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Abstract 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.

Recently, a new species of eukaryotic endoparasites of amoebae was described as Nucleophaga striatae, clustering within the previously established Rozellomycota on the basis of DNA sequence data. As a supplement to the molecular phylogenetic identification, this paper focuses on the EM description of this new species in the experimental host Thecamoeba foliovenanda. The spores of Nucleophaga are engulfed by the amoeba by phagocytosis, and then the endoparasites reach the nucleus of the amoeba, where one of the first detectable changes is the enlargement of the circularly arranged pieces of the nucleolar substance. Subsequently, the parasites grow and develop into multinucleate plasmodia, causing a 2-3-fold enlargement of the host amoeba's nucleus. As a result of multiple nuclear divisions within the plasmodia (endogenous sporogony), sporoblasts are forming which contain a network of endoplasmic reticulum and mitochondria. The sporoblasts, which more and more resemble spheres, form a wall and mature by becoming spores. These nearly spherical spores have diameters of 1.4 µm to 1.5 µm and a thick cell wall of ~50 nm. Other free-living amoebae such as Saccamoeba and Vannella (Amoebozoa), and the more distant Naegleria and Willaertia (Heterolobosea, class within phylum Percolazoa), have proven to be resistant to infection, showing that these intranuclear parasites are infectious only for members of the genus Thecamoeba (Amoebozoa). [ABSTRACT FROM AUTHOR]

Various microbial eukaryotes have been reported as endocytobionts in a wide range of protists, primarily as parasites. Those which parasitize the nucleus (Nucleophaga-like) or the cytoplasm (Sphaerita-like) of free-living amoebae, described since the end of the 19th century, have been the subject of recent studies by our team, revealing that they belong to the same group, the Rozellomycota, linked to fungi and ancestors of Microsporidia. We report here the observation of an amoeba isolated from a sycamore tree near Koblenz, Germany, and found to be infected by an intranuclear parasite. By light microscopy, the host amoeba was identified as Thecamoeba striata while the intranuclear parasite looks like previously studied Nucleophaga species. The parasite proliferates in the enlarged nucleus of the host amoeba filling it with its spores, ultimately causing its death by rupture of the membranes. Compared to other Nucleophaga spp., the current parasite grows more slowly. The 18S rDNA sequence of the parasite was thus obtained to perform biomolecular analyses. Phylogenetic analysis confirms that the parasite belongs to Nucleophaga, differing from the other described species in sequence similarity values. We propose to consider this strain as a new species, Nucleophaga striatae sp. nov. [ABSTRACT FROM AUTHOR]

Although aquatic and parasitic fungi have been well known for more than 100 years, they have only recently received increased awareness due to their key roles in microbial food webs and biogeochemical cycles. There is growing evidence indicating that fungi inhabit a wide range of marine habitats, from the deep sea all the way to surface waters, and recent advances in molecular tools, in particular metagenome approaches, reveal that their diversity is much greater and their ecological roles more important than previously considered. Parasitism constitutes one of the most widespread ecological interactions in nature, occurring in almost all environments. Despite that, the diversity of fungal parasites, their ecological functions, and, in particular their interactions with other microorganisms remain largely speculative, unexplored and are often missing from current theoretical concepts in marine ecology and biogeochemistry. In this review, we summarize and discuss recent research avenues on parasitic fungi and their ecological potential in marine ecosystems, e.g., the fungal shunt, and emphasize the need for further research.

Plasma membrane-located transport proteins are key adaptations for obligate intracellular Microsporidia parasites, because they can use them to steal host metabolites the parasites need to grow and replicate. However, despite their importance, the functions and substrate specificities of most Microsporidia transporters are unknown. Here, we provide functional data for a family of transporters conserved in all microsporidian genomes and also in the genomes of related endoparasites. The universal retention among otherwise highly reduced genomes indicates an important role for these transporters for intracellular parasites. Using Trachipleistophora hominis, a Microsporidia isolated from an HIV/AIDS patient, as our experimental model, we show that the proteins are ATP and GTP transporters located on the surface of parasites during their intracellular growth and replication. Our work identifies a new route for the acquisition of essential energy and nucleotides for a major group of intracellular parasites that infect most animal species including humans.

The Rozellomycota form a lineage basal or sister to the Fungi, ancestor of Microsporidia. Their biodiversity is very rich but remains poorly characterized. The few known species are all parasites, whether of water molds and algae (Rozella), crustaceans (Mitosporidium), or as endonuclear parasites of amoebae (Nucleophaga, Paramicrosporidium). Since the nineteenth century, intracytoplasmic parasites of various protozoa have been described as species of the same genus Sphaerita. However, it was later thought possible to separate these parasites into at least two distinct groups, those forming flagellated zoospores, prevalent in Euglena and other flagellates, and those forming immobile spores, found mainly in free-living and endozoic amoebae. Herein, we report the recovery of a strain of the free-living amoeba species Saccamoeba lacustris, naturally infected by an intracytoplasmic parasite, which under light microscope has a morphology consistent with that of Sphaerita. Biomolecular analyses were thus performed. Our results show that the intracytoplasmic parasite of Saccamoeba belongs to the same subgroup of Mitosporidium and that it forms a new genus within Rozellomycota, Morellospora, that corresponds to the former spore-forming Sphaerita-like parasites of amoebae. [ABSTRACT FROM AUTHOR]

