Thomas Læssøe

Phylogenetics of the Pezizaceae, with an emphasis on Peziza

Phylogenetic relationships among mem- bers of the Pezizaceae were studied using 90 partial LSU rDNA sequences from 51 species of Peziza and 20 species from 8 additional epigeous genera of the Pezizaceae, viz. Boudiera, Iodophanus, Iodowynnea, Kimbropezia, Pachyella, Plicaria, Sarcosphaera and Sca- bropezia, and 5 hypogeous genera, viz. Amylascus, Ca- zia, Hydnotryopsis, Ruhlandiella and Tirmania. To test the monophyly of the Pezizaceae and the rela- tionships to the genera Marcelleina and Pfistera (Py- ronemataceae), 6 species from the families Ascobo- laceae, Morchellaceae and Pyronemataceae were in- cluded. Maximum parsimony and maximum likeli- hood analyses of these sequences suggest that the Pezizaceae is paraphyletic, because the non-amyloid Marcelleina is nested within it. If Marcelleina were transferred to the Pezizaceae, then the family would be monophyletic. Although the Pezizaceae is tradi- tionally characterized by amyloid asci, our results in- dicate that the amyloid reaction is a symplesiomor- phy, which has been lost in some lineages, e.g., in those including Marcelleina and Cazia. Nodes deep in the tree could not be resolved, but 7 groups of species (I-VII) are generally well supported or pre- sent in all trees. Peziza species, which constitute the core of the family, are present in all groups except group III, confirming the non-monophyly of the ge- nus. The analyses suggest that the other included genera of the Pezizaceae are all nested within Peziza, the placement of lodophanus being unresolved. The morphologically distinct Peziza gerardii, which forms a clade with Marcelleina, appears to be the sister group to the rest of the Pezizaceae. Morphological features were studied and evaluated in the context of the phylogeny. Distinct types of ascus amyloid reac- tions were found to support different rDNA lineages, e.g., a distinct amyloid ring zone at the apex is a syn- apomorphy for group IV, an intense and unrestricted amyloid reaction of the apex is mostly found in group VI, and asci that are weakly or diffusely amy- loid in the entire length are present in group II. Oth- er morphological features, such as spore surface re- lief, guttulation, excipulum structure and pigments, while not free from homoplasy, do support the groupings. Anamorphs likewise provide clues to high- er-order relationships within the Pezizaceae. Several macro- and micromorphological features, however, appear to have evolved several times independently, including ascomatal form and habit (epigeous, se- mihypogeous or hypogeous), spore discharge mech- anisms, and spore shape. Parsimony-based optimiza- tion of character states on our phylogenetic trees sug- gested that transitions to truffle and truffle-like forms evolved at least three times within the Pezizaceae (in group III, V and VI). The 9 hypogeous species in- cluded are nested in lineages with epigeous peziza- ceous taxa. Species with apothecia of various shapes and with forcible spore discharge are spread among all groups and the apothecium is suggested to be symplesiomorphic in the Pezizaceae. The results in- dicate that the apothecia forming Pezizaceae have given rise to at least 3 different forms of hypogeous ascomata without forcible spore discharge: ptychoth- ecia, stereothecia and exothecia.

A polyphasic taxonomy of Daldinia (Xylariaceae)

