The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Chytrids, often overshadowed by their other fungal counterparts, take center stage as we unravel the mysteries surrounding new species within Rhizophydiales and explore their unique characteristics. In the broader spectrum of chytrids, their significance lies not only in their roles as decomposers but also as key players in nutrient cycling within aquatic ecosystems as parasites and saprobes. Baited soil and aquatic samples collected from various provinces of Thailand, yielded new species of the Rhizophydiales (Chytridiomycota), some of which expanded previously single species genera. Our investigation incorporated a combination of morphological and phylogenetic approaches, enabling us to identify these isolates as distinct taxa. The novel isolates possess distinguishing features, such as variations in size and shape of the sporangium and zoospores, that somewhat differentiate them from described taxa. To confirm the novelty of the species, we employed robust phylogenetic analyses using maximum likelihood and bayesian methods. The results provided strong support for the presence of eight distinct lineages within the Rhizophydiales, representing our newly discovered species. Furthermore, we employed Poisson Tree Processes to infer putative species boundaries and supplement evidence for the establishment of our new Rhizophydiales species. By meticulously exploring their morphological characteristics and genetic makeup, we expand the known catalogue of fungal diversity by describing Alphamyces thailandicus, Angulomyces ubonensis, Gorgonomyces aquaticus, G. chiangraiensis, G. limnicus, Pateramyces pingflumenensis, Terramyces aquatica, and T. flumenensis and also provide valuable insights into the intricacies of this order. This newfound knowledge not only enriches our understanding of Rhizophydiales but also contributes significantly to the broader field of mycology, addressing a critical gap in the documentation of fungal species. The identification and characterization of these eight novel species mark a noteworthy stride towards a more comprehensive comprehension of fungal ecosystems and their vital role.

The Chytridiomycota is a phylum of zoosporic eufungi that inhabit terrestrial, freshwater, and oceanic habitats. Within the phylum, the Rhizophydiales contains several monotypic families theorized to hold a diverse assemblage of fungi yet to be discovered and properly described. Based on morphology alone, many species in this order are difficult or impossible to identify. In this study, we isolated three chytrids from northern Thailand. Phylogenetic analyses placed the isolates in three monotypic genera within Rhizophydiales. Intrageneric genetic distances in the internal transcribed spacer (ITS) ranged between 1.5 and 8.5%. Angulomyces solicola sp. nov. is characterized by larger sporangia, spores, and fewer discharge papilla than A.argentinensis; Gorgonomyces thailandicus sp. nov. has larger zoospores and fewer discharge papillae in culture compared to G. haynaldii; Terramyces chiangraiensis sp. nov. produces larger sporangia than T. subangulosum. We delimited species of Angulomyces, Gorgonomyces and Terramyces using a tripartite approach that employed phylogeny, ITS genetic distances and Poisson tree processes (PTP). Results of these approaches suggest more than one species in each genus. This study contributes to the knowledge of chytrids, an understudied group in Thailand and worldwide.

Cladochytriales comprises a taxonomic order and lineage of chytrid fungi that are primarily saprobes of organic matter in aquatic and terrestrial ecosystems. The order contains species from 12 genera, though molecular confirmations of almost all type species are lacking, and many genera are considered polyphyletic. We conducted phylogenetic analyses of ribosomal RNA genes and developmental morphology of Cladochytriales isolates from the Collection of Zoosporic Eufungi at the University of Michigan and noted some strains that were distinct from described genera. In our phylogenetic analysis, Endochytriaceae represented by only one strain of Endochytrium ramosum was sister to Septochytriaceae while the incertae sedis lineage formed by the new species Thomasia carolinae was sister to Nowakowskiellaceae . Additionally, the phylogenetic relatedness of the other two new species, Dogmamyces elongatus and Allochytrium aureum , was also highly supported. A thorough revision of the order is needed because some genera, such as Catenochytridium and Nephrochytrium , remain polyphyletic or paraphyletic. In addition to indicating these problems, our updated phylogeny supports the description of two new genera and three new species and thereby begins to bring the knowledge of the Cladochytriales up to date.

