Genomic analysis reveals cryptic diversity in aphelids and sheds light on the emergence of Fungi.

Over the past decade, molecular phylogenetics has reshaped our understanding of the fungal tree of life by unraveling a hitherto elusive diversity of the protistan relatives of Fungi. Aphelida constitutes one of these novel deep branches that precede the emergence of osmotrophic fungal lifestyle and hold particular significance as the pathogens of algae. Here, we obtain and analyze the genomes of aphelid species Amoeboaphelidium protococcarum and Amoeboaphelidium occidentale. Genomic data unmask the vast divergence between these species, hidden behind their morphological similarity, and reveal hybrid genomes with a complex evolutionary history in two strains of A. protococcarum. We confirm the proposed sister relationship between Aphelida and Fungi using phylogenomic analysis and chart the reduction of characteristic proteins involved in phagocytic activity in the evolution of Holomycota. Annotation of aphelid genomes demonstrates the retention of actin nucleation-promoting complexes associated with phagocytosis and amoeboid motility and also reveals a conspicuous expansion of receptor-like protein kinases, uncharacteristic of fungal lineages. We find that aphelids possess multiple carbohydrate-processing enzymes that are involved in fungal cell wall synthesis but do not display rich complements of algal cell-wall-processing enzymes, suggesting an independent origin of fungal plant-degrading capabilities. Aphelid genomes show that the emergence of Fungi from phagotrophic ancestors relied on a common cell wall synthetic machinery but required a different set of proteins for digestion and interaction with the environment.

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.

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota).

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.

A taxonomic summary of Aphelidiaceae.

Aphelids are parasitoids of various algae and diatoms, and in a recent classification are contained in family Aphelidiaceae, phylum Aphelidiomycota, kingdom Fungi. Family Aphelidiaceae (the only family in the phylum) is composed of four genera: Aphelidium, Paraphelidium, Amoeboaphelidium, and Pseudaphelidium. All species are known morphologically, and most have been illustrated. Few have been examined ultrastructurally, and even fewer have been sequenced for molecular comparisons. Recent studies in molecular phylogenetics have revealed an abundance of related environmental sequences that indicate unrealized biodiversity within the group. Herein, we briefly summarize the history of aphelids and acknowledge the controversy of placement of the group with related organisms. With light microscopic images and transmission electron micrographs, we illustrate typical life cycle stages for aphelids, provide updated descriptions and taxonomy for all described species, and provide a key to the species.

Cytochemical Localization of Polyphenol Oxidase Activity in K2-Bodies of Saprolegnia ferax Secondary Zoospores.

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K-bodies at the onset of attachment to substrates. To understand more fully how K-bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB-staining characteristic of catalase activity was detected in the granular matrix regions of K-bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal-containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K-bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat-inactivation of enzymes) all supported the presence of PPO in K-bodies. This study highlights potential roles for K-body PPO in stabilization of adhesion bodies by: cross-linking matrix phenolic proteins or glycoproteins as K-bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.

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.

Morphology, zoospore ultrastructure, and molecular position of taxa in the Asterophlyctis lineage (Chytridiales, Chytridiomycota).

The purpose of our research is to investigate morphology, zoospore ultrastructure, and molecular placement of six strains in the Asterophlyctis (Chytridiales) lineage. In previous molecular analyses strain JEL 186, putatively Asterophlyctis sarcoptoides, placed as basal in family Chytriomycetaceae. Recent sampling for chytrids resulted in isolation of five strains (WJD 209, MP 058, JEL 524, JEL 857, and JEL 885) molecularly related to strain JEL 186. Our morphological evaluations reveal that strains JEL 186 and WJD 209 are members of Asterophlyctis. Strain WJD 209 is considered representative of the type, A. sarcoptoides, and strain JEL 186 a new species, Asterophlyctis michiganensis. The four strains MP 058, JEL 524, JEL 857, and JEL 885 are distinct from Asterophlyctis, and we consider them as members of a new genus, Wheelerophlyctis, composed of two species, Wheelerophlyctis interior and Wheelerophlyctis interiexterior. Asterophlyctis and Wheelerophlyctis are sister taxa and we demarcate that lineage as Asterophlyctaceae. The two genera also have similar zoospore ultrastructure, which is unique among strains in Chytridiales. In consideration of their molecular position and zoospore ultrastructure, we hypothesize that Asterophlyctis and Wheelerophlyctis represent a bridge between Chytriomycetaceae and Chytridiaceae. This research expands our concepts of systematics and zoospore ultrastructural variation in Chytridiales.

