Meredith Blackwell

The Fungi: 1, 2, 3 … 5.1 million species?

PREMISE OF THE STUDY Fungi are major decomposers in certain ecosystems and essential associates of many organisms. They provide enzymes and drugs and serve as experimental organisms. In 1991, a landmark paper estimated that there are 1.5 million fungi on the Earth. Because only 70000 fungi had been described at that time, the estimate has been the impetus to search for previously unknown fungi. Fungal habitats include soil, water, and organisms that may harbor large numbers of understudied fungi, estimated to outnumber plants by at least 6 to 1. More recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist. METHODS Technological advances make it possible to apply molecular methods to develop a stable classification and to discover and identify fungal taxa. KEY RESULTS Molecular methods have dramatically increased our knowledge of Fungi in less than 20 years, revealing a monophyletic kingdom and increased diversity among early-diverging lineages. Mycologists are making significant advances in species discovery, but many fungi remain to be discovered. CONCLUSIONS Fungi are essential to the survival of many groups of organisms with which they form associations. They also attract attention as predators of invertebrate animals, pathogens of potatoes and rice and humans and bats, killers of frogs and crayfish, producers of secondary metabolites to lower cholesterol, and subjects of prize-winning research. Molecular tools in use and under development can be used to discover the worlds unknown fungi in less than 1000 years predicted at current new species acquisition rates.

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.

Primer -- The Fungi

The Kingdom Fungi, home to molds, mushrooms, lichens, rusts, smuts and yeasts, comprises eukaryotes with remarkably diverse life histories that make essential contributions to the biosphere, human industry, medicine and research. With the aim of enticing biologists to include fungi in their research, we note that many fungi have haploid genetics, and that those in cultivation are essentially immortal, two features that make it easier to associate traits with genotype, even for complex traits, than with Drosophila or Arabidopsis. The typical fungal genome size of 30–40 Mb is small by eukaryotic standards, which is why fungi have led the way as models for eukaryote genome sequencing with over 100 assembled genome sequences available [1, 2]. For some fungi, DNA transformations, gene knockouts and knockdowns are routine. Species of Ascomycota and Basidiomycota show simple, multicellular development with differentiated tissues. In many species these tissues are large enough to support studies of transcription and translation in the lab and even in nature. About a billion years ago, give or take 500 million years [3], a population of aquatic, unicellular eukaryotes making sporangia containing zoospores each with a single posterior flagellum split into two lineages: one eventually gave rise to animals, the other to fungi. Here we shall summarize the major diversifications of the Fungi by introducing each major fungal branch in the order that it is thought to have diverged (Figure 1) and presenting salient facts about fungal modes of nutrition, reproduction, communication and interaction with other life. Our views are strongly influenced by the Fungal Tree of Life Project [4–6]. Readers interested in learning more about fungi are encouraged to consult any of a number of comprehensive texts [7, 8]. Figure 1 The fungi. Phylogenetic tree, based on [4], showing relationships of many of the fungal lineages fit to geologic time using the program r8s [73] and considering Paleopyrenomycites to be a member of the Ascomycota [3]. Arrows depict changes in morphology ... The exact order of divergence in deep regions of the eukaryotic tree is controversial. On the lineage that leads to the Fungi there are thought to be two other groups; the first to diverge are the nucleariid amoebae [9], and the next the Microsporidia. Microsporidia are either the sister group to the Fungi, or lie within the Fungi, (Figure 1), and they should be included in studies of fungi. They are unculturable, obligate parasites of animals, including humans. They have extremely reduced eukaryotic genomes — with a genome size of ~2.6 Mb and ~2000 genes [10, 11] — remnant mitochondria, and unique morphologies related to parasitism, including a very frightening polar tube used to initiate infection [10].

MOLECULAR SYSTEMATICS OF UNITUNICATE PERITHECIAL ASCOMYCETES: THE CLAVICIPITALES-HYPOCREALES CONNECTION

Cladistic analysis of partial sequences (840 nucleotide positions) from the nuclear encoded small subunit ribosomal DNA was performed to infer the higher taxonomic placement of the Clavicipitales within unitunicate perithecial ascomycetes. Two major classifications exist concerning the placement of this order of ascomycetes; one places it as a sister group to the Hypocreales and the other, as a member of or a near relative to the Xylariales. A strict consensus of 10 equally most parsimonious trees was in agreement with the placement of the Clavicipitales as a monophyletic sister group to the Hypocreales; relationships within the Clavicipitales were not fully resolved in the strict consensus. The taxa sampled from the Hypocreales comprised a paraphyletic lineage in the strict consensus. Characters derived from anamorphs, stromata, centrum anatomy and nutritional modes were reviewed with respect to their level of congruence with the results inferred from the molecular data.

