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The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Generalist pathogens maintain infectivity in numerous hosts; how this broad ecological niche impacts host-pathogen coevolution remains to be widely explored. Batrachochytrium dendrobatidis (Bd) is a highly generalist pathogenic fungus that has caused devastating declines in hundreds of amphibian species worldwide. This review examines amphibian chytridiomycosis host-pathogen interactions and available evidence for coevolution between Bd and its numerous hosts. We summarize recent evidence showing that Bd genotypes vary in geographic distribution and virulence, and that amphibian species also vary in Bd susceptibility according to their geographic distribution. How much variation can be explained by phenotypic plasticity or genetic differences remains uncertain. Recent research suggests that Bd genotypes display preferences for specific hosts and that some hosts are undergoing evolution as populations rebound from Bd outbreaks. Taken together, these findings suggest the potential for coevolution to occur and illuminate a path for addressing open questions through integrating historical and contemporary genetic data., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Model organisms are crucial in research as they can provide key insights applicable to other species. This study proposes the use of the amphibian species Hymenochirus boettgeri , widely available through the aquarium trade, as a model organism for the study of chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd) and linked to amphibian decline and extinction globally. Currently, no model organisms are used in the study of chytridiomycosis, particularly because of the lack of availability and nonstandardized methods. Thus, laboratories around the world use wild local species to conduct Bd infection experiments, which prevents comparisons between studies and reduces reproducibility. Here, we performed a series of Bd infection assays that showed that H. boettgeri has a dose- and genotype-dependent response, can generalize previous findings on virulence estimates in other species, and can generate reproducible results in replicated experimental conditions. We also provided valuable information regarding H. boettgeri husbandry, including care, housing, reproduction, and heat treatment to eliminate previous Bd infections. Together, our results indicate that H. boettgeri is a powerful and low-ecological-impact system for studying Bd pathogenicity and virulence.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium -like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota., Competing Interests: The authors declare no conflict of interest.

Habitat fragmentation and infectious diseases threaten wildlife globally, but the interactions of these threats are poorly understood. For instance, while habitat fragmentation can impact genetic diversity at neutral loci, the impacts on disease-relevant loci are less well-studied. We examined the effects of habitat fragmentation in Brazil's Atlantic Forest on amphibian genetic diversity at an immune locus related to antigen presentation and detection (MHC IIB Exon 2). We used a custom high-throughput assay to sequence a fragment of MHC IIB and quantified Batrachochytrium dendrobatidis (Bd) infections in six frog species in two Atlantic Forest regions. Habitat fragmentation was associated with genetic erosion at MHC IIB Exon 2. This erosion was most severe in forest specialists. Significant Bd infections were detected only in one Atlantic Forest region, potentially due to relatively higher elevation. In this region, forest specialists showed an increase in both Bd prevalence and infection loads in fragmented habitats. Reduced population-level MHC IIB diversity was associated with increased Bd infection risk. On the individual level, MHC IIB heterozygotes exhibited a trend toward reduced Bd infection risk, although this was marginally non-significant. Our results suggest that habitat fragmentation increases Bd infection susceptibility in amphibians, mediated at least in part through erosion of immunogenetic diversity. Our findings have implications for management of fragmented populations in the face of emerging infectious diseases., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Molecular and ultrastructural investigations of the Chytridiomycota during the last 20 years have led to the separation of new orders, including the Rhizophydiales. Most species in this order are morphologically similar, possessing monocentric, eucarpic, spherical thalli. Here, based on analysis of nuc 18S and 28S rDNA, we add the new genus and species Polyrhizophydium stewartii to the order. This saprobe of moribund aquatic plant leaves is the first known rhizomycelial species in the order. In our molecular phylogeny, P. stewartii groups with the amphibian pathogens Batrachochytriuim dendrobatidis and B. salamandrivorans , making it of particular interest to investigators studying evolutionary pathways associated with host-switching and morphological adaptation.

