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Fungi evolution revisited: application of the penalized likelihood method to a Bayesian fungal phylogeny provides a new perspective on phylogenetic relationships and divergence dates of Ascomycota groups.

The depiction of evolutionary relationships within phylum Ascomycota is still controversial because of unresolved branching orders in the radiation of major taxa. Here we generated a dataset of 166 small subunit (18S) rDNA sequences, representative of all groups of Fungi and used as input in a Bayesian phylogenetic analysis. This phylogeny suggests that Discomycetes are a basal group of filamentous Ascomycetes and probably maintain ancestor characters since their representatives are intermingled among other filamentous fungi. Also, we show that the evolutionary rate heterogeneity within Ascomycota precludes the assumption of a global molecular clock. Accordingly, we used the penalized likelihood method, and for calibration we included a 400 million-year-old Pyrenomycete fossil considering two distinct scenarios found in the literature, one with an estimated date of 1576 Myr for the plant-animal-fungus split and the other with an estimated date of 965 Myr for the animal-fungus split. Our data show that the current classification of the fossil as a Pyrenomycete is not compatible with the second scenario. Estimates under the first scenario are older than dates proposed in previous studies based on small subunit rDNA sequences but support estimates based on multiprotein analysis, suggesting that the radiation of the major Ascomycota groups occurred into the Proterozoic era.

Ascomycota↗

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota).

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.

Ascomycota↗

Molecular analysis of the split cox1 gene from the Basidiomycota Agrocybe aegerita: relationship of its introns with homologous Ascomycota introns and divergence levels from common ancestral copies.

The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527aa (58377Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility. When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc). This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions. When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes. The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences. The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences. These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF. The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e. the highest values reported to date between introns of two phylogenetically distant species. This low divergence argues for a recent lateral transfer between the two species. On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences. The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns. This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.

Animals↗

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.

Ascomycota↗

A phylogenomic analysis of the Ascomycota.

An automated procedure was developed to extract orthologous sequences from fungal genomes and incorporate them into phylogenomic analyses in a timely and efficient manner. This approach involves parsing an all versus all BLASTP search of 17 proteomes and creating a similarity matrix from e-values, which is then used to cluster proteins into related groups by means of a Markov Clustering algorithm. After performing this analysis at different stringency levels, 854 single copy protein clusters, which were ubiquitously distributed in all 17 proteomes, were identified. These clusters were culled to include only those clusters where all proteins had best hits to and received hits from a protein within the same cluster. The final data set included gapless alignments for 781 clusters of orthologous sequences that were concatenated into one super alignment containing 195,664 amino acid characters. Neighbor-joining distance and maximum likelihood analyses resulted in identical topologies and all except one node received 100% bootstrap support. The node supporting Stagonospora nodorum's position received 83% support or higher; it was also the only taxon differentially resolved in the maximum parsimony analyses. All analyses resolved the two derived subphyla Pezizomycotina and Saccharomycotina, and Schizosaccharomyces pombe as an early diverging lineage of the Ascomycota. Importantly, these analyses resolved the Leotiomycetes as the sister group to the Sordariomycetes, a region of the Ascomycota phylogeny that has remained problematic in molecular phylogenetic studies of more limited character sampling. Additional phylogenetic analyses which included orthologous sequences from an unannotated ascomycotan genome (e.g., Coccidioides immitis) and subsets of orthologs with different characteristics supported this topology. Phylogenetic analyses of the 595 orthologs which included C. immitis resulted in an identical topology to the previous 781 ortholog analysis and correctly placed C. immitis in the Eurotiomycetes. This demonstrated the correct identification of orthologs and the ability to incorporate unannotated genomic data into a common phylogenetic analysis.

Ascomycota↗

Phylogeny of the Gyalectales and Ostropales (Ascomycota, Fungi): among and within order relationships based on nuclear ribosomal RNA small and large subunits.

Despite various morphological and anatomical similarities, the two orders Gyalectales (lichenized ascomycetes) and Ostropales (lichenized and non-lichenized ascomycetes) have been considered to be distantly related to each other and their position within the Ascomycota was unsettled. To estimate relationships within these groups and their respective phylogenenetic placement within the Ascomycota, we analyzed DNA sequences from the nuclear small and large subunit ribosomal RNA genes using Maximum Parsimony, Maximum Likelihood, and Bayesian statistics with Markov chain Monte Carlo algorithms. Support for internal branches estimated with bootstrap was compared to Bayesian posterior probabilities. We report here that the Ostropales, in their current circumscription, are paraphyletic, and that the Ostropales s.l. include the Gyalectales and Trapeliaceae. The Unitunicate Ascohymenials are redelineated to include the Ostropales s.l., as defined here, and the Baeomycetaceae. Dimerella and Coenogonium are congeneric, and Petractis thelotremella and P. hypoleuca are reunited with members of the genus Gyalecta. In addition to requiring less computational time, Bayesian inference of phylogeny recovered the same topology as a conventional heuristic search using Maximum Likelihood as the optimization criterion and seems superior to bootstrapping in estimating support for short internal branches.