The internal transcribed spacer (ITS) is used often in DNA metabarcoding of fungi. One disadvantage of its high variability may be a failure to classify operational taxonomic units (OTUs) when no similar reference sequence exists. We tested whether the 5.8S region, often sequenced with ITS2 but discarded before analysis, could provide OTU classifications when ITS2 fails.We used in silico evaluation to compare classification success of 5.8S and ITS2 from the UNITE database when reference sequences of the same species, genus, or family were removed. We then developed an automated pipeline for a combined 5.8S–ITS2 analysis and applied it to mixed environmental samples containing many lineages that are underrepresented in databases.ITS was clearly superior for species‐level classifications with a complete reference database, but 5.8S outperformed ITS at higher level classifications with an incomplete database. Our combined 5.8S‐ITS2 pipeline classified 3× more fungal OTUs compared to ITS2 alone, particularly within Chytridiomycota (27×) and Rozellomycota (6×).Missing reference sequences led to the failure of ITS to classify many fungal OTUs at all, and to a significant underestimation of environmental fungal diversity. Using 5.8S to complement ITS classification will likely provide better estimates of diversity in lineages for which database coverage is poor. [ABSTRACT FROM AUTHOR]

Microsporidia are intracellular eukaryotic parasites of animals, characterized by unusual morphological and genetic features. They can be divided in three main groups, the classical microsporidians presenting all the features of the phylum and two putative primitive groups, the chytridiopsids and metchnikovellids. Microsporidia originated from microsporidia-like organisms belonging to a lineage of chytrid-like endoparasites basal or sister to the Fungi. Genetic and genomic data are available for all members, except chytridiopsids. Herein, we filled this gap by obtaining the rDNA sequence (SSU-ITS-partial LSU) of Chytridiopsis typographi (Chytridiopsida), a parasite of bark beetles. Our rDNA molecular phylogenies indicate that Chytridiopsis branches earlier than metchnikovellids, commonly thought ancestral, forming the more basal lineage of the Microsporidia. Furthermore, our structural analyses showed that only classical microsporidians present 16S-like SSU rRNA and 5.8S/LSU rRNA gene fusion, whereas the standard eukaryote rRNA gene structure, although slightly reduced, is still preserved in the primitive microsporidians, including 18S-like SSU rRNA with conserved core helices, and ITS2-like separating 5.8S from LSU. Overall, our results are consistent with the scenario of an evolution from microsporidia-like rozellids to microsporidians, however suggesting for metchnikovellids a derived position, probably related to marine transition and adaptation to hyperparasitism. The genetic and genomic data of additional members of Chytridiopsida and Rozellomycota will be of great value, not only to resolve phylogenetic relationships but also to improve our understanding of the evolution of these fascinating organisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

High-throughput sequencing studies generate vast amounts of taxonomic data. Evolutionary ecological hypotheses of the recovered taxa and Species Hypotheses are difficult to test due to problems with alignments and the lack of a phylogenetic backbone. We propose an updated phylum- and class-level fungal classification accounting for monophyly and divergence time so that the main taxonomic ranks are more informative. Based on phylogenies and divergence time estimates, we adopt phylum rank to Aphelidiomycota, Basidiobolomycota, Calcarisporiellomycota, Glomeromycota, Entomophthoromycota, Entorrhizomycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota and Olpidiomycota. We accept nine subkingdoms to accommodate these 18 phyla. We consider the kingdom Nucleariae (phyla Nuclearida and Fonticulida) as a sister group to the Fungi. We also introduce a perl script and a newick-formatted classification backbone for assigning Species Hypotheses into a hierarchical taxonomic framework, using this or any other classification system. We provide an example of testing evolutionary ecological hypotheses based on a global soil fungal data set. [ABSTRACT FROM AUTHOR]

Intracellular parasitism often results in gene loss, genome reduction, and dependence upon the host for cellular functioning. Rozellomycota is a clade comprising many such parasites and is related to the diverse, highly reduced, animal parasites, Microsporidia. We sequenced the nuclear and mitochondrial genomes of Paramicrosporidium saccamoebae [Rozellomycota], an intranuclear parasite of amoebae. A canonical fungal mitochondrial genome was recovered from P. saccamoebae that encodes genes necessary for the complete oxidative phosphorylation pathway including Complex I, differentiating it from most endoparasites including its sequenced relatives in Rozellomycota and Microsporidia. Comparative analysis revealed that P. saccamoebae shares more gene content with distantly related Fungi than with its closest relatives, suggesting that genome evolution in Rozellomycota and Microsporidia has been affected by repeated and independent gene losses, possibly as a result of variation in parasitic strategies (e.g. host and subcellular localization) or due to multiple transitions to parasitism.

