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Abstract: Comparisons were made between two commonly used methods for the extraction of ancient DNA from charred plant remains. Using artificially charred wheat seeds, we show that silica-binding is the most efficient method for extraction of DNA. We describe a improved silica-binding procedure, including pre-incubation with N-phenacylthiazolium bromide and increased washing of bound DNA, which yields amplifiable DNA from seeds heated at 200°C for up to 8h, conditions which promote the formation of Maillard products which often copurify with aDNA and inhibit subsequent PCRs. We believe that this method will be effective in ancient DNA extraction with most types of charred archaeobotanical material. Both cold- and hot-start PCR procedures gave good amplicon yields with extracts prepared in this way, but cold-start PCRs also resulted in synthesis of short artefact products. Addition of bovine serum albumin to PCRs, an inert carrier substance thought to enhance amplification efficiency by binding contaminants, had no advantageous effect and in fact reduced amplicon synthesis. [Copyright &y& Elsevier]

Abstract: To determine whether ancient DNA (aDNA) can be used to study the palaeopathology of venereal syphilis, we carried out a comprehensive analysis of the preservation of human and pathogen DNA in a set of 46 bones of various ages, most of which displayed osteological indications of the disease. Bones came from seven English cemetery sites that were in use during the 9th–19th centuries. Twelve of the 46 bones consistently yielded mitochondrial DNA (mtDNA) sequences after replicate polymerase chain reactions (PCRs), and a further 13 bones yielded mtDNA sequences with less reproducibility. The sequence data enabled tentative mitochondrial haplogroups to be assigned to nine of the bones, and the identities and frequencies of these haplogroups were compatible with the geographical origins of the bones. Twenty-one bones consistently gave negative results with all mtDNA PCRs, indicating that at least these bones were not contaminated with modern human DNA, and those bones that gave positive results only yielded one sequence each, again suggesting that widespread modern contamination had not occurred. Mycobacterium tuberculosis sequences were obtained from seven bones, including three of five bones with tuberculous lesions. The cloned and direct sequences obtained from both the mtDNA and M. tuberculosis PCR products showed features typical of degraded aDNA. All of these results suggest that at least some of the 46 bones that we studied were suitable for aDNA analysis. All 46 bones were tested with nine different treponemal PCRs, each optimised to give a detection limit of ≤5 genomes. Although various bones gave PCR products of the expected size with one or more of these PCRs, sequencing showed that none of these products were authentic treponemal amplicons. Our failure to detect treponemal DNA in bones that were suitable for aDNA analysis, using highly sensitive PCRs, suggests that treponemal DNA is not preserved in human bone and that it is therefore not possible to use aDNA analysis to study venereal syphilis. Any past or future paper claiming detection of treponemal aDNA should therefore be accompanied by a detailed justification of the results. [Copyright &y& Elsevier]

To mimic damage occurring during preservation of ancient DNA, we heated wheat DNA at 95 °C for 2–21 days. Samples were amplified by PCRs directed at a 181-bp segment of the mitochondrial atpA gene. PCR products were cloned and multiple sequences obtained. We anticipated that nucleotide changes indicating damage would be randomly distributed, but this was not the case. Of the 124 cloned sequences, 107 displayed the same adenine to guanine change, this change being seen in clones from all of the eight heated samples. Fifteen of the cloned sequences, from six samples, showed the same apparent deletion at position 92. If non-random sequence changes are a widespread feature of DNA degradation then current strategies for analysis of ancient DNA sequences must be revised, as these could lead to nucleotide variations that are artefacts resulting from DNA diagenesis being mistaken for genuine sequence features of the ancient DNA. [Copyright &y& Elsevier]

In this paper we describe artificial heating experiments that were used as a model for the events that give rise to archaeological assemblages of charred grains. Samples of Triticum aestivum L. seeds were heated at 150, 200, 225 and 250 °C for periods up to 5 h. There were two distinct patterns of weight loss during heating, one due to expulsion of water and the other involving more substantial organic changes. Blue fluorescing material, typical of the intermediate compounds in the Maillard reaction, was observed after longer periods of heating and/or exposure to the higher temperatures. At each temperature there was a sharp initial decrease in the DNA content of the seeds followed by a gradual reduction in the rate of decay. At 150 and 200 °C, the DNA decay appeared to level off with very little additional breakdown occurring after 2 h, but at 225 and 250 °C the DNA content of the seeds reached undetectable levels at some point during the heating period. Polymerase chain reactions (PCRs) were used to study the degradation of the nuclear and mitochondrial genomes. PCRs were successful with seeds heated at 150 and 200 °C for all time points up to and including 5 h. At 225 and 250 °C, both PCRs failed after shorter periods of heating. The mitochondrial product was more resistant to heating, as expected as the mitochondrial genome is present at a higher copy number than the nuclear genome. PCR products from the mitochondrial locus were cloned and sequenced to determine if heating had any effect on the accuracy of sequence information obtained from charred wheat seeds. The frequencies of sequencing errors were similar, and low, in all six samples that were studied. All aspects of seed and DNA degradation were delayed in low-oxygen samples compared with the equivalent aerobic material. [Copyright &y& Elsevier]

