Michael Weiß

Phylogenetic relationships in Cortinarius , section Calochroi , inferred from nuclear DNA sequences

BackgroundSection Calochroi is one of the most species-rich lineages in the genus Cortinarius (Agaricales, Basidiomycota) and is widely distributed across boreo-nemoral areas, with some extensions into meridional zones. Previous phylogenetic studies of Calochroi (incl. section Fulvi) have been geographically restricted; therefore, phylogenetic and biogeographic relationships within this lineage at a global scale have been largely unknown. In this study, we obtained DNA sequences from a nearly complete taxon sampling of known species from Europe, Central America and North America. We inferred intra- and interspecific phylogenetic relationships as well as major morphological evolutionary trends within section Calochroi based on 576 ITS sequences, 230 ITS + 5.8S + D1/D2 sequences, and a combined dataset of ITS + 5.8S + D1/D2 and RPB1 sequences of a representative subsampling of 58 species.ResultsMore than 100 species were identified by integrating DNA sequences with morphological, macrochemical and ecological data. Cortinarius section Calochroi was consistently resolved with high branch support into at least seven major lineages: Calochroi, Caroviolacei, Dibaphi, Elegantiores, Napi, Pseudoglaucopodes and Splendentes; whereas Rufoolivacei and Sulfurini appeared polyphyletic. A close relationship between Dibaphi, Elegantiores, Napi and Splendentes was consistently supported. Combinations of specific morphological, pigmentation and molecular characters appear useful in circumscribing clades.ConclusionOur analyses demonstrate that Calochroi is an exclusively northern hemispheric lineage, where species follow their host trees throughout their natural ranges within and across continents. Results of this study contribute substantially to defining European species in this group and will help to either identify or to name new species occurring across the northern hemisphere. Major groupings are in partial agreement with earlier morphology-based and molecular phylogenetic hypotheses, but some relationships were unexpected, based on external morphology. In such cases, their true affinities appear to have been obscured by the repeated appearance of similar features among distantly related species. Therefore, further taxonomic studies are needed to evaluate the consistency of species concepts and interpretations of morphological features in a more global context. Reconstruction of ancestral states yielded two major evolutionary trends within section Calochroi: (1) the development of bright pigments evolved independently multiple times, and (2) the evolution of abruptly marginate to flattened stipe bulbs represents an autapomorphy of the Calochroi clade.

Phylogenetic relationships of the downy mildews (Peronosporales) and related groups based on nuclear large subunit ribosomal DNA sequences

In order to investigate phylogenetic relationships of the Peronosporomycetes (Oomycetes), nuclear large subunit ribosomal DNA sequences containing the D1 and D2 region were analyzed of 92 species belonging to the orders Peronosporales, Pythiales, Leptomitales, Rhipidiales, Saprolegniales and Sclerosporales. The data were analyzed applying methods of neighbor-joining as well as maximum parsimony, both statistically supported using the bootstrap method. The results confirm the major division between the Pythiales and Peronosporales on the one hand and the Saprolegniales, Leptomitales, and Rhipidiales on the other. The Sclerosporales were shown to be polyphyletic; while Sclerosporaceae are nested within the Peronosporaceae, the Verrucalvaceae are merged within the Saprolegniales. Within the Peronosporomycetidae, Pythiales as well as Peronosporales as currently defined are polyphyletic. The well supported Albugo clade appears to be the most basal lineage, followed by a Pythium-Lagenidium clade. The third, highly supported clade comprises the Peronosporaceae together with Sclerospora, Phytophthora, and Peronophythora. Peronophythora is placed within Phytophthora, indicating that both genera should be merged. Bremiella seems to be polyphyletic within the genus Plasmopara, suggesting a transfer to Plasmopara. The species of Peronospora do not appear as a monophyletic group. Peronospora species growing on Brassicaceae form a highly supported clade.

Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus‐avis (L.) L.C.M. Rich. and neighbouring tree ectomycorrhizae

Several achlorophyllous orchids associate with ectomycorrhizal hymenomycetes deriving carbon from surrounding trees for the plant. However, this has not been shown for achlorophyllous orchids associating with species of Rhizoctonia, a complex of basal lineages of hymenomycetes that are the most common orchid partners. We analysed Neottia nidus‐avis, an achlorophyllous orchid symbiotic with a Rhizoctonia, to identify its symbionts by internal transcribed spacer (ITS) sequencing. Analysis of 61 root systems from 23 French populations showed that N. nidus‐avis associates highly specifically with a group of species of Sebacinaceae. Their diversity emphasizes the need for further investigations in the Sebacinaceae systematics. Sebacinoid ITS sequences were often identical within orchid populations and a trend to regional variation in symbionts was observed. Using ITS and intergenic spacer (IGS) polymorphism, we showed that each root system harboured a single species, but that several genets colonized it. However, no polymorphism of these markers was found among portions of each root: this is consistent with the putative mode of entry of the fungus, i.e. from the rhizome into roots but not repeatedly from the soil. In addition, ectomycorrhizae were always found within the N. nidus‐avis root systems: 120 of the 144 ectomycorrhizae typed by ITS sequencing were colonized by a sebacinoid fungus identical in ITS sequence to the respective orchid symbiont (even for the IGS polymorphism in some cases). Because sebacinoids were demonstrated recently to be ectomycorrhizal, the orchid is likely to derive its resources from surrounding trees, a mycorrhizal cheating strategy similar to other myco‐heterotrophic plants studied to date.

Sebacinales Everywhere: Previously Overlooked Ubiquitous Fungal Endophytes

Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as ‘endophytes’ have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Ladys slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.