Inventory of chytrid diversity in two temporary forest ponds using a multiphasic approach.

Food webs in temporary forest ponds are driven by decomposition of terrestrial inputs. Chytrid fungi are important components of the fungal community, degrading leaf litter in streams reliant on terrestrial inputs and in lake ecosystems where they may stabilize the food web. However, little is known about chytrid fungi in temporary forest ponds. We inventoried the chytrid diversity present in two temporary forest ponds via light microscopy of baited samples and ion semiconductor (Ion Torrent) sequencing of environmental DNA. We quantified trends of chytrid alpha and beta diversity as a function of spatial and temporal factors. A total of 59 chytrid taxa were detected throughout the study. Beta diversity exhibited variation across the sampled months for both the entire fungal community as well as for chytrids alone. Shifts in community composition were also apparent, although diversity metrics and composition patterns did not meet adjusted P values. The results of this study highlight the diversity of chytrid fungi in temporary forest ponds and the need for further studies on the spatial and temporal dynamics of chytrid species.

A taxonomic summary and revision of Rozella (Cryptomycota).

Rozella is a genus of endoparasites of a broad range of hosts. Most species are known by their morphology and host specificity, while only three have been examined ultrastructurally and had portions of their genome sequenced. Determined in molecular phylogenies to be the earliest diverging lineage in kingdom Fungi, Rozella currently nests among an abundance of environmental sequences in phylum Cryptomycota, superphylum Opisthosporidia. Here we briefly summarize a history of Rozella, provide descriptions of all species, and include a key to the species of Rozella.

Morphology, Ultrastructure, and Molecular Phylogeny of Rozella multimorpha, a New Species in Cryptomycota.

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.

Ultrastructural characterization of the host-parasite interface between Allomyces anomalus (Blastocladiomycota) and Rozella allomycis (Cryptomycota).

Rozella allomycis is an obligate endoparasite of the water mold Allomyces and a member of a clade (= Opisthosporidia) sister to the traditional Fungi. Gaining insights into Rozella's development as a phylogenetically pivotal endoparasite can aid our understanding of structural adaptations and evolution of the Opisthosporidia clade, especially within the context of genomic information. The purpose of this study is to characterize the interface between R. allomycis and Allomyces anomalus. Electron microscopy of developing plasmodia of R. allomycis in host hyphae shows that the interface consists of three-membrane layers, interpreted as the parasite's plasma membrane (inner one layer) and a host cisterna (outer two layers). As sporangial and resting spore plasmodia develop, host mitochondria typically cluster at the surface of the parasite and eventually align parallel to the three-membrane layered interface. The parasite's mitochondria have only a few cristae and the mitochondrial matrix is sparse, clearly distinguishing parasite mitochondria from those of the host. Consistent with the expected organellar topology if the parasite plasmodia phagocytize host cytoplasm, phagocytic vacuoles are at first bounded by three-membrane layers with host-type mitochondria lining the inner membrane. Thus, Rozella's nutrition, at least in part, is phagotrophic in contrast to osmotrophic nutrition of traditional fungi.

Integrating chytrid fungal parasites into plankton ecology: research gaps and needs.

Chytridiomycota, often referred to as chytrids, can be virulent parasites with the potential to inflict mass mortalities on hosts, causing e.g. changes in phytoplankton size distributions and succession, and the delay or suppression of bloom events. Molecular environmental surveys have revealed an unexpectedly large diversity of chytrids across a wide range of aquatic ecosystems worldwide. As a result, scientific interest towards fungal parasites of phytoplankton has been gaining momentum in the past few years. Yet, we still know little about the ecology of chytrids, their life cycles, phylogeny, host specificity and range. Information on the contribution of chytrids to trophic interactions, as well as co-evolutionary feedbacks of fungal parasitism on host populations is also limited. This paper synthesizes ideas stressing the multifaceted biological relevance of phytoplankton chytridiomycosis, resulting from discussions among an international team of chytrid researchers. It presents our view on the most pressing research needs for promoting the integration of chytrid fungi into aquatic ecology.

