Phylogenomics including new sequence data of phytoplankton-infecting chytrids reveals multiple independent lifestyle transitions across the phylum.

Parasitism is the most common lifestyle on Earth and has emerged many times independently across the eukaryotic tree of life. It is frequently found among chytrids (Chytridiomycota), which are early-branching unicellular fungi that feed osmotrophically via rhizoids as saprotrophs or parasites. Chytrids are abundant in most aquatic and terrestrial environments and fulfil important ecosystem functions. As parasites, they can have significant impacts on host populations. They cause global amphibian declines and influence the Earth's carbon cycle by terminating algal blooms. To date, the evolution of parasitism within the chytrid phylum remains unclear due to the low phylogenetic resolution of rRNA genes for the early diversification of fungi, and because few parasitic lineages have been cultured and genomic data for parasites is scarce. Here, we combine transcriptomics, culture-independent single-cell genomics and a phylogenomic approach to overcome these limitations. We newly sequenced 29 parasitic taxa and combined these with existing data to provide a robust backbone topology for the diversification of Chytridiomycota. Our analyses reveal multiple independent lifestyle transitions between parasitism and saprotrophy among chytrids and multiple host shifts by parasites. Based on these results and the parasitic lifestyle of other early-branching holomycotan lineages, we hypothesise that the chytrid last common ancestor was a parasite of phytoplankton.

Diversity, multifaceted evolution, and facultative saprotrophism in the European Batrachochytrium salamandrivorans epidemic.

While emerging fungi threaten global biodiversity, the paucity of fungal genome assemblies impedes thoroughly characterizing epidemics and developing effective mitigation strategies. Here, we generate de novo genomic assemblies for six outbreaks of the emerging pathogen Batrachochytrium salamandrivorans (Bsal). We reveal the European epidemic currently damaging amphibian populations to comprise multiple, highly divergent lineages demonstrating isolate-specific adaptations and metabolic capacities. In particular, we show extensive gene family expansions and acquisitions, through a variety of evolutionary mechanisms, and an isolate-specific saprotrophic lifecycle. This finding both explains the chytrid's ability to divorce transmission from host density, producing Bsal's enigmatic host population declines, and is a key consideration in developing successful mitigation measures.

Phylogenomics of a new fungal phylum reveals multiple waves of reductive evolution across Holomycota.

Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids' phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages.

Two new species of Chytriomycetaceae: Morphological, phylogenetic, and ultrastructural characterization.

Systematics of Chytridiales has been deeply influenced by analyses of molecular loci and zoospore ultrastructure. Even though the Chytridiales is the largest order within Chytridiomycota, Brazilian isolates of this clade have been poorly integrated. Here, we isolated seven species and documented their morphology, including zoospore ultrastructure for Siphonaria aurea, and phylogenetic positions for all based on analyses of nuc 18S and 28S rDNA. Phylogenetic results support the placement of these species in Chytriomycetaceae and Chytridiaceae, with two new species described, Rhizidium crepaturum and Siphonaria aurea, and Rodmanochytrium sphaericum recorded for the first time from Brazil.

The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdom.

Cells from across the eukaryotic tree use actin polymer networks for a wide variety of functions, including endocytosis, cytokinesis, and cell migration. Despite this functional conservation, the actin cytoskeleton has undergone significant diversification, highlighted by the differences in the actin networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast) and therefore provide a unique opportunity to study actin cytoskeletal evolution. Chytrids have two life stages: zoospore cells that can swim with a flagellum and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here, we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures resembling those of animal cells, including an actin cortex, pseudopods, and filopodia-like spikes. In contrast, Bd sporangia assemble perinuclear actin shells and actin patches similar to those of yeast. The use of specific small-molecule inhibitors indicate that nearly all of Bd's actin structures are dynamic and use distinct nucleators: although pseudopods and actin patches are Arp2/3 dependent, the actin cortex appears formin dependent and actin spikes require both nucleators. Our analysis of multiple chytrid genomes reveals actin regulators and myosin motors found in animals, but not dikaryotic fungi, as well as fungal-specific components. The presence of animal- and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss.

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts.

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota-a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

A historical overview of Batrachochytrium dendrobatidis infection from specimens at the National Zoological Collection Suriname.

