Donald H. Pfister

Reconstructing the early evolution of Fungi using a six-gene phylogeny

The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to members of the extant phylum Chytridiomycota (chytrids). Current classifications assume that chytrids form an early-diverging clade within the kingdom Fungi and imply a single loss of the spore flagellum, leading to the diversification of terrestrial fungi. Here we develop phylogenetic hypotheses for Fungi using data from six gene regions and nearly 200 species. Our results indicate that there may have been at least four independent losses of the flagellum in the kingdom Fungi. These losses of swimming spores coincided with the evolution of new mechanisms of spore dispersal, such as aerial dispersal in mycelial groups and polar tube eversion in the microsporidia (unicellular forms that lack mitochondria). The enigmatic microsporidia seem to be derived from an endoparasitic chytrid ancestor similar to Rozella allomycis, on the earliest diverging branch of the fungal phylogenetic tree.

The ascomycota tree of life: A phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Fungi evolved right on track.

Dating of fungal divergences with molecular clocks thus far has yielded highly inconsistent results. The origin of fungi was estimated at between 660 million and up to 2.15 billion y ago, and the divergence of the two major lineages of higher fungi, Ascomycota and Basidiomycota, at between 390 million y and up to 1.5 billion y ago. Assuming that these inconsistencies stem from various causes, we reassessed the systematic placement of the most important fungal fossil, Paleopyrenomycites, and recalibrated internally unconstrained, published molecular clock trees by applying uniform calibration points. As a result the origin of fungi was re-estimated at between 760 million and 1.06 billion y ago and the origin of the Ascomycota at 500–650 million y ago. These dates are much more consistent than previous estimates, even if based on the same phylogenies and molecular clock trees, and they are also much better in line with the fossil record of fungi and plants and the ecological interdependence between filamentous fungi and land plants. Our results do not provide evidence to suggest the existence of ancient protolichens as an alternative to explain the ecology of early terrestrial fungi in the absence of land plants.

Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage.

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.

Castor, Pollux and life histories of fungi

The literature on teleomorph-anamorph connections in the Orbiliaceae and the position of the family in the Leotiales is reviewed. 18S data show that the Orbiliaceae occupies an isolated position in relationship to the other members of the Leotiales which have so far been studied. The following form genera have been studied in cultures derived from ascospores of Orbiliaceae: Anguillospora, Arthrobotrys, Dactylella, Dicranidion, Helicoon, Monacrosporium, Trinacrium and conidial types that are referred to as being Idriella-like. Characteristics of the anamorphs are discussed and illustrated. Analyses of the ITS region of several of the isolates indicate that there are several well-supported clades within the Orbiliaceae. These clades can be recognized based on the anamorphs produced. They are: an Arthrobotrys-Monacrosporium clade, a Dicranidion clade, and a Helicoon clade. Outside of these clades is a well-supported clade which contains two Arthrobotrys isolates which were derived from conidia produced on natural substrates. The taxonomic and phylogenetic implications of this information are discussed. The Orbiliaceae occur in nature on substrates that are either continually wet or on substrates that periodically dry out. Field observations indicate that those taxa which occur on wet substrates produce perennial mycelia. Some discussion is provided on the way in which scientific information is viewed and can be used.

Promotion of Mn(II) Oxidation and Remediation of Coal Mine Drainage in Passive Treatment Systems by Diverse Fungal and Bacterial Communities

ABSTRACT Biologically active, passive treatment systems are commonly employed for removing high concentrations of dissolved Mn(II) from coal mine drainage (CMD). Studies of microbial communities contributing to Mn attenuation through the oxidation of Mn(II) to sparingly soluble Mn(III/IV) oxide minerals, however, have been sparse to date. This study reveals a diverse community of Mn(II)-oxidizing fungi and bacteria existing in several CMD treatment systems.

