nucLSU rDNA Research Articles

A simple method for the cultivation of the “unculturable” asterinaceous fungi (Asterinales/Dothideomycetes)

Although asterinaceous fungi have been studied for many years, all previous attempts to isolate, cultivate, and propagate these fungi in vitro have failed. This paper provides the first reports of in vitro isolation of representative strains of species belonging to five fungi from different genera belonging to Asterinales. To confirm if the sequences of DNA obtained from the mycelia are the same obtained in the direct extraction, a phylogenetic analysis of nuc LSU rDNA was performed. This paper reports for the first time the success of in vitro culturing of asterinaceous fungi using the ascospores ejection technique, opening perspectives of studies of genetics, physiology, among other aspects of the biology for this very understudied group of fungi.

Hydnaceous fungi of China 8. Morphological and molecular identification of three new species of Sarcodon and a new record from southwest China.

Three new stipitate hydnoid fungi, Sarcodon coactus, S. grosselepidotus and S. lidongensis, are described and illustrated, based on morphological characteristics and nuc ITS rDNA + nuc LSU rDNA sequence analyses and a new record, S. leucopus, from China is reported. S. coactus is characterised by ellipsoid to round basidiocarps, reddish-brown to dark brown, felted pileal surface with white and incurved margins, simple-septate and partly short-celled generative hyphae and irregular subglobose, thin-walled, brown basidiospores with tuberculate ornamentation (tuberculi up to 1 μm long). S. grosselepidotus is characterised by infundibuliform to round, occasionally deeply fissured pileus, pale orange to dark ruby pileal surface with ascending and coarse scales, simple-septate generative hyphae and irregular ellipsoid to globose, thin-walled, brown basidiospores with tuberculate ornamentation (tuberculi up to 0.7 μm long). S. lidongensis is characterised by plano-convex to somewhat depressed and regular orbicular pileus, light brown to dark brown pileal surface with adhering squamose and purplish-brown, incurved and occasionally incised margin, cylindrical or broadened below stipe, simple-septate generative hyphae and irregular ellipsoid to subglobose, thin-walled basidiospores with tuberculate ornamentation (tuberculi up to 1 μm long). The absence of the clamp connection is the common morphological characteristic of these three new species; however, S. leucopus, a new record from China, has frequently clamped generative hyphae. Molecular analyses confirm the phylogenetic positions of three new and the new record species. The discriminating characters of these three new species and closely related species are discussed and a key to the species of Sarcodon from China is provided.

Two Rare Phallales Recorded from São Tomé

Two little known species of Phallales, Mutinus zenkeri and Blumenavia angolensis, were collected on the volcanic island of Sao Tome in the Gulf of Guinea, Africa. Descriptions and in situ photographs are provided for both species. Inferences of their phylogenetic relationships within the Phallales are provided based on partial nucLSU rDNA sequence data.

Phylogenetic taxonomy of Hymenochaete and related genera (Hymenochaetales)

Species delimitation in Hymenochaete sensu lato was evaluated based on partial nucLSU rDNA sequences, isoenzyme analyses and morphological data. Analyses of LSU data revealed Hymenochaete sensu stricto and Pseudochaete as distinct monophyletic genera. Transfer of Hydnochaete olivacea into genus Pseudochaete and Dichochaete setosa into genus Hymenochaete (as H. resupinata) was supported by the results of molecular analyses. A new species Hymenochaete koeljalgii from Tanzania was described. The species of genus Hymenochaete sensu stricto were divided into four well-supported clades possessing no distinctive morphological characters.

The Polychytriales ord. nov. contains chitinophilic members of the rhizophlyctoid alliance

During the past 5 y the Rhizophydiales, Cladochytriales and Lobulomycetales have been segregated from the formerly recognized Chytridiales. Descriptions of new chytridiomycete orders are based on molecular and ultrastructural characters, which have been phylogenetically mutually supportive. The Polychytrium clade has consisted of a few chitinophilic, soil and aquatic chytrids that clustered in phylogenetic hypotheses but have not been placed in a new order. We isolated additional putative members of this clade, sequenced their nucSSU and nucLSU rDNA and examined zoospores of some of the isolates with TEM. Our isolates are in a well supported clade with previous Polychytrium clade members, but zoospore ultrastructural types vary within the clade, with characters that often are conserved within other orders (e.g. flagellar plug, rumposome) being either present or absent. Based on the isolates in culture we describe the Polychytrium clade as the Polychytriales. This order contains Polychytrium, Lacustromyces, Karlingiomyces, two new genera (Arkaya and Neokarlingia) and additional undescribed taxa.

