these lineages 311 species status and to name recombinants ‘hybrids’. Accordingly, the most biologically 312 accurate description of the genomic mosaics that are increasingly being described are 313 ‘recombinants’. 314 315 Finding that Bd is a recombining species is not only academically interesting. The 316 process of recombination through secondary contact is likely important in an 317 epidemiological context. Outcrossing can purge deleterious alleles and generate 318 variation that may facilitate host exploitation, exacerbating epizootics. Theory and 319 experimentation have shown that interactions between diverse genotypes can lead to 320

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competitive interactions that result in increased transmission and may exacerbate 321 infection dynamics 63,64. Coinfections of Bd lineages have been observed in South 322 Africa where BdGPL and BdCAPE co-occur65, and in absence of a defined 323 environmental developmental stage, coinfection is when recombination events will 324 occur. That recombination can affect the virulence of Bd was demonstrated in a 325 study49 that showed that BdGPL and BdASIA-2/BRAZIL recombinant genotypes were 326 more aggressive than those of both parents in two amphibian species. Their result 327 suggests that outcrossing in Bd results in genetic dominance and enhanced fitness. 328 Whereas these hybrids were inferred to be F1, an F2 backcross in Brazil has been 329 observed suggesting that recombinants can survive beyond their immediate F1 330 genesis 48. 331 332 [H1] Batrachochytrid virulence 333 334 Infection of amphibians by Bd and Bsal is a remarkably complex process that can have 335 markedly different outcomes, ranging from mild or no symptoms to death (Figure 3). 336 Here we discuss the genetic factors that underpin the expression of the 337 batrachochytrids intrinsic ability to infect the amphibian dermis, alongside the biotic 338 and abiotic factors that modify the outcome of these host-pathogen interactions. 339 340 [H2] Genetic factors 341 342 The identification of significant variation in virulence both within and amongst 343 lineages has raised more questions than have been answered. We and others have 344 shed some light on which intrinsic genetic factors underpin virulence in 345 batrachochytrids (Figure 4). 346 347 Comparisons with the genomes of free-living saprobic chytrids have shown greater 348 secreted protein repertoires and extensive gene-family radiations in the pathogenic 349 batrachochytrids 66,67. Metalloproteases in the M36 metalloprotease fungalysin family 350 are important pathogenicity determinants in a number of skin-infecting fungi, and are 351 strikingly expanded in both Bsal and Bd with 110 and 35 of theses proteases, 352 respectively, compared to the free-living saprobic chytrids Spizellomyces punctatus 353 and Homolaphlyctis polyrhiza, which have 2 and 3, respectively 24 66. That the M36 354 metalloproteases are highly expressed in vivo and in vitro is in line with their role as 355 virulence factors, however differences in the number of copies and timing of their 356 expression between Bsal and Bd suggest different roles in pathogenicity 66. 357 Carbohydrate-binding modules (CBM) are markedly expanded in both 358 batrachochytrids compared to free-living Chytridiomycota and may be important in 359 host recognition and adhesion 66,68. There is pronounced divergence in other gene-360 families that could explain the substantial variation in the host range and 361

