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.

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.

Genus Nucleophaga is a parasite of the nucleus of various protozoa, i.e., a number of amoebae (free-living and parasitic forms), types of flagellates, and allegedly certain ciliates. Nucleophaga was, since its original description (Dangeard, 1895), considered to be fungal (a Chytridiomycete). Within Chytridiomycetes, the genus was included in family Olpidiaceae (Sparrow, 1960; Karling, 1977) along with a similar genus, Sphaerita (occurring in the cytoplasm rather than the nucleus), and with Olpidium (some species of which have been more recently determined, cf. James et al. 2006, to have membership in groups other than Chytridiomycota). Recent work (e.g., Corsaro et al., 2014, 2016) has indicated, however, that closer relationships of Nucleophaga are with Cryptomycota (‘Rozellomycota’ of some), and perhaps even more so with Microsporidia (Bass et al., 2018). In any case, only two species of Nucleophaga (N. amoebae and N. terricolae) have been examined in molecular and electron-microscopic studies. And, no single publication has yet fully accounted for all named species of the genus and a number of putative species remaining unnamed. Part of the reason for the relatively poorly known status of potential taxa of Nucleophaga, among mycologists, is that much early publication on the genus was in protozoological (rather than mycological, or botanical) literature--related to the fact that early observers of Nucleophaga were often interested in protozoa as primary subjects of investigation. The purpose of our study is to review literature (including that of protozoology) and bring named species of Nucleophaga together, and, importantly, provide discussion of each. A further goal is to assemble an annotated list of potential (unnamed) additional species. We exclude forms once considered to possibly belong to Nucleophaga, but which, in all probability, do not. We hope our collation of information will encourage additional finds (and molecular and electron microscopic investigations) of the genus--now understood, along with related genera, to be significant in understanding early fungal/protist (holomycotan) evolution. Published on-line www.phytologia.org Phytologia 101(1): 1-18 (March 21, 2019). ISSN 030319430.

Rhizoclosmatium globosum, the type for the genus Rhizoclosmatium, is commonly isolated from freshwater aquatic systems, especially on chitin-containing substrates. The genus Rhizoclosmatium currently includes three additional described species: R. aurantiacum and R. hyalinum, which can grow on chitin, and R. marinum reported growing on a marine green alga. Our study employed analyses of ribosomal gene sequences, zoospore ultrastructure, and thallus morphology and development to identify chytrid strains related to Rhizoclosmatium. Phylogenetic analyses of ribosomal genes of strains revealed three major lineages within a clade that includes the type, R. globosum. Based on these results, we describe four new species and one new variety of Rhizoclosmatium, all capable of growth on chitin: R. sparsum, R. umbonatum, R. umbonatum var. sphaericum, R. persicum, and R. pessaminum. We have also found that R. globosum strains can grow on a range of substrates, including chitin, cellulose, keratin, and pollen. This information is herein assimilated into the existing taxonomy of the genus, which is also summarized and nomenclaturally updated. Published on-line www.phytologia.org Phytologia 101(2): 139-163 (June 21, 2019). ISSN 030319430.

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.

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.

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.

The chytrid Gaertneriomyces was first delineated based on a distinct constellation of zoospore ultrastructural characters when the new order Spizellomycetales was erected. Later phylogenetic analysis of small ribosomal, large ribosomal and ITS 1-5.8S-ITS2rRNA gene sequences demonstrated that Gaertneriomyces was polyphyletic, with G. tenuis placing in a separate linage from the type of the genus, G. semiglobifer. In this study we characterize 22 strains placing in the Gaertneriomyces clade and revise the genus. Within Gaertneriomyces, a new species (G. palmatus) is described and two new combinations (G. spectabilis, G. californicus) are made. Additionally, detailed ultrastructure of the G. tenuis zoospore is characterized for the first time, distinguishing its zoospores from zoospores of G. semiglobifer and other described members of the Spizellomycetales. Because its zoospore ultrastructure is unique and in molecular phylogeny it stands alone from the other Gaertneriomyces strains, a new genus, Barromyces, is erected for G. tenuis.

Spizellomycetaceae is a family of soil-inhabiting chytrids that presently contains four genera - Spizellomyc.es, Triparticalcar, Kochiomyces, and Gaertneriomyces - that were delineated on the basis of unique zoospore ultrastructure. A recent molecular phylogeny of Spizellomycetaceae revealed Triparticalcar and Kochiomyces to be monophyletic, Spizellomyces and Gaertneriomyces to occur in multiple lineages, and several lineages of unidentified strains. To define better the uncharacterized strains, here we closely replicate that recent phylogeny and incorporate several new strains. Molecular phylogenetic and ultrastructural analyses revealed three lineages with unique zoospore ultrastructure, which we delineate as three new genera Brevicalcar. Bulbosomyces, and Gallinipes. Our results demonstrate greater molecular and ultrastructural diversity in Spizellomycetaceae than was previously realized and reveal ultrastructural features not exhibited in other taxa in 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.

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.

Cultivation of algae in outdoor ponds is a cost-effective method to produce algal bioproducts for various economic sectors including agriculture, fuel and cosmetics. However, like any agricultural system, the algal crop is susceptible to a variety of pests, many of which are not described in the literature. This knowledge gap precludes the development of effective treatments and, in turn, limits the viability of the algal cultivation industry. Here we describe a novel pleomorphic bacterial predator (henceforth FD111) of the eukaryotic alga Nannochloropsis, which we identified as a causal agent of culture loss in outdoor ponds. Transmission electron microscopy revealed that FD111 has an endobiotic life cycle similar to described predatory bacteria, but previously undescribed for a eukaryotic host. A putative sequence for FD111 shows similarity to bacteria within the order Bdellovibrionales. Primers were developed for this sequence allowing the monitoring of infection by qPCR. Sodium hypochlorite is presented as a potential treatment to prevent culture loss caused by FD111 infection. FD111 is an amenable model organism for studying algae-bacteria interactions and is relevant to disciplines from evolutionary biology to biotechnology.

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1–20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced.

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.

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.

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.

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.

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.

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.

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.