Phylogeny of Powellomycetaceae fam. nov. and description of Geranomyces variabilis gen. et comb. nov.

The genus Powellomyces was described to accommodate two monocentric chytrid species from soil that develop exogenously and possess zoosporic ultrastructure similar to other members of the order Spizellomycetales. Despite Powellomyces-like chytrids being commonly observed in gross culture, the genus contained only two species. To determine diversity in this group I amassed 49 isolates of Powellomyces-like chytrids, including the cultures upon which species types were based and new isolates from pollen-baited water cultures of soils, plant detritus and manure. I sequenced portions of nucSSU and nucLSU rDNA regions and the EF-1α-like gene from each isolate to produce a molecular phylogeny. This phylogeny supports monophyly of spizellomycetalean chytrids with exogenous development and suggests that multiple distinct lineages exist within this group. This phylogeny, along with a reevaluation of the ultra-structural features of the two described species, supports the recognition of a new family, Powellomycetaceae, and genus, Geranomyces, which contains 31 isolates of G. variabilis comb. nov.

Periconiella species (anamorphic Mycosphaerellaceae) from Taiwan

Three Periconiella species were collected in Taiwan and investigated with light and scanning electron microscopy and nuc LSU rDNA analysis. A new species of Periconiella is proposed for a hyphomycete with apically branched conidiophores and relationship to Mycosphaerellaceae found on living leaves of Machilus zuihoensis and an unidentified species of Lauraceae. Two further species, Periconiella lygodii and P. perseae-macranthae, are recorded outside India for the first time and on new host species. Hadrotrichum laurinum is transferred to Passalora.

Multi-locus phylogeny of Pleosporales: a taxonomic, ecological and evolutionary re-evaluation.

Five loci, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, are used for analysing 129 pleosporalean taxa representing 59 genera and 15 families in the current classification of Pleosporales. The suborder Pleosporineae is emended to include four families, viz. Didymellaceae, Leptosphaeriaceae, Phaeosphaeriaceae and Pleosporaceae. In addition, two new families are introduced, i.e. Amniculicolaceae and Lentitheciaceae. Pleomassariaceae is treated as a synonym of Melanommataceae, and new circumscriptions of Lophiostomataceae s. str., Massarinaceae and Lophiotrema are proposed. Familial positions of Entodesmium and Setomelanomma in Phaeosphaeriaceae, Neophaeosphaeria in Leptosphaeriaceae, Leptosphaerulina, Macroventuria and Platychora in Didymellaceae, Pleomassaria in Melanommataceae and Bimuria, Didymocrea, Karstenula and Paraphaeosphaeria in Montagnulaceae are clarified. Both ecological and morphological characters show varying degrees of phylogenetic significance. Pleosporales is most likely derived from a saprobic ancestor with fissitunicate asci containing conspicuous ocular chambers and apical rings. Nutritional shifts in Pleosporales likely occured from saprotrophic to hemibiotrophic or biotrophic.

A class-wide phylogenetic assessment of Dothideomycetes

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

Bellojisia, a new sordariaceous genus for Jobellisia rhynchostoma and a description of Jobellisiaceae fam. nov.

The phylogenetic analyses of partial nucLSU rDNA sequence data of three Jobellisia species indicate that J. rhynchostoma is distinct from the core species of Jobellisia. Jobellisia luteola, the type species of the genus, and J. fraterna reside as a strongly supported monophyletic clade in a basal position in a grouping containing the Diaporthales, the Calosphaeriales and the Togniniaceae, while all phylogenies confirm the placement of J. rhynchostoma within the Sordariales. The new family Jobellisiaceae (incertae sedis) is described for Jobellisia. A new perithecial ascomycete genus, Bellojisia (Lasiosphaeriaceae, Sordariales), is introduced for J. rhynchostoma. The fungus produces nonstromatic, long-necked perithecia with a superficial to semi-immersed pyriform venter and carbonaceous three-layered perithecial wall, 1-septate, hyaline, later brown, reniform to navicular ascospores with a polar germ pore formed in unitunicate asci. The fungus was not observed to produce a conidial anamorph in vitro. Both morphological and molecular data suggest Corylomyces selenosporus of the Sordariales is the closest relative of J. rhynchostoma. The other relatives of Bellojisia (viz. Cercophora, Lasiosphaeria and Podospora) recruit from the Lasiosphaeriaceae (Sordariales). Cercophora and Podospora are shown as polyphyletic within the Sordariales, which is in agreement with previous molecular studies.