There are many reports of ancient DNA from bacteria of the Mycobacterium tuberculosis complex (MTBC) being present in skeletons with and without osteological indications of tuberculosis. A possible complication in these studies is that extracts might also contain DNA from the microbiome of the individual whose remains are being analysed and/or from environmental bacteria that have colonised the skeleton after death. These contaminants might include ‘mycobacteria other than tuberculosis’ (MOTT), which are common in the environment, but which are not normally associated with clinical cases of tuberculosis. In this paper we show that MOTT of various types, as well as bacteria of related genera, are present in most if not all archaeological remains. Our results emphasise the complications inherent in the biomolecular study of archaeological human tuberculosis. The specificity of any polymerase chain reaction directed at the MTBC cannot be assumed and, to confirm that an amplification is authentic, a sequencing strategy must be applied that allows characterisation of the PCR product. Any variations from the reference MTBC sequence must then be checked against sequence data for MOTT and other species to ensure that the product does actually derive from MTBC. Our results also illustrate the challenges faced when assembling MTBC genome sequences from ancient DNA samples, as misidentification of MOTT sequence reads as MTBC would lead to errors in the assembly. Identifying such errors would be particularly difficult, if not impossible, if the MOTT DNA content is greater than that of the authentic MTBC. The difficulty in identifying and excluding MOTT sequences is exacerbated by the fact that many MOTT are still uncharacterized and hence their sequence features are unknown. [ABSTRACT FROM AUTHOR]

Abstract: A microfluidic device has been developed for the sex identification of ancient DNA samples and works by manipulating liquids within an environment of micrometer dimensions. In this work a range of microfluidic DNA extraction methods were evaluated for their compatibility with ancient DNA samples, and the use of streptavidin-coated super paramagnetic particles to isolate biotin-labeled abasic sites within damaged DNA was shown to be the most reproducible. Polymerase chain reaction-based DNA amplification was possible on the microfluidic device when less than 50 pg of template DNA was added. As a proof-of-principle, powdered bone samples were analysed using the integrated methodology developed. Following conventional capillary gel electrophoresis, two out of the three samples produced positive amplification results and were successfully identified as female. These sex identifications were corroborated by independent Amelogenin, anthropological and Y chromosome analysis. The work reported here is the first step in the development of a complete miniaturized microfluidic system that would enable on-site ancient DNA analysis. [Copyright &y& Elsevier]

Abstract: We investigated whether ‘next generation’ methods can be used to sequence ancient DNA molecules in charred cereal grains. We prepared a DNA extract from a mixed sample of barley, einkorn, emmer and broomcorn millet, taken from a 3300-year-old assemblage of charred cereal grains from Assiros Toumba, Greece. Using the SOLiD 5500 system, we obtained 21,112,844 unique sequence reads. Of these, 178,779 had a 75% or greater nucleotide sequence similarity with one or more entries in the full nucleotide sequence database; 496 of these matches were to previously reported sequences from barley, einkorn, emmer, broomcorn millet or related species such as hexaploid wheat. The unique reads were also compared with a database comprising only wheat sequences. This analysis identified 1658 charred grain sequences that had 90% or greater similarity with segments of the wheat genome. The presence of barley, wheat and millet sequences in the next generation dataset confirms the presence of ancient DNA in this charred grain assemblage. Enrichment of extracts by hybridization capture or equivalent methods is likely to enable sequences to be obtained for entire genes and other genomic regions of interest. [Copyright &y& Elsevier]

The pre-colonial 6th–14th century terracotta forms of Koma Land, Northern Ghana, contain cavities which may have been intended to hold liquids. These have been linked to traditional African libation, but the specific nature of their contents is unclear. We used generic polymerase chain reactions that would amplify DNA from a range of plant and fungal species in order to identify remains of libations applied to fourteen terracotta items. We anticipated difficulties in distinguishing genuine ancient DNA sequences from those resulting from contaminating material, and therefore also carried out a series of control experiments to assess the extent to which the samples had become contaminated with exogenous DNA during burial, excavation and downstream analysis. Taking account of the results of the control experiments, as well as the difficulties in assigning matches between ancient DNA sequences and database entries, we provide evidence for the use of three different types of plant – plantain/banana, pine and grasses – in libations associated with the terracotta items. We also identified DNA from Coniochaeta yeast within the mouth cavity of one figurine, suggesting that this structure was burnt prior to deposition. [ABSTRACT FROM AUTHOR]