Ascomycota↗

Phylogenetic relationships of Lecanoromycetes (Ascomycota) as revealed by analyses of mtSSU and nLSU rDNA sequence data.

The phylogeny of Lecanoromycetes (Ascomycota, Fungi) is investigated utilizing parsimony and Bayesian Markov Chain Monte Carlo analyses, of combined nLSU rDNA and mtSSU rDNA sequence datasets. The results suggest that Acarosporaceae, Candelariaceae, Phlyctis and Pycnora are not members of the monophyletic Lecanorales, and that Timdalia and Pleopsidium are members of a monophyletic Acarosporaceae. Pycnora, Candelariaceae and Acarosporaceae form a monophyletic group. Umbilicariaceae, Hypocenomyce scalaris, H. friesii, Ophioparmaceae, Boreoplaca, Elixia and Fuscidea form either a basal paraphyletic assemblage in Lecanoromycetes, or a monophyletic group which is the sister-group to Lecanorales and the rest of Lecanoromycetes (excluding Acarosporaceae). The Acarosporaceae forms a group with Pycnora and Candelariaceae, which may be outside the Lecanoromycetes. Chaetothyriales, Verrucariales, Eurotiales, Lichinales and Mycocaliciales form a monophyletic group, but with low support. We briefly discuss incongruence between datasets from different genetic markers, comparing the differences between the separate parsimony analyses, where the ILD test indicated a very significant incongruence. The phylogenetic significance of ascus-types that have influenced most recent Ascomycota classifications heavily is also discussed, and we finally point out risks with formalizing classifications too early.

Ascomycota↗

Accelerated evolutionary rate may be responsible for the emergence of lineage-specific genes in ascomycota.

The evolutionary origin of "orphan" genes, genes that lack sequence similarity to any known gene, remains a mystery. One suggestion has been that most orphan genes evolve rapidly so that similarity to other genes cannot be traced after a certain evolutionary distance. This can be tested by examining the divergence rates of genes with different degrees of lineage specificity. Here the lineage specificity (LS) of a gene describes the phylogenetic distribution of that gene's orthologues in related species. Highly lineage-specific genes will be distributed in fewer species in a phylogeny. In this study, we have used the complete genomes of seven ascomycotan fungi and two animals to define several levels of LS, such as Eukaryotes-core, Ascomycota-core, Euascomycetes-specific, Hemiascomycetes-specific, Aspergillus-specific, and Saccharomyces-specific. We compare the rates of gene evolution in groups of higher LS to those in groups with lower LS. Molecular evolutionary analyses indicate an increase in nonsynonymous nucleotide substitution rates in genes with higher LS. Several analyses suggest that LS is correlated with the evolutionary rate of the gene. This correlation is stronger than those of a number of other factors that have been proposed as predictors of a gene's evolutionary rate, including the expression level of genes, gene essentiality or dispensability, and the number of protein-protein interactions. The accelerated evolutionary rates of genes with higher LS may reflect the influence of selection and adaptive divergence during the emergence of orphan genes. These analyses suggest that accelerated rates of gene evolution may be responsible for the emergence of apparently orphan genes.

Ascomycota↗

Stictis s. lat. (Ostropales, Ascomycota) in northern Scandinavia, with a key and notes on morphological variation in relation to lifestyle.

The closely related genera Carestiella, Schizoxylon and Stictis (Stictidaceae, Ostropales, Ascomycota) are revised in northern Scandinavia, and a revised phylogenetic hypothesis, including all seven species of this group in the area, is presented. A key to the species of Stictis s. lat. (including Carestiella, Schizoxylon and Stictis) in Northern Scandinavia is presented. S. albescens and S. confusum are described as new to science, and the new name S. brunnescens is introduced for S. mollis ssp. populorum, when treated as a distinct species. C. socia and S. albescens are the only representatives of their genera in northern Scandinavia, but a total of five species of Stictis are present in the area (S. brunnescens, S. confusum, S. mollis, S. populorum and S. radiata). Substantial within-species variation in ascoma morphology and wall pigmentation is revealed and discussed, and found to correlate with fungal lifestyles; optional lichenization apparently affects several details in fungal anatomy and morphology.

Ascomycota↗

Resolving evolutionary relationships in the lichen-forming genus Porpidia and related allies (Porpidiaceae, Ascomycota).