Phylogenetic relationship of Marasmius mbalmayoensis sp. nov. to the tropical African Marasmius bekolacongoli complex based on nuc-LSU rDNA sequences

Marasmius mbalmayoensis (Basidiomycotina, Marasmiaceae) growing on decayed leaves of Canarium schweinfurthii in the Mbalmayo Forest Reserve, Cameroon, is described. The species is remarkable due to the large basidiomata with shallow orange yellow umbilicus, long central stipe accompanied by similar long rhizomorphs on thickened basal mycelium and large lacrymiform to sigmoid basidiospores. The coarsely plicate pilei with lilac, violet to dark violaceous tints, and large distant adnate lamellae are reminiscent macroscopically of the tropical African species M. bekolacongoli. The phylogenetic relationship among M. mbalmayoensis and M. bekolacongoli was assessed, extended to other species of sects. Globulares and Sicci based on DNA sequences. Phylogenetic analysis based on nuc-LSU rDNA sequence data of selected Marasmiaceae taxa confirmed the placement of M. mbalmayoensis within the Marasmius spp. and its phylogenetic separation from M. bekolacongoli. Amyloflagellula inflata, which a BLAST analysis closely related to M. mbalmayoensis, clustered in the same clade with M. mbalmayoensis and M. bekolacongoli. The findings also indicated the complexity of M. bekolacongoli.

Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure

Traditional classifications of agaric fungi involve gross morphology of their fruit bodies and meiospore print-colour. However, the phylogeny of these fungi and the evolution of their morphological and ecological traits are poorly understood. Phylogenetic analyses have already demonstrated that characters used in traditional classifications of basidiomycetes may be heavily affected by homoplasy, and that non-gilled taxa evolved within the agarics several times. By integrating molecular phylogenetic analyses including domains D1–D3 and D7–D8 of nucLSU rDNA and domains A–C of the RPB1 gene with morphological and chemical data from representative species of 88 genera, we were able to resolve higher groups of agarics. We found that the species with thick-walled and pigmented basidiospores constitute a derived group, and hypothesize that this specific combination of characters represents an evolutionary advantage by increasing the tolerance of the basidiospores to dehydration and solar radiation and so opened up new ecological niches, e.g. the colonization of dung substrates by enabling basidiospores to survive gut passages through herbivores. Our results confirm the validity of basidiospore morphology as a phylogenetic marker in the agarics.

Polyphyly and two emerging lineages in the rust genera Puccinia and Uromyces

The phylogenetic validity of Puccinia and Uromyces, Pucciniaceae, and closely related genera was evaluated using nucLSU rDNA sequences. Using a wide range of rust species with different life cycles and with different host specificities, Puccinia and Uromyces were shown to be highly polyphyletic and to also include representatives of the genera Aecidium, Cumminsiella, Dietelia, Endophyllum, Miyagia, and Uredo. Furthermore, the structure of the phylogenetic data did not reflect previous sub-generic delimitations based on teliospore pedicel structure, but rather suggests that at least two major lineages have evolved within Puccinia/ Uromyces: Rusts with telial states on Poaceae were exclusively found in one of these groupings and those with telial states on Cyperaceae resided in the other lineage. This might suggest that the two lineages evolved in close association with these host groups in different biomes.

A multigene phylogeny of the Dothideomycetes using four nuclear loci

We present an expanded multigene phylogeny of the Dothideomycetes. The final data matrix consisted of four loci (nuc SSU rDNA, nuc LSU rDNA, TEF1, RPB2) for 96 taxa, representing five of the seven orders in the current classification of Dothideomycetes and several outgroup taxa representative of the major clades in the Pezizomycotina. The resulting phylogeny differentiated two main dothideomycete lineages comprising the pseudoparaphysate Pleosporales and aparaphysate Dothideales. We propose the subclasses Pleosporomycetidae (order Pleosporales) and Dothideomycetidae (orders Dothideales, Capnodiales and Myriangiales). Furthermore we provide strong molecular support for the placement of Mycosphaerellaceae and Piedraiaceae within the Capnodiales and introduce Davidiellaceae as a new family to accommodate species of Davidiella with Cladosporium anamorphs. Some taxa could not be placed with certainty (e.g. Hysteriales), but there was strong support for new groupings. The clade containing members of the genera Botryosphaeria and Guignardia resolved well but without support for any relationship to any other described orders and we hereby propose the new order Botryosphaeriales. These data also are consistent with the removal of Chaetothyriales and Coryneliales from the Dothideomycetes and strongly support their placement in the Eurotiomycetes.

