Cristina Olivieri

In the last years several phylogeographic studies of both extant and extinct red deer populations have been conducted. Three distinct mitochondrial lineages (western, eastern and North-African/Sardinian) have been identified reflecting different glacial refugia and postglacial recolonisation processes. However, little is known about the genetics of the Alpine populations and no mitochondrial DNA sequences from Alpine archaeological specimens are available. Here we provide the first mitochondrial sequences of an Alpine Copper Age Cervus elaphus. DNA was extracted from hair shafts which were part of the remains of the clothes of the glacier mummy known as the Tyrolean Iceman or Ötzi (5,350–5,100 years before present). A 2,297 base pairs long fragment was sequenced using a mixed sequencing procedure based on PCR amplifications and 454 sequencing of pooled amplification products. We analyzed the phylogenetic relationships of the Alpine Copper Age red deer's haplotype with haplotypes of modern and ancient European red deer. The phylogenetic analyses showed that the haplotype of the Alpine Copper Age red deer falls within the western European mitochondrial lineage in contrast with the current populations from the Italian Alps belonging to the eastern lineage. We also discussed the phylogenetic relationships of the Alpine Copper Age red deer with the populations from Mesola Wood (northern Italy) and Sardinia.

Sheep (Ovis aries) were domesticated in the Fertile Crescent region about 9,000-8,000 years ago. Currently, few mitochondrial (mt) DNA studies are available on archaeological sheep. In particular, no data on archaeological European sheep are available. Here we describe the first portion of mtDNA sequence of a Copper Age European sheep. DNA was extracted from hair shafts which were part of the clothes of the so-called Tyrolean Iceman or Ötzi (5,350 - 5,100 years before present). Mitochondrial DNA (a total of 2,429 base pairs, encompassing a portion of the control region, tRNA^Phe, a portion of the 12S rRNA gene, and the whole cytochrome B gene) was sequenced using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products. We have compared the sequence with the corresponding sequence of 334 extant lineages. A phylogenetic network based on a new cladistic notation for the mitochondrial diversity of domestic sheep shows that the Ötzi's sheep falls within haplogroup B, thus demonstrating that sheep belonging to this haplogroup were already present in the Alps more than 5,000 years ago. On the other hand, the lineage of the Ötzi's sheep is defined by two transitions (16147, and 16440) which, assembled together, define a motif that has not yet been identified in modern sheep populations.

Abstract:  Consuming honey can result in adverse effects owing to poisoning by bacterial (botulism) or plant toxins. We have devised a method to extract polymerase chain reaction (PCR) amplifiable DNA of up to c. 400 bp in length based on dialysis of a 15‐mL honey sample for 18 h against deionized water followed by sequential extraction using phenol, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol, and ether. Sequence analysis of PCR products obtained using “universal” plant, fungal, and bacterial primers targeted to the ribosomal RNA genes has allowed us to identify six different orders of plants (Apiales, Fabales, Asterales, Solanales, Brassicales, and Sapindales), two orders of fungi (Entylomatales and Saccharomycetales), and six orders of bacteria (Sphingomonadales, Burkholderiales, Pseudomonadales, Enterobacteriales, Actinomycetales, and Bifidobacteriales) in a single honey specimen.

The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation (“miscoding lesions”) has been the object of extensive investigations. To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350–5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Ötzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences. This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine→thymine/guanine→adenine) transitions and that type 1 transitions are essentially PCR artifacts.

Starting from specimens of the intestinal contents of the so-called Tyrolean Iceman or Ötzi (5,350–5,100 years before present), it was possible by polymerase chain reaction to amplify fragments of the human mitochondrial DNA (mtDNA) control region that correspond to the sequence found in 1994 at the Munich and Oxford laboratories and which had been attributed to the original DNA of the mummy. The particularly favorable condition of the specimens, showing very low contamination levels, made it easier to extend the analyses to the coding region, which had not previously been considered. The mtDNA of the European population is currently divided into nine (H, T, U, V, W, X, I, J, and K) main groups (haplogroups). The K haplogroup, in particular, is composed of two (K1 and K2) subclusters. The results demonstrate that the Iceman's mtDNA belongs to the K1 subcluster, yet it does not fit any of the three known branches (a, b, and c) into which the K1 subcluster is presently divided. In addition, some other sites, reported to be linked to environmental adaptation or pathologies, were investigated. Am J Phys Anthropol, 2006.

Analysis of ancient microorganism DNA represents one of the newest and most promising branches of molecular archaeology. In particular, microbial DNA associated with human remains can provide direct evidence of the occurrence and frequency of infectious diseases in historic times. Human mummies represent very interesting subjects for palaeomicrobiological investigations as they retain soft tissues. Recently reports on the identification of ancient bacterial pathogens in human mummies by DNA analysis are steadily becoming more numerous. However, despite this favourable trend, the analysis of ancient microbial DNA is still a contentious issue. As a model system, we studied the preservation of the intestinal microbiota's 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 (3.350-3.100 BC).