Aquaculture companies grow the green alga Haematococcus pluvialis (Chlorophyta) to extract the carotenoid astaxanthin to sell, which is used as human and animal dietary supplements. We were requested to identify an unknown pathogen of H. pluvialis from an alga growing facility in the southwestern United States. To identify this zoosporic fungus and determine its phylogenetic placement among other chytrids, we isolated it into pure culture, photographed its morphology and zoospore ultrastructure, and sequenced and analyzed portions of nuc rDNA 18S and 28S genes. The organism belongs in the Chytridiomycota, but a comparison of rDNA with available representatives of the phylum did not convincingly place it in any described order. The unique zoospore ultrastructure supports its indeterminate ordinal position, and the morphology, as determined by light microscopy, did not match any described species. Consequently, we have placed this chytrid in the new genus, Quaeritorhiza , and described it as the new species Q. haematococci in the family Quaeritorhizaceae but otherwise incertae sedis in the Chytridiomycetes. This new taxon is important because it increases the known diversity of Chytridiomycota and the organism has the ability to disrupt agricultural production of an algal monoculture.

Hybridization is increasingly recognized as an important force impacting adaptation and evolution in many lineages of fungi. During hybridization, divergent genomes and alleles are brought together into the same cell, potentiating adaptation by increasing genomic plasticity. Here, we review hybridization in fungi by focusing on two fungal pathogens of animals. Hybridization is common between the basidiomycete yeast species Cryptococcus neoformans × Cryptococcus de neoformans , and hybrid genotypes are frequently found in both environmental and clinical settings. The two species show 10-15% nucleotide divergence at the genome level, and their hybrids are highly heterozygous. Though largely sterile and unable to mate, these hybrids can propagate asexually and generate diverse genotypes by nondisjunction, aberrant meiosis, mitotic recombination, and gene conversion. Under stress conditions, the rate of such genetic changes can increase, leading to rapid adaptation. Conversely, in hybrids formed between lineages of the chytridiomycete frog pathogen Batrachochytrium dendrobatidis ( Bd ), the parental genotypes are considerably less diverged (0.2% divergent). Bd hybrids are formed from crosses between lineages that rarely undergo sex. A common theme in both species is that hybrids show genome plasticity via aneuploidy or loss of heterozygosity and leverage these mechanisms as a rapid way to generate genotypic/phenotypic diversity. Some hybrids show greater fitness and survival in both virulence and virulence-associated phenotypes than parental lineages under certain conditions. These studies showcase how experimentation in model species such as Cryptococcus can be a powerful tool in elucidating the genotypic and phenotypic consequences of hybridization., Competing Interests: The authors declare no conflict of interest.

Outbreaks of emerging infectious diseases are becoming more frequent as climate changes wildlife communities at unprecedented rates, driving population declines and raising concerns for species conservation. One critical disease is the global pandemic of chytridiomycosis in frogs, which can be caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although there is clear evidence for Bd-induced mortality across high-elevation frog communities, little attention is given to the role of lowlands in Bd's persistence and spread because low elevations are assumed to be too warm to harbor significant levels of Bd. Here, we report widespread Bd infection across 80 frog species from three sites in the lowland Peruvian Amazon, an area with no documented Bd-related amphibian declines. Despite observing no clinical signs of infection in the field, we found that 24-46% of individuals were infected per site (up to ≈105,000 zoospore equivalents per frog) by three Bd strains from the global pandemic lineage (Bd-GPL). We also found collection site and seasonal effects to be only weak predictors of Bd prevalence and load, with lower elevation and drier habitats marginally decreasing both prevalence and load. We found no further effect of host phylogeny, ecotype, or body size. Our results showing high and widespread prevalence across a lowland tropical ecosystem contradict the expectations based on the global pattern of pathogenicity of Bd that is largely restricted to higher elevations and colder temperatures. These findings imply that the lowlands may play a critical role in the spread and persistence of Bd over time and space., Competing Interests: The authors have declared that no competing interests exist.

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

Phenotypes are the target of selection and affect the ability of organisms to persist in variable environments. Phenotypes can be influenced directly by genes and/or by phenotypic plasticity. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has a global distribution, unusually broad host range, and high genetic diversity. Phenotypic plasticity may be an important process that allows this pathogen to infect hundreds of species in diverse environments. We quantified phenotypic variation of nine Bd genotypes from two Bd lineages (Global Pandemic Lineage [GPL] and Brazil) and a hybrid (GPL-Brazil) grown at three temperatures (12, 18 and 24°C). We measured five functional traits including two morphological traits (zoospore and zoosporangium sizes) and three life history traits (carrying capacity, time to fastest growth and exponential growth rate) in a phylogenetic framework. Temperature caused highly plastic responses within each genotype, with all Bd genotypes showing phenotypic plasticity in at least three traits. Among genotypes, Bd generally showed the same direction of plastic response to temperature: larger zoosporangia, higher carrying capacity, longer time to fastest growth and slower exponential growth at lower temperatures. The exception was zoospore size, which was highly variable. Our findings indicate that Bd genotypes have evolved novel phenotypes through plastic responses to temperature over very short timescales. High phenotypic variability likely extends to other traits and may facilitate the large host range and rapid spread of Bd., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