Phylogenetics of Saccharomycetales, the ascomycete yeasts

Ascomycete yeasts (phylum Ascomycota: subphylum Saccharomycotina: class Saccharomycetes: order Saccharomycetales) comprise a monophyletic lineage with a single order of about 1000 known species. These yeasts live as saprobes, often in association with plants, animals and their interfaces. A few species account for most human mycotic infections, and fewer than 10 species are plant pathogens. Yeasts are responsible for important industrial and biotechnological processes, including baking, brewing and synthesis of recombinant proteins. Species such as Saccharomyces cerevisiae are model organisms in research, some of which led to a Nobel Prize. Yeasts usually reproduce asexually by budding, and their sexual states are not enclosed in a fruiting body. The group also is well defined by synapomorphies visible at the ultrastructural level. Yeast identification and classification changed dramatically with the availability of DNA sequencing. Species identification now benefits from a constantly updated sequence database and no longer relies on ambiguous growth tests. A phylogeny based on single gene analyses has shown the order to be remarkably divergent despite morphological similarities among members. The limits of many previously described genera are not supported by sequence comparisons, and multigene phylogenetic studies are under way to provide a stable circumscription of genera, families and orders. One recent multigene study has resolved species of the Saccharomycetaceae into genera that differ markedly from those defined by analysis of morphology and growth responses, and similar changes are likely to occur in other branches of the yeast tree as additional sequences become available.

Phylogenetic distribution of fungal sterols.

Background Ergosterol has been considered the “fungal sterol” for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation. Methodology/Principal Findings The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Δ5 sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Δ5 sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28)-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis. Conclusions/Significance Five dominant end products of sterol biosynthesis (cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl cholesterol, brassicasterol), and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs (e.g., azoles) target reactions in the synthesis of ergosterol. These findings also invalidate use of ergosterol as an indicator of biomass of certain fungal taxa (e.g., Glomeromycota). Data from this study are available from the Assembling the Fungal Tree of Life (AFTOL) Structural and Biochemical Database: http://aftol.umn.edu.

Comparative genomics of biotechnologically important yeasts

Significance The highly diverse Ascomycete yeasts have enormous biotechnological potential. Collectively, these yeasts convert a broad range of substrates into useful compounds, such as ethanol, lipids, and vitamins, and can grow in extremes of temperature, salinity, and pH. We compared 29 yeast genomes with the goal of correlating genetics to useful traits. In one rare species, we discovered a genetic code that translates CUG codons to alanine rather than canonical leucine. Genome comparison enabled correlation of genes to useful metabolic properties and showed the synteny of the mating-type locus to be conserved over a billion years of evolution. Our study provides a roadmap for future biotechnological exploitations. Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.

Convergent Origins of Ambrosia Fungi

The genus Ambrosiella appears to be non- monophyletic based on evidence from cladistic anal- ysis of characters derived from nuclear encoded small subunit (18S) rDNA sequences. The conclusion also is supported by differential sensitivity to cyclohexi- mide. Six species of Ambrosiella (A. brunnea, A. sul- cati, A. gnathotrichi, A. sulfurea, A. macrospora, and A. ips) are closely related to species of Ophiostoma (Ophiostomatales) and show cycloheximide toler- ance. Three other species (A. xylebori, A. ferruginea, and A. hartigii) are related to Ceratocystis (Microas- cales) and are intolerant of cycloheximide. The dif- ficulty in determining monophyletic groupings based on morphological characters among the asexual Am- brosiella species parallels that of their close sexual rel- atives Ophiostoma or Ceratocystis that also had been considered closely related in the past. This finding emphasizes the role of strong selection pressure for insect dispersal features, not only for sexual states, but for conidial and yeast forms as well.

A Group I intron in the nuclear small subunit rRNA gene of Cryptendoxyla hypophloia, an ascomycetous fungus: evidence for a new major class of Group I introns.

Abstract. The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns.

Convergent coevolution in the domestication of coral mushrooms by fungus-growing ants.

Comparisons of phylogenetic patterns between coevolving symbionts can reveal rich details about the evolutionary history of symbioses. The ancient symbiosis between fungus–growing ants, their fungal cultivars, antibiotic–producing bacteria and cultivar–infecting parasites is dominated by a pattern of parallel coevolution, where the symbionts of each functional group are members of monophyletic groups. However, there is one outstanding exception in the fungus–growing ant system, the unidentified cultivar grown only by ants in the Apterostigma pilosum group. We classify this cultivar in the coral–mushroom family Pterulaceae using phylogenetic reconstructions based on broad taxon sampling, including the first mushroom collected from the garden of an ant species in the A. pilosum group. The domestication of the pterulaceous cultivar is independent from the domestication of the gilled mushrooms cultivated by all other fungus–growing ants. Yet it has the same overall assemblage of coevolved ant–cultivar–parasite–bacterium interactions as the other ant–grown fungal cultivars. This indicates a pattern of convergent coevolution in the fungus–growing ant system, where symbionts with both similar and very different evolutionary histories converge to functionally identical interactions.