Phylogenetic relationships and convergence of helicosporous fungi inferred from ribosomal DNA sequences.

Helicosporous fungi form elegant, coiled, and multicellular mitotic spores (conidia). In this paper, we investigate the phylogenetic relationships among helicosporous fungi in the asexual genera Helicoma, Helicomyces, Helicosporium, Helicodendron, Helicoon, and in the sexual genus Tubeufia (Tubeufiaceae, Dothideomycetes, and Ascomycota). We generated ribosomal small subunit and partial large subunit sequences from 39 fungal cultures. These and related sequences from GenBank were analyzed using parsimony, likelihood, and Bayesian analysis. Results showed that helicosporous species arose convergently from six lineages of fungi in the Ascomycota. The Tubeufiaceae s. str. formed a strongly supported monophyletic lineage comprising most species from Helicoma, Helicomyces, and Helicosporium. However, within the Tubeufiaceae, none of the asexual genera were monophyletic. Traditional generic characters, such as whether conidiophores were conspicuous or reduced, the thickness of the conidial filament, and whether or not conidia were hygroscopic, were more useful for species delimitation than for predicting higher level relationships. In spite of their distinctive, barrel-shaped spores, Helicoon species were polyphyletic and had evolved in different ascomycete orders. Helicodendron appeared to be polyphyletic although most representatives occurred within Leotiomycetes. We speculate that some of the convergent spore forms may represent adaptation to dispersal in aquatic environments.

Ribosomal DNA and resolution of branching order among the ascomycota: how many nucleotides are enough?

Molecular phylogenies for the fungi in the Ascomycota rely heavily on 18S rRNA gene sequences but this gene alone does not answer all questions about relationships. Particularly problematical are the relationships among the first ascomycetes to diverge, the Archiascomycetes, and the branching order among the basal filamentous ascomycetes, the Euascomycetes. Would more data resolve branching order? We used the jackknife and bootstrapping resampling approach that constitutes the "pattern of resolved nodes" method to address the relationship between number of variable sites in a DNA sequence alignment and support for taxonomic clusters. We graphed the effect of increasing sizes of subsamples of the 18S rRNA gene sequences on bootstrap support for nodes in the Ascomycota tree. Nodes responded differently to increasing data. Some nodes, those uniting the filamentous ascomycetes for example, would still have been well supported with only two thirds of the 18S rRNA gene. Other nodes, like the one uniting the Archiascomycetes as a monophyletic group, would require about double the number of variable sites available in the 18S gene for 95% neighbor-joining bootstrap support. Of the several groups emerging at the base of the filamentous ascomycetes, the Pezizales receive the most support as the first to diverge. Our analysis suggests that we would also need almost three times as much sequence data as that provided by the 18S gene to confirm the basal position for the Pezizales and more than seven times as much data to resolve the next group to diverge. If more data from other genes show the same pattern, the lack of resolution for the filamentous ascomycetes may indicate rapid radiation within this clade.

Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors.

In most fungal ascomycetes, mating is controlled by a single locus (MAT). Fungi requiring a partner to mate are heterothallic (self-sterile); those not requiring a partner are homothallic (self-fertile). Structural analyses of MAT sequences from homothallic and heterothallic Cochliobolus species support the hypothesis that heterothallism is ancestral. Homothallic species carry both MAT genes in a single nucleus, usually closely linked or fused, in contrast to heterothallic species, which have alternate MAT genes in different nuclei. The structural organization of MAT from all heterothallic species examined is highly conserved; in contrast, the organization of MAT in each homothallic species is unique. The mechanism of conversion from heterothallism to homothallism is a recombination event between islands of identity in otherwise dissimilar MAT sequences. Expression of a fused MAT gene from a homothallic species confers self-fertility on a MAT-null strain of a heterothallic species, suggesting that MAT alone is sufficient to change reproductive life style.

Phylogenetic origins of the asexual mycorrhizal symbiont Cenococcum geophilum Fr. and other mycorrhizal fungi among the ascomycetes.

