Trond Schumacher

High diversity of root associated fungi in both alpine and arctic Dryas octopetala.

BackgroundDryas octopetala is a widespread dwarf shrub in alpine and arctic regions that forms ectomycorrhizal (ECM) symbiotic relationships with fungi. In this study we investigated the fungal communities associated with roots of D. octopetala in alpine sites in Norway and in the High Arctic on Svalbard, where we aimed to reveal whether the fungal diversity and species composition varied across the Alpine and Arctic regions. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was used to identify the fungal communities from bulk root samples obtained from 24 plants.ResultsA total of 137 operational taxonomic units (OTUs) were detected (using 97% similarity cut off during sequence clustering) and well-known ECM genera such as Cenococcum, Cortinarius, Hebeloma, Inocybe and Tomentella occurred frequently. There was no decrease in fungal diversity with increasing latitude. The overall spatial heterogeneity was high, but a weak geographical structuring of the composition of OTUs in the root systems was observed. Calculated species accumulation curves did not level off.ConclusionsThis study indicates that the diversity of fungi associated with D. octopetala does not decrease in high latitude arctic regions, which contrasts observations made in a wide spectrum of other organism groups. A high degree of patchiness was observed across root systems, but the fungal communities were nevertheless weakly spatially structured. Non-asymptotical species accumulation curves and the occurrence of a high number of singletons indicated that only a small fraction of the fungal diversity was detected.

MOLECULAR PHYLOGENY AND EVOLUTION OF MONILINIA (SCLEROTINIACEAE) BASED ON CODING AND NONCODING RDNA SEQUENCES

The nuclear internal transcribed spacers, the 5.8S subunit, ~560 bp of the small subunit, and ~320 bp of the large subunit of the nuclear ribosomal DNA repeat from 17 species of Monilinia and eight species of closely related genera were sequenced. Phylogenies were constructed using maximum parsimony. The results support the hypothesis that Monilinia is not monophyletic. A fundamental distinction was found between the section Junctoriae and the section Disjunctoriae. Four evolutionary lineages were identified within the Disjunctoriae: one species on Crataegus, one group of species on dry stone fruits of rosaceous hosts, one group of species on capsular fruits of ericaceous hosts, and one group of species on sweet berry fruits of ericaceous hosts. Comparisons between branching topologies of hosts and Monilinia species suggest that although cospeciation among hosts and parasites has been the rule, several host jumps have taken place. Sclerotinia pirolae was determined to be a true member of the Disjunctoriae. The closest taxon groups to the Junctoriae were found to be Botrytis and Sclerotinia, with Ciborinia being the closest taxon group to the Disjunctoriae. There is evidence of an increased rate of ssrRNA evolution in the lineage of species that attack ericaceous berries.

Multiple gene genealogies and AFLPs suggest cryptic speciation and long-distance dispersal in the basidiomycete Serpula himantioides (Boletales).

Serpula himantioides (Boletales, Basidiomycota) produces thin resupinate basidiocarps on dead coniferous wood worldwide and causes damage in buildings as well. In this study, we present evidence for the existence of at least three phylogenetically defined cryptic species (referred to as Sib I–III) within the morphospecies S. himantioides, a conclusion based on analyses of sequence data from four DNA regions and amplified fragment length polymorphisms (AFLPS). A low degree of shared sequence polymorphisms was observed among the three lineages indicating a long‐lasting separation. The AFLPs revealed two additional subgroups within Sib III. Results from mating studies were consistent with the molecular data. In Sib III, no correspondence between genetic and geographical distance was observed among isolates worldwide, presumably reflecting recent dispersal events. Our results indicate that at least two of the lineages (Sib II and Sib III) have wide sympatric distributions. A population genetic analysis of Sib III isolates, scoring sequence polymorphisms as codominant SNP markers, indicates that panmictic conditions exist in the Sib III group. This study supports the view that cryptic speciation is a common phenomenon in basidiomycete fungi and that phylogenetic species recognition can be a powerful inference to detect cryptic species. Furthermore, this study shows that AFLP data are a valuable supplement to DNA sequence data in that they may detect a finer level of genetic variation.

