Merlin M. White

A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

Using a five-gene phylogeny to test morphology-based hypotheses of Smittium and allies, endosymbiotic gut fungi (Harpellales) associated with arthropods

Smittium, one of the first described genera of gut fungi, is part of a larger group of endosymbiotic microorganisms (Harpellales) that live predominantly in the digestive tracts of aquatic insects. As a diverse and species-rich taxon, Smittium has helped to advance our understanding of the gut fungi, in part due to the relative success of attempts to culture species of Smittium as compared to other members of Harpellales. Approximately 40% of the 81 known species of Smittium have been cultured. This is the first Smittium multigene dataset and phylogenetic analysis, using the 18S and 28S rRNA genes, as well as RPB1, RPB2, and MCM7 translated protein sequences. Several well-supported clades were recovered within Smittium. One includes the epitype S. mucronatum (the True Smittium clade), and another contains many species including S. simulii and S. orthocladii (the Parasmittium clade). Ancestral states were reconstructed for holdfast shape, thallus branching type, as well as asexual (trichospore) and sexual (zygospore) spore morphology. Two of these characters, holdfast shape and trichospore morphology, supported the split of the two main clades revealed by the molecular phylogeny, suggesting these are natural clades and these traits may have evolutionary and perhaps ecological significance.

Overview of 75 years of Smittium research, establishing a new genus for Smittium culisetae, and prospects for future revisions of the ‘Smittium’ clade

The Harpellales includes 38 genera of endosymbiotic microfungi associated with various Arthropoda. Smittium, the second genus to be described, is now also the most species rich of the order. Species of Smittium inhabit the digestive tracts of larval aquatic insects, especially lower Diptera, worldwide. During the 75 y since the type, Smittium arvernense, was described a number of advances in our understanding of the gut fungi have unfolded, in whole or in part, with Smittium as a model for the fungal trichomycetes. This in part relates to the high number of successful isolation attempts, with about 40% of known species having been cultured, a total number that far exceeds any other genus of gut fungus. Many isolates of Smittium have been used in laboratory studies for ultrastructural, physiological, host feeding, serological, as well as isozyme, and now ongoing molecular systematic studies. Molecular studies have shown that Smittium is polyphyletic but with consistent separation of Smittium culisetae, one of the most common and widespread species, from the remainder of Smittium. A brief overview of Smittium research is provided. Zygospore and trichospore morphology and molecular evidence (immunological, isozyme, DNA sequences and phyiogenetic analyses) are used to establish Zancudomyces and to accommodate Smittium culisetae. For the latter evidence, we include the first two-gene phylogenetic analysis, using combined 18S and 28S rRNA gene sequence data to show a cluster of Zancudomyces culisetae separate from Smittium. As the broadest taxon sampling of Smittium to date, this also serves a molecular systematic update toward revisionary syntheses of this and other Harpellales taxa.

Trifoliellum bioblitzii, a new genus of trichomycete from mayfly nymphs in Nova Scotia, Canada

Trichomycetes are an ecological group of fungi and protists that colonize the gut lining of invertebrates in aquatic and moist terrestrial habitats. The diversity of this group appears to be high with many new species discovered each year. A new genus of fungal trichomycete, Trifoliellum (Harpellales), is described here with the type species T. bioblitzii. This genus is characterized by having unique, trefoil-shaped asexual spores (trichospores). Another new species, Legeriosimilis halifaxensis, also is described from the same mayfly host, Eurylophella temporalis, collected from the same site near Halifax, Nova Scotia.

Challenges and Future Perspectives in the Systematics of Kickxellomycotina, Mortierellomycotina, Mucoromycotina, and Zoopagomycotina

This review presents a phylogenetically based classification of four subphyla of the early-diverging fungi: Kickxellomycotina, Mortierellomycotina, Mucoromycotina, and Zoopagomycotina. The Kickxellomycotina contains four orders: Asellariales (symbionts with isopods and Collembola), Dimargaritales (haustorial mycoparasites), Harpellales (symbionts of insect larvae), and Kickxellales (saprobes). Mortierellomycotina contains a single order, Mortierellales, that includes both saprobes and root endophytes. Zoopagomycotina also has one order, Zoopagales, that contains species of obligate animal parasites or mycoparasites. Mucoromycotina has two orders, Endogonales (saprobes and ectomycorrhizal fungi) and Mucorales (primarily saprobes). The Mucorales is by far the most diverse order and includes 14 families and two distinct but unnamed clades. In addition to providing a phylogenetic and taxonomic overview of these subphyla, we provide information on growth and axenic cultivation of these fungi. We also discuss DNA barcoding, environmental sampling, genome sequencing, and phylogenetic analyses as they relate to these subphyla and other early-diverging fungal lineages. Evidence suggests that genome sequencing from a wide array of taxa in these four subphyla combined with innovative environmental sampling approaches is likely to revolutionize our understanding of these fungi and also the fungal tree of life.