Molecular Phylogeny and Ultrastructure of Aphelidium desmodesmi, a New Species in Aphelida (Opisthosporidia).

Aphelids are a diverse group of intracellular parasitoids of algae and diatoms, and are sister to true fungi. Included in four genera, the 14 described species utilize phagocytosis as their mode of nutrition, and the life cycles of these taxa are remarkably similar. However, their putative specificity of host, morphological and ultrastructural features, and genetic divergence have been considered in taxon delineation. Here, we examine the host specificity, morphology, ultrastructure, and molecular 18S gene sequence of a new species in Aphelida, Aphelidium desmodesmi sp. nov. This taxon is in a well-supported clade with two other species of Aphelidium, and this lineage is sister to Amoeboaphelidium and Paraphelidium. Of interest, the mitochondrial structure of Aph. desmodesmi is more like that of Paraphelidium than that of Aphelidium aff. melosirae, the only other species of Aphelidium to have been examined ultrastructurally. This research examines and expands our understanding of host range, morphological diversity, and genetic divergence of the aphelids.

Morphological, molecular, and ultrastructural characterization of Rozella rhizoclosmatii, a new species in Cryptomycota.

Rozella is a genus of unwalled endoparasites of a variety of hosts including Oomycota (Stramenopiles), Blastocladiomycota and Chytridiomycota (Fungi), and one green alga (Coleochaete, Chlorophyceae). It currently includes more than 20 formally described species, and no new species of Rozella have been described since 1987. We discovered a new Rozella species parasitizing Rhizoclosmatium globosum (Chytridiales, Chytridiomycota) and investigated its morphology, ultrastructure, and phylogenetic position. Herein named as Rozella rhizoclosmatii sp. nov., the organism induces hypertrophy of the host. Its zoospore is ultrastructurally similar to that of Rozella allomycis, although it has a unique zoospore ultrastructural feature, a lattice of perpendicular rods about the nucleus. The 18S rDNA molecular sequence of R. rhizoclosmatii is similar to that of the previously sequenced 'Rozella ex Rhizoclosmatium'. This is the first study to inclusively characterize a new species of Rozella with morphological, ultrastructural and molecular data. As this is only the second Rozella species to be examined ultrastructurally, and because it is parasitic on a member of Chytridiomycota and not Blastocladiomycota, this research supports the conservative nature of zoospore ultrastructure to help define the genus.

Characterization of a new chytrid species parasitic on the dinoflagellate, Peridinium gatunense.

Only a few chytrid fungi have been reported as parasites of dinoflagellates. Among these reports, chytrids are periodically observed growing on the dinoflagellate, Peridinium gatunense, in Lake Kinneret (Sea of Galilee), Israel. Because of the distinctive roles of parasitic chytrid fungi in decreasing phytoplankton populations and in transforming inedible algae into chytrid biomass which zooplankton grazers can eat, characterizing dinoflagellate parasites contributes to our understanding of the sustainability of this important water resource. An undescribed chytrid parasite of P. gatunense from Lake Kinneret has recently been brought into pure culture (KLL_TL-060613), facilitating exploration of its infection process. To evaluate the ability of this chytrid to affect host populations, we determined the effect of: (1) temperature and light (or dark) on prevalence of infection and (2) host growth phase and parasite:host ratio on percentage of infection. The greatest amplification in host infection occurred in cultures grown in the dark at 25 C. The percentage of host cells infected increased as the availability of host cells compared to parasite cells increased. These results demonstrate that environmental factors influence the chytrid's potential to affect Peridinium gatunense populations. Because this chytrid had not been described taxonomically, we characterized its thallus morphology, development, zoospore ultrastructure and phylogenetic relationships. Zoospore ultrastructure was compatible with the Group II type zoospore characteristic of the family Chytridiaceae in the Chytridiales. Consistent with this observation, phylogenetic analyses of nuc 28S rDNA D1-D3 domains (28S) placed the chytrid in a clade among described taxa in the Chytridiaceae. Because thallus morphology was distinct from these other taxa, as well as other described parasites of dinoflagellates, this chytrid is described as a new genus and species, Dinochytrium kinnereticum.