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epidemiology of Bd and Bsal. Bd’s significantly smaller genome (23.7 Mb versus 32.6 362 Mb for Bsal) contains regions of low gene density characterised by a proliferation of 363 crinkler-and-necrosis (CRN-like) genes, which are expressed during the early stages of 364 infection, whereas the Bsal genome contains two expanded secreted tribes of genes 365 of unknown function, which are highly expressed during infection. Clearly, although 366 mining the genomes of the batrachochytrids has identified features that are linked to 367 infection, further exploration is needed to understand the role of these diverse 368 expanded gene-families in infection. The development of new models of infection is 369 needed to increase understanding of batrachochytrids biology. Recent advances, such 370 as amphibian cell culture and skin-explant models 69, and in vivo zebrafish Bd infection 371 models 70, are exciting developments. 372 373 The observation that different genotypes and lineages show some variation in plastic 374 morphological traits such as the number and size of infectious zoospores suggests that 375 virulence traits may be to some extent governed by simple parameters such as growth 376 rate and fecundity 71,72. Genetic factors that modify growth rate and investment in 377 zoospores may be found in the large number of genes that are upregulated during 378 infection. Additionally, putative, secreted virulence factors affect host colonization 379 rates, the first step in the pathogen life cycle. Despite its recent evolutionary history, 380 the virulence of BdGPL genotypes is highly variable under controlled experimental 381 settings and virulence is to a large extent determined by how rapidly Bd establishes 382 infection 25,33. Moreover, within the laboratory, passaged isolates show high 383 evolvability 26, attenuation 73,74 and phenotypic variation 75. As described above, 384 genome architecture is highly plastic across short time-scales, involving large scale 385 rearrangements that should affect traits involved in host damage 26,76. The plasticity 386 in virulence observed in BdGPL seems to be mirrored by other lineages, with 387 substantial lethality observed in experimental exposures (eg. 39,49). Although less is 388 known about variation of virulence in Bsal owing to all isolates currently stemming 389 from a single epizootic clone, the discovery of an environmentally-persistent encysted 390 zoospore suggests that this species also may manifest phenotypically-plastic life-391 history traits that affect virulence 10. 392 393 [H2] Abiotic factors 394 395 Batrachochytrids may carry a diverse and variable array of genetic traits that influence 396 virulence, but the global emergence of chytridiomycosis is a radically novel 397 epidemiological event, affecting hundreds of host species near-simultaneously and 398 interacting intimately with the diverse environments that they occupy (Figure 3). 399 Despite overwhelming evidence that batrachochytrids are invasive outside of East 400 Asia, once established, environmental factors have an important role for disease 401 outcomes and infection dynamics may map more closely with the predictions of the 402

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EPH. Indeed, ecological factors have been identified as important determinants of 403 disease outcomes, such as climate and altitude 77,78, seasonality 79-82, ultraviolet 404 exposure 83,84 and agrochemicals 85. Combining field observations with experiments 405 has illustrated the processes through which the environment affects infection and 406 disease. These processes include the importance of reservoirs of transmission 86,87, 407 how the environment affects the survival and abundance of infectious zoospores 81,88-408 90 and how increasing zoospore density drives host mortality through increasing 409 burdens of infection 91,92. Trophic interactions can also affect the density of the 410 infectious zoosporic stages in the environment 10,93. A note of caution here, laboratory 411 measurements of virulence that disregard ecological variation identified in field 412 studies can have limited predictive utility. For instance, repeated experimental 413 observations that virulence of BdGPL exceeds that of BdCAPE 25,33 do not explain why 414 the two lineages are equally likely to be associated with chytridiomycosis and 415 amphibian declines in nature 33. Even the endemic Korean BdASIA-1 has been shown 416 to be virulent in non-Korean amphibians 39, showing that its long coevolutionary 417 history has not blunted this lineages virulence. 418 419 Extrapolating environmentally-driven processes to global change has been 420 predominantly a macroecological exercise 94 and changing climates have been shown 421 to force patterns of chytridiomycosis. For instance, although early analyses suggesting 422 climate change drove patterns of chytridiomycosis in Costa Rica 95 were only weakly 423 statistically supported 94, associations between El Niño events and chytridiomycosis 424 have been demonstrated 96. Increasingly, studies are attempting to incorporate 425 environmental factors into epidemiological models that attempt to predict the 426 outcome of infection at the population level, with a focus on single, highly susceptible, 427 host species such as midwive toads 97 37,80. In these studies the host species that were 428 infected during seasonal ‘outlier’ events experienced mass mortality events not 429 occurring after colder, longer winters, and included a species previously predicted to 430 be qt low risk of disease by a macroecological analysis 98. Less disconcerting, a 16-year 431 time-series99 disentangled the impacts of Bd and climate warming on nine montane 432 species in Iberia. Surprisingly, only a small subset of the host community appeared 433 affected by chytridiomycosis, and regional warming promoted range expansions of 434 some species into the region where disease had decimated one host species decades 435 previously; only a single species showed reasonably tight links between temperature 436 fluctuations and infection dynamics 99. 437 438 [H2] Biotic factors 439 440 Host responses to chytridiomycosis vary on different levels, ranging from individual to 441 population and host community structure (Figure 3). At the individual level, evidence 442 exists for both resistance and tolerance strategies that may involve adaptive and 443

