Eric Kuhnert

The Xylariaceae as model example for a unified nomenclature following the “One Fungus-One Name” (1F1N) concept

The introduction of a One Fungus-One Name (1F1N) concept has led to intensive discussions among taxonomists. Based on the Xylariaceae, a hyperdiverse family of Ascomycota with over 1300 species, the advantages and pitfalls of these nomenclatural changes and their consequences for taxonomy and applied mycology are discussed. Historically, in the Xylariaceae, an 1F1N concept had already been realised: the types of all important genera are conserved. Most teleomorphs were discovered long before the anamorphic states and the latter did not receive separate names, hence no drastic taxonomic changes are expected. The new nomenclature calls for abandonment of some ill-defined anamorph genera, such as Muscodor. Other anamorph genera will be retained because their names refer to morphological symplesiomorphies that occur in several teleomorph genera. Various important taxa are only known from ancient specimens, and no DNA-based data are available. Much work still remains to be done to recollect these fungi, select epitypes, and settle their phylogenetic relationships. Until then, we recommend that taxonomic changes be applied at the suprageneric ranks whenever possible to maintain nomenclatural stability.

New Hypoxylon species from Martinique and new evidence on the molecular phylogeny of Hypoxylon based on ITS rDNA and β-tubulin data

Three new species of Hypoxylon (Xylariaceae) collected from Martinique in the French Caribbean are recognised by new combinations of morphological characters. Their status as undescribed taxa was supported by secondary metabolite profiling based on High performance liquid chromatography with diode array and mass spectrometric detection (HPLC/DAD-MS) as well as by comparison of ITS and partial ß-tubulin DNA sequences with related taxa. In the course of this study, the teleomorph of Nodulisporium griseobrunneum was found, and this species could be transferred to Hypoxylon. Moreover, several names in Hypoxylon are epitypified by selecting recently collected specimens from the same geographic areas as the holotypes came from. Despite the fact that our study used the hitherto most extensive taxon sampling, the phylogenetic analyses inferred from ITS and ß-tubulin sequences remain contradictory to each other, and neither genealogy was found fully in agreement with phenotype-derived traits. We conclude that the right gene (or multi-gene genealogies) to reflect the phylogeny and evolution of Hypoxylon still remains to be found. For the time being, we recommend that the application of polyphasic taxonomic concepts should be continued in taxonomic studies of Hypoxylon.

Lenormandins A—G, new azaphilones from Hypoxylon lenormandii and Hypoxylon jaklitschii sp. nov., recognised by chemotaxonomic data

Seven new azaphilone pigments named lenormandins A–G were isolated from stromata (fruiting bodies) of the xylariaceous fungus, Hypoxylon lenormandii using preparative High Performance Liquid Chromatography (HPLC) and their structures were elucidated by NMR spectroscopy, high resolution mass spectrometry and other spectral methods. Their occurrence in herbarium specimens (including various type materials collected in the 19th and early 20th century) and several fresh collections was studied by analytical HPLC with diode array and mass spectrometric detection (HPLC-DAD/MS), revealing that this group of pigments is specific for Hypoxylon lenormandii from various geographic regions and otherwise only occurs in closely related taxa, i.e. H. sublenormandii and an undescribed fungus from Sri Lanka, for which we propose the name H. jaklitschii. The status of the new species was established by using molecular phylogenetic data based on ITS and partial beta-tubulin sequences, and by detailed morphological studies.

Resurrection and emendation of the Hypoxylaceae, recognised from a multigene phylogeny of the Xylariales

A multigene phylogeny was constructed, including a significant number of representative species of the main lineages in the Xylariaceae and four DNA loci the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, the second largest subunit of the RNA polymerase II (RPB2), and beta-tubulin (TUB2). Specimens were selected based on more than a decade of intensive morphological and chemotaxonomic work, and cautious taxon sampling was performed to cover the major lineages of the Xylariaceae; however, with emphasis on hypoxyloid species. The comprehensive phylogenetic analysis revealed a clear-cut segregation of the Xylariaceae into several major clades, which was well in accordance with previously established morphological and chemotaxonomic concepts. One of these clades contained Annulohypoxylon, Hypoxylon, Daldinia, and other related genera that have stromatal pigments and a nodulisporium-like anamorph. They are accommodated in the family Hypoxylaceae, which is resurrected and emended. Representatives of genera with a nodulisporium-like anamorph and bipartite stromata, lacking stromatal pigments (i.e. Biscogniauxia, Camillea, and Obolarina) appeared in a clade basal to the xylarioid taxa. As they clustered with Graphostroma platystomum, they are accommodated in the Graphostromataceae. The new genus Jackrogersella with J. multiformis as type species is segregated from Annulohypoxylon. The genus Pyrenopolyporus is resurrected for Hypoxylon polyporus and allied species. The genus Daldinia and its allies Entonaema, Rhopalostroma, Ruwenzoria, and Thamnomyces appeared in two separate subclades, which may warrant further splitting of Daldinia in the future, and even Hypoxylon was divided in several clades. However, more species of these genera need to be studied before a conclusive taxonomic rearrangement can be envisaged. Epitypes were designated for several important species in which living cultures and molecular data are available, in order to stabilise the taxonomy of the Xylariales.

