DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.     

A bioarchaeological study of a Western Basin tradition cemetery on the Detroit River

Archaeological monitoring of construction in a Windsor city park on the Detroit River led to the discovery of an isolated cemetery containing the remains of eight individuals assigned to the Late Woodland Western Basin Tradition. At the request and consent of the contemporary First Nation community, tissue samples from five individuals were subjected to radiocarbon dating, mtDNA, and stable isotope analysis to confirm cultural affiliation and further understand the subsistence practices of these people. Radiocarbon dating placed the cemetery at the transition from the Younge phase (AD 900–1200) to the Springwells phase (AD 1200–1400). The stable carbon and nitrogen isotope results provide an unexpected but fuller understanding of Late Woodland Western Basin Tradition subsistence. All individuals were as enriched in carbon as those found on Iroquoian horticulturalist sites to the east, suggesting a very high reliance on maize. Nitrogen isotope values indicate that the protein component of the diet was comprised largely of high trophic level food sources, likely fish. An in situ osteological analysis identified a high number of carious lesions in the visible teeth, also suggesting a diet high in carbohydrates. The mtDNA findings support the antiquity of the Western Basin presence in Northeast North America through genetic links with the Hind Site, an Archaic site in southern Ontario. These results underscore the importance of such studies for providing novel insight into the archaeological histories and lifeways of this distinct Late Woodland tradition. This study also emphazises the need to work with descendant communities to provide them with information on the past that reflects their distinct heritage in the lower Great Lakes region.     

Assessing the effects of conservation treatments on short sequences of DNA in vitro

Little is known about what effects conservation treatments used to preserve human and animal hard and soft tissues have on DNA preservation. We have developed a method to assess quantitatively the extent of lesions or strand breakage caused by conservation treatments on short sequences of DNA in vitro. The method developed enables the determination of the percentage of DNA preserved following exposure to a conservation treatment solution relative to control samples, thereby allowing the direct comparison of treatments based upon their preserving/damaging effects on a DNA sequence. Forty-three chemicals commonly used in the preparation and/or conservation of human and/or animal remains were examined. We found that the majority were damaging, in particular and as expected, acidic treatments and treatments carried out at elevated temperatures. A few, primarily organic solvents, were not damaging. The approach we have adopted can be applied to screen other treatments either used in the past or for future conservation applications as they are developed to assess their effects on DNA. How these results should be interpreted in terms of conservation and sampling is also discussed.     

Amplification and sequencing of Trichuris trichiura ancient DNA extracted from archaeological sediments

Despite high prevalence of Trichuris trichiura infection, PCR-based analysis on T. trichiura from archaeological samples has not been established so far. In the present study, we sought to perform PCR-based amplification of T. trichiura aDNA using the sediments from medieval tomb of Korea. The presence of Trichuris eggs were first detected by microscopic observation; then confirmed by PCR-based aDNA analysis. Obtained sequence showed 100% homology to that of the small subunit ribosomal RNA (SSUrRNA) gene of T. trichiura but distinct from that of other Trichuris species. PCR-based aDNA analysis in this study can serve as effective method to confirm the presence of T. trichiura eggs in the soils or coprolites collected from archaeological sites.     

Ultrastructure and DNA sequence analysis of single Concentricystis cells from Alta Val Tiberina Holocene sediment

We successfully combined ultrastructure and chloroplast DNA analysis for single specimens of Concentricystis isolated from Holocene sediment by a palynology method based on filtration, progressive sieving and percoll-gradient sedimentation to concentrate fossil cells. We compared our method with the traditional harsh chemical treatment commonly used to isolate fossil pollen and spores. With our method, the cytoplasm of Concentricystis was sufficiently preserved to distinguish several cell structures by light and electron microscopy. DNA analysis of Concentricystis involved PCR amplification using specific primers for a spacer region of the chloroplast genome. Significant homologies were found with the Angiosperm chloroplast genome by BLAST DNA search for PCR product sequences.     

A modified sampler for uncontaminated DNA cores from soft sediments

A piston-type mud sampler is described. It is a development of the Livingstone corer and was designed to extract cores for DNA analysis of sediments. Prevention of contamination was paramount. As the entire system is self-contained and the sample is retained in the corer, it was found ideal for extracting very soft sediments which could not be captured by existing corers.     

