Eric D. Tretter

Examining new phylogenetic markers to uncover the evolutionary history of early-diverging fungi: comparing MCM7, TSR1 and rRNA genes for single- and multi-gene analyses of the Kickxellomycotina

The recently recognised protein-coding genes MCM7 and TSR1 have shown significant promise for phylogenetic resolution within the Ascomycota and Basidiomycota, but have remained unexamined within other fungal groups (except for Mucorales). We designed and tested primers to amplify these genes across early-diverging fungal clades, with emphasis on the Kickxellomycotina, zygomycetous fungi with characteristic flared septal walls forming pores with lenticular plugs. Phylogenetic tree resolution and congruence with MCM7 and TSR1 were compared against those inferred with nuclear small (SSU) and large subunit (LSU) rRNA genes. We also combined MCM7 and TSR1 data with the rDNA data to create 3- and 4-gene trees of the Kickxellomycotina that help to resolve evolutionary relationships among and within the core clades of this subphylum. Phylogenetic inference suggests that Barbatospora, Orphella, Ramicandelaber and Spiromyces may represent unique lineages. It is suggested that these markers may be more broadly useful for phylogenetic studies among other groups of early-diverging fungi.

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.

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.

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.