A multigene phylogeny of the Dothideomycetes using four nuclear loci

We present an expanded multigene phylogeny of the Dothideomycetes. The final data matrix consisted of four loci (nuc SSU rDNA, nuc LSU rDNA, TEF1, RPB2) for 96 taxa, representing five of the seven orders in the current classification of Dothideomycetes and several outgroup taxa representative of the major clades in the Pezizomycotina. The resulting phylogeny differentiated two main dothideomycete lineages comprising the pseudoparaphysate Pleosporales and aparaphysate Dothideales. We propose the subclasses Pleosporomycetidae (order Pleosporales) and Dothideomycetidae (orders Dothideales, Capnodiales and Myriangiales). Furthermore we provide strong molecular support for the placement of Mycosphaerellaceae and Piedraiaceae within the Capnodiales and introduce Davidiellaceae as a new family to accommodate species of Davidiella with Cladosporium anamorphs. Some taxa could not be placed with certainty (e.g. Hysteriales), but there was strong support for new groupings. The clade containing members of the genera Botryosphaeria and Guignardia resolved well but without support for any relationship to any other described orders and we hereby propose the new order Botryosphaeriales. These data also are consistent with the removal of Chaetothyriales and Coryneliales from the Dothideomycetes and strongly support their placement in the Eurotiomycetes.

The halotolerant fungus Glomerobolus gelineus is a member of the Ostropales

Glomerobolus gelineus is a halotolerant species with a unique method of ballistic propagation. The absence of both sexual and asexual spores made reliable placement of this species, based on morphology alone, within the current fungal classification problematical. A phylogenetic analysis of the large and small nuclear ribosomal subunit and the second largest subunit of RNA polymerase II placed this fungus within the Ostropales, an order comprising lichenized and saprobic species, with good statistical support. Subsequently, a more detailed analysis that combined the nuc LSU rDNA and the mt SSU rDNA confirmed a close relationship to the Stictidaceae. The phylogenetic placement of G. gelineus is also supported by morphological characters. We postulate that the hyphoma lobes of Glomerobolus correspond to the periphysoidal layer in the apothecium of Stictis, and the propagule to the hymenium. Moreover, the presence of crystals in the outer lobes of G. gelineus is another indication of its relationship with Ostropales, which have characteristic crystalliferous hyphae. The placement of Glomerobolus within the Ostropales further expands the ecological diversity exhibited by this order. It also provides a phylogenetic hypothesis for assessing the homology of the enigmatic hyphomal morphology with apothecia-forming Ascomycota.

Phylogenetic relationships of cyphelloid homobasidiomycetes

The homobasidiomycetes includes the mushroom-forming fungi. Members of the homobasidiomycetes produce the largest, most complex fruiting bodies in the fungi, such as gilled mushrooms (“agarics”), boletes, polypores, and puffballs. The homobasidiomycetes also includes species that produce minute, cup- or tube-shaped “cyphelloid” fruiting bodies, that rarely exceed 1–2 mm diameter. The goal of this study was to estimate the phylogenetic placements of cyphelloid fungi within the homobasidiomycetes. Sequences from the nuclear large subunit (nuc-lsu) ribosomal DNA (rDNA), 5.8S rDNA, and internal transcribed spacers (ITS) 1 and 2 were obtained for 31 samples of cyphelloid fungi and 16 samples of other homobasidiomycetes, and combined with published sequences. In total, 71 sequences of cyphelloid fungi were included, representing 16 genera. Preliminary phylogenetic analyses of a 1477-sequence data set and BLAST searches using sequences of cyphelloid forms as queries were used to identify taxa that could be close relatives of cyphelloid forms. Subsequent phylogenetic analyses of one data set with 209 samples represented by nuc-lsu rDNA sequences (analyzed with parsimony) and another with 38 samples represented by nuc-lsu and 5.8S rDNA sequences (analyzed with parsimony and maximum likelihood) indicated that cyphelloid forms represent a polyphyletic assemblage of reduced agarics (euagarics clade, Agaricales). Unconstrained tree topologies suggest that there have been about 10–12 origins of cyphelloid forms, but evaluation of constrained topologies with the Shimodaira–Hasegawa test suggests that somewhat more parsimonious scenarios cannot be rejected. Whatever their number, the multiple independent origins of cyphelloid forms represent striking cases of parallel evolutionary reduction of complex fungal morphology.

