Persistence and decay of the intestinal microbiota’s DNA in glacier mummies from the Alps Franco Rollo * , Stefania Luciani, Isolina Marota, Cristina Olivieri, Luca Ermini Department of Molecular Cellular and Animal Biology, Laboratorio di Archeo-antropologia Molecolare/DNA Antico, University of Camerino, UNICAM, via Filippo Camerini 2, I-62032 Camerino, Italy Received 3 July 2006; received in revised form 19 September 2006; accepted 20 October 2006 Abstract The study of the DNA of ancient microorganisms in human remains represents one of the newest and most promising branches of molecular archaeoanthropology. Despite the growing number of papers addressing this subject, however, the analysis of ancient bacterial DNA is still a con- tentious issue. The indigenous microbiota of the gastrointestinal tract of man represents a community characterized by relative constancy in spe- cies composition and proportion. As a model system, we studied the preservation of the intestinal microbiota DNA in two naturally freeze-dried human mummies found on the Alps. This kind of mummy is an ideal subject for ancient DNA investigations. The first is a male body historically dated 1918 A.D. while the second is the famous Tyrolean Iceman (3.350e3.100 B.C.). The screening of bacterial 16S rRNA gene libraries from colon samples of the two mummies (49 clones for the 1918 mummy, 119 clones for the Iceman) showed that the characteristic composition of the intestinal microbiota of man (Alpha-, Beta-, Gammaproteobacteria, Bacteroides, Clostridia) is still kept in the library from the recent mummy while the Iceman’s library is almost entirely composed by the DNA of clostridia. Comparison of the intestinal data with those from the literature describing the screening of 16S rRNA gene libraries from other parts of the Iceman’s body and from permafrost specimens indicates that the changes in library composition may partly be attributed to the proliferation of clostridia inside the corpses, as described in forensic literature, and partly to the differential persistence of the DNA of gram-negative bacteria and endospore-former low-GC gram-positive bacteria. The present results contribute to the issue of the authentication of claims of pathogen DNA identification in archaeological human remains. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Ancient DNA; Natural mummies; Intestinal microbiota; DNA decay; Authentication criteria 1. Introduction The analysis of the DNA of ancient microorganisms repre- sents one of the newest and most promising branches of mo- lecular archaeology. Because of their impact on present-day human health and of possible clues to palaeopathological and palaeopopulational issues, pathogens have been the object of the vast majority of investigations. First reports on the identification of pathogens using DNA analysis in ancient human remains (skeleton, mummies) date back to the early nineties (Salo et al., 1994; Spigelman and Lemma, 1993). For the following ten years, as reported in a recent review (Drancourt et al., 2004), the number of papers dealing with ancient bacterial DNA has increased almost exponentially. In the course of time, two main methods of search have been proposed. The first directly stems from the methods of identi- fication set up for clinical purposes. It is basically centered on a pair of polymerase chain reaction (PCR) primers designed to selectively bind to a specific sequence of the relevant bac- terium (for example IS6110, TPP15 gene, pla gene in the case of, respectively, Mycobacterium tuberculosis, Treponema pallidum, and Yersinia pestis). This approach has been used to screen bone and soft tissue specimens dated at from 17,000 to 200 years before present for the presence of M. tuberculosis DNA (Arriaza et al., 1995; Crubezy et al., 1998; Mays et al., * Corresponding author. Tel.: þ39 737 403 219; fax: þ39 737 636 216. E-mail address: francougo.rollo@unicam.it (F. Rollo). 0305-4403/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jas.2006.10.019 Journal of Archaeological Science 34 (2007) 1294e1305 http://www.elsevier.com/locate/jas