Competition between genotypes is likely to be a key driver of pathogen evolution, particularly following a geographical invasion by distant strains. Theory predicts that competition between disease strains will result in the most virulent strain persisting. Despite its evolutionary implications, the role of strain competition in shaping populations remains untested for most pathogens. We experimentally investigated the in vivo competitive differences between two divergent lineages of the amphibian-killing chytrid fungus ( Batrachochytrium dendrobatidis, Bd). These Bd lineages are hypothesized to have diverged in allopatry but been recently brought back into secondary contact by human introduction. Prior studies indicate that a panzootically-distributed, global lineage of Bd was recently introduced into southern Brazil, and is competitively excluding enzootic lineages in the southern Atlantic Forest. To test for differences in competitive ability between invasive and enzootic Brazilian Bd isolates, we coinfected a model host frog system which we developed for this study ( Hymenochirus curtipes). We tracked isolate-specific zoospore production over the course of the coinfection experiment with chip-based digital PCR (dPCR). The globally invasive panzootic lineage had a competitive advantage in spore production especially during the first one to four weeks of infection, and on frogs that eventually succumbed to Bd infection. Our study provides new evidence that competitive pressure resulting from the human movement of pathogen strains can rapidly alter the genetics, community dynamics and spatial epidemiology of pathogens in the wild.

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

Amphibians suffer from a number of factors that make them the most threatened group of vertebrates. One threat is the fungal disease chytridiomycosis caused by the emerging pathogen Batrachochytrium dendrobatidis (Bd), which has rapidly spread and caused the loss of massive amphibian biodiversity worldwide. Recently, Bd was associated with a few amphibian population declines and extinctions in some areas of the Brazilian Atlantic Forest. However, the mechanisms underlying such declines are not fully understood. Therefore, it is essential to improve our knowledge of abiotic factors that can possibly influence Bd prevalence and chytridiomycosis disease severity. Herein we tested the hypothesis that water availability (such as in perennial streams, where Bd is frequently present in larvae) and rainfall would increase the prevalence of Bd. To test this, we sampled frogs from 6 transects with different numbers of perennial waterbodies, and we report that the more water available in the area, the higher the probability of Bd infection on anurans. Seasonality also influenced both the Bd prevalence in the area and the intensity of infection in infected frogs. However, Bd prevalence was higher during the rainy months whereas the infection burden was lower. We suggest that Bd is likely spread during the summer, when most anuran species gather near the water for spawning and when rainfall overfills ephemeral wetlands. On the other hand, during the drier months, a higher infection burden may be explained by increased disease susceptibility.

Host-generalist pathogens sporadically infect naive hosts, potentially triggering epizootics. The waterborne fungus Batrachochytrium dendrobatidis (Bd) is linked to declines of hundreds of amphibian species with aquatic larvae. Although several population declines and extinctions attributed to Bd have been reported among cryptic species undergoing direct development away from water, epidemiological studies focused on these terrestrial frogs are lacking. Our field data support that terrestrial direct-developing hosts are less exposed to Bd during their ontogeny than species with aquatic larvae, and thus they might lack adaptive responses against waterborne chytrids. Using controlled laboratory experiments, we exposed wild-caught amphibian species with terrestrial and aquatic life histories to Bd and found that direct developers showed more rapid increases in infection loads and experienced higher mortality rates than species with aquatic larvae. Our findings provide novel information about host responses to generalist pathogens and specifically show that our focal direct developing species have low resistance to Bd infections. Finally, our results underscore that we should not ignore Bd as a potential threat to direct developing species simply because they are less exposed to Bd in nature; instead future amphibian conservation plans should include efforts to safeguard hundreds of direct-developing amphibian species globally.

