Identification of fungal fossils and novel azaphilone pigments in ancient carbonised specimens of Hypoxylon fragiforme from forest soils of Châtillon-sur-Seine (Burgundy)
Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC–DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.
KeywordsBiodiversity Chemotaxonomy Phylogeny Sordariomycetes Xylariales Structure elucidation
We wish to thank Prof. D. L. Hawksworth for establishing contact between the working groups in France and Germany. AN is indebted for a grant of the Iranian government for a research stay in Germany. LW is grateful for a PhD grant from the province government of Lower Saxony (HSBDR graduate school). KB and MS are grateful for a grant from the Deutsche Forschungsgemeinschaft (DFG) in the Priority Programme “Taxon-Omics: New Approaches for Discovering and Naming Biodiversity” (SPP 1991). Christel Kakoschke, Cäcilia Schwager, Aileen Gollasch, Anke Skiba and Vanessa Stiller are thanked for expert technical assistance. We are grateful to Annelise Binois for her helpful comments on the manuscript.
- Daranagama DA, Hyde KD, Sir EB, Thambugala KM, Tian Q, Samarakoon MC, McKenzie EHC, Jayasiri SC, Tibpromma S, Bhat JD, Liu X, Stadler M (2018) Towards a natural classification and backbone tree for Graphostromataceae, Hypoxylaceae, Lopadostomataceae and Xylariaceae. Fungal Divers 88:1–165CrossRefGoogle Scholar
- Hashimoto T, Asakawa Y (1998) Biologically active substances of Japanese inedible mushrooms. Heterocycles 2(47):1067–1110Google Scholar
- Kuhnert E, Surup F, Sir EB, Lambert C, Hyde KD, Hladki AI, Romero AI, Stadler M (2014c) Lenormandins A-G, new azaphilones from Hypoxylon lenormandii and Hypoxylon jaklitschii sp. nov., recognised by chemotaxonimic data. Fungal Divers 71:165–184. https://doi.org/10.1007/s13225-014-0318-1 CrossRefGoogle Scholar
- Provost M (2009) Carte archéologique de la Gaule, 21, La Côte-d’Or. Vol. 3, De Nuits-Saint-Georges à Voulaines-les-Templiers. Paris, France : Académie des inscriptions et belles-lettres: Ministère de l’éducation nationale : Ministère de la rechercheGoogle Scholar
- Quang DN, Hashimoto T, Nomura Y, Wollweber H, Hellwig V, Fournier J, Stadler M, Asakawa Y (2005b) Cohaerins A and B, azaphilones from the fungus Hypoxylon cohaerens, and comparison of HPLC-based metabolite profiles in Hypoxylon sect. Annulata. Phytochemistry 66:797–809CrossRefPubMedCentralGoogle Scholar