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innate immune responses 100. Bd can evade lymphocyte responses as part of adaptive 444 immunity101, but evidence exists that hosts can, to some degree, improve repressed 445 immune responses over time 102. Whether or not chytridiomycosis has exerted 446 selective pressure on these and other components of adaptive immunity is uncertain, 447 but at least for some host species evidence supports this scenario, or alternatively the 448 pre-existence of host genetic variation that preceded the emergence of the global 449 panzootic and facilitated tolerance to infection when the initial outbreak occurred 103. 450 Equally, or possibly even more, important, is the innate arm of the amphibian immune 451 response, which has been predominantly explored through investigations of secreted 452 antimicrobial peptides (AMPs)100. An example of the importance of AMPs is the 453 threatened European salamander genus Speleomantes, all species of which secrete 454 skin peptides that decrease zoospore survival and thereby prevent infection 104. As 455 with adaptive immunity, the innate immunity afforded by AMPs is influenced by 456 exposure to batrachochytrids. Adaptation of AMPs appears to be the primary driver 457 behind the recovery of some anurans that experienced catastrophic declines due to 458 the emergence of chytridiomycosis in Central America 105. For most amphibians, 459 adaptive and innate immunity vary substantially across host life history stages and age 460 classes, and as a result, so does host susceptibility to infection and disease. This means 461 that, within a single population, one species can simultaneously be a infection-462 tolerant, often larval, reservoir whilst being at risk of decline due to chytridiomycosis 463 in its mature stages 86 84. 464 465 A particularly topical vein of research is exploring how transkingdom interactions 466 between commensal fungi and bacteria of the amphibian skin microbiota may limit 467 batrachochytrid infections 93,106-109. An extension of this question is to understand how 468 pathogen competition can alter batrachochytrid infection dynamics and virulence. 469 Although at very early stages, experiments have illustrated how intraspecific 470 competition amongst B. dendrobatidis lineages may be in part responsible for the 471 emergence of the global pandemic lineage BdGPL 110 and coinfections may be a 472 precursor for the patterns of recombination we have discussed above 33. Furthermore, 473 batrachochytrids will interact with other amphibian pathogens such as the emerging 474 ranavirus, and field data suggest that host declines to cocirculating pathogens exceed 475 what would be predicted if interactions were additive 111. Whether this is attributable 476 to shifts in batrachochytrid virulence is uncertain, and a more likely explanation is that 477 sublethal B. dendrobatidis exposures are facilitating the invasion of a viral pathogen 478 (TWJ Garner, unpubl. data). In either case, interactions between batrachochytrids and 479 other pathogens can shift epidemiological patterns, either through dynamical 480 processes, natural selection, or both. 481 482 [H1] Mitigating batrachochytrid threats 483 484