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Cladochytriales comprise multiple monocentric and polycentric lineages of chytrids, which are commonly found as saprobes in algae and decaying plant material in aquatic and terrestrial ecosystems. Although monocentric species are more common among the Chytridiomycota, additional polycentric chytrids have been described in recent years. During our ongoing effort to add to the knowledge of the diversity of chytrids, we found two polycentric chytrids on onion skin bait that produce rhizomycelia like none of described cladochytrialean genera or like those of genera in other orders. The rhizomycelia of these two newly found isolates consist of isodiametric filaments that lack swellings such as those found in either Cladochytrium or Nowakowskiella. Both zoosporangia and resting spores develop on short, right-angled offshoots from these filaments. Our analyses of the small and large subunits of rDNA indicate that these two isolates belong to separate branches of a predominantly monocentric clade of the Cladochytriales. Phytochytrium stagnum gen. et sp. nov. groups with two monocentric strains and Sparrowiella insolita gen. et sp. nov. are in an independent lineage distantly related to a monocentric species. We have expanded the concept of Septochytriaceae to include this clade. The ancestral state reconstruction allowed us to hypothesize for the first time a possible growth pattern of the common ancestral that gave origin to the order, besides to corroborate the unreliable nature of thalli's development as a taxonomic character.

Batrachochytrium dendrobatidis is a zoospore-forming aquatic fungus and the etiological agent of chytridiomycosis, a skin infection affecting all orders of amphibians. B. dendrobatidis emerged worldwide during the 20th century, causing a panzootic that has contributed to widespread declines in the abundance of at least 501 amphibian species and 90 presumed extinctions. The pathogen has a two-stage life cycle, whereby flagellate-motile zoospores seek out amphibians by means of chemotaxis before attaching, encysting, and developing intracellularly into zoosporangia, which gestate and discharge new zoospores.

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.

We formed the Collection of Zoosporic Eufungi at the University of Michigan (CZEUM) in 2018 as a cryopreserved fungal collection consolidating the University of Maine Culture Collection (UMCC, or JEL), the University of Alabama Chytrid Culture Collection (UACCC), and additional zoosporic eufungal accessions. The CZEUM is established as a community resource containing 1045 cryopreserved cultures of Chytridiomycota, Monoblepharidomycota, and Blastocladiomycota, with 52 cultures being ex-type strains. We molecularly characterized 431 cultures by amplifying the majority of the rDNA operon in a single reaction, yielding an average fragment length of 4739 bp. We sequenced multiplexed samples with an Oxford Nanopore Technology MinION device and software, and demonstrate the method is accurate by producing sequences identical to published Sanger sequences. With these data, we generated a phylogeny of 882 zoosporic eufungi strains to produce the most comprehensive phylogeny of these taxa to date. The CZEUM is thus largely characterized by molecular data, which can guide instructors and researchers on future studies of these organisms. Cultures from the CZEUM can be purchased through an online portal.

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.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions. IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Donald John Stoddart Barr (FIG. 1) was born on September 18, 1937, and grew up in wartime England near Guildford in Surrey. With a large garden, forests, and fields around his country home, Donald,...

An account is provided of the world's ten most feared fungi. Within areas of interest, we have organized the entries in the order of concern. We put four human pathogens first as this is of concern to most people. This is followed by fungi producing mycotoxins that are highly harmful for humans; Aspergillus flavus, the main producer of aflatoxins, was used as an example. Problems due to indoor air fungi may also directly affect our health and we use Stachybotrys chartarum as an example. Not everyone collects and eats edible mushrooms. However, fatalities caused by mushroom intoxications often make news headlines and therefore we include one of the most poisonous of all mushrooms, Amanita phalloides, as an example. We then move on to the fungi that damage our dwellings causing serious anxiety by rotting our timber structures and flooring. Serpula lacrymans, which causes dry rot is an excellent example. The next example serves to represent all plant and forest pathogens. Here we chose Austropuccinia psidii as it is causing devastating effects in Australia and will probably do likewise in New Zealand. Finally, we chose an important amphibian pathogen which is causing serious declines in the numbers of frogs and other amphibians worldwide. Although we target the top ten most feared fungi, numerous others are causing serious concern to human health, plant production, forestry, other animals and our factories and dwellings. By highlighting ten feared fungi as an example, we aim to promote public awareness of the cost and importance of fungi.