The amphibian skin disease chytridiomycosis, caused by the pathogenetic fungus Batrachochytrium dendrobatidis (Bd) has become one of the major contributors to global amphibian population declines and extinctions. This fungus has spread globally and has caused mortalities in nearly every continent. In South America, Suriname, Guyana and Paraguay are among the remaining three countries where Bd has not been detected to date. To complete the assessment of the possible presence of Bd in Suriname, 205 specimens from the Zoological Collection of Suriname, compromising 6 frog families and 15 genera were sampled for chytrid fungus. No specimens were found to be infected by this fungus and as such the outcome strengthens the previous result of field sampling that there is no support that Bd has spread to Suriname.

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains.

Wildlife diseases are contributing to the current Earth's sixth mass extinction; one disease, chytridiomycosis, has caused mass amphibian die-offs. While global spread of a hypervirulent lineage of the fungus Batrachochytrium dendrobatidis (BdGPL) causes unprecedented loss of vertebrate diversity by decimating amphibian populations, its impact on amphibian communities is highly variable across regions. Here, we combine field data with in vitro and in vivo trials that demonstrate the presence of a markedly diverse variety of low virulence isolates of BdGPL in northern European amphibian communities. Pre-exposure to some of these low virulence isolates protects against disease following subsequent exposure to highly virulent BdGPL in midwife toads (Alytes obstetricans) and alters infection dynamics of its sister species B. salamandrivorans in newts (Triturus marmoratus), but not in salamanders (Salamandra salamandra). The key role of pathogen virulence in the complex host-pathogen-environment interaction supports efforts to limit pathogen pollution in a globalized world.

New species of aquatic chytrids from Oman.

Oman is a desert country in the south of the Middle East. Springs and other water sources that harbor aquatic organisms can be separated by hundreds of kilometers. In Oct 2019, we isolated four freshwater aquatic fungi (Chytridiomycota) from benthic detritus baited with pine pollen on a general nutrient medium near Salalah, Oman. Database queries of nuc 28S rRNA (28S) and internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) revealed that one of these strains was Dinochytrium kinnereticum, a recently described algal pathogen from the Sea of Galilee. The other three strains had low molecular identity to available ITS sequences. These unknown strains varied in size and released endogenously swarming zoospores through papillae from mature zoosporangia. Zoospore ultrastructure was consistent with described species in the Rhizophydiales, and molecular phylogenetic results grouped these three strains into a clade in the genus Rhizophydium. We circumscribe these three strains as a sp. nov., thereby expanding the diversity within Rhizophydium described as the new species R. jobii. In doing so, we provide the first report of Chytridiomycota from Oman.

Quaeritorhiza haematococci is a new species of parasitic chytrid of the commercially grown alga, Haematococcus pluvialis.

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.

Chytrid fungi and global amphibian declines.

Discovering that chytrid fungi cause chytridiomycosis in amphibians represented a paradigm shift in our understanding of how emerging infectious diseases contribute to global patterns of biodiversity loss. In this Review we describe how the use of multidisciplinary biological approaches has been essential to pinpointing the origins of amphibian-parasitizing chytrid fungi, including Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, as well as to timing their emergence, tracking their cycles of expansion and identifying the core mechanisms that underpin their pathogenicity. We discuss the development of the experimental methods and bioinformatics toolkits that have provided a fuller understanding of batrachochytrid biology and informed policy and control measures.

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Recent Findings of Potentially Lethal Salamander Fungus Batrachochytrium salamandrivorans.

The distribution of the chytrid fungus Batrachochytrium salamandrivorans continues to expand in Europe. During 2014-2018, we collected 1,135 samples from salamanders and newts in 6 countries in Europe. We identified 5 cases of B. salamandrivorans in a wild population in Spain but none in central Europe or the Balkan Peninsula.

The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

Chytrid infecting the bloom-forming marine diatom Skeletonema sp.: Morphology, phylogeny and distribution of a novel species within the Rhizophydiales.