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Studies 80 99 showing species-specific and variable responses illustrate how we cannot 485 generalize the impacts of batrachochytrids. The emergence of lethal chytridiomycosis 486 can be persistent or transient and the effects on host communities can in themselves 487 modify the virulence of batrachochytrids 4,105. Nevertheless, the global increase in 488 incidence of new fungal infections alongside those that have evolved to evade control 489 has led to the recognition that we urgently need to strengthen detection, monitoring 490 and control of fungal disease 112,113. The identification of East Asia as a hotspot of 491 batrachochytrid diversity alongside its relatively unsurveyed status suggests 492 undiscovered chytrid biodiversity in this region that requires urgent investigation. Our 493 finding that all known lineages of Bd are circulating in globally-traded amphibians 494 proved that, despite listing by the World Organisation for Animal Health, trade is still 495 disseminating amphibian vectors 33 (Figure 2). Stage-specific goals and management 496 actions can theoretically be deployed to prevent and/or manage wildlife disease114. 497 Before the emergence of wildlife pathogens, biosecurity is a first line of defence and 498 therefore needs strengthening through import controls and establishment of an 499 infection-free trade 115. Motivated by the discovery of Bsal, the European Union has 500 implemented health protection measures for the trade of salamanders 116, and similar 501 measures have been adopted by the USA 117 and Canada 118. These pre-emergence 502 ‘prezootic’ biosecurity-oriented strategies remain the best option for avoiding disease 503 emergence and should be urgently adopted across uninfected regions and countries. 504 505 Combating wildlife diseases after invasion is extremely challenging with only one 506 partially successful example for chytridiomycosis. In this example a chemical-led 507 approach using the antifungal itraconazole and the environmental disinfectant virkon 508 was applied to eradicate Bd from Mallorca, which only partially succeeded. However, 509 this approach is not likely applicable to more ecologically complex settings 45,119. 510 Bioaugmentation of amphibian cutaneous microbiota and vaccination have been 511 proposed as methods to strengthen the resilience of amphibians against invasive 512 chytrids. However, despite promising in vivo studies (reviewed by 115,120), this 513 approach has yet to be successfully implemented (but see 121). In situations in which 514 species are highly threatened by the pathogen, their safeguarding through 515 establishing ex situ captive breeding programs currently remains the only active 516 conservation method to avoid species loss after invasion. Amphibian Arks [G] maintain 517 the possibility for selective breeding or genetic modification of amphibians for 518 resistance, and it is likely that advances in gene-editing will be used to augment 519 amphibian immune responses to batrachochytrids in the future115. Clearly, the factors 520 discussed above do not operate in isolation. Interactions between chytridiomycosis 521 and other threatening processes are well-described, and we are beginning to explore 522 how pathogen genotype, host immunity and environmental conditions generate non-523 linear patterns of infection and disease. There is every possibility that strategies for 524 mitigating chytridiomycosis in nature will involve largely ignoring the pathogen and 525

14

focussing on mitigating other threats or modifying environments and host 526 communities so that host responses may operate more effectively. Whatever our 527 responses, the main lesson from the panzootic of chytridiomycosis has been that 528 biodiversity is far less resilient against emerging infections than was previously 529 believed 122. This has been further confirmed in other systems as microorganisms 530 continue to cross continental barriers—the devastating emergence of bat white nose 531 syndrome is a case in point 123. The fragility of wildlife health in the face of 532 globalisation eroding geographical constraints to pathogen spread is exemplified by 533 panzootic chytridiomycosis. It is heartening to see that rapid policy measures enacted 534 following scientific advances are on the rise now that the consequences of failing to 535 prevent batrachochytrid introductions are more widely realised. Although we believe 536 that research will eventually yield the means to mitigate the emergence of wildlife 537 diseases, for research to have its impact reinforcing links between science, policy and 538 the public will be key to success. 539 540 References 541 1 Berger,L.etal.Chytridiomycosiscausesamphibianmortalityassociated542

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985 986 Acknowledgments 987 988 We acknowledge funding from NERC (NE/E006701/1, NE/E006841/1, NE/G002193/1, 989 NE/K014455/1, NE/K012 509/1, NE/M000591/1, NE/N009800/1, NE/N009967/1, 990 NE/S000844/1, NE/S000992/1), The Morris Animal Foundation (D12ZO-002 and 991 D16ZO-022) and the Leverhulme Trust (RPG-2014-273). We thank Dr Simon O’Hanlon 992 and Dr Pria Ghosh who assisted with drafting figures. Matthew C. Fisher is a Fellow in 993 the CIFAR ‘Fungal Kingdom’ Program 994 995

Author contributions 996 Both authors wrote the article. 997

Competing interests 998 The authors declare no competing interests. 999

Publisher's note 1000

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Springer Nature remains neutral with regard to jurisdictional claims in published maps 1001 and institutional affiliations. 1002

