Chemotaxonomy of Hypoxylon


Chemotaxonomy of Hypoxylon

An obvious character of the members of the genus Hypoxylon is that stromata are more or less coloured, at least when young, and usually contain coloured granules just beneath the stromatal surface and between perithecia. These granules yield variously coloured pigments in 10% KOH, which appeared to be a highly reliable criterion for segregation of related species, when combined with other morphological features. The revision of the genus Hypoxylon by Ju and Rogers (1996) was largely based on these pigment data, which allow for grouping species with similar pigments and segregation of species formerly considered very closely related or identical. A good example in European species is that of H. perforatum which has long been considered identical to H. rubiginosum s. l. (Miller, 1961) or merely a variety of H. rubiginosum (Petrini & Müller, 1986), and was separated by Ju & Rogers (1996) on the basis of yellow pigments in KOH instead of orange in H. rubiginosum. Most of tropical species included in H. rubiginosum s. l. as conceived by Miller (1961) were likewise separated primarily by means of KOH-extractable pigments (Ju & Rogers, 1996).

The taxonomic importance of secondary metabolites produced in culture or present in stromata of Hypoxylon was revealed by the pioneer works of Steglich et al.,(1974), Whalley & Greenhalgh (1971), Whalley & Whalley (1977) and Whalley & Edwards (1995). When studying chemotaxonomy of Daldinia, Stadler et al. (2001a, 2001b) included some Hypoxylon spp. from Europe to the samples submitted to HPLC and observed significant differences between both genera as to secondary metabolites present either in stromata or in cultures. Further studies focusing on Hypoxylon (Mühlbauer et al., 2002; Quang et al., 2003 b, 2004; Stadler et al., 2001a, 2001b,2004b; Hellwig et al., 2005) illustrated how "extremely creative is this genus with regard to secondary metabolite production" (Stadler et al., 2001b), how the colours of KOH-extractable pigments are usually consistent with the secondary metabolites present in the stromatal granules, and how HPLC fingerprinting can be efficient in segregating closely related species. Current ongoing researches on Hypoxylon metabolites still prove able to notably improve our knowledge of relationships between the members of this genus.

The current knowledge of the chemotaxonomy in Hypoxylon, and in Daldinia as well, shows that for a given species, secondary metabolites appear to make up a mix of constant constituents, but in varying proportions. Each constituent having a specific coloured reaction in KOH, the result of the KOH reaction reflects which one is prevailing in the mix, and therefore explains why colours of KOH reactions may vary with the age of stromata, or why species belonging to different chemotypes can display a same colour in KOH. Both techniques prove useful, but HPLC provides more accurate results than KOH reaction.

HPLC is an analytical chromatographic method which, when coupled with UV-visual and mass spectrometric detection, is able to discriminate between very close compounds and to draw accurate metabolites profiles from very small amounts of material. For a comprehensive description of this chemical methodology and subsequent works of elucidation of chemical structure and purification, the reader is referred to Stadler et al. (2001a; 2001b) and further publications of Quang et al. (2003 b, 2004).

The main known compounds found in Hypoxylon of both sections can be classified as follows in five groups: the combination of BNT and derivatives and absence of mitorubrin-like compounds seem a reliable character of the members of section Annulata (Quang et al. 2005), supporting the separation from the section Hypoxylon suggested by Ju & Rogers (1996) based on morphological characters.

Binaphtalènetetrol (BNT) and derivatives daldinones and truncatone.

Azaphilones, including daldinins, cohaerins and mitorubrin-like compounds including mitorubrin and derivatives, rubiginosins and hypomiltin.

Orsellinic acid


Rubiginosic acid : fatty acid

Data on secondary metabolites in Hypoxylon are still incomplete, but allow for a provisional presentation of different chemotypes within this genus, based on the combination of the above compounds. This presentation is inspired by the results of further mentioned authors. European Hypoxylon species with known metabolites profile are included.

Chemotype of section Annulata: BNT as major compound, with naphtalene derivatives :H. stygium var. annulatum, or azaphilones of the cohaerin type: H. cohaerens, H. cohaerens var. microsporum, H. multiforme, H. michelianum. (Quang et al. 2005).

Chemotypes of section Hypoxylon:

fuscum chemotype : BNT and daldinins: H. fuscum, H. fuscopurpureum (Mühlbauer et al., 2002; Quang et al., 2003b)

macrocarpum chemotype : BNT and macrocarpones: H. macrocarpum. (Mühlbauer et al., 2002)

petriniae chemotype: BNT and rubiginosins A & C. Mitorubrin absent: H. petriniae (Stadler et al., 2004b)

fragiforme chemotype : mitorubrins and orsellinic acid; BNT absent: H. fragiforme, H. howeianum, H. ticinense, H. ferrugineum (Hellwig et al., 2004)

rubiginosum chemotype: mitorubrins and orsellinic acid plus specific compounds; BNT absent : H. rubiginosum, H. julianii, H. rutilum, H. crocopeplum, H. laschii, H. subticinense (Hellwig et al., 2004; Stadler et al., 2004b).

hypomiltum chemotype : hypomiltin and orsellinic acid; BNT absent: H. perforatum, H. intermedium (Hellwig et al., 2004)

Other chemotypes: BNT plus other unknown azaphilones: H. carneum and several additional tropical taxa. (Hellwig et al., 2004)