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Identification of fungal fossils and novel azaphilone pigments in ancient carbonised specimens of Hypoxylon fragiforme from forest soils of Châtillon-sur-Seine (Burgundy)

  • Frank Surup
  • Abolfazl Narmani
  • Lucile Wendt
  • Sebastian Pfütze
  • Robin Kretz
  • Kevin Becker
  • Clément Menbrivès
  • Alain Giosa
  • Michelle Elliott
  • Christophe Petit
  • Manfred Rohde
  • Marc Stadler
Article

Abstract

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.

Keywords

Biodiversity Chemotaxonomy Phylogeny Sordariomycetes Xylariales Structure elucidation 

Notes

Acknowledgements

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.

Supplementary material

13225_2018_412_MOESM1_ESM.pdf (1.1 mb)
Supplementary material 1 (PDF 1103 kb)

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Copyright information

© School of Science 2018

Authors and Affiliations

  1. 1.Dept. Microbial DrugsHelmholtz-Zentrum für Infektionsforschung GmbHBraunschweigGermany
  2. 2.German Centre for Infection Research (DZIF), Partner Site Hannover-BraunschweigBraunschweigGermany
  3. 3.Department of Plant Protection, Faculty of AgricultureUniversity of TabrizTabrizIran
  4. 4.University of Paris 1 Panthéon-Sorbonne, UMR 7041, Archéologies EnvironnementalesParisFrance
  5. 5.Helmholtz-Zentrum für Infektionsforschung GmbH, Central Facility for MicroscopyBraunschweigGermany

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