Borealophlyctis nickersoniae, a new species in Rhizophlyctidales.

Zoospore ultrastructural characters combined with molecular phylogenetic hypotheses have been used to revise the taxonomy of zoosporic true fungi. An example is the reclassification of Rhizophlyctis rosea-like fungal strains into four new families and three new genera within the order Rhizophlyctidales. One genus was Borealophlyctis, which included a Canadian isolate, DAOMC 229843. A recent survey of chytrid diversity in Alabama (USA) yielded additional strains (WJD 170, WJD 171) in the Borealophlyctis lineage. With light and transmission-electron microscopy we examined strains DAOMC 229843, WJD 170 and WJD 171. We also analyzed partial nuc 28S rDNA D1-D3 domains (28S) and nuc rDNA region encompassing the internal transcribed spacers 1 and 2 and 5.8S (ITS) sequences to determine the phylogenetic placement of the strains within Rhizophlyctidales. Based on molecular divergence and morphological differences from the type Borealophlyctis paxensis, we recognize DAOMC 229843, WJD 170 and WJD 171 as representatives of the new species Borealophlyctis nickersoniae.

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.

An ultrastructural study of Paraphysoderma sedebokerense (Blastocladiomycota), an epibiotic parasite of microalgae.

Successful algal cultivation for biofuel production is one path in the transition to a renewable energy economy. The green alga Scenedesmus dimorphus is a candidate for biofuel production, but is subject to parasitism and subsequent population crash when cultivated in open ponds. From an open pond cultivating S. dimorphus for biofuel production in New Mexico, USA, an amoeboid parasite was isolated, designated as isolate FD61, and its rDNA operon sequenced. A BLAST search for nuc 18S rDNA (18S) sequence similarity identified the parasite as Paraphysoderma sedebokerense (Blastocladiomycota). Here, we examine the ultrastructure of P. sedebokerense and compare it with that of a sister taxon, Physoderma maydis. The parasite has thin-walled vegetative sporangia and thick-walled resting sporangia. Our observations indicate that amoeboid swarmers are produced in the vegetative phase, while either amoeboid swarmers or zoospores are the product of meiosis in resting sporangia. Meiosis is confirmed by the presence of synaptonemal complexes in resting sporangia nuclei. Notably, P. sedebokerense has a Golgi apparatus with stacked cisternae, a feature reported for P. maydis, but which is absent in all other examined taxa in Blastocladiomycota. This report furthers our knowledge of the life cycle of P. sedebokerense.

A new family and four new genera in Rhizophydiales (Chytridiomycota).

Many chytrid phylogenies contain lineages representing a lone taxon or a few organisms. One such lineage in recent molecular phylogenies of Rhizophydiales contained two marine chytrids, Rhizophydium littoreum and Rhizophydium aestuarii. To better understand the relationship between these organisms, we increased sampling such that the R. littoreum/R. aestuarii lineage included 10 strains of interest. To place this lineage in Rhizophydiales, we constructed a molecular phylogeny from partial nuc 28S rDNA D1-D3 domains (28S) of these and 80 additional strains in Rhizophydiales and examined thallus morphology and zoospore ultrastructure of our strains of interest. We also analyzed sequences of the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS) of our 10 strains of interest to assess sequence similarity and phylogenetic placement of strains within the lineage. The 10 strains grouped together in three well supported clades: (i) Rhizophydium littoreum+Phlyctochytrium mangrovei, (ii) three strains of Rhizophydium aestuarii and (iii) five previously unidentified strains. Light microscopic observations revealed four distinct thallus morphologies, and zoospore ultrastructural analyses revealed four distinct constellations of ultrastructural features. On the bases of morphological, ultrastructural and molecular evidence we place these strains in the new family Halomycetaceae and four new genera (Halomyces, Paludomyces, Ulkenomyces, Paranamyces) in Rhizophydiales.