Phylogenetics of the Pezizaceae, with an emphasis on Peziza

Phylogenetic relationships among mem- bers of the Pezizaceae were studied using 90 partial LSU rDNA sequences from 51 species of Peziza and 20 species from 8 additional epigeous genera of the Pezizaceae, viz. Boudiera, Iodophanus, Iodowynnea, Kimbropezia, Pachyella, Plicaria, Sarcosphaera and Sca- bropezia, and 5 hypogeous genera, viz. Amylascus, Ca- zia, Hydnotryopsis, Ruhlandiella and Tirmania. To test the monophyly of the Pezizaceae and the rela- tionships to the genera Marcelleina and Pfistera (Py- ronemataceae), 6 species from the families Ascobo- laceae, Morchellaceae and Pyronemataceae were in- cluded. Maximum parsimony and maximum likeli- hood analyses of these sequences suggest that the Pezizaceae is paraphyletic, because the non-amyloid Marcelleina is nested within it. If Marcelleina were transferred to the Pezizaceae, then the family would be monophyletic. Although the Pezizaceae is tradi- tionally characterized by amyloid asci, our results in- dicate that the amyloid reaction is a symplesiomor- phy, which has been lost in some lineages, e.g., in those including Marcelleina and Cazia. Nodes deep in the tree could not be resolved, but 7 groups of species (I-VII) are generally well supported or pre- sent in all trees. Peziza species, which constitute the core of the family, are present in all groups except group III, confirming the non-monophyly of the ge- nus. The analyses suggest that the other included genera of the Pezizaceae are all nested within Peziza, the placement of lodophanus being unresolved. The morphologically distinct Peziza gerardii, which forms a clade with Marcelleina, appears to be the sister group to the rest of the Pezizaceae. Morphological features were studied and evaluated in the context of the phylogeny. Distinct types of ascus amyloid reac- tions were found to support different rDNA lineages, e.g., a distinct amyloid ring zone at the apex is a syn- apomorphy for group IV, an intense and unrestricted amyloid reaction of the apex is mostly found in group VI, and asci that are weakly or diffusely amy- loid in the entire length are present in group II. Oth- er morphological features, such as spore surface re- lief, guttulation, excipulum structure and pigments, while not free from homoplasy, do support the groupings. Anamorphs likewise provide clues to high- er-order relationships within the Pezizaceae. Several macro- and micromorphological features, however, appear to have evolved several times independently, including ascomatal form and habit (epigeous, se- mihypogeous or hypogeous), spore discharge mech- anisms, and spore shape. Parsimony-based optimiza- tion of character states on our phylogenetic trees sug- gested that transitions to truffle and truffle-like forms evolved at least three times within the Pezizaceae (in group III, V and VI). The 9 hypogeous species in- cluded are nested in lineages with epigeous peziza- ceous taxa. Species with apothecia of various shapes and with forcible spore discharge are spread among all groups and the apothecium is suggested to be symplesiomorphic in the Pezizaceae. The results in- dicate that the apothecia forming Pezizaceae have given rise to at least 3 different forms of hypogeous ascomata without forcible spore discharge: ptychoth- ecia, stereothecia and exothecia.

Powdery mildew pathogenesis of Arabidopsis thaliana

A powdery mildew (Erysiphales) infection of Arabidopsis thaliana was found in the greenhouse of the Department of Molecular Biology at Massachu- setts General Hospital (MGH isolate). We studied the structure of the anamorph, the sequence and chro- nology of its development, and its virulence on A. thaliana and other hosts. The teleomorph of the fun- gus was not found on the material we examined. The MGH isolate caused severe powdery mildew symp- toms on some A. thaliana accessions, infecting ro- sette and cauline leaves, stems and siliques, but was practically avirulent on others. Previously, two Erysi- phales have been reported to infect A. thaliana. These were identified as Erysiphe cichoracearum (UCSC isolate) and E. cruciferarum (UEA isolate). The MGH isolate was found to be a member of the E. cichoracearum group in the section Golovinomyces. It can be distinguished from both of these previously described powdery mildews of A. thaliana based on the morphology, host range and virulence. It might be best referred to E. orontii following the system pro- posed by Braun.

High diversity and widespread occurrence of mitotic spore mats in ectomycorrhizal Pezizales

Fungal mitospores may function as dispersal units and/ or spermatia and thus play a role in distribution and/or mating of species that produce them. Mitospore production in ectomycorrhizal (EcM) Pezizales is rarely reported, but here we document mitospore production by a high diversity of EcM Pezizales on three continents, in both hemispheres. We sequenced the internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA from 292 spore mats (visible mitospore clumps) collected in Argentina, Chile, China, Mexico and the USA between 2009 and 2012. We collated spore mat ITS sequences with 105 fruit body and 47 EcM root sequences to generate operational taxonomic units (OTUs). Phylogenetic inferences were made through analyses of both molecular data sets. A total of 48 OTUs from spore mats represented six independent EcM Pezizales lineages and included truffles and cup fungi. Three clades of seven OTUs have no known meiospore stage. Mitospores failed to germinate on sterile media, or form ectomycorrhizas on Quercus, Pinus and Populus seedlings, consistent with a hypothesized role of spermatia. The broad geographic range, high frequency and phylogenetic diversity of spore mats produced by EcM Pezizales suggests that a mitospore stage is important for many species in this group in terms of mating, reproduction and/or dispersal.

Two Arthrobotrys anamorphs from Orbilia auricolor

Cultures derived from ascospores of two collections both referable to Orbilia auricolor produced anamorphs which were assigned to Arthrobotrys cladodes var. macroides and A. oligospora var. oligospora. These morphologically distinct isolates formed nematode- capturing hyphal networks when nematodes were present. Descriptions of the Arthrobotrys isolates are given. At least one other nematophagous hyphomy- cete is connected with a teleomorph that can be re- ferred to 0. auricolor suggesting that 0. auricolor is not a single entity but a species complex.