Mushroom poisoning caused by species of the genus Cortinarius fries

Symptomatology, clinical characteristics and pathogenesis of mushroom poisoning caused by Cortinarius species are surveyed. The isolation of a bipyridilium — orellanine — from Cortinarius orellanus is held to be responsible for the nephrotoxicity of this species as well as the closely related C. speciosissimus. The present knowledge on the toxicity of structurally related and well-known bipyridines such as paraquat and diquat is brought up and found comparable to orellanine toxicity. Pharmacokinetic experiments on the nephrotoxic bipyridines suggest that haemoperfusion is a rational therapy of intoxicated persons, even several days after mushroom ingestion.

Outcrossing or inbreeding: DNA markers provide evidence for type of reproductive mode in Phellinus nigrolimitatus ( Basidiomycota )

The aim of this study was to clarify the reproductive mode in the wood-rotting fungus Phellinus nigrolimitatus , using molecular markers and pairing experiments of cultural isolates. A 511 bp segment of the translation elongation factor 1a (EF1a) gene and the internal and intergenic transcribed spacer sequences (ITS and IGS) of the nuclear ribosomal DNA (nrDNA), were amplified by PCR from somatic cultures derived directly from basidiomes and single spore cultures of the fungus. Sequence analyses of the partial EF1a sequence (511 bp) and ITS (732 bp) of single spore cultures gave five and fourteen variable sites, respectively. Five somatic isolates yielded double ITS sequences, which suggested the existence of different ITS types within the same isolate. Restriction digestion of the partial EF1a and the complete ITS and IGS (3.4 ± 0.2 kbp) sequences with Hinf l and Hae lll gave restriction fragment length polymorphisms (RFLPs) among the isolates. The sum of fragments exceeded the lengths of the initial PCR product in several somatic isolates, which indicated the existence of EF1a and nrDNA heterotypes within isolates. Segregation of ITS and IGS RFLPs in single spore isolates suggested heterokaryosis in somatic isolates of P. nigrolimitatus. The polymorphic EF1a and ITS restriction sites were used as codominant markers in a test for Hardy-Weinberg expectations. The molecular data suggests a predominant outcrossing reproductive mode in P. nigrolimitatus. Results from cultural pairing experiments with single spore isolates corresponded poorly with the molecular data, suggesting that laboratory pairing experiments do not necessarily provide reliable information of the mode of reproduction in natural populations of fungi.

Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

Towards a phylogenetic classification of Cordyceps : ITS nrDNA sequence data confirm divergent lineages and paraphyly

The ascomycetous genus Cordyceps accommodates endoparasitic species that attack arthropods or other fungi. Analyses of ITS nrDNA sequence data of 72 taxa from the teleomorph genera Cordyceps, Claviceps, Epichloë, and the anamorph genera Akanthomyces, Beauveria, Metarhizium, Hirsutella, Hymenostilbe, Paecilomyces, Polycephalomyces, and Tolypocladium assigned the taxa to four main evolutionary lineages not reflected in the current classification of Cordyceps. Ten subclades were recognized from separate analyses of data subsets. Judged from the ITS phylogenies, Cordyceps spp. with branched stromata were highly supported as a divergent lineage. Host specificity was found to be of limited phylogenetic significance, and several host shifts are suggested to have occurred during the evolution of Cordyceps. Similar ascospore morphology was not reflected in the phyletic groups, and closely related taxa showed large interspecific variation with respect to the number of segments in which the ascospores are divided. However, combinations of selected characters were found to delimitate some lineages, e.g. all Cordyceps spp. that attack hosts in the insect orders Coleoptera and Lepidoptera, and with non-immersed perithecia and clavate to acicular, brightly yellowish to reddish stromata, constituted a separate clade. Furthermore, all Cordyceps spp. with perithecia obliquely immersed in the stroma were recognized as a distinct monophyletic group. This clade is additionally characterized by the formation of anamorphs ascribable to the genus Hymenostilbe. The mycogenous Cordyceps spp. grouped in a separate subclade, interspersed by two cicadaen parasites and all Tolypocladium spp. except T. parasiticum. Tolypocladium and Beauveria were found to be polyphyletic. The included Claviceps and Epichloë taxa appeared to be derived within Cordyceps, thus making Cordyceps paraphyletic as suggested in other studies.

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.

Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum

The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1α showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall FST = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.

Genetic structure of Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota)

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR-RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy-Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.