The lichen-forming genus Porpidia (Porpidiaceae, Ascomycota) provides excellent opportunities for evolutionary, reproductive, and ecological studies of crustose epilithic lichen symbioses. However, despite the fact that the genus itself seemed to be clearly delimited, the group was thought to be a hopeless case with regard to intrageneric relationships and species delimitations due to apparently rampant homoplasy throughout most character systems. Contrary to the situation for non-molecular data, a robust and generally well-resolved phylogeny was recovered based on DNA-sequence data. Separate and combined analyses of nuclear ribosomal RNA large subunit and nuclear beta-tubulin gene fragments were performed using maximum parsimony, maximum likelihood, and Bayesian approaches. Branch support was estimated using non-parametic bootstrapping and posterior probabilities, while monophyly of a priori defined groups was tested using posterior probabilities. The results reveal a highly supported "Porpidia sensu lato," however, Porpidia itself is not monophyletic. Several smaller genera of the Porpidiaceae and probably the large genus Lecidea (Lecideaceae) are nested within the group. Most taxa analyzed fall into one of four major subgroups within Porpidia s.l., though the basal relationships among these subgroups could not be supported. This phylogeny will make it possible to re-evaluate morphological and chemical characters in the group, and to conduct detailed studies of species delimitations within the monophyletic subgroups.

Ascomycota↗

Galiellalactone and its biogenetic precursors as chemotaxonomic markers of the Sarcosomataceae (Ascomycota).

(-)-Galiellalactone is a hexaketide metabolite with interesting pharmacological activities which was detected in four strains of Galiella rufa (Sarcosomataceae, Ascomycota) and in two unidentified fungi shown by their 18S rDNA sequences also to belong to the Sarcosomataceae. These were a wood-inhabiting apothecial species from Chile and an endophytic isolate from Cistus salviifolius (Sardinia). Other members of the family (Urnula helvelloides, one Strumella coryneoidea isolate) produced no galiellalactone but merely hexaketides structurally related to galiellalactone precursors, whereas a third group of species (Sarcosoma latahensis, Strumella griseola, one S. coryneoidea isolate) lacked hexaketide production altogether. Despite thorough screening programmes, galiellalactone and its precursors have not yet been found in any fungus outside the Sarcosomataceae and may thus be a chemotaxonomic marker of the family, supporting its current phylogenetic definition. Two pentaketide derivatives of the 6-pentyl-alpha-pyrone type were found in all G. rufa strains as well as in A111-95 and the hexaketide-producing S. coryneoidea isolate.

Ascomycota↗

Seasonality of Laboulbenia phaeoxanthae (Ascomycota, Laboulbeniales) and its host Phaeoxantha aequinoctialis (Coleoptera, Carabidae) at a central Amazonian blackwater floodplain.

Terrestrial invertebrates in central Amazonian floodplains must cope with annual long-term inundation. Parasites should be affected mainly indirectly through the specific life-cycles of their hosts. We studied the temporal structure of a beetle-fungus system at a central Amazonian blackwater floodplain (Rio Negro, Brazil). The host species Phaeoxantha aequinoctialis (Coleoptera, Carabidae, Cicindelinae) showed a seasonal, univoltine life-cycle triggered by the annual flood pulse. Infestation frequency of its fungal parasite, Laboulbenia phaeoxanthae (Ascomycota, Laboulbeniales), varied seasonally. However, the seasonality was opposed in host and parasite: the lowest infestation frequencies were observed during periods of highest beetle abundance and vice versa. Periods of lowest beetle abundance coincided with the end of the old generation, those with highest abundance with the appearance of new adults. The resulting annual patterns of a slow spread in the host population resembled the few records of temporal patterns from temperate regions. It is explicitly demonstrated that older adult female beetles are more frequently infested than younger ones. Future studies may reveal whether this is simply the result of specific host life-cycles (driven by a flood pulse, winter, or other factors), or might also be related to potentially easier infestation in older individuals.

Animals↗

Taxonomy of the non-yellow species of Rhizocarpon (Rhizocarpaceae, lichenized Ascomycota) in the Nordic countries, with hyaline and muriform ascospores.

The taxonomy of the non-yellow species of the genus Rhizocarpon (Rhizocarpaceae, lichenized Ascomycota) occurring in the Nordic countries, with hyaline and muriform ascospores, has been revised. Rhizocarpon amphibium, R. anaperum, R. distinctum, R. furfurosum, R. lavatum, R. petraeum, R. postumum, R. reductum, R. roridulum, R. rubescens, R. subgeminatum, R. sublavatum (reported from the study area for the first time), R. subpostumum, R. suomiense, R. timdalii, and R. umbilicatum are recognized. Their morphology, anatomy, secondary chemistry, ecology, and distribution in the Nordic countries are investigated and discussed. Distribution maps and a key to the species are provided. The most important characters for separating the treated species are pruinose/epruinose thallus, number of ascospores in asci, ascospore size and number of cells per ascospore in optical view, insoluble lichen pigments of the epihymenium and the proper excipulum, and lichen substances. Seven names are lectotypified and two are neotypified.