For a monograph based on a polythetic concept, several thousands of herbarium specimens, and several hundreds of freshly collected and cultured specimens of Daldinia and allied Xylariaceae, originating from around the world, were studied for morphological traits, including by SEM, and chemically by HPLC profiles using UV-visible and mass spectrometric detection. Emphasis was given to tropical material, and importantly, ancient specimens, including as many types as possible, were tracked and studied to review earlier taxonomic concepts. An epitype of D. eschscholtzii was selected as representative of the morphochemotype that is most widely distributed in the tropics. Six new species of Daldinia from the tropics and the southern Hemisphere are described. Daldinia asphalatum is resurrected, and D. cudonia is regarded as its synonym. In addition, the following binomials are epi-, iso-, neo- and/or lectotypified: Daldinia asphalatum, D. caldariorum, D. clavata, D. cuprea, D. durissima, D. eschscholtzii, D. grandis, D. loculata, and D. vernicosa. Annellosporium and Versiomyces are regarded as synonyms of Daldinia. Many new synonymies in Daldinia are proposed, and some previously published names are rejected. In total, 47 taxa in Daldinia are recognised and a key is provided. Their biogeography, chorology, and ecology, as well as the importance of their secondary metabolites, are also discussed. The previous definition of the genus is emended. The species concept is based mainly on morphological and other phenotype-derived characters because, despite diligent search, no molecular data or cultures of several of the accepted species could be obtained. Daldinia is segregated into five major groups, based on phenotypic characteristics. Some unnamed but aberrant specimens were not found in good condition and are therefore not formally described as new species. However, they are illustrated in detail in a hope that this will facilitate the discovery of fresh material in future. A preliminary molecular phylogeny based on 5.8S/ITS nrDNA including numerous representatives of all hitherto described taxa for which cultures are extant, was found basically in agreement with the above mentioned segregation of the genus, based on morphological and chemotaxonomic evidence. In the rDNA based phylogenetic tree, Daldinia appears clearly distinct from members of the genera Annulohypoxylon and Hypoxylon; nevertheless, representatives of small genera of predominantly tropical origin (Entonaema, Phylacia, Ruwenzoria, Rhopalostroma, Thamnomyces) appear to have evolved from daldinioid ancestors and are nested inside the Daldinia clade. Interestingly, these findings correlate with chemotaxonomic characters to a great extent, especially regarding the distribution of marker metabolites in their mycelial cultures. Hence, the current study revealed for the first time that fungal secondary metabolite profiles can have taxonomic value beyond the species rank and even coincide with phylogenetic data. Taxonomic novelties: Daldinia andina sp. nov., D. australis sp. nov., D. hausknechtii sp. nov., D. rehmii sp. nov., D. starbaeckii sp. nov., D. theissenii sp. nov., D. cahuchosa comb. nov., D. nemorosa comb. nov.

Phylogenetic relationships of Termitomyces and related taxa

Phylogenetic relationships of termitophilic fungi were estimated with Bayesian as well as other phylogenetic methods from partial sequences of the nuclear encoded large subunit ribosomal DNA (nLSU-rDNA) and the mitochondrial encoded small subunit ribosomal DNA (mtSSU-rDNA). Sequences were obtained from basidiomes covering the morphological, taxonomical, and geographical span of termitophilic mushroom-forming fungi, and analysed together with sequences from termite nests and termite guts from most known genera of fungus growing termites from geographically diverse regions. Topologies of trees resulting from the combined analyses of the two ribosomal genes generally show no positive conflicts with those obtained from separate analyses. We show that termitophilic fungi constitute a strongly supported monophyletic group within lyophylloid species. The genera Sinotermitomyces and Podabrella are derived within Termitomyces, and do not form monophyletic groups. Identical sequences were frequently found among samples of basidiomes from the same continents and among fungi utilized by termites from the same continent. However, only two sequences were identical between basidiome samples and termite nest/gut samples suggesting fruiting species do not form a representative sample of termitophilic fungi. No sequences were identical between samples from Asia and Africa indicating some geographic differentiation between these continents.

Molecular phylogeny, morphology, pigment chemistry and ecology in Hygrophoraceae (Agaricales)

Molecular phylogenies using 1–4 gene regions and information on ecology, morphology and pigment chemistry were used in a partial revision of the agaric family Hygro- phoraceae. The phylogenetically supported genera we recognize here in the Hygrophoraceae based on these and previous analyses are: Acantholichen, Ampulloclitocybe, Arrhenia, Cantharellula, Cantharocybe, Chromosera, Chrysomphalina, Cora, Corella, Cuphophyllus, Cyphellostereum, Dictyonema, Eonema, Gliophorus, Haasiella, Humidicutis, Hygroaster, Hygrocybe, Hygrophorus, Lichenomphalia, Neohygrocybe, Porpolomopsis and Pseudoarmillariella. A new genus that is sister to Chromosera is described as Gloioxanthomyces. Revisions were made at the ranks of subfamily, tribe, genus, subgenus, section and subsection. We present three new subfamilies, eight tribes (five new), eight subgenera (one new, one new combination and one stat. nov.), 26 sections (five new and three new combinations and two stat. nov.) and 14 subsections (two new, two stat. nov.). Species of Chromosera, Gliophorus, Humidicutis, and Neohygrocybe are often treated within the genus Hygrocybe; we therefore provide valid names in both classification systems. We used a minimalist approach in transferring genera and creating new names and combinations. Consequently, we retain in the Hygrophoraceae the basal cuphophylloid grade comprising the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe, despite weak phylogenetic support. We include Aeruginospora and Semiomphalina in Hygrophoraceae based on morphology though molecular data are lacking. The lower hygrophoroid clade is basal to Hygrophoraceae s.s., comprising the genera Aphroditeola, Macrotyphula, Phyllotopsis, Pleurocybella, Sarcomyxa, Tricholomopsis and Typhula.