The Monoblepharidomycetes is the sister class to the Chytridiomycetes in the phylum Chytridiomycota. The six known genera have thalli that are either monocentric and without rhizoids or produce hyphae with an independent evolutionary origin from the hyphae of higher fungi. On the basis of morphological characters and phylogenetic evidence from the small and large subunits of nuclear ribosomal RNA, we established two new genera, Sanchytrium and Telasphaerula, each with a single species. We re-analyzed intergeneric relationships within the monoblephs, and established two new families. The new genera significantly expand the known morphological and ecological diversity of the Monoblepharidomycetes by adding a monocentric, epibiotic, algal parasitic species and a rhizomycelial, saprotrophic species. Based on the presence of environmental sequences related to Sanchytrium strains, the Monoblepharidomycetes contain previously unsuspected diversity. The ribosomal DNA of the new genera contains an unusually high density of group I introns. We found 20 intron insertion positions including six that are new for rRNA genes (S1053, L803, L829, L961, L1844, and L2281)., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

To understand the evolutionary pathways that lead to emerging infections of vertebrates, here we explore the genomic innovations that allow free-living chytrid fungi to adapt to and colonize amphibian hosts. Sequencing and comparing the genomes of two pathogenic species of Batrachochytrium to those of close saprophytic relatives reveals that pathogenicity is associated with remarkable expansions of protease and cell wall gene families, while divergent infection strategies are linked to radiations of lineage-specific gene families. By comparing the host-pathogen response to infection for both pathogens, we illuminate the traits that underpin a strikingly different immune response within a shared host species. Our results show that, despite commonalities that promote infection, specific gene-family radiations contribute to distinct infection strategies. The breadth and evolutionary novelty of candidate virulence factors that we discover underscores the urgent need to halt the advance of pathogenic chytrids and prevent incipient loss of biodiversity.

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has recently emerged as a primary factor behind declining global amphibian populations. Much about the basic biology of the pathogen is unknown, however, such as its true ecological niche and life cycle. Here we evaluated invertebrates as infection models by inoculating host species that had previously been suggested to be parasitized in laboratory settings: crayfish (Procambarus alleni) and nematodes (Caenorhabditis elegans). We found neither negative effects on either host nor evidence of persistent infection despite using higher inoculum loads and more pathogen genotypes than tested in previous studies. In contrast, addition of Bd to C. elegans cultures had a slight positive effect on host growth. Bd DNA was detected on the carapace of 2/34 crayfish 7 weeks post-inoculation, suggesting some means of persistence in the mesocosm. These results question the role of invertebrates as alternative hosts of Bd and their ability to modulate disease dynamics.

Fungi grow within their food, externally digesting it and absorbing nutrients across a semirigid chitinous cell wall. Members of the new phylum Cryptomycota were proposed to represent intermediate fungal forms, lacking a chitinous cell wall during feeding and known almost exclusively from ubiquitous environmental ribosomal RNA sequences that cluster at the base of the fungal tree [1, 2]. Here, we sequence the first Cryptomycotan genome (the water mold endoparasite Rozella allomycis) and unite the Cryptomycota with another group of endoparasites, the microsporidia, based on phylogenomics and shared genomic traits. We propose that Cryptomycota and microsporidia share a common endoparasitic ancestor, with the clade unified by a chitinous cell wall used to develop turgor pressure in the infection process [3, 4]. Shared genomic elements include a nucleotide transporter that is used by microsporidia for stealing energy in the form of ATP from their hosts [5]. Rozella harbors a mitochondrion that contains a very rapidly evolving genome and lacks complex I of the respiratory chain. These degenerate features are offset by the presence of nuclear genes for alternative respiratory pathways. The Rozella proteome has not undergone major contraction like microsporidia; instead, several classes have undergone expansion, such as host-effector, signal-transduction, and folding proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Understanding the evolutionary history of microbial pathogens is critical for mitigating the impacts of emerging infectious diseases on economically and ecologically important host species. We used a genome resequencing approach to resolve the evolutionary history of an important microbial pathogen, the chytrid Batrachochytrium dendrobatidis (Bd), which has been implicated in amphibian declines worldwide. We sequenced the genomes of 29 isolates of Bd from around the world, with an emphasis on North, Central, and South America because of the devastating effect that Bd has had on amphibian populations in the New World. We found a substantial amount of evolutionary complexity in Bd with deep phylogenetic diversity that predates observed global amphibian declines. By investigating the entire genome, we found that even the most recently evolved Bd clade (termed the global panzootic lineage) contained more genetic variation than previously reported. We also found dramatic differences among isolates and among genomic regions in chromosomal copy number and patterns of heterozygosity, suggesting complex and heterogeneous genome dynamics. Finally, we report evidence for selection acting on the Bd genome, supporting the hypothesis that protease genes are important in evolutionary transitions in this group. Bd is considered an emerging pathogen because of its recent effects on amphibians, but our data indicate that it has a complex evolutionary history that predates recent disease outbreaks. Therefore, it is important to consider the contemporary effects of Bd in a broader evolutionary context and identify specific mechanisms that may have led to shifts in virulence in this system.

Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd-GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd-GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd-Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd-GPL and Bd-Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd-GPL and Bd-Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd-Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization., (© 2012 Blackwell Publishing Ltd.)

Batrachochytrium dendrobatidis (Bd), the cause of a fatal fungal skin disease of amphibians that has led to massive die-offs, global declines and extinctions, has spread internationally as a pandemic clone with low genetic diversity. A need exists to develop highly polymorphic markers to determine centers of origin and patterns of spread to assist in the development of management strategies. Comparison of paralogous sequences, obtained from the 2 sequenced Bd genomes, indicates useful levels of inter-strain polymorphism in repetitive fragments. We assessed 6 repetitive loci for variation within and among Australian isolates using standard fragment analysis and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis. Confirmation of inter-isolate polymorphism was achieved for 2 marker systems, highlighting the potential of repetitive loci for the development of polymorphic markers in Bd. In addition, we found that repetitive loci in Bd include possible orthologs of virulence-related genes from pathogenic fungi.

Ten years after the first discovery of the chytrid pathogen Batrachochytrium dendrobatidis (Bd), the catastrophic effect of Bd on wild amphibian populations is indisputable. However, a number of persistent questions remain about Bd's origin and mechanisms of pathogenicity. Here we discuss the promise of genetic and genomic tools for answering these previously intractable questions about the biology and evolutionary history of Bd. Full genomes of 2 Bd strains have recently been sequenced, and Bd research on this species using population genetics, phylogenetics, proteomics, comparative genomics and functional genomics is already underway. We review some of the insights gleaned from the first studies using these genome-scale approaches focusing particularly on Bd's genomic architecture, patterns of global genetic variation, virulence factors and genetic interactions with hosts. Avenues of future research promise to be particularly fruitful and highlight the need for integrative studies that unite genetic, ecological and spatial data in both Bd and its amphibian hosts.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.

The Chytridiales, one of the four orders in the Chytridiomycetes (Chytridiomycota), is polyphyletic, but contains several well-supported clades. One of these clades is referred to as the Chytriomyces angularis clade, and the phylogenetic placement of this group within the Chytridiomycetes is uncertain. The morphology and zoospore ultrastructure of C. angularis have been studied using LM and were shown to differ from those of the type species of Chytriomyces, which is in the Chytridiaceae and is phylogenetically distinct from the C. angularis clade. In this study, chytrids with morphologies or rDNA sequences similar to C. angularis, including two isolates of the morphologically similar C. poculatus, were isolated and their phylogenetic relationships determined using molecular sequence data. Results of Bayesian and MP analyses of nuSSU and partial nuLSU rDNA sequences grouped the new isolates and the type isolate of C. angularis in a monophyletic clade within the Chytridiomycota but distinct from the Chytridiaceae. Zoospores of isolates examined using TEM had ultrastructural features similar to those of C. angularis. Genetic analyses, ultrastructural data, and morphology support the establishment of a new order Lobulomycetales, placement of C. angularis and C. poculatus in a new genus (Lobulomyces), and description of additional taxa, which we have named Clydaea vesicula and Maunachytrium keaense.

A parasite was found in cultures of the green microalga Haematococcus pluvialis that grew epibiotically on algal cells and caused epidemics resulting in damage to the host cultures. The parasite was isolated into axenic culture on solid and liquid media. It was demonstrated to be the sole causative agent of the epidemics. According to its life cycle and phylogenetic analysis based on 18S ribosomal DNA sequences, the pathogen appears to represent a novel chytrid fungus closely related to the vascular plant pathogen Physoderma (Blastocladiomycota), although it differs from all other known chytrids by its infective stage, a wall-less propagule endowed with amoeboid motion and lacking the group's typical flagellum.