Peer review information 1003 Nature Reviews Microbiology thanks Timothy James and the other, anonymous, 1004 reviewer(s) for their contribution to the peer review of this work 1005 1006 Figure 1: Global distribution of Batrachochytrium. As of 2019, Batrachochytrium 1007 dendrobatidis (Bd) has invaded and caused chytridiomycosis in six regions globally: 1008 western Australia, the Mesoamerican peninsula, South America, the western United 1009 States, Africa and Europe. Five lineages of Bd, as well as recombinants, have been 1010 identified. In addition, another species, Batrachochytrium salamandrivorans (Bsal), 1011 was discovered in 2010. Batrachochytrids cause severe amphibian declines. The figure 1012 shows declines that match Scheele et al4 category 3 or above (3, extreme decline with 1013 >90% of individuals lost; 4, presumed extinct in the wild (no known extant populations, 1014 and no individuals detected at known historical locations, but some reasonable doubt 1015 that the last individual has died); 5, confirmed extinct in the wild (as per IUCN listing). 1016 Maps adapted from 65 1017 1018 Figure 2. Global spread of Batrachochytrium dendrobatidis and amphibian trade. A 1019 | Intercontinental movements of Batrachochytrium dendrobatidis (Bd) was inferred 1020 from geographically separated isolates that form closely related phylogenetic clades 1021 with high bootstrap support (≥90%). Numbers show where isolates of Bd have been 1022 recovered from traded amphibians, with pictures of the species involved shown on 1023 the left hand side of the figure. B | The movement of CITES-listed amphibians is listed 1024 and the figure shows their global movements owing to trade. Part B adapted from ref 1025 131 (map from https://science.sciencemag.org/content/363/6434/1386 & permission 1026 is needed or should be redrawn). 1027 1028 Figure 3: Factors influencing the virulence of batrachochytrids. The host response to 1029 batrachochytrid ranges from resistance to lethal infection and several factors have 1030 been identified that contribute to this variability. For one, pathogen lineages vary in 1031 their genetic repertoire of proven and suspected virulence factors, including 1032 proteases, carbohydrate-binding modules, Crinkler-like proteins and other secreted 1033 proteins, such as tribes of expanded gene families. The genomic potential for virulence 1034 is influenced by the genome plasticity of batrachochytrids, which has contributed to 1035 the expansion and radiation of gene families with potential roles in pathogenicity. 1036 Host susceptibility also varies greatly, depending on the host immune responses, prior 1037 exposure to chytrids and/or other pathogens, the host microbiota and the host life 1038 history (for example, developmental stage). Amphibian larva, as well as other 1039 alternative hosts such as crayfish, can function as pathogen reservoirs. Finally, abiotic, 1040

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environmental variables, such as climate, water system properties, pesticides, 1041 fertilizers and others, also influence the outcome of batrachochytrid exposure. 1042 1043 Figure 4. Pathogenic potential of batrachocytrids. (A) Genome alignments show 1044 gene-family expansions that discriminate pathogenic batrachochytrids 1045 (Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal)) 1046 from non-pathogenic chytrids (Homolaphlyctis polyrhiza (Hp) and Spizellomyces 1047 punctatus (Sp)) (B) For example, the M36 metalloproteases, a gene family involved in 1048 infection, have been amplified in the genomes of pathogenic batrachochytrid lineages, 1049 and especially in the genome of Batrachochytrium salamandrivorans (Bsal). (C) Bd 1050 growing on explanted amphibian skin secretes proteases, which cause extensive skin 1051 digestion (far right), whereas the non-pathogenic Hp (middle) leaves the skin intact. 1052 (D) Bd but not Bsal zoospores show high concentrations of proteases prior to infection 1053 suggesting that the proteases have a role in the initial establishment of infection for 1054 Bd but not Bsal. Part A adapted from ref 66, part B and D reproduced from ref 66 and 1055 part C reproduced from ref 24. 1056 1057 Box 1. Dating the emergence of Batrachochytrium dendrobatidis. Sequence data is 1058 increasingly being used to time epidemiological events ranging across different 1059 infections (for example, the emergence of HIV-1 124, the spread and diversification of 1060 plague 125 and the emergence of Cryptococcus gattii in North America 126). For 1061 microbial species with rapidly evolving genomes or short generation times, genetic 1062 lineages may measurably diverge over observable timespans, allowing substitution 1063 rates to be directly calculated rather than assumed 127. Calculation is based on known 1064 dates of isolation to determine the rate of evolution. For example, the amount of 1065 sequence change that has occurred between cultures of Batrachochytrium 1066 dendrobatidis (Bd) isolated from Xenopus and Litoria frogs together with the date of 1067 isolation (T(X) and T(L) in figure, part A) can be used to estimate an evolutionary rate 1068 and thus the time at which the pathogen lineages in the two frogs most recently 1069 shared a common ancestor (TMRCA). This method is known as tip dating 128 and several 1070 computational packages exist to carry out such analyses (reviewed in 129). Measurable 1071 molecular evolution has occurred between T(X) and T(L), which, together with data from 1072 other isolates (figure, part B) can be used to estimate the rate of evolution. A core 1073 assumption of tip dating is that sequences are not recombining, as this introduces 1074 additional divergence that is not linearly related to TMRCA. To avoid this bias, genome 1075 sequences can be statistically ‘cleaned’ of recombining sites using programs such as 1076 Gubbins 130, or can focus on recombination-free genomic regions such the 1077 mitochondrial genome. Attempts to date the emergence of Bd either assumed a rate 1078 of molecular evolution extrapolated from other eukaryotic species 27, or used tip 1079 dating on nuclear genomes in which major recombination breakpoints had been taken 1080 into account 25. The former method dated the origin of Bd in the region of 26,400 years 1081