Recognition of hypoxyloid and xylarioid Entonaema species and allied Xylaria species from a comparison of holomorphic morphology, HPLC profiles, and ribosomal DNA sequences

The genus Entonaema comprises Xylariaceae with hollow, gelatinous stromata that accumulate liquid. Some of its species, including the type species, appear related to Daldinia from a polyphasic approach, comprising morphological studies, comparisons of ribosomal DNA sequences, and high performance liquid chromatography (HPLC) profiles with diode array and mass spectrometric detection (HPLC-DAD-MS). This methodology was used to study Entonaema pallidum. Its major stromatal constituent was identified as xylaral, a secondary metabolite known from Xylaria polymorpha. This compound was detected in several Xylaria spp., including the tropical X. telfairii and morphologically similar taxa, whose stromata may also become hollow and filled with liquid. Cultures of E. pallidum resembled those of Xylaria, substantially differing from other Entonaema spp., in their morphology, 5.8S/ITS nrDNA sequences, and HPLC profiles. The type specimen of E. mesentericum was located in the spirit collection of the herbarium B and found to agree morphologically with the nomenclatorily younger E. pallidum. Traces of xylaral were even detected by HPLC-DAD-MS in the spirit in which the fungus had been preserved. Entonaema pallidum is thus regarded as a later synonym of E. mesentericum. Therefore, the latter name is transferred to Xylaria. A key to entonaemoid Xylariaceae is provided. Colour reactions (NH3, KOH) of the ectostroma were applied to a limited number of Xylaria spp., but metabolite profiles of cultures appear more promising as chemotaxonomic traits to segregate this genus. As xylaral was also found in Nemania and Stilbohypoxylon spp., while being apparently absent in Hypoxylon and allied genera, it may be a chemotaxonomic marker for Xylariaceae with Geniculosporium-like anamorphs.

Molecular and morphological investigation of Daldinia in northern Europe

A molecular and morphological investigation of 35 collections of Daldinia from 11 plant species revealed five taxonomic entities of Daldinia in northern Europe. These include Daldinia concentrica, D. cf. fissa, D. grandis, D. loculata and D. cf. petriniae. D. cf. fissa, D. grandis and D. loculata were found exclusively on burnt wood, but they do not form a monophyletic unit in a phylogenetic tree derived from analysis of the ITS sequence of the ribosomal DNA.

Phylogenetic diversity in the core group of Peziza inferred from ITS sequences and morphology

Species delimitation within the core group of Peziza is highly controversial. The group, typified by P. vesiculosa ,i s morphologically coherent and in previous analyses of LSU rDNA sequences it formed a highly supported clade. Phylogenetic diversity and species limits were investigated within the group using sequences from the complete ITS region (ITS1-5‐8S-ITS2). Eighty-three specimens were selected for molecular study from a larger sample of material studied morphologically to explore the intra- and interspecific variation of each putative species. The sister group taxon, P. ampelina was used as the outgroup and two specimens of P. subcitrina were additionally included. Seven independent lineages of rDNA were identified (I‐VII), each representing one to several species. These lineages form two larger clades, A (II, and I or III) and B (IV‐VII), supported by macromorphology: small (generally ! 2 cm), shallowly cup- to disc-shaped apothecia (A) and large (up to 15 cm), deeply cup-shaped to expanded apothecia (B). The overall exciple structure (a stratified or non-stratified medullary layer) and to some degree spore surface relief, likewise support the groupings. Clade A contains taxa with smooth or nearly smooth spores (except for P. lohjaeX nsis), while clade B contains taxa with a range of spore ornamentations, from smooth, finely warty to distinctly warty, and spiny. The position of groups I (P. vesiculosa and P. ammophila) and III (P. lohjaeX nsis) are uncertain, and these taxa also deviate morphologically from the other clade A members. The following species are recognized based on morphology and ITS rDNA analyses: P. ammophila and P. vesiculosa (I); P. alcis, P. ampliata, P. domiciliana, P. fimeti, P. nivalis, and a number of putative species or intraspecific entities (II); P. lohjaeX nsis (III); P. sp. c (IV); P. arvernensis (V); P. echinispora and P. sp. d (VI); and P. varia (VII). The nomenclature of these species is analyzed and taxa are typified as necessary. Based on ITS and morphology, we found no justification for recognizing more than one species in the ‘P. varia complex’, including 27 specimens that have been referred to under the names P. cerea, P. micropus and P. repanda, from an array of substrates and dierent geographical areas. Morphological characters previously used to delimit species within this complex, such as colour variation of the apothecia, presence or absence of a stipe, stratified or non-stratified medullary exciple (or thickness of the excipular layers), cell types in the outermost exciple and moniliform vs filiform paraphyses were not correlated with the subgroups supported by ITS analyses and appeared to be plastic. Therefore, P. cerea and P. micropus are placed in synonymy with P. varia. The name P. repanda is rejected. Levels of sequence divergence were low within group II, comprising 33 small apothecial specimens. Twelve fine-scale lineages were identified, but the analyses did not resolve relationships among these. P. granulosa sensu Boudier is considered a synonym of P. fimeti. These have previously been distinguished mainly by occurrence on various soil types, including burnt soil and soil mixed with sawdust or woodchips vs on dung. The substrate and habitat have been much emphasized in the taxonomy of Peziza, but the results obtained here indicate that populations on a diverse array of substrates may be closely related, or indeed, conspecific.

