Hartmund Wollweber's research while affiliated with Tokushima Bunri University and other places

Azaphilones, named cohaerins A and B were isolated from stromata of the xylariaceous ascomycete Hypoxylon cohaerens. Their absolute structures were determined by spectroscopic methods (2D NMR, MS, IR, UV CD), and subsequently confirmed by acetylation. Stromatal metabolite profiles of several taxa of Hypoxylon sect. Annulata were also generated using analytical HPLC with diode array and MS detection. The cohaerins were neither found in other Hypoxylon spp., nor in other Xylariaceae. However, they were present even in holotype material of H. cohaerens, collected over 200 years ago. The binaphthalene BNT was also omnipresent in sect Annulata, and its derivatives, the benzo[j]fluoranthenes daldinone A and truncatone, as well as presumably related compounds. These fungi were found devoid of other types of azaphilone pigments of the Xylariaceae, such as mitorubrins and daldinins, the latter of which are widespread in certain groups of Hypoxylon sect. Hypoxylon. Hence, chemotaxonomic data largely support the current generic concept. The original source of truncatone was identified as Hypoxylon annulatum.

The present molecular study focuses on the phylogenetic position of,the genera Daldinia, Entonaema, and Hypoxylon. 28 double-stranded new 5.8S/ITS nrDNA sequences of species representing these genera were obtained and aligned with all 351 publicly available Xylariales sequences. In contrast to earlier works on ITS phylogeny, the phylogenetic analysis presented here was limited to the less variable, reliably alignable parts of the ITS region. This approach resulted in a tree topology that is largely in accordance with the current classification. The tree revealed four monophyletic families, Amphisphaeriaceae, Apiosporaceae, Diatrypaceae, and Hyponectriaceae, within the Xylariales. The large family Xylariaceae, however, split into three major groups. The genera with geniculosporium-like anamorphs (e.g., Entoleuca, Kretzschmaria, Nemania, Rosellinia, Xylaria) formed one clade, while genera with nodulisporium-like conidial stages (Biscogniauxia/Camillea, and Daldinia/Entonaema/Hypoxylon, respectively) were found in two clades. Within one of these clades, Daldinia and Entonaema appeared closely related, while Hypoxylon split into several subclades along an unresolved backbone. A monophyletic origin of Hypoxylon could therefore neither be confirmed nor rejected, and Hypoxylon species cluster in several well-supported groups, most of which are in accordance with the current classification at the species or species-group levels. Most of these groups exhibit a unique sequence of the initial base pairs of the ITS I region. Additionally, signature sequences could be inferred. The molecular studies indicate that the widely distributed species H. fuscum is not divided into host-specific entities, but a geographic distinction should be more closely investigated. Aside from certain dubious sequences, H. fragiforme formed a monophyletic group and the sequences of H. cohaerens and H. multiforme clustered together. The phylogenetic position of H. fuscopurpureum remained unclear. The relationships among Daldinia, Entonaema and Hypoxylon are discussed with respect to morphological and chemotaxonomical characters.

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.

Hypoxylon petriniae sp. nov. is described from a new combination of morphological characters of its teleomorph and anamorph. It was found in France and other countries of Western and Central Europe, mostly colonising Fraxinus excelsior. Its status is supported by a chemotaxonomic study by analytical HPLC profiling with diode array and mass spectrometric detection of secondary metabolites in several Hypoxylon spp. These analyses revealed the presence of binaphthalene tetrol (BNT) as major stromatal metabolite of H. petriniae. This widespread purple stromatal pigment of Xylariaceae was not found in H. rubiginosum, H. cercidicolum, and further, presumably related species. In contrast to H. rubiginosum and H. cercidicolum, H. petriniae was found devoid of mitorubrin. Orsellinic acid and rubiginosins, which are recently identified azaphilone pigments chemically related to mitorubrin were found as common stromatal metabolites of many of the aforementioned taxa, and the occurrence of particular azaphilones appears to be specific for some species. A key to European Hypoxylon spp. is provided.

Daldinia macaronesica (from the Canary Islands and Madeira), D. palmensis (from the Canary Islands), D. martinii and D. raimundi (from Sicily), and D. vanderguchtiae (from Jersey, Channel Islands) spp. nov., are described, based on new combinations of teleomorphic and anamorphic characters. They all resemble the pantropical D. eschscholzii and/or the European D. concentrica with regard to teleomorphic characters and secondary metabolite profiles generated by analytical HPLC. The status of the newly described taxa was established by SEM of ascospores and microscopic studies of their anamorphs in comparison with various materials of the aforementioned known species. HPLC and SEM studies on the holotype of D. bakeri confirmed its relationships to D. fissa and D. loculata. Yellowish pigments contained in the type specimen of D. bakeri are probably artificial.

