Phylogenetic and Chemotaxonomic Studies Confirm the Affinities of Stromatoneurospora phoenix to the Coprophilous Xylariaceae

Abstract

The genus Stromatoneurospora was erected in 1973 by Jong and Davis to accommodate the pyrophilic pyrenomycete Sphaeria phoenix and has traditionally been placed in the family Xylariaceae based on morphological features. However, no living culture of this genus has so far been available in the public domain. Molecular data were restricted to an internal transcribed spacer (ITS) sequence that only confirmed the familial position, and was generated from a strain that is not deposited in a public culture collection. We have recently collected fresh material and were able to culture this fungus from Thailand. The secondary metabolites of this strains were analysed after fermentation in multiple media. The the prominent components of these fermentation were purified, using preparative chromatography. Aside from two new eremophilane sesquiterpenoids named phoenixilanes A-B (1-2), four other components that are known from species of the xylariaceous genera Xylaria and Poronia were identified by spectral methods (nuclear magnetic resonance spectroscopy and high resolution mass spectrometry). Notably, (-)-(R)-6-hydroxy-3-methyl-4-dihydroisocoumarin-5-carboxylic acid (6) has not been reported as a natural product before. Moreover, DNA sequences of Stromatoneurospora phoenix clustered with members of the genera Poronia and Podosordaria in a multi-locus molecular phylogeny. These results confirmed that the genus belongs to the same evolutionary lineage as the coprophilic Xylariaceae. The results also suggest that this lineage has evolved independently from the plant-inhabiting saprotrophs and endophytes that are closely related to the genus Xylaria. These findings are discussed in relation to some theories about the endophytic vs. the pyrophilic/coprophilic fungal life style.

Keywords: Sordariomycetes; Xylariales; secondary metabolites; sesquiterpenoids; structure elucidation.

Figure 1

Morphological characteristics of Stromatoneurospora phoenix (specimen BBH 42282). (a,b): stromata in the natural habitat; (c): stromatal surface and ostioles; (d): longitudinal section of stroma showing perithecia and the tissue below the perithecial layer; (e): asci with apical apparatus bluing in Melzer’s reagent (black arrow); (f,g): ascospores by scanning electron microscopy (SEM); (h–k): ascospores by light microscopy. Scale is indicated by bars ((a): 2 mm. (b): 1 mm. (d): 500 µm; (e): 20 µm, (f–k): 5 µm).

Figure 2

Culture characteristics and lindquistia–like anamorph of Stromatoneurospora phoenix strain BCC82040; (a): mature synnema in culture; (b): young synnema in culture; (c–f): conidiogenous cells (indicated by black arrows) and conidia; conidiophore indicated in d by white arrow; (g,h): colony on OA after one month. Scale is indicated by bars: (a) scale bar = 2 mm; (c–f) scale bar = 10 µm; (g,h) scale bar = 2 cm).

Figure 3

Phylogenetic relationships inferred from RAxML of Stromatoneurospora phoenix and other selected Xylariales based on ribosomal internal transcribed spacer (ITS) DNA sequence data. Support values of via MP, ML, and Bayesian (MB) analyses higher than 50% (MP, ML) and 0.95 (MB) and are given above (MP/ML) and below (MB) the respective branches. The black arrow indicates the sequences of S. phoenix (in orange font) and the clade comprising the sequences of the coprophilic and pyrophilic Xylariaceae is marked by a grey rectangle.

Figure 4

Phylogenetic relationships inferred from RAxML on multi-locus alignment of Stromatoneurospora phoenix and other selected Xylariales based on concatenated ribosomal (ITS and LSU) and proteinogenic (TUB2 and RPB2) DNA sequence data. Support values of via MP, ML and Bayesian (MB) analyses higher than 50% (MP, ML) and 0.95 (MB) and are given above (MP/ML) and below (MB) the respective branches. Branches of significant support (BS ≥ 95% and PP ≥ 0.95) are thickened. The black arrow indicates the sequences of S. phoenix (in orange font) and the clade comprising the sequences of the coprophilic and pyrophilic Xylariaceae is marked by a grey rectangle.

Figure 5

Chemical structures of secondary metabolites isolated from cultures of Stromatoneurospora phoenix. Phoenixilanes A–B (1,2) punctaporonin B (3), 8,9-dehydroxylarone (4), (−)-(R)-6 hydroxy-3-methyl-4-dihydroisocoumarin-5-carboxylic acid (5), and 3-methoxycarbonyl indole (6).

Figure 6

Key nuclear magnetic resonance (NMR) correlations of phoenixilane A (1). Left: structure with ¹H/¹H Correlation Spectroscopy-(COSY; bold bonds) and ¹H/¹³C Heteronuclear Multiple Bond Correlation (HMBC) correlations (blue arrows). Right: relative conformation with Rotating Frame Nuclear Overhauser Effect Spectroscopy (ROESY) correlations (dashed, pink arrows); conformation of ring substituents: a: axial, e: equatorial.