A chytrid isolate (JEL 221) we identified as the rarely reported species, Rhizidium endosporangiatum Karling, was cultured axenically for the first time. The purposes of this study are to characterize the developmental morphology of isolate JEL 221 and to elucidate its zoospore ultrastructural features. Thallus development and morphology of isolate JEL 221 are characteristic of R. endosporangiatum as it was originally described. However, thallus morphology of R. endosporangiatum is not entirely typical of the genus Rhizidium, especially that of the type R. mycophilum. The presence of an endosporangium, a layer of material encapsulating the edges of the protoplast protruding through multiple discharge pores, makes this a distinctive species. Consistent with its published molecular-based phylogenetic placement, we found that isolate JEL 221 shared ultrastructural features with the two major zoospore types described for the Chytridiales but had distinct zoospore architecture. A new genus, Pseudorhizidium, is erected for this chytrid based on its thallus morphology, molecular phylogenetic placement and unique zoospore ultrastructure. This new genus does not fit into either of the described families (Chytridiaceae or Chytriomycetaceae) in the Chytridiales because of its unique zoospore ultrastructure, especially the two-layered nature of the electron-opaque plug in the base of the flagellum.

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.

Zoosporic fungi are key saprotrophs and parasites of plants, animals and other fungi, playing important roles in ecosystems. They comprise at least three phyla, of which two, Chytridiomycota and Blastocladiomycota, developed a range of thallus morphologies including branching hyphae. Here we describe Retesporangicus lyonii gen. et sp. nov., an exceptionally well preserved fossil, which is the earliest known to produce multiple sporangia on an expanded hyphal network. To better characterize the fungus we develop a new method to render surfaces from image stacks generated by confocal laser scanning microscopy. Here, the method helps to reveal thallus structure. Comparisons with cultures of living species and character state reconstructions analysed against recent molecular phylogenies of 24 modern zoosporic fungi indicate an affinity with Blastocladiomycota. We argue that in zoosporic fungi, kinds of filaments such as hyphae, rhizoids and rhizomycelium are developmentally similar structures adapted for varied functions including nutrient absorption and anchorage. The fossil is the earliest known type to develop hyphae which likely served as a saprotrophic adaptation to patchy resource availability. Evidence from the Rhynie chert provides our earliest insights into the biology of fungi and their roles in the environment. It demonstrates that zoosporic fungi were already diverse in 407 million-year-old terrestrial ecosystems.This article is part of a discussion meeting issue 'The Rhynie cherts: our earliest terrestrial ecosystem revisited'.

Three-dimensional reconstruction models of Fungi from a 407 million year old blastocladiomycota-like fossil showing multiple sporangia and an extensive hyphal network in SPIERSView and VAXML format. 2D and 3D (Red/Cyan) images also provided as a PDF. Notes: SPIERSView file (.SPV) models can conveniently be viewed using the SPIERSView software, freely available in both Windows and Mac versions from http://www.spiers‐software.org. However, note that low-performance computers may not possess a sufficiently powerful graphics card to render and rotate the model. VAXML file format models are saved as a ZIP-compressed VAXML datasets. VAXML uses one or more .STL files to define the geometry of objects that comprise the dataset, together with one .VAXML file that provides metadata on the dataset as a whole, and specifies how the .STL files should be put together. We recommend using the free SPIERS software to view this model format (http://spiers-software.org/). However, .STL files can be opened independently in several freely available software programs (e.g. MeshLab, Blender). Additional information on the VAXML format can be found here: http://spiers-software.org/VAXML.htm.

Increasing numbers of sequences of basal fungi from environmental DNA studies are being deposited in public databases. Many of these sequences remain unclassified below the phylum level because sequence information from identified species is sparse. Lack of basic biological knowledge due to a dearth of identified species is extreme in Cryptomycota, a new phylum widespread in the environment and phylogenetically basal within the fungal lineage. Consequently, we are attempting to fill gaps in the knowledge of Rozella, the best-known genus in this lineage. Rozella is a genus of unwalled, holocarpic, endobiotic parasites of hosts including Chytridiomycota, Blastocladiomycota, Oomycota, Basidiomycota, and a green alga, with most species descriptions based on morphology and host specificity. We found a Rozella parasitizing a Pythium host that was a saprobe on spruce pollen bait placed with an aquatic sample. We characterized the parasite with light microscopy, TEM of its zoospores and sporangia, and its 18S/28S rDNA. Comparison with other Rozella species indicates that the new isolate differs morphologically, ultrastructurally, and genetically from Rozella species for which we have data. Features of the zoospore also differ from those of previously studied species. Herein we describe the Rozella as a new species, R. multimorpha.