Chytridiomycota (chytrids) is the only phylum of true Fungi that reproduces with motile spores (zoospores). Chytrids currently are classified into five orders based on habitat, zoospore characters and life cycles. In this paper we estimate the phylogeny of the chytrids with DNA sequences from the ribosomal RNA operon (18S+5.8S+28S subunits). To our surprise the morphologically reduced parasites Olpidium and Rozella comprise two entirely new, and separate, lineages on the fungal tree. Olpidium brassicae groups among the Zygomycota, and Rozella spp. are the earliest branch to diverge in the fungal kingdom. The phylogeny also suggests that Chytridiomycota is not monophyletic and there are four major lineages of chytrids: Rozella spp., Olpidium brassicae, the Blastocladiales and a "core chytrid clade" containing the remaining orders and families and the majority of flagellated fungi. Within the core chytrid group 11 subclades can be identified, each of which correlates well with zoospore ultrastructure or morphology. We provide a synopsis of each clade and its morphological circumscription. The Blastocladiales appears to be the sister taxon of most nonflagellated fungi. Based on molecular phylogenetic and ultrastructural characters this order is elevated to a phylum, the Blastocladiomycota.

Chytridiomycosis is a recently identified fungal disease associated with global population declines of frogs. Although the fungus, Batrachochytrium dendrobatidis, is considered an emerging pathogen, little is known about its population genetics, including the origin of the current epidemic and how this relates to the dispersal ability of the fungus. In this study, we use multilocus sequence typing to examine genetic diversity and relationships among 35 fungal strains from North America, Africa and Australia. Only five variable nucleotide positions were detected among 10 loci (5918 bp). This low level of genetic variation is consistent with the description of B. dendrobatidis as a recently emerged disease agent. Fixed (i.e. 100%) or nearly fixed frequencies of heterozygous genotypes at two loci suggested that B. dendrobatidis is diploid and primarily reproduces clonally. In contrast to the lack of nucleotide polymorphism, electrophoretic karyotyping of multiple strains demonstrated a number of chromosome length polymorphisms.

Harmful algal blooms (HABs) impose major costs on aquatic ecosystems worldwide, including the Laurentian Great Lakes. Microbial consumers, including fungi, can have important interactions with bloom-forming algae and cyanobacteria, although relatively few studies have investigated the relationship between fungi and HABs. We examined changes in the aquatic fungal community coincident with the occurrence of large cyanobacterial blooms in two areas of the Great Lakes (western Lake Erie and Saginaw Bay, Lake Huron). We collected water samples over the course of bloom development, peak, and decline from 3 sites in western Lake Erie on 11 dates and 2 sites in Saginaw Bay on 4 dates. Single molecule sequencing (PacBio RS II) with two molecular markers (the internal transcribed spacer (ITS) of the rRNA locus using fungal-specific primers and the 18S rRNA with primers targeting early-diverging lineages of fungi) was used to estimate fungal community composition. Results indicate a diverse fungal community within the lakes, including several major fungal phyla. The Chytridiomycota were particularly well-represented (54.8% and 45.4% of ITS and 18S sequences, respectively), and we also found representation from both Cryptomycota and Aphelidiomycota, which are putatively obligate intracellular parasites. Further, we found associations between the fungal community (alpha diversity; community composition) and measures of bloom magnitude (chlorophyll a , phycocyanin, and microcystin concentrations) in western Lake Erie. Our results suggest potentially important spatial and temporal heterogeneity in the fungal community that motivates further research on functional importance of fungi in the Great Lakes and consequences for HABs and freshwater ecosystems more broadly. [ABSTRACT FROM AUTHOR]

Our knowledge of zoosporic fungal phylogeny, physiology, and ecological functions, in particular their role in aquatic food web dynamics and biogeochemistry, is limited. The recent discovery of numerous dark matter fungi (DMF), i.e., uncultured and poorly known taxa belonging to early diverging branches of the fungal tree (namely the Rozellomycota and Chytridiomycota) calls for reconsideration of the phylogeny and ecology of zoosporic fungi. In this opinion paper, we summarize the exploration of new, recently discovered lineages of DMF and their implications for the ecology, evolution, and biogeography of the rapidly growing fungal tree. We also discuss possible ecological roles of zoosporic fungi in relation to recent methodological developments including single cell genomics and cultivation efforts. Finally, we suggest linking explorative with experimental research to gain deeper insights into the physiology and ecological functioning of zoosporic fungi DMF in aquatic habitats. [ABSTRACT FROM AUTHOR]