Representative specimens of fifteen Daldinia spp. were studied for ultrastructural characteristics of their ascospores by scanning electron microscopy (SEM). The ornamentation of their outermost spore layers was found to be species-consistent, confirming the results of concurrent studies on the morphology of their teleomorphs and anamorphs, secondary metabolite profiles and PCR-based genetic fingerprints. Daldinia spp. may either show smooth or transversally striated ascospores. The spores of the species within the latter group are always ellipsoid-equilateral to ellipsoid-inequilateral with narrowly rounded ends. Smooth, broadly ellipsoid to cylindrical ascospores were observed in all species (D. caldariorum, D. fissa and D. loculata) that are known to produce their stromata on substrates damaged by fire. The ascospores of D. concentrica differed from those of D. childiae (i.e., the cosmopolitan taxon previously regarded as D. concentrica ss. auct.) and other Daldinia spp. in showing a very faint ornamentation, which only became visible at 10000× magnification by SEM. A specimen collected on the isle of Jersey (Channel Islands, UK) showed morphological similarities to the pantropical D. eschscholzii, but its ascospores appeared smooth by SEM, and it may therefore represent a previously undescribed species.

Several species of the genus Hypoxylon (Xylariaceae) were studied for morphological characters and HPLC-based secondary metabolite profiles. It was confirmed that Hypoxylon is divided into two groups of species, containing either Mitorubrin type azaphilones or binaphthyls, respectively, as main metabolites. In Hypoxylon species of the latter group, some metabolites that are known from the allied genus Daldinia (i.e., Daldinal A in H. fuscum and Daldinin C in H. fuscopurpureum), were encountered for the first time. Moreover, three novel aromatic polyketides, for which the trivial names Macrocarpones A, B and C are proposed, were isolated from ascostromata of Hypoxylon macrocarpum and identified by high resolution mass spectrometry and 2D-NMR spectroscopy. Orsellinic Acid was also identified from ascostromata of H. howeianum and detected by HPLC-MS and HPLC-UV/Vis fingerprinting methodology in H. fragiforme and H. rubiginosum, but not in other examined Hypoxylon species. Due to these studies, H. fuscopurpureum sensu Y.-M. Ju & J.D. Rogers 1996 was identified as a new record for Germany.

Daldinia albofibrosa, Daldinia pyrenaica, and Daldinia steglichii, three species with yellowish KOH extractable stromatal pigments, are newly described, based on previously undescribed combinations of teleomorphic and anamorphic characters. Their status as separate species is confirmed by HPLC-based secondary metabolite profiles, ultrastructural examination of ascospores by SEM, and genetic fingerprints.

Daldinia decipiens sp. nov., associated with Betula, and the known species D. lloydii were both collected and identified from several locations in Germany. The anamorph of D. loculata is also described. SEM studies of ascospores and further observations on Daldinia spp. associated with Betulaceae in Europe (D. fissa, D. lloydii, D. loculata and D. petriniae) are given.

In a polyphasic classi®cation approach, stromata and cultures of Daldinia and allied Xylariaceae from around the world were studied for : (1) morphology of teleomorphs and anamorphs; (2) metabolite patterns in stromata and cultures, employing analytical HPLCUV- visible (diode array) detection and mass spectrometric detection ; (3) ampli®ed 18S rDNA restriction patterns (ARDRA); and (4) PCR ampli®ed minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identi®ed in Europe and other locations for the ®rst time. The results point towards the existence of further undescribed species. Stromata of a given species never contained the same major metabolites as corresponding cultures. Most cultures of Daldinia spp. produced naphthalene and chromane derivatives, differing from allied genera by the absence of mellein. Stromata of Daldinia spp. did not produce mitorubrin, but generally contained binaphthyls. Metabolite pro®les were correlated with colours of KOH-extractable stromatal pigments. The yellow azaphilones and benzophenones found in D. childiae were lacking in species with purple stromatal pigments. Cytochalasins were found in stromata of D. eschscholzii. Genetic ®ngerprints helped to distinguish morphologically closely related taxa. ARDRA gave speci®c results for species whose 18S rDNA contained insertions, while minisatellite PCR provided speci®c genetic ®ngerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, re¯ecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite pro®les.