Genetic analysis of archaeological wood remains: first results and prospects

A total of 51 ancient oak wood samples originating from various European archaeological sites, dating from the Neolithic period to the 18th century, were assayed for the presence of reproducible chloroplast (cp) DNA sequences. Five polymorphic chloroplast fragments were targeted. Only five of the samples could be fully genetically characterised, revealing four different oak cpDNA haplotypes. In all cases, the haplotypes detected on ancient woods and the haplotypes characterised from fresh samples from the same localities matched. Overall, this congruence is consistent with a genetic continuity between ancient and modern European oaks, confirming the hypothesis that the mapped genetic patterns largely reflect the original structure that established during the post-glacial. This stability of the genetic structure implies that, in the future, the technique could be used to infer or confirm the transport of wood by man, providing interesting perspectives for the genetic analysis of ancient woods.     

Variable nucleotide tandem repeat (VNTR) typing of two palaeopathological cases of lepromatous leprosy from Mediaeval England

Using ancient DNA methods, we have examined in detail two archaeological cases of leprosy from Mediaeval England. The first was a child skeleton with rhino-maxillary changes typical of lepromatous leprosy (LL). The second case was the skeleton of a male adult who showed both typical rhino-maxillary changes and osteitis/periostitis on the leg and foot bones. Bone powder was sampled from both cases and DNA extracts were prepared. These were subjected to a series of polymerase chain reactions (PCRs) specific for regions on the Mycobacterium leprae genome. The repetitive element RLEP was used for confirmation of M. leprae DNA and then three polymorphic regions were successfully amplified and sequenced to determine the number of variable nucleotide tandem repeats (vntr) at these loci. These were the microsatellite regions ML2344 and ML2172 and the minisatellite region ML0058. Genotyping data from the strains preserved within the skeletal remains were compared with those obtained for a reference strain of M. leprae. Variation at these three loci was found between both burials and the reference strain, indicating that vntr typing of LL cases from the archaeological record is a useful way of confirming disease and an additional means of authenticating aDNA data. This demonstrates the feasibility of targeting multiple loci for phylogenetic studies of leprosy strains from archival sources.     

A Mediaeval Case of Lepromatous Leprosy from 13–14th Century Orkney, Scotland

Erosion in the 1960s resulted in exposure of human skeletal remains from a Norse Christian cemetery at Newark Bay, Orkney, Scotland. One set of remains showed osteological evidence of advanced lepromatous leprosy, but the absence of bones from the lower limbs precluded definitive diagnosis. The aim of the present study was to determine whether Mycobacterium leprae could be detected in bone extracts, as a means of confirming the diagnosis of leprosy. Bone samples were examined from the suspected leprosy case and from a second contemporary burial thought to be free of disease. DNA was amplified by polymerase chain reaction (PCR) using primers specific for a repetitive element (RLEP) characteristic of M. leprae. Additional PCR tests specific for Mycobacterium tuberculosis and for amelogenin (a human gene suitable for sex determination) were also applied to the samples. M. leprae DNA was detected only in the skull sample from the suspected leprosy case. The DNA sequence was identical to that found in present day isolates of M. leprae. Positive results were obtained only using a PCR reaction designed to amplify relatively short stretches of DNA (<175 bp), suggesting the microbial DNA had undergone extensive fragmentation. There was no evidence of M. tuberculosis DNA in bones from the leprosy suspect or control individual. The ability to recover ancient samples of DNA provides an opportunity to study long-term evolutionary changes that may affect the epidemiology of microbial pathogens.     

Addressing seasonal site use through ancient DNA species identification of Pacific salmon at Dionisio Point,Galiano Island,British Columbia

We use ancient DNA analysis to identify Pacific salmon vertebrae to species in order to provide an important line of evidence that helps to establish the timing of seasonal residence at a Pacific Northwest Coast village site. Ancient DNA results from House 2 at Dionisio Point allow a characterization of the salmon fishery. Ten of eleven randomly selected smaller-sized salmon vertebrae were positively identified as sockeye salmon (Oncorhynchus nerka) while only a single pink salmon (Oncorhynchus gorbuscha) was identified. Of the 322 whole salmon vertebrae identified from House 2 occupation deposits during zooarchaeological analysis, 58 percent measure less than 8.0 mm and 70 percent are less than 8.5 mm in maximum transverse diameter. Together with documented aspects of the material record from Dionisio Point, most notably the vertebrate fauna from House 2, the indication that sockeye was the primary focus of the Dionisio Point salmon fishery suggests the site was inhabited during the spring and summer. This approach to the identification of season-specific site occupation has the potential for application over much of the Northeast Pacific.