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ago, whereas the latter method estimated a more recent origin 35–257 years ago. At 1082 299,707 bp Bd has the largest mitochondrial genome of any fungus 33 and contains 1083 substantial diversity. Tip dating based on the mitochondrial DNA of Bd estimated a 1084 TMRCA for the emergence of BdGPL as 1962 (1859–1988), substantiating earlier 1085 estimates based on nuclear DNA and matching the onset of global amphibian declines 1086 4 (figure part C; arrow indicates when Bd was discovered; severity of declines is shown 1087 as the cumulative number of lost individuals). Part C adapted from ref4. 1088 1089 Glossary 1090 1091 Panzootic: global outbreak of an infectious disease in animals. 1092 1093 Multilocus sequence typing: Matching DNA sequences of fragments of multiple 1094 housekeeping genes to assay genetic diversity 1095 1096 Epizootic: local outbreak of an infectious disease in animals 1097 1098 Bayesian-based haplotype clustering: population assignment using large numbers of 1099 resequenced genomes 1100 1101 Mutation-drift equilibrium: where the rate at which variation is lost through genetic 1102 drift is equal to the rate at which new variation is created by mutation 1103 1104 Tajima’s D statistic: population genetic test statistic to distinguish between DNA 1105 sequences evolving neutrally (at mutation-drift equilibria) to those evolving under a 1106 non-random process such as demographic change or natural selection 1107 1108 Phased: assigning alleles to the paternal and maternal chromosomes 1109 1110 Crossovers: segregation of alleles between homologous chromosomes through DNA 1111 breaks and reconnections 1112 1113 Meiosis: sexual recombination resulting in crossovers 1114 1115 Mating type-alleles: genes regulating compatability leading to meiosis in fungi, also 1116 called mating type ‘idiomorphs’ 1117 1118 Chromosomal copy number variation: where the number of copies of a haplotype 1119 varies between one individual and another, also known as ‘aneuploidy’ 1120 1121 Amphibian Arks: ex situ breeding of threatened species in biocontainment facilities 1122

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1123 Related links 1124 AmphibiaWeb https://amphibiaweb.org 1125 North American Bsal Task Force http://www.salamanderfungus.org/about-bsal/ 1126 The Amphibian Disease Portal https://amphibiandisease.org 1127 Epicollect https://five.epicollect.net/project/bd-global-isolation-protocol 1128 1129 ToC blurb 1130 1131 Worldwide amphibian declines caused by pathogenic chytrid fungi are emblematic of 1132 emerging infectious diseases driven by globalisation. Fisher and Garner discuss how 1133 these wildlife pathogens emerged to drive global declines in amphibian biodiversity 1134 and the implications for policy and control measures. 1135

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