Assessing the Diversity of Arbuscular Mycorrhizal Fungi in Semiarid Shrublands Dominated by Artemisia tridentata ssp. wyomingensis

Variation in the abiotic environment and host plant preferences can affect the composition of arbuscular mycorrhizal (AMF) assemblages. This study analyzed the AMF taxa present in soil and seedlings of Artemisia tridentata ssp. wyomingensis collected from sagebrush steppe communities in southwestern Idaho, USA. Our aims were to determine the AMF diversity within and among these communities and the extent to which preferential AMF–plant associations develop during seedling establishment. Mycorrhizae were identified using molecular methods following DNA extraction from field and pot culture samples. The extracted DNA was amplified using Glomeromycota specific primers, and identification of AMF was based on phylogenetic analysis of sequences from the large subunit-D2 rDNA region. The phylogenetic analyses revealed seven phylotypes, two within the Claroideoglomeraceae and five within the Glomeraceae. Four phylotypes clustered with known species including Claroideoglomus claroideum, Rhizophagus irregularis, Glomus microaggregatum, and Funneliformis mosseae. The other three phylotypes were similar to several published sequences not included in the phylogenetic analysis, but all of these were from uncultured and unnamed glomeromycetes. Pairwise distance analysis revealed some phylotypes with high genetic variation. The most diverse was the phylotype that included R. irregularis, which contained sequences showing pairwise differences up to 12xa0%. Most of the diversity in AMF sequences occurred within sites. The smaller genetic differentiation detected among sites was correlated with differences in soil texture. In addition, multiplication in pot cultures led to differentiation of AMF communities. Comparison of sequences obtained from the soil with those from A. tridentata roots revealed no significant differences between the AMF present in these samples. Overall, the sites sampled were dominated by cosmopolitan AMF taxa, and young seedlings of A. tridentata ssp. wyomingensis were colonized in relation to the abundance of these taxa in the soil.

An Eight-Gene Molecular Phylogeny of the Kickxellomycotina, Including the First Phylogenetic Placement of Asellariales

Kickxellomycotina is a recently described subphylum encompassing four zygomycete orders (Asellariales, Dimargaritales, Harpellales, Kickxellales). These fungi are united by the formation of disciform septal pores containing lenticular plugs. Morphological diversification and life history evolution has made the relationships within and among the four orders difficult to resolve on those grounds alone. Here we infer the phylogeny of the Kickxellomycotina based on an eight-gene supermatrix including both ribosomal rDNA (18S, 28S, 5.8S) and protein sequences (MCM7, TSR1, RPB1, RPB2, β-tubulin). The results of this study demonstrate that Kickxellomycotina is monophyletic and related to members of the Zoopagomycotina. Eight unique clades are distinguished in the Kickxellomycotina, including the four defined orders (Asellariales, Dimargaritales, Harpellales, Kickxellales) as well as four genera previously placed within two of these orders (Barbatospora, Orphella, Ramicandelaber, Spiromyces). Dimargaritales and Ramicandelaber are the earliest diverging members of the subphylum, although the relationship between these taxa remains uncertain. The remaining six clades form a monophyletic group, with Barbatospora diverging first. The next split divides the remaining members of the subphylum into two subclades: (i) Asellariales and Harpellales and (ii) Kickxellales, Orphella and Spiromyces. Estimation of ancestral states for four potentially informative morphological and ecological characters reveals that arthropod endosymbiosis might have been an important factor in the early evolution of the Kickxellomycotina.

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

Typification of Smittium , an Important Genus in the Taxonomy of Harpellales

The Harpellales genus Smittium is based on a type species, S. arvernense, which was described by Poisson in 1937 without designation of a type specimen. Smittium arvernense has not been reported since its originalPUBLICation. Because the other 79 species of Smittium cannot be compared to the type species, a lectotype is proposed as well as an epitype for that lectotype that is also the holotype of S. mucronatum. Because Smittium is believed to be polyphyletic these type designations will provide stable application of names and, as well resolved phylogenetic analyses of member species emerge based on morphological and DNA sequence characters, they will provide a foundation for a more robust and revised classification.

Extended studies of Baltomyces styrax in Idaho and expanded distribution of this isopod gut fungus in USA.

Trichomycetes or gut fungi are currently considered to be an ecological group consisting of both fungi and protists that inhabit the guts of arthropods from varied environments. Baltomyces styrax has been included as a member of the Asellariales, a small and understudied order of true gut fungi. Baltomyces styrax has remained monotypic and not been reported formally since it was first described by Cafaro in 1999 from one collection in Louisiana. Herein we report on subsequent collections of this fungus, from both lentic and lotic locations, over the past several years from four other states, most recently Idaho, where we have records over a 4 y span. We repeatedly collected at one lentic site in Idaho for 5 mo, which allowed an extended study of not only the life history of B. styrax but also the discovery of morphological characteristics that were not reported earlier. Therefore we are emending the species description and providing additional information on the ecology and host range of B. styrax as well as providing a context for comparison to Asellaria, the type genus of the Asellariales. We also extend the known host range and suggest that the fungus is much more widespread than current records indicate.