Lepraric acid derivatives as chemotaxonomic markers in Hypoxylon aeruginosum, Chlorostroma subcubisporum and C. cyaninum, sp. nov.

Hypoxylon aeruginosum (Xylariaceae), an infrequently encountered predominantly tropical pyrenomycete, of which two varieties are known to science, is characterised by having a cyan blue stromatal surface or subsurface. In the course of our ongoing chemotaxonomic evaluation of the Xylariaceae, specific profiles of H. aeruginosum were observed by high performance liquid chromatography, coupled with diode array detection and mass spectrometry (hplc-DAD/MS). By comparison with an authentic standard, lepraric acid and several yet unidentified metabolites with similar hplc-DAD/MS characteristics were detected in the stromata of the type material and other specimens of this species. Interestingly, lepraric acid was hitherto only known from lichenised ascomycetes. Hypoxylon aeruginosum, which is here reported first from Africa and Asia, contained none of the metabolites that were previously detected in other Xylariaceae, except for stromata growing hyperparasitically on other Hypoxylon species. A different lepraric acid derivative was also detected in the type specimen of Chlorostroma subcubisporum, which differs from H. aeruginosum by having a green stromatal surface, cuboid ascospores, and in lacking an amyloid ascal apical apparatus. A second species of Chlorostroma, which showed essentially the same metabolite profile as H. aeruginosum, is described from Thailand. We conclude that Chlorostroma and H. aeruginosum are closely related. However, no taxonomic conclusions are drawn from these findings because no cultures have so far become available to study their anamorphic morphology, their secondary metabolites in culture, and their molecular phylogeny. Taxonomic novelty: Chlorostroma cyaninum Læssøe, Srikitikulchai & J. Fournier, sp. nov.

Ruwenzoria, a new genus of the Xylariaceae from Central Africa

During a foray to the mountain rainforests of the Democratic Republic of the Congo, a peculiar species of Xylariaceae was found, which could not be accommodated in any of the existing genera. It is recognised as representative of a new genus, named Ruwenzoria, owing to the presence of a new combination of teleomorphic and anamorphic characters that are regarded as significant for generic segregation within the Xylariaceae. Studies on its secondary metabolites in stromata and cultures by high performance liquid chromatography, coupled with diode array and mass spectrometric detection (HPLC-DAD/MS), and on its phylogenetic affinities based on 5.8S/ITS rDNA sequence data, respectively, revealed a close relationship of the new taxon to the genera Daldinia and Entonaema, from which it differs by having neither a hollow, gelatinous nor a conspicuously zonate stromatal interior, and an anamorph featuring enteroblastic rather than holoblastic conidiogenesis. A specimen from the same geographic region, previously identified as Daldinia bakeri by R.W.G. Dennis was found to constitute a mixture of stromata of Ruwenzoria and an additional, undescribed Daldinia species. The latter fungus is not formally described due to the scantiness of the material, but its morphological characteristics are illustrated.

A survey of Daldinia species with large ascospores

Specimens of Daldinia (Xylariaceae) from around the world possessing large ascospores were studied for teleomorphic and anamorphic morphological characters and compared with authentic material of D. grandis. A culture made from a specimen of D. grandis collected from Ecuador produced stromatic structures, but no conidiogenous structures referable to known xylariaceous anamorphs were observed. D. grandis is reconsidered and viewed as a species of warmer climates in the Americas. Three new species are recognised from new combinations of anamorphic and teleomorphic characters: (1) D. novaezelandiae sp. nov. from New Zealand, a fungus obviously related to D. bakeri, which is also reported from that country; (2) D. dennisii sp. nov., from Australia and New Zealand, of which two varieties are erected; and (3) D. loculatoides sp. nov., from the UK and Canada with affinities to D. loculata. The type of Sphaeria durissima was identified as D. loculata. Notes on further Daldinia spp. are included.