Isolina Marota

Ötzi's last meals: DNA analysis of the intestinal content of the Neolithic glacier mummy from the Alps

Samples of the intestinal content were collected from the ileum and colon of the Neolithic glacier mummy popularly known as the Tyrolean Iceman, or Ötzi. DNA was extracted from the samples and PCR amplified, using a variety of primer pairs designed to bind to different genes (mammal mitochondrial 12S ribosomal RNA gene, plant/fungal nuclear 18S ribosomal RNA gene, plant chloroplast ribulose bisphosphate carboxylase large subunit gene). This made it possible to distinguish between animal and plant food residues (macroremains) and pollen (microremains). According to the DNA reconstruction, the mans last meal was composed of red deer (Cervus elaphus) meat, and, possibly, cereals; this meal had been preceded by another one based on ibex (Capra ibex), different species of dicots, and cereals. The DNA spectrum corresponding to pollen residues in the colon, on the other hand, fits with the hypothesis that the last journey of the Neolithic hunter/warrior was made through a subalpine coniferous forest to the site at over 3,200 m above sea level, where his mummified body was to be discovered 5,000 years later.

Complete Mitochondrial Genome Sequence of the Tyrolean Iceman

The Tyrolean Iceman was a witness to the Neolithic-Copper Age transition in Central Europe 5350-5100 years ago, and his mummified corpse was recovered from an Alpine glacier on the Austro-Italian border in 1991 [1]. Using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products, we have retrieved the first complete mitochondrial-genome sequence of a prehistoric European. We have then compared it with 115 related extant lineages from mitochondrial haplogroup K. We found that the Iceman belonged to a branch of mitochondrial haplogroup K1 that has not yet been identified in modern European populations. This is the oldest complete Homo sapiens mtDNA genome generated to date. The results point to the potential significance of complete-ancient-mtDNA studies in addressing questions concerning the genetic history of human populations that the phylogeography of modern lineages is unable to tackle.

Phylogenetic analysis of Veneridae (Bivalvia): Comparison of molecular and palaeontological data

An approximately 400-by-long portion of the 16s rRNA gene sequence has been determined for the venerid clamsChamelea gallina (Chioninae),Dosinia lupinus (Dosiniinae),Pitar rudis,Callista chione (Pitarinae),Tapes decussatus,T. philippinarum,Venerupis (=Paphia)aurea (Tapetinae), andVenus verrucosa (Venerinae). Neighbor-joining and maximum parsimony trees support the results of traditional classification methods at the subfamily level but do not support the concept of a genusTapes. The transversion divergence rate estimated on the basis of the palaeontological record for theC. gallina/V. verrucosa separation and for the Pitarinae is very close (0.14–0.16% per Myr, respectively) to that of ungulates and cetaceans, while the Tapetinae exhibit a much higher (0.36% per Myr) rate.

Sequence analysis of bacterial DNA in the colon of an Andean mummy.

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.

Analysis of bacterial DNA in skin and muscle of the Tyrolean iceman offers new insight into the mummification process.

About 80 sequences (16s ribosomal RNA gene) of bacterial DNA in samples of skin and muscle taken directly from the Tyrolean iceman (3350-3100 years B.C.) or recovered during the 1992 archaeological expedition at the Alpine site were analyzed to obtain clues to the natural mummification process that allowed the corpse of the Neolithic shepherd/hunter to be preserved for more than 5,000 years. The investigation was made more complex by the fact that the surface of the mummy had been swabbed with phenol soon after the discovery (September 19, 1991). Our results show that no trace of microbial DNA is left on the actual surface of the body, while the untreated skin still bears the remains of large numbers of bacteria belonging to the genera Sphingomonas, Afipia, Curtobacterium, Microbacterium, Agromyces, and others. Compared to the untreated skin, the icemans muscle is also very rich in bacterial DNA. However, this DNA comes, with few exceptions, from the species Clostridium algidicarnis. The sharp difference in the bacterial DNA composition of skin and muscle suggests that the remains of the original cadaveric microflora of the latter have not disappeared during the icemans taphonomic history. On the other hand, the massive presence of C. algidicarnis, a cold-adapted sporigenous, the DNA of which was previously (Ubaldi et al. [1998] Am. J. Phys. Anthropol. 107:285-295) found in the soft tissue of a naturally desiccated Andean mummy, indicates that the hypothesis that the icemans corpse underwent rapid dehydration by the effect of a warm wind (föhn) is no longer plausible. The results best fit with the hypothesis (Bereuter et al. [1997] Chem. Eur. J. 7:1032-1038) that the body was first covered by snow and ice, and then underwent thawing and, finally, desiccation.

