Unravelling the ancient fungal DNA from the Iceman's gut

Here, we explore the possible ancient fungal species in the gut of Otzi, the Iceman, a naturally mummified human found in the Tyrolean Alps (border between Italy and Austria). While ancient DNA (aDNA) has been extensively used to study human, animal, and plant evolution, this research focuses on ancient microbial diversity, specifically fungi. Fungal DNA is often underestimated in metagenomic samples, however here we hypothesise the possibility of retrieving ancient fungal sequences from Otzis gut. A robust bioinformatic pipeline has been developed to detect and authenticate fungal aDNA from stomach, small intestine, and large intestine samples. We revealed the presence of Pseudogymnoascus genus, with P. destructans and P. verrucosus as possible species, that were particularly abundant in the stomach and small intestine. We suggest that Otzi may have consumed these fungi accidentally, likely in association with other elements of his diet, and they thrived in his gut after his death due to their adaptability to harsh and cold environments. This research provides insight into the coexistence of ancient humans with specific fungal species and proposes and validates a conservative bioinformatic approach for detecting fungal aDNA in historical metagenomic samples. Significance statementDespite their essential interactions with all kingdoms of life, limited molecular studies have focused on ancient fungi. Here, we developed a thorough bioinformatic pipeline that allowed us to detect the presence of ancient DNA likely belonging to Pseudogymnoascus destructans and P. verrucosus in the gut of Otzi, a human naturally mummified over 3,000 years ago in the Tyrolean Alps. Both species can survive harsh environmental conditions, and P. destructans is known for its pathogenicity, suggesting that Otzi may have accidentally ingested them and providing valuable insights into how ancient humans coexisted with specific fungal species. We propose a highly reliable methodology for detecting ancient fungal DNA in metagenomic studies of historical samples that can have broader applications to understand ancient ecosystems and their interactions.