Abstract: Previous analysis of the perinatal infant burials from Romano-British Yewden villa, Hambleden, indicated the practice of infanticide at that site. We attempted to determine whether this practice was specifically targeted at one sex or other by determining the sex of the infants using analysis of fragments of the amelogenin gene. We also analysed mtDNA in order to shed light on aspects of kinship. Thirty-three infants were analysed, and sex was successfully identified in 12. Seven were female, five male. No two infants shared identical mtDNA polymorphisms, indicating that all came from different mothers. Taken together with previous DNA results from perinatal remains from Romano-British sites where infanticide has been identified, they provide no evidence that manipulation of the sex ratio was a motivation for infanticide in Roman Britain. [Copyright &y& Elsevier]

Abstract: Excavations at Cladh Hallan, a Bronze Age-Iron Age settlement on South Uist in the Outer Hebrides off the west coast of Scotland, revealed the skeletons of two adults, a sub-adult and a child buried beneath the foundations of three roundhouses. Osteological and isotopic evidence has shown that the male adult skeleton is a composite made up of parts of at least three different individuals. To test the hypothesis that the female skeleton was also a composite we examined ancient DNA from four of its components: the skull, mandible, right humerus and right femur. Seven polymerase chain reactions (PCRs) were attempted, these covering positions 15,996–16,420, 16–132 and 232–368 of the mitochondrial DNA hypervariable I and II regions. Three PCRs were successful for each sample and a total of 55 sequences were obtained from the cloned products. After exclusion of possible contaminating sequences, the remaining 34 were compared. It was concluded that the mandible, humerus and femur come from different individuals. Insufficient data were obtained to draw conclusions regarding the origin of the skull. The presence of two composite skeletons at Cladh Hallan indicates that the merging of identities may have been a deliberate act, perhaps designed to amalgamate different ancestries into a single lineage. [Copyright &y& Elsevier]

Abstract: We typed five microsatellite loci in 52 landraces of Italian emmer wheat to determine if genetic analysis of cereals can provide information relevant to the spread of agriculture. Each of the five loci was polymorphic with 43 allele combinations identified in the 52 landraces. The allele combinations fell into two groups. Group 1 comprised 27 genotypes found in 42 landraces and Group 2 comprised 15 genotypes found in 10 landraces. The landraces with Group 1 genotypes showed a strong correlation between geographical and genetic distances (r = 0.601, p < 0.001) but those with Group 2 genotypes did not (r = 0.116, p = 0.244). We inferred that the Group 1 landraces might therefore retain a phylogeographical structure that reflects ancient events. We present a phylogeographical model for the spread of agriculture that enables the point of origin of crop cultivation to be predicted by comparison between the genetic and geographical distances between landraces. We applied this model to the Group 1 landraces by positioning 131 hypothetical points of origin around the coastline and northern border of Italy. The highest correlation coefficients between genetic and geographical distances were seen for hypothetical points of origin located on the coast of northern Puglia. We repeated the analysis with 1040 hypothetical points of origin located within the Italian peninsula. Again, the highest correlation coefficients were located in northern Puglia. These predicted points of origin correspond with the location of the earliest agricultural sites in Italy. The results show that plant genetics can be used to study the spread of agriculture. [Copyright &y& Elsevier]

Abstract: We explore the standards of research and reporting needed to justify the destructive analysis of archaeological human bone for biomolecular studies of ancient tuberculosis (TB). Acceptable standards in osteological interpretation have been met in some biomolecular papers, but there are also cases where insufficient care has been taken in distinguishing between pathognomonic lesions and those that are ‘consistent with’ a diagnosis of TB. Some biomolecular studies have failed to recognize that archaeological bones might be contaminated with environmental mycobacteria whose DNA could give rise to false positives in polymerase chain reactions directed at members of the Mycobacterium tuberculosis complex. The difficulties of applying spoligotyping to ancient DNA have also been underestimated and conclusions drawn from such analyses are often weakly supported. Assumptions that mycobacterial DNA preserves better than human DNA, and that contamination with modern DNA is less of a problem, has led in some cases to a laxity in research standards with insufficient attention paid to the need to authenticate ancient DNA results. We illustrate our concerns by reference to a recent paper reporting biomolecular detection of ancient TB DNA in skeletons from the eastern Mediterranean Neolithic settlement of Atlit-Yam. We are unconvinced that the skeletal evidence presented in this paper gives sufficient indication of TB to warrant destructive analysis, and we are concerned that during the biomolecular part of the project inadequate attention was paid to the possibility that results might be due to laboratory cross-contamination or to amplification of environmental mycobacterial DNA present in the bones. [Copyright &y& Elsevier]