The small-subunit rRNA gene sequences of Venerids and the phylogeny of Bivalvia

Abstract. The complete nucleotide sequence of the 18S subunit of ribosomal DNA (rDNA) was determined for the venerid clams Callista chione (Pitarinae) and Venus verrucosa (Venerinae). Comparison of the new sequences with the published sequences of 1 annelid, 2 gastropods, 2 polyplacophorans, and 19 bivalves showed that when the annelids are used as outgroup the gastropods diverge from the bivalves, which form a cluster including the polyplacophorans. When the gastropods alone were compared with the bivalves, the latter split in two groups corresponding to the two subclasses of Heterodonta and Pteriomorpha. The former include two taxa that diverged early, Galeomma and Tridacna, while the Veneridae and Mactridae form two sister groups. In contrast to previous reports and in line with morphological data, the Ostreidae are included in the Pteriomorphia and form a monophyletic group.

Molecular ecology of a Neolithic meadow: The DNA of the grass remains from the archaeological site of the Tyrolean Iceman

The paper reports on the molecular analysis of samples of approximately 5,300-year-old grass found at the alpine archaeological site where the so-called Tyrolean Iceman was discovered. The grass comes from a ‘cloak’ made of long grass blades and/or the stuffing of the ‘snow footwear’ worn by the Iceman. The results show that while the largest fraction of the DNA extractable from the grass is of ‘foreign’ origin, a much smaller part belongs to the original genetic material of the grass itself, and can be used as a valuable taxonomic clue to the plant species utilized by neolithic men to manufacture their equipment. On the other hand, the ‘foreign’ DNA, or at least a portion of it, comes from microorganisms-mainly filamentous fungi and unicellular algae-which seem to have been associated with the grass since the time the grass was harvested.

Taxonomic and predicted metabolic profiles of the human gut microbiome in pre-Columbian mummies.

Characterization of naturally mummified human gut remains could potentially provide insights into the preservation and evolution of commensal and pathogenic microorganisms, and metabolic profiles. We characterized the gut microbiome of two pre-Columbian Andean mummies dating to the 10-15th centuries using 16S rRNA gene high-throughput sequencing and metagenomics, and compared them to a previously characterized gut microbiome of an 11th century AD pre-Columbian Andean mummy. Our previous study showed that the Clostridiales represented the majority of the bacterial communities in the mummified gut remains, but that other microbial communities were also preserved during the process of natural mummification, as shown with the metagenomics analyses. The gut microbiome of the other two mummies were mainly comprised by Clostridiales or Bacillales, as demonstrated with 16S rRNA gene amplicon sequencing, many of which are facultative anaerobes, possibly consistent with the process of natural mummification requiring low oxygen levels. Metagenome analyses showed the presence of other microbial groups that were positively or negatively correlated with specific metabolic profiles. The presence of sequences similar to both Trypanosoma cruzi and Leishmania donovani could suggest that these pathogens were prevalent in pre-Columbian individuals. Taxonomic and functional profiling of mummified human gut remains will aid in the understanding of the microbial ecology of the process of natural mummification.

Tracking Plant, Fungal, and Bacterial DNA in Honey Specimens*

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.