Hartmund Wollweber

Molecular chemotaxonomy of Daldinia and other Xylariaceae

In a polyphasic classification 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 HPLC-UV-visible (diode array) detection and mass spectrometric detection; (3) amplified 18S rDNA restriction patterns (ARDRA); and (4) PCR amplified minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identified in Europe and other locations for the first 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 profiles 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 fingerprints helped to distinguish morphologically closely related taxa. ARDRA gave specific results for species whose 18S rDNA contained insertions, while minisatellite PCR provided specific genetic fingerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, reflecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite profiles.

Concentricol, a taxonomically significant triterpenoid from Daldinia concentrica

In the course of a chemotaxonomic study of xylariaceous Ascomycetes, a major metabolite was isolated and identified from the ascostromata of Daldinia concentrica. The compound, for which the name concentricol is proposed, constitutes a highly oxidised squalene derivative. A survey of several Daldinia spp. from around the world (including several type materials), employing analytical HPLC-UV/Vis (with diode array detection) and positive electrospray HPLC-MS of stromatal MeOH extracts revealed that concentricol was omnipresent in the stromata of D. concentrica, as well as in those of several collections of the pantropical Daldinia eschscholzii. All other investigated Daldinia spp. were found devoid of concentricol but contained binaphthalenes, benzophenones and/or azaphilones as further taxonomically relevant main metabolites.

Macrocarpones, novel metabolites from stromata of Hypoxylon macrocarpum, and new evidence on the chemotaxonomy of Hypoxylon 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.

Cryptic species related to Daldinia concentrica and D. eschscholzii, with notes on D. bakeri.

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.

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.

Importance of ascospore ornamentation in the taxonomy of Daldinia

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.