Ascomycota↗

The postfire discomycete Geopyxis carbonaria (Ascomycota) is a biotrophic root associate with Norway spruce (Picea abies) in nature.

The hypothesis that the postfire discomycete Geopyxis carbonaria (Ascomycota, Pezizales, Pyronemataceae) has a biotrophic association with roots of Norway spruce (Picea abies) in nature was tested by isolation of fungal strains from fresh, brown, smooth mycorrhiza-like root tips of Norway spruce collected from below the depth of detrimental heat penetration in a postfire site. The morphology of seven culture isolates originating from the smooth mycorrhiza-like root tips of two different spruce trees was congruent with the morphology of axenic culture isolates obtained from ascospores of G. carbonaria. DNA sequences of the nuclear ribosomal internal transcribed spacers ITS1 and ITS2 from these root-derived cultures and the ascosporic G. carbonaria culture isolates were found to be identical, further supporting the conclusion that the isolates were conspecific. The extensive ascocarp and ascospore formation of G. carbonaria which succeeds a forest fire may be explained in terms of a fungal escape from a moribund tree associate. Possible ecological adaptations of G. carbonaria to the pre- and postfire community are discussed.

Ascomycota↗

Unusual compact rDNA gene arrangements within some members of the Ascomycota: evidence for molecular co-evolution between ITS1 and ITS2.

The internal transcribed spacers of the ribosomal DNA tandem repeat were examined in members of the ascomycetous genus Sphaeronaemella. Species of Sphaeronaemella and its mitotic counterpart Gabarnaudia, have a compact rDNA gene arrangement due to unusually short internal transcribed spacer (ITS) regions. Examination of these regions from phylogenetically related taxa, Cornuvesica, Gondwanamyces, and Ceratocystis, showed that their ITS1 and ITS2 regions could be folded into central hairpin-like structures with the size reduction in species of Sphaeronaemella being due to length reduction of the main-hairpin and the loss of smaller hairpin-like structures that emanate from the main hairpin. A databank compilation, combined with newly obtained sequences, provided an ITS data set that includes sequences of 600 species belonging to the Ascomycota. Correlation analysis revealed that the sizes of ITS1 and ITS2 show a strong positive correlation, suggesting that the 2 rDNA regions have co-evolved. This supports biochemical evidence indicating that the ITS1 and ITS2 segments interact to facilitate the maturation of the rRNA precursor.

Ascomycota↗

A phylogenetic study of the genus Haligena (Halosphaeriales, Ascomycota).

The genus Haligena (Halosphaeriales, Ascomycota), with two accepted species, is encountered frequently in marine habitats, especially on wood in temperate regions. Phylogenetic analyses of Haligena elaterophora (type species) and H. salina were undertaken, with partial large subunit ribosomal DNA sequences, to determine their relationships with other closely related genera in the order. The genus was shown to be polyphyletic within the Halosphaeriales with the type species forming a basal clade to the order. Haligena salina constituted a sister clade with weak support of Neptunella longirostris in all analyses. Haligena elaterophora and H. salina differ significantly in the nature of their ascospore appendages: wider, more sticky and strap-like in H. elaterophora and spoon-shaped at the point of attachment; in H. salina they are longer and narrower, finely drawn out filaments. A new genus, Morakotiella, is introduced to accommodate H. salina.

Ascomycota↗

First record of Clonostachys rosea (Ascomycota: Hypocreales) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina.

Clonostachys rosea (Link: Fries) Schroers, Samuels, Seifert, and Gams (Ascomycota: Hypocreales) has been reported as a mycoparasite of fungi and nematodes and as saprobe in soils, but this fungus has not been reported previously to be entomopathogenic. Many species of cicadellid leafhoppers cause economic damage to crops as vectors of plant pathogens. In the present work, we report the first record of C. rosea as an entomopathogenic fungus of two leafhoppers pest, Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae), in Argentina and evaluate the pathogenicity of C. rosea against them.

Animals↗

Conservation of protein-protein interactions - lessons from ascomycota.

Interacting proteins from Saccharomyces cerevisiae are evolutionarily conserved and their likelihood of having an ortholog in other ascomycota species correlates with the number of interaction partners. Moreover, interacting proteins show a clear preference to be conserved as a pair, indicating that nature maintains selection pressure on the interaction links between proteins. The conservation of interacting protein pairs between different organisms does not exhibit any bias with respect to protein functional roles.

Binding Sites↗