Chytrids have long been recognised as important parasites of microalgae in freshwater systems, able to shape the dynamics of blooms, the gene pool of their host and phytoplankton succession. In the sea however, where the presence of these organisms is erratic and ephemeral, studies concerning chytrids are sparse and confined to metabarcoding surveys or microscopy observations. Despite the scarcity of data, chytrid epidemics are supposed to play an important role in marine biogeochemical cycles, being one of the drivers of phytoplankton dynamics. Here we combine microscopy observations and in silico mining of a single-cell whole genome to molecularly and morphologically characterise a novel chytrid parasite of the dominant diatom genus Skeletonema. Morphological observations highlight features of the thallus and ascertain the parasitic nature of the interaction whilst the genetic markers obtained allows for a phylogenetic reconstruction, placing the new species in the order Rhizophydiales. Thanks to the molecular data obtained we are also able to provide a first investigation of the global distribution of this organism by screening the Ocean Sampling Day (OSD) dataset, highlighting a northern transatlantic dissemination.

Novel taxa in Cladochytriales (Chytridiomycota): Karlingiella (gen. nov.) and Nowakowskiella crenulata (sp. nov.).

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.

New Member of Gromochytriales (Chytridiomycetes)-Apiochytrium granulosporum nov. gen. et sp.

Molecular phylogenetic analysis of 18S rRNA gene sequences of nearly any species of Chytridiomycota has typically challenged traditional classification and triggered taxonomic revision. This has often led to the establishment of new taxa which, normally, appears well supported by zoospore ultrastructure, which provides diagnostic characters. To construct a meaningful and comprehensive classification of Chytridiomycota, the combination of molecular phylogenies and morphological studies of traditionally defined chytrid species is needed. In this work, we have studied morphological and ultrastructural features based on light and transmission electron microscopy as well as molecular phylogenetic analysis of a parasite (strain X-124 CCPP ZIN RAS) morphologically similar to Rhizophydium granulosporum living on the yellow-green alga Tribonema gayanum. Phylogenetic analysis of the 18S rRNA gene sequence of this strain supports that it represents a new genus and species affiliated to the recently established order Gromochytriales. The ultrastructure of X-124 confirms its phylogenetic position sister to Gromochytrium and serves as the basis for the description of the new genus and species Apiochytrium granulosporum. The 18S rRNA gene of A. granulosporum contains a S943 group I intron that carries a homing endonuclease pseudogene.

Morphology, zoospore ultrastructure, and phylogenetic position of Polyphlyctis willoughbyi, a new species in Chytridiales (Chytridiomycota).

The purpose of our research is to investigate the morphology, zoospore ultrastructure, and molecular phylogenetic placement of a chytrid from Australia. From a survey of chytrid fungi in New South Wales, Australia, we isolated strain PL AUS 026 and putatively identified it as Polyphlyctis unispina. Light microscopic evaluation determined strain PL AUS 026 to be similar to two other strains of P. unispina characterized in the literature but to have a more complex thallus than that of the type. Molecular phylogenetic analyses placed our strain as sister of or basal to Chytridiaceae, Chytridiales. Ultrastructural analysis of the zoospore of strain PL AUS 026 revealed unique features. On the basis of our analyses we designate strain PL AUS 026 as a new species, Polyphlyctis willoughbyi. This research extends our concept of Chytridiaceae systematics and ultrastructural variation in the Chytridiales zoospore.

Zopfochytrium is a new genus in the Chytridiales with distinct zoospore ultrastructure.

While surveying chytrid diversity in lakes and streams, we found on cellulosic bait a chytrid that had both monocentric and polycentric thallus forms. We brought this chytrid into axenic culture from three sites in eastern North America, studied its thallus development and zoospore ultrastructure, and compared its 28S rDNA sequence with those of other members of the Chytridiomycota. Thallus morphology matched that described for the rare chytrid, Cladochytrium polystomum Zopf. Sporangia were spherical and produced numerous long discharge tubes. After discharge, zoospores remained in spherical clusters at the tips of the inoperculate openings of discharge tubes. After 10-30 min zoospores either swam away or encysted in place. Zoospore ultrastructural features included a cell coat, flagellar plug, and paracrystalline inclusion, features typical of members of the Chytridiales. However, the flagellar apparatus structure and organellar organization differed from that of zoospores previously described. Based on its molecular phylogeny and its zoospore ultrastructural features, we classify C. polystomum as a member of the Chytridiaceae in the Chytridiales. Because its thallus development and its ribosomal DNA sequences diverged decidedly from those of Cladochytrium tenue Nowak, the type species of Cladochytrium, we erected Zopfochytrium as a new genus for this chytrid.