Intragenomic Variation of Fungal Ribosomal Genes Is Higher than Previously Thought

Nuclear ribosomal genes in most eukaryotes are present in multiple copies and often used for taxonomic and phylogenetic analyses. We comprehensively examined intragenomic polymorphism levels of three nuclear ribosomal loci for four important plant pathogenic fungi by polymerase chain reaction amplification and cloning. Here, we show that single nucleotide polymorphisms are present in an unexpectedly high amount. This might have implications for studies of fungal evolution, phylogenetics, and population genetics. Furthermore, our work demonstrates that the majority of all ribosomal sequences obtained from one individual and gene is identical to the majority rule consensus sequence of all detected sequence variants. Due to the large number of polymorphisms found and the fact that the polymorphism level differed markedly even between ribosomal genes of one and the same individual, we assume that nuclear ribosomal genes might not always evolve in a strictly concerted manner.

Phylogenetic relationships in Auriculariales and related groups—hypotheses derived from nuclear ribosomal DNA sequences

In order to estimate phylogenetic relationships in the Auriculariales sensu Bandoni (1984) and allied groups we have analysed a representative sample of species by comparison of nuclear coded ribosomal DNA sequences, applying models of neighbour joining, maximum parsimony, conditional clustering, and maximum likelihood. Analyses of the 5’ terminal domain of the gene coding for the 28 S ribosomal large subunit supported the monophyly of the Dacrymycetales and Tremellales, while the monophyly of the Auriculariales was not supported. The Sebacinaceae, including the genera Sebacina, Efibulobasidium, Tremelloscypha, and Craterocolla, was confirmed as a monophyletic group, which appeared distant from other taxa ascribed to the Auriculariales. Within the latter the following subgroups were significantly supported: (1) a group of closely related species containing members of the genera Auricularia, Exidia, Exidiopsis, Heterochaete, and Eichleriella; (2) a group comprising species of Bourdotia and Ductifera; (3) a group of globose-spored species of the genus Basidiodendron; (4) a group that includes the members of the genus Myxarium and Hyaloria pilacre; (5) a group consisting of species of the genera Protomerulius, Tremellodendropsis, Heterochaetella, and Protodontia. Additional analyses of the internal transcribed spacer (ITS) region of the species contained in group (1) resulted in a separation of these fungi due to their basidial types.

Phylogenetic studies of Saprolegniomycetidae and related groups based on nuclear large subunit ribosomal DNA sequences

To reveal phylogenetic relationships within the Peronosporomycetes (Oomycetes), we sequenced a part of the nuclear rDNA coding for the ribosomal large subunit of 46 Peronosporomycetes species and one representative of the Xanthophyta. The main emphasis of our study was put on the phylogenetic relationships within the Saprolegniomycetidae. We supplemented our data with a sequence of Phytophthora megaspermaDrechsler from GenBank. Two sets of sequences were analysed using the neighbor-joining method, statistically supported by the bootstrap method, as well as the maximum parsimony method. Our results are well compatible with the tripartite subclassification of the Peronosporomycetes into Saprolegniomycetidae, Rhipidiomycetidae and Peronosporomycetidae, as well as with the placement of the orders Saprolegniales and Leptomitales in the Saprolegniomycetidae. Pachymetra chaunorhiza Croft & Dick, which has been placed in the Sclerosporales, was grouped within the Saprolegniales. Within the Peronosporomycetidae, the orders Peronosporales and Pythiales could not be separated. There are indications that Phytophthora de Bary and the Peronosporales form a common natural group. The genus Achlya Nees proved to be a heterogeneous group.

The systematic relevance of conidiogenesis modes in the gilled Agaricales

Dikaryotic and haploid mycelia of more than 150 gilled species of euagarics were studied morphologically and by molecular phylogenetic methods. The morphological investigations revealed anamorphs in more than 90 species that were often specific at the genus or family level. Thallic conidiogenesis dominated and varied from fragmentation of normally branched hyphae to the formation of differentiated sympodially branched conidiophores. Secession modes, coiling of the conidiogenous hyphae or the swelling of the conidia were additional distinguishing features. Phylogenetic analysis of the D1-D3 domains of the nuclear gene for the ribosomal large subunit using a Bayesian Markov chain Monte Carlo approach resulted in several well-supported groups that are consistent with anamorph morphology. These results indicate that the anamorphs provide valuable characters for a natural classification of the Agaricales.

Taxonomic studies in the Microbotryomycetidae: Leucosporidium golubevii sp. nov., Leucosporidiella gen. nov. and the new orders Leucosporidiales and Sporidiobolales

The subclass Microbotryomycetidae (Basidiomycota, Urediniomycetes) comprises a remarkably diverse assemblage of fungi. This group includes phytoparasites, mycoparasites and probably also saprobes that show a wide range of ecological preferences. In order to study the phylogenetic relationships within the Microbotryomycetidae, and to develop a more natural classification system, mitosporic and meiosporic taxa were investigated using an integrated approach. Sequence data of 26S rDNA D1/D2 domains were analyzed using several procedures, including the Bayesian Markov chain Monte Carlo method of phylogenetic inference. Ultrastructural markers such as the type of septal pore and presence / absence of colacosomes were investigated and micromorphological and nutritional properties were compared. In this study the current concept of the genus Leucosporidium and its apparent polyphyletic nature were addressed, as well as the relationships of this genus with the Microbotryales and Mastigobasidium. The classification of the anamorphic species closely related to Leucosporidium, and the concepts of the order Sporidiales and family Sporidiobolaceae were also reviewed.