Abstract: Between ca. 6000 BC and ca. 500 BC, barley cultivation spread across the continent of Europe from the extreme south to the extreme north. Carbon-dating would suggest that this spread, and indeed the spread of crop cultivation generally, varied in its pace, with ‘delays’ at certain points along its route. Such delays in the spread of agriculture have been explained as resulting from the slow assimilation of agricultural practices by existing indigenous human populations or as the time taken for the crops to adapt to novel climatic conditions, such as altered temperature regimes and day-lengths. A mutant form of the photoperiod response gene, Ppd-H1, causes barley to be non-responsive to long days, while the wild-type responsive form allows plants to flower in response to long days. We sequenced this gene in 65 ‘historic’ barley accessions, from the late 19th and early 20th centuries, in order to explore the potential role of environmental adaptation in the spread of agriculture. We chose to use ‘historic’ material, to complement the richer patterns in extant genetic lines, by spreading the data range in both time and space. Our ‘historic’ barley data shows a latitudinal divide in the Ppd-H1 gene similar to that found in extant lines, but with clearer geographical resolution, and extending northwards into the Arctic Circle. We discuss the implications of our results in relation to the dynamics of agricultural spread across Europe. [Copyright &y& Elsevier]

Abstract: Attempts were made to detect ancient DNA (aDNA) in samples of 88 human skeletons from eight Neolithic and Bronze Age sites in Greece and Crete. Ancient DNA was absent in specimens from Nea Nikomedia, Lerna, Karaviádena (Zakro), Antron Grave Circle A and Mycenae Grave Circle A. For each of three skeletons from Antron Grave Circle B that were sampled, polymerase chain reactions (PCRs) gave products for nuclear but not mitochondrial DNA, but amplicon yield was low and inconsistent with replicate PCRs failing to give reproducible results. With specimens from Mycenae Grave Circle B, evidence for mitochondrial aDNA was obtained for four of the 22 skeletons that were studied, and at Kouphovouno evidence for mitochondrial and/or nuclear aDNA was obtained with eight of the 20 skeletons that were examined. We conclude that, although aDNA might be present in some Eastern Mediterranean skeletons from later centuries of the Bronze Age, it is not commonly found in material from this period and is likely to be absent from older material. [Copyright &y& Elsevier]

Abstract: The richness of the burials in Grave Circle B at Mycenae, Greece indicates that the 35 people interred there held elite status during their lifetimes 3500 years ago. It has been speculated that the groups of burials represent different dynasties or branches of the same family. To test this hypothesis, we carried out an exhaustive ancient DNA (aDNA) study of 22 of the skeletons. We were unable to identify nuclear aDNA in any specimen, but we obtained authentic mitochondrial aDNA sequences for four individuals. The results were compared with facial reconstructions and interpreted within the archaeological context represented by the organisation of the graves and the positions of the burials within the graves. We conclude that the contemporaneous male Γ55 and female Γ58 skeletons, which both possess the UK mitochondrial haplogroup, were brother and sister. The implication is that Γ58 was buried in Grave Circle B not because of a marital connection but because she held a position of authority by right of birth. The results illustrate the difficulty in using aDNA to study kinship relationships between archaeological specimens, but also show that aDNA can advance understanding of kinship when used to test hypotheses constructed from other evidence. [Copyright &y& Elsevier]

To study the origins of South American maize, without the complicating factors introduced by the extensive movement of genotypes during the post-Columbian period, we made a genetic analysis of primitive landraces and preserved maize remains. Fifteen alcohol dehydrogenase 2 (Adh2) allele sequences were obtained from the landraces and 11 from the archaeological specimens. When these and six previously published Adh2 sequences were aligned, three allele groups were seen, distinguished by the structure of a dinucleotide repeat sequence. The three allele groups had distinct distributions within South America, the distributions supporting a model in which the two Central American agricultural systems—highland and lowland—generated separate expansions of maize cultivation into South America. One expansion centred on a highland culture that spread from Central America through the Panama highlands into the Andean regions on the western side of South America, and the second expansion centred on a lowland culture which spread along the lowlands of the northeast coast of South America, entering the continent through the river systems. The Adh2 biogeography was consistent with limited cultural contact across the Andes between northern Chile and Paraguay. From the diversity of the Adh2 allele sequences, we deduced that maize has undergone a rapid rate of evolution since domestication. [Copyright &y& Elsevier]