The phylogenetic relationship of the asexual mycorrhizal fungus Cenococcum geophilam Fr. among sexual ascomycetes was examined by phylogenetic analysis of nucleotide sequence data from the nuclear small subunit (18S) ribosomal RNA genie region. A specific focus of this study was to test the hypothesis that the genus Elaphomyces is the closest sexual relative of C. geophilum. Thus nucleotide sequence data of five C. geophilum isolates, three Elaphomyces species, and 44 additional genera of ascomycetes were included in the phylogenetic analyses. The percentage of similarity among the 18S rDNA sequences of the C. geophilum isolates examined was 99.8 to 100%, indicating that C. geophilum is monophyletic. Percent similarity of nucleotide sequence among the three Elaphomyces species was also high and ranged from 99.4 to 99.5%. DNA parsimony and distance analysis of the sequence data separated these 2 genera on distant clades when sequence from 44 additional genera of ascomycetes was included. Parsimony and distance analyses positioned C. geophilum as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothidiales, and strongly supported Elaphomyces to be of Plectomycete origin. Among the sexual Ascomycetes examined, which included representative taxa from four classes of filamentous Ascomycetes (Plectomycetes, Pyrenomycetes, Discomycetes, and Loculoascomycetes), no close sexual relative to C. geophilum was identified. At least four independent lineages of mycorrhizal fungi were identified among the ascomycetes examined.

Loculoascomycete origins and evolution of filamentous ascomycete morphology based on 18S rRNA gene sequence data.

The fungal subclass Loculoascomycetes is characterized by asci or sexual spore sacks with two separable wall layers. At maturity, the inner wall layer protrudes out beyond the outer wall as in a jack-in-the-box. If the Loculoascomycetes were monophyletic and their jack-in-the-box type asci evolved once, then taxa from diverse loculoascomycete lineages would cluster together in a DNA sequence-based tree. To evaluate the phylogenetic history of the two-walled asci, I sequenced the 18S nuclear rRNA genes of 16 species from seven families in the loculoascomycete orders Pleosporales, Dothideales, and Chaetothyriales. Within the Loculoascomycetes, the Pleosporales form a monophyletic group in 99% of the bootstrapped parsimony trees. The Dothideales usually appear as a monophyletic group but without statistical support. Capronia pilosella (Herpotrichiellaceae, Order Chaetothyriales) clusters with plectomycete members of the subclass Euascomycetes rather than the other Loculoascomycetes in 99% of parsimony and neighbor-joining bootstrap replicates. Although the jack-in-the-box-type ascus is a good marker for large, monophyletic loculoascomycete orders, it must have evolved at least twice or been lost at least once.

18S rDNA sequences and the holometabolous insects.

The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence.

Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release. Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms. The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes. The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently. Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge. In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates. A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test. Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge. Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA. The transition-to-transversion ratio was 1.9. Of all 12 possible substitutions, 29% were from U to C. At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4). In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).

Two ascomycete classes based on fruiting-body characters and ribosomal DNA sequence.

Traditional fruiting body-based classification of ascomycetes has been under attack for 2 decades. Fruiting-body types can converge, and few researchers now assume that either the closed fruiting bodies (cleistothecia) characterizing the class Plectomycetes or the flask-shaped fruiting bodies (perithecia) characterizing the class Pyrenomycetes are stable, unifying characters. Unless we identify characters uniting major ascomycete groups, orders of ascomycetes remain narrowly defined, and supraordinal classification is impossible. We sequenced both strands of 18s rDNA from nine ascomycete fungi, adding three sequences from GenBank into our analysis. The phylogeny, inferred from 162 informative sites in 1,700 bp of DNA sequence data and using yeast as an outgroup, divided the fungi into two groups correlating well both with fruiting-body type and with the traditional classes Plectomycetes and Pyrenomycetes. Each group received strong statistical support. Genera producing cleistothecia, such as Talaromyces (with a Penicillium asexual state) and the human pathogen Ajellomyces capsulatus (causing histoplasmosis), fall within the plectomycete group. Plectomycetes also includes Eremascus albus and the bee pathogen Ascosphaera apis, although both lack typical fruiting bodies. The Dutch elm disease fungus groups with pyrenomycetes such as Neurospora, in spite of its confusing mixture of class-level characters.

Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.

For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.