An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence.

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.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

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.

Diploid-dominant life cycles characterize the early evolution of Fungi.

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.

Polyrhizophydium stewartii, the first known rhizomycelial genus and species in the Rhizophydiales, is closely related to Batrachochytrium.

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.

The Collection of Zoosporic Eufungi at the University of Michigan (CZEUM): introducing a new repository of barcoded Chytridiomyceta and Blastocladiomycota cultures.

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.

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.

Adaptation by Loss of Heterozygosity in Saccharomyces cerevisiae Clones Under Divergent Selection.

Loss of heterozygosity (LOH) is observed during vegetative growth and reproduction of diploid genotypes through mitotic crossovers, aneuploidy caused by nondisjunction, and gene conversion. We aimed to test the role that LOH plays during adaptation of two highly heterozygous Saccharomyces cerevisiae genotypes to multiple environments over a short time span in the laboratory. We hypothesized that adaptation would be observed through parallel LOH events across replicate populations. Using genome resequencing of 70 clones, we found that LOH was widespread with 5.2 LOH events per clone after ∼500 generations. The most common mode of LOH was gene conversion (51%) followed by crossing over consistent with either break-induced replication or double Holliday junction resolution. There was no evidence that LOH involved nondisjunction of whole chromosomes. We observed parallel LOH in both an environment-specific and environment-independent manner. LOH largely involved recombining existing variation between the parental genotypes, but also was observed after de novo, presumably beneficial, mutations occurred in the presence of canavanine, a toxic analog of arginine. One highly parallel LOH event involved the ENA salt efflux pump locus on chromosome IV, which showed repeated LOH to the allele from the European parent, an allele originally derived by introgression from S. paradoxus Using CRISPR-engineered LOH we showed that the fitness advantage provided by this single LOH event was 27%. Overall, we found extensive evidence that LOH could be adaptive and is likely to be a greater source of initial variation than de novo mutation for rapid evolution of diploid genotypes.

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 Raffaelea species (Ophiostomatales) from the USA and Taiwan associated with ambrosia beetles and plant hosts.

Raffaelea (Ophiostomatales) is a genus of more than 20 ophiostomatoid fungi commonly occurring in symbioses with wood-boring ambrosia beetles. We examined ambrosia beetles and plant hosts in the USA and Taiwan for the presence of these mycosymbionts and found 22 isolates representing known and undescribed lineages in Raffaelea. From 28S rDNA and ß-tubulin sequences, we generated a molecular phylogeny of Ophiostomatales and observed morphological features of seven cultures representing undescribed lineages in Raffaelea s. lat. From these analyses, we describe five new species in Raffaelea s. lat.: R. aguacate, R. campbellii, R. crossotarsa, R. cyclorhipidia, and R. xyleborina spp. nov. Our analyses also identified two plant-pathogenic species of Raffaelea associated with previously undocumented beetle hosts: (1) R. quercivora, the causative agent of Japanese oak wilt, from Cyclorhipidion ohnoi and Crossotarsus emancipatus in Taiwan, and (2) R. lauricola, the pathogen responsible for laurel wilt, from Ambrosiodmus lecontei in Florida. The results of this study show that Raffaelea and associated ophiostomatoid fungi have been poorly sampled and that future investigations on ambrosia beetle mycosymbionts should reveal a substantially increased diversity.

Synchytrium microbalum sp. nov. is a saprobic species in a lineage of parasites.

The diversity of the Chytridiomycota is poorly known and sequence information is not well represented in databases, often preventing identification of chytrid sequences retrieved from environmental samples. We found an unknown, saprobic chytrid, related to Synchytrium, which heretofore has been considered a lineage of parasites. Because of its phylogenetic relationship, and ecological dissimilarity to other Synchytrium species, we considered this fungus of scientific interest and describe it herein. We based our study on an analysis of 18S rDNA, light microscopic morphology, and ultrastructural characters of the zoospores. The fungus is tiny and produces filamentous projections 4-5 times as long as the diameter of the sporangium. The small zoospores are longer than wide and their ultrastructure is similar to that of Synchytrium endobioticum, except that they lack a microtubule root. Our molecular analysis placed the organism within a clade of Synchytrium species and we name the new species Synchytrium microbalum.

Ophiocordyceps myrmicarum, a new species infecting invasive Myrmica rubra in Maine.

Myrmica rubra L. (Formicidae: Hymenoptera), an invasive ant species in Maine, has been problematic in coastal communities over the past 15years and the subject of studies on its ecology, effects, and management. In recent years we observed a fungus growing from moribund M. rubra (in vitro) and have isolated multiple cultures of this previously unreported fungus over 2 y. We analyzed morphology and DNA molecular sequences of this fungus and identified it as a member of the genus Hirsutella Pat. (Ophiocordycipitaceae; Hypocreales), the use of which is suppressed in favor of a monophyletic Ophiocordyceps clade. Molecular and morphological characters support the description of this fungus as a new species, O. myrmicarum, which is the first species in this genus to be isolated from North American M. rubra.

Phylogeny of Hirsutella species (Ophiocordycipitaceae) from the USA: remedying the paucity of Hirsutella sequence data.