Nephridiophagids are poorly known unicellular eukaryotes, previously of uncertain systematic position, that parasitize the Malpighian tubules of insects. Their life cycle includes merogony with multinucleate plasmodia and sporogony leading to small, uninucleate spores. We examined the phylogenetic affiliations of three species of Nephridiophaga, including one new species, Nephridiophaga maderae, from the Madeira cockroach (Leucophaea maderae). In addition to the specific host, the new species differs from those already known by the size of the spores and by the number of spores within the sporogenic plasmodium. The inferred phylogenetic analyses strongly support a placement of the nephridiophagids in the fungal kingdom near its root and with a close, but unresolved, relationship to the chytids (Chytridiomycota). We found evidence for the nephridiophagidean speciation as being strongly coupled to host speciation. [ABSTRACT FROM AUTHOR]

Fungi in the phylum Cryptomycota have been recovered in numerous environmental DNA (eDNA) surveys but are only known from five described genera of intracellular parasites. These fungi are common in aquatic and soil habitats, but little is known about their relative diversity and specificity among particular habitats. We surveyed Cryptomycota from 80 eDNA samples including freshwater, soil, and marine habitats using Cryptomycota-preferential primers coupled with long-amplicon PacBio sequencing (1.2 kb of the 18S rRNA gene region). We found that freshwater samples were the most diverse, comprising 175 operational taxonomic units (OTUs) of Cryptomycota and also showed a high abundance of the related algae-parasitic group Aphelidiomycota, while marine samples were the least diverse with 25 OTUs. The composition of Cryptomycota communities was influenced by habitat, with freshwater and soil showing statistically distinct communities. Phylogenetic analyses showed that the present survey recovered most previously sampled major clades of Cryptomycota, but most (61%) OTUs were novel to this study, indicative of an extensive diversity of the group that remains largely uncharacterized. [ABSTRACT FROM AUTHOR]

The phylum Rozellomycota has been proposed for a group of early-branching holomycotan lineages representing obligate parasites and hyperparasites of zoosporic fungi, oomycotes or phytoplankton. Given their predominantly intracellular lifestyle, rozellids are typically known from environmental ribosomal DNA data, except for the well-studied Rozella species. To date, the phylogenetic relationship between rozellids and microsporidians (Microsporidia) is not fully understood and most reliable hypotheses are based on phylogenomic analyses that incorporate the only publicly available rozellid genome of Rozella allomycis . Here, we provide genomic data of three new rozellid lineages obtained by single-cell sequencing from environmental samples and show with a phylogenomic approach that rozellids form a monophyletic group that is sister to microsporidians, corroborating the previously proposed phylum Rozellomycota. Whereas no mitochondrial genes coding for the respiratory Complex I could be found, we discovered a gene coding for a nucleotide phosphate transporter in one of the three draft genomes. The scattered absence of Complex I genes and scattered presence of nucleotide transporter genes across diverse microsporidian and rozellid lineages suggest that these adaptations to a parasitic lifestyle, which reduce the parasite's capability to synthesize ATP but enables it to steal ATP from its host, evolved independently in microsporidians and rozellids.

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar. [ABSTRACT FROM AUTHOR]

Our knowledge of zoosporic fungal phylogeny, physiology, and ecological functions, in particular their role in aquatic food web dynamics and biogeochemistry, is limited. The recent discovery of numerous dark matter fungi (DMF), i.e., uncultured and poorly known taxa belonging to early diverging branches of the fungal tree (namely the Rozellomycota and Chytridiomycota) calls for reconsideration of the phylogeny and ecology of zoosporic fungi. In this opinion paper, we summarize the exploration of new, recently discovered lineages of DMF and their implications for the ecology, evolution, and biogeography of the rapidly growing fungal tree. We also discuss possible ecological roles of zoosporic fungi in relation to recent methodological developments including single cell genomics and cultivation efforts. Finally, we suggest linking explorative with experimental research to gain deeper insights into the physiology and ecological functioning of zoosporic fungi DMF in aquatic habitats. [ABSTRACT FROM AUTHOR]

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota. [ABSTRACT FROM AUTHOR]

Although aquatic and parasitic fungi have been well known for more than 100 years, they have only recently received increased awareness due to their key roles in microbial food webs and biogeochemical cycles. There is growing evidence indicating that fungi inhabit a wide range of marine habitats, from the deep sea all the way to surface waters, and recent advances in molecular tools, in particular metagenome approaches, reveal that their diversity is much greater and their ecological roles more important than previously considered. Parasitism constitutes one of the most widespread ecological interactions in nature, occurring in almost all environments. Despite that, the diversity of fungal parasites, their ecological functions, and, in particular their interactions with other microorganisms remain largely speculative, unexplored and are often missing from current theoretical concepts in marine ecology and biogeochemistry. In this review, we summarize and discuss recent research avenues on parasitic fungi and their ecological potential in marine ecosystems, e.g., the fungal shunt, and emphasize the need for further research. [ABSTRACT FROM AUTHOR]