Ancient DNA Analysis of 5,000 Year Old Wheat and Barley Seeds from Armenia Reveal Insights into Fungal Pathogens and Wine-Making Culture in the Late Chalcolithic

As the first Neolithic farmers abandoned their hunter-gatherer lifestyle, the founding crops growing throughout the Near East became a valuable food source and commodity to trade. Domestication of the cereals Emmer wheat (Triticum turgidum), Einkorn wheat (Triticum boeoticum), millet (Setaria italica) and barley (Hordeum vulgare), along with legumes and fruit (such as grape), supplied sufficient energy to allow the development of complex structured societies and urbanisation, which underpins much of modern civilization. In this study, we use next-generation sequencing technology to sequence 5,000-year-old wheat and barley grains found in an Armenian cave Areni-1, which was found to contain the world’s oldest shoe (Pinhasi et al. 2010), and oldest winemaking vessel (Barnard et al. 2011). Archaeological and anecdotal evidence from historians suggests that wheat and barley grains were added to early wine ferments. Using next-generation amplicon and shotgun sequencing and ancient DNA damage patterns, we show that fungal species such as Aspergillus oryzae and Saccharomyces cerevisiae are present on the surface of wheat and barley seeds, supporting previous reports of winemaking in Areni-1. Additionally, trace amounts of cereal-specific fungal pathogens such as Stagonospora nodorum, Pyrenophora tritici-repentis and Fusarium complex species were also identified and compared to modern sub-types. Domesticated and wild, pathogenic fungal species identified in this study provide important insights into early cultural and agricultural systems in the Near East, as well as revealing how fungal species have evolved over the past five millennia.