Hirsutella (Ophiocordycipitaceae: Hypocreales) is a genus of insect, mite, and nematode pathogens with an asexual morph, which generally produce a mucilaginous cluster of one or several conidia on phialides that are basally subulate and taper to a fine neck. The generic name Hirsutella has been proposed for suppression in favour of Ophiocordyceps as a consequence of the ending of dual nomenclature for different morphs of pleomorphic fungi in 2011. Though the generic name is well established, geographically dispersed, and speciose, exceptionally few sequences are available in online databases. We examined 46 isolates of 23 Hirsutella species from the USA, curated by the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF Culture Collection), that previously had not been molecularly characterized and produced a phylogeny of these organisms; we included previously published Hirsutella and Ophiocordyceps taxa. In producing the largest phylogeny of Hirsutella isolates so far, we provide: (1) context for discussing previously-hypothesized relationships; (2) evidence for revisions as taxonomic transitions move forward; and (3) available molecular data to be incorporated into further evolutionary studies of Ophiocordycipitaceae.

Thoreauomyces gen. nov., Fimicolochytrium gen. nov. and additional species in Geranomyces.

Powellomycetaceae (Spizellomycetales) contain a diverse group of exogenously developing chytrids found by baiting water preparations of soils and manure with pollen. A previous molecular phylogenetic study indicated that some lineages within this family represent undescribed genera and species. Description of genera within the Spizellomycetales traditionally has relied on ultrastructural characters of zoospores, whereas species have been based on thallus development and morphology. We analyzed Powellomycetaceae chytrids that had not yet had ultrastructural and thallus morphologies determined. Because these chytrids vary little morphologically, we used a linear discriminant function analysis of thallus characters to determine features most useful for separating species when grown in pure culture on identical media. Zoosporic ultrastructures of two groups of isolates differed from those of the two described genera in the family, and we describe the new genera Thoreauomyces with one new species and Fimicolochytrium with two new species. Also, we describe three new species within Geranomyces. Linear discriminant function analysis, although helpful for determining more stable morphological characters, was not completely accurate in assigning chytrids to the correct genus or species, thus emphasizing the importance of molecular characters for identifying these taxa.

The Polychytriales ord. nov. contains chitinophilic members of the rhizophlyctoid alliance.

During the past 5 y the Rhizophydiales, Cladochytriales and Lobulomycetales have been segregated from the formerly recognized Chytridiales. Descriptions of new chytridiomycete orders are based on molecular and ultrastructural characters, which have been phylogenetically mutually supportive. The Polychytrium clade has consisted of a few chitinophilic, soil and aquatic chytrids that clustered in phylogenetic hypotheses but have not been placed in a new order. We isolated additional putative members of this clade, sequenced their nucSSU and nucLSU rDNA and examined zoospores of some of the isolates with TEM. Our isolates are in a well supported clade with previous Polychytrium clade members, but zoospore ultrastructural types vary within the clade, with characters that often are conserved within other orders (e.g. flagellar plug, rumposome) being either present or absent. Based on the isolates in culture we describe the Polychytrium clade as the Polychytriales. This order contains Polychytrium, Lacustromyces, Karlingiomyces, two new genera (Arkaya and Neokarlingia) and additional undescribed taxa.

Alogomyces tanneri gen. et sp. nov., a chytrid in Lobulomycetales from horse manure.

The order Lobulomycetales contains chytrids from soil, freshwater and marine habitats; environmental DNA sampling has indicated that representatives of this order might be found in deep ocean localities. We describe Alogomyces tanneri as the first lobulomycetalean chytrid isolated from horse manure; A. tanneri is also the first species in the order to possess a rumposome in its zoospore. This species widens the range of habitats, ultrastructural variation and thallus morphology for Lobulomycetales.

Phylogeny of Powellomycetaceae fam. nov. and description of Geranomyces variabilis gen. et comb. nov.

The genus Powellomyces was described to accommodate two monocentric chytrid species from soil that develop exogenously and possess zoosporic ultrastructure similar to other members of the order Spizellomycetales. Despite Powellomyces-like chytrids being commonly observed in gross culture, the genus contained only two species. To determine diversity in this group I amassed 49 isolates of Powellomyces-like chytrids, including the cultures upon which species types were based and new isolates from pollen-baited water cultures of soils, plant detritus and manure. I sequenced portions of nucSSU and nucLSU rDNA regions and the EF-1α-like gene from each isolate to produce a molecular phylogeny. This phylogeny supports monophyly of spizellomycetalean chytrids with exogenous development and suggests that multiple distinct lineages exist within this group. This phylogeny, along with a reevaluation of the ultra-structural features of the two described species, supports the recognition of a new family, Powellomycetaceae, and genus, Geranomyces, which contains 31 isolates of G. variabilis comb. nov.

Cladochytriales--a new order in Chytridiomycota.

Recently, molecular and ultrastructural analyses have resulted in revised phylogenetic hypotheses in the phylum Chytridiomycota. The order Chytridiales, once considered monophyletic, has been subdivided into several new orders. However, the most recent analyses indicate that the emended Chytridiales is also polyphyletic. One monophyletic lineage in Chytridiales includes Cladochytrium, Nowakowskiella, and five other genera. Many of the chytrids in this clade have often been observed growing on decaying plant tissue and other cellulosic substrates from aquatic habitats and moist soils. In this study we analysed combined nu-rRNA gene sequences (partial SSU and LSU) of 30 isolates from North American aquatic and soil samples. Based on molecular monophyly and zoospore ultrastructure, we designate this clade as a new order, Cladochytriales, which includes four families: Cladochytriaceae, Nowakowskiellaceae, Septochytriaceae fam. nov., and Endochytriaceae.

Lobulomycetales, a new order in the Chytridiomycota.

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.