Rickenyls A–E, antioxidative terphenyls from the fungus Hypoxylon rickii (Xylariaceae, Ascomycota)

Our screening efforts for new natural products with interesting bioactivity have revealed the neotropical ascomycete Hypoxylon rickii as a prolific source. We isolated five secondary metabolites with a p-terphenyl backbone from the mycelial extract of a fermentation of this fungus in 70 l scale by using RP-HPLC, which were named rickenyls A-E (1-5). Their structures were elucidated by X-ray crystallography and NMR spectroscopy, complemented by HRESIMS. Two of the compounds contained a quinone core structure in ortho (2) and para-position (5), respectively. We obtained 2 spontaneously and by lead tetraacetate oxidation from 1. All compounds were screened for antimicrobial, antioxidative and cytotoxic activities. Rickenyl A (1) exhibited strong antioxidative effects and moderate cytotoxic activity against various cancer cell lines.

Deconins A-E: Cuparenic and Mevalonic or Propionic Acid Conjugates from the Basidiomycete Deconica sp. 471.

Bioassay-guided fractionation of antibacterial extracts from cultures of a basidiomycete from Northern Thailand, which represents a new species of the genus Deconica, yielded the terpenoid deconin A (1), whose structure was elucidated by spectral methods (NMR, HRMS) as a cuparenic/mevalonic acid conjugate. The absolute configuration of 1 was determined after saponification and comparison of specific rotations of the resulting cuparenic acid and mevalonolactone with authentic standards and literature data. Six minor congeners (2-7) were isolated and identified, and their antimicrobial and cytotoxic effects are reported. Compounds 1-4 are the first natural products featuring an unmodified mevalonic acid residue as a building block.

Gymnopalynes A and B, chloropropynyl-isocoumarin antibiotics from cultures of the basidiomycete Gymnopus sp.

A chlorinated isocoumarin with an acetylenyl side chain and its 3,4-dihydro derivative, named gymnopalynes A (1) and B (2), were isolated from cultures of a basidiomycete originating from the rain forest of Northern Thailand. The producing organism was identified as a species of Gymnopus (Marasmiaceae). Their structures were elucidated by spectroscopic methods including UV/vis and NMR spectroscopy as well as high-resolution mass spectrometry as 3-(3-chloroprop-1-yn-1-yl)-1H-isochromen-1-one (1) and 3-(3-chloroprop-1-yn-1-yl)-3,4-dihydro-1H-isochromen-1-one (2). The absolute stereochemistry of 2 was assigned as S by CD spectroscopy. Both compounds showed weak to moderate antimicrobial and pronounced cytotoxic activities.

Hypoxyvermelhotins A–C, new pigments from Hypoxylon lechatii sp. nov

A new species of Hypoxylon was discovered, based on material collected in French Guiana and recognised on the basis of new combination of morpholological characters in comparison with type and authentic material of macroscopically similar taxa. These findings were corroborated by the rather isolated positions of its ITS-nrDNA and beta-tubulin DNA sequences in molecular phylogenies. However, the most salient feature of this fungus only became evident by a comparison of its stromatal HPLC profile, revealing several secondary metabolites that were hitherto not observed in stromata of any other member of the Xylariaceae. Part of the stromata were subsequently extracted to isolate these apparently specific components, using preparative chromatography. Five metabolites were obtained in pure state, and their chemical structures were elucidated by means of high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. They turned out to be tetramic acid derivatives of the so-called vermelhotin type. Aside from vermelhotin, previously isolated from cultures of endophytic fungi, we identified three novel congeners, for which the trivial names hypoxyvermelhotins A-C were proposed. Like vermelhotin, they constitute orange-red pigments and a preliminary biological characterisation revealed them to have rather strong cytotoxic and moderate to weak antimicrobial effects. These results further illustrate the high diversity of unique secondary metabolites in stromata of the hypoxyloid Xylariaceae, a family in which biological diversity seems to parallel the chemical diversity of their bioactive principles to a great extent.

Sporothriolide derivatives as chemotaxonomic markers for Hypoxylon monticulosum

During the course of a screening for novel anti-infective agents from cultures of tropical Xylariaceae originating from French Guiana and Thailand, pronounced antifungal activity was noted in extracts of cultures of Hypoxylon monticulosum. A bioassay-guided fractionation led to the known metabolite sporothriolide as active principle. In addition, three new derivatives of sporothriolide were isolated, for which we propose the trivial names sporothric acid, isosporothric acid and dihydroisosporothric acid. Their chemical structures were elucidated by high-resolution electrospray mass spectrometry in conjunction with two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. From earlier studies on the biogenesis of the chemically similar canadensolides, we postulate that the new compounds were shunt products, rather than biogenetic precursors of sporothriolide. Interestingly, this compound class, as well as strong antifungal activities, was only observed in multiple cultures of H. monticulosum, but not in several hundreds of Hypoxylon cultures studied previously or concurrently. Therefore, sporothriolide production may constitute a species-specific feature with respect to Hypoxylon and the Xylariaceae, although the compound was previously reported from non-related fungal taxa.

Botryane, noreudesmane and abietane terpenoids from the ascomycete Hypoxylon rickii.

In the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (1-3) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A-C (7-9); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.