Ascobotryozyma cognata sp. nov., a new ascomycetous yeast associated with nematodes from wood-boring beetle galleries

A new species of Ascobotryozyma, A. cognata sp. nov. (anamorph Botryozyma cognata), was isolated from beetle galleries in Idaho, USA. A. cognata was found on the surface of free-living nematodes, Panagrellus dubius, collected from galleries created by the long-horned beetle Saperda calcarata in Populus (aspen), and the weevil Cryptorhynchus lapathi in Salix (willow). A. cognata isolates were collected from similar habits and in relatively close proximity to those of A. americana, the only species described from North America. The recognition of A. cognata as a distinct species was supported by morphological and molecular data. Thallus cells of A. cognata were significantly shorter than those of A. americana. Low DNA reassociation values, notably different randomly amplified polymorphic DNA (RAPD), inter-sequence simple repeat (ISSR), and amplified fragment-length polymorphic (AFLP) fingerprints, and sequence divergence in both the D1/D2 domain of the nuc-LSU rDNA and an additional unidentified region were all consistent with the recognition of a new species.

Proceedings towards a natural classification of the worldwide taxa Phellinus s.l. and Inonotus s.l., and phylogenetic relationships of allied genera

The classification of Phellinus s.l., Inonotus s.l. and the phylogenetic relationships of allied genera were studied using nuc-lsu rDNA sequence data. The worldwide taxon sampling comprised 107 species, 99 of them belonging to the Hymenochaetales. The phylogenetic trees were discussed in relation to morphological and anatomical features of the fruit bodies. The Hymenochaetales formed no monophyletic group and several non-Hymenochaetales appeared as intermingled with the Hymenochaetales. Trichaptum abietinum and Oxyporus populinus showed no certain affinities within the Hymenochaetales, whereas Basidioradulum radula was closely related to Phellopilus nigrolimitatus, and Hyphodontia quercina and Schizopora paradoxa were related to Coltricia, Coltriciella and Pyrrhoderma adamantinum. Phellinus s.l. and Inonotus s.l. formed no monophyletic groups, and a subdivision in the following genera is accepted: Phellinus s.s., Inonotus s.s., Inocutis, Fomitiporella, Aurificaria, Phylloporia, Fulvifomes, Mensularia, Pseudoinonotus, Fomitiporia, Porodaedalea, Onnia, Fuscoporia, and Inonotopsis. Coltricia and Coltriciella were confirmed as seperate genera. The taxonomic status of Phellinidium and Pyrrhoderma remained uncertain. 16 new combinations are proposed.

Proceedings Towards a Natural Classification of the Worldwide Taxa Phellinus s.l. and Inonotus s.l., and Phylogenetic Relationships of Allied Genera

The classification of Phellinus s.l., Inonotus s.l. and the phylogenetic relationships of allied genera were studied using nuc-lsu rDNA sequence data. The worldwide taxon sampling comprised 107 species, 99 of them belonging to the Hymenochaetales. The phylogenetic trees were discussed in relation to morphological and anatomical features of the fruit bodies. The Hymenochaetales formed no monophyletic group and several non-Hymenochaetales appeared as intermingled with the Hymenochaetales. Trichaptum abietinum and Oxyporus populinus showed no certain affinities within the Hymenochaetales, whereas Basidioradulum radula was closely related to Phellopilus nigrolimitatus, and Hyphodontia quercina and Schizopora paradoxa were related to Coltricia, Coltriciella and Pyrrhoderma adamantinum. Phellinus s.l. and Inonotus s.l. formed no monophyletic groups, and a subdivision in the following genera is accepted: Phellinus s.s., Inonotus s.s., Inocutis, Fomitiporella, Aurificaria, Phylloporia, Fulvifomes, Mensularia, Pseudoinonotus, Fomitiporia, Porodaedalea, Onnia, Fuscoporia, and Inonotopsis. Coltricia and Coltriciella were confirmed as seperate genera. The taxonomic status of Phellinidium and Pyrrhoderma remained uncertain. 16 new combinations are proposed.