Pick the Right Pocket. Sub‐sampling of Bone Sections to Investigate Diagenesis and DNA Preservation

Many archaeological bones display a heterogeneous degradation pattern. Highly degraded bones could contain pockets of well‐preserved bone, harbouring good quality DNA. This dichotomy may explain why the relationships between global bone preservation parameters such as histological integrity, bone mineral crystallinity or collagen yield, and bulk DNA preservation/amplification success rate have been found to be at best, weak to moderate. In this pilot study, we explore whether or not a more localised approach will highlight a stronger relationship between diagenetic parameters and DNA preservation. This study includes a detailed histological characterisation of bone diagenesis in sub‐areas of three bone samples. Regions of the same bone, which displayed differential degrees of preservation or type of diagenesis were sampled for further analysis and both genetic (small scale Illumina MiSeq sequencing) and chemical (Fourier‐transform infrared spectrometric analysis) analyses were performed. The aim was to investigate how bone diagenetic processes relate to DNA preservation at a higher resolution than in previous studies. This is key in order to improve DNA analytical success rates. The expected relationship between bone and DNA preservation (retrieved endogenous DNA) was observed and the results corroborate previous work that DNA preservation is linked to the integrity of bone collagen and mineral. The results further suggest that non‐biological diagenetic alterations such as etching and the presence of mineral infiltrations and inclusions have a negative effect on DNA preservation/extraction. Copyright © 2016 John Wiley & Sons, Ltd.     

An improved PCR method for endogenous DNA retrieval in contaminated Neandertal samples based on the use of blocking primers

Neandertal skeletal remains are usually contaminated with modern human DNA derived from handling and washing of the specimens during excavation. Despite the fact that the distinct Neandertal haplotypes allow the design of specific primer pairs, for instance in most of the mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1), the human contaminants can often outnumber the endogenous DNA, thus preventing a successful retrieval of Neandertal sequences. We have developed a novel PCR method, based on the use of blocking primers that preferentially bind to modern human contaminant DNA and block their amplification, and greatly improve the efficiency of Neandertal DNA retrieval. We tested the method in four El Sidrón Neandertal samples (two teeth and two bone fragments) with different contamination levels and taphonomic conditions, and we have been able to significantly increase the Neandertal yield from figures around 25.23% (5–69.6%) up to 90.18% (75.3–100%).     

Biochemical and physical correlates of DNA contamination in archaeological human bones and teeth excavated at Matera,Italy

The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In this study the levels of endogenous contamination (i.e. present within the sample prior to sampling for the DNA analysis) are assessed within human bone and teeth specimens sampled from the cemetery of Santa Lucia alle Malve, Matera, Italy. This site is of exceptional interest, because the samples have been assayed for 18 measures of biochemical and physical preservation, and it is the only one identified in a study of more than 107 animal and 154 human bones from 43 sites across Europe, where a significant number of human bones was well preserved. The findings demonstrate several important issues: (a) although teeth are more resilient to contamination than bone, both are readily contaminated (presumably through handling or washing), and (b) once contaminated in this way, both are difficult (if not impossible) to decontaminate. Furthermore, although assessed on bone samples, several of the specific biochemical and physical characteristics that describe overall sample preservation, levels of microbial attack and related increases in sample porosity directly correlate with the presence of observable contamination in both bone and teeth samples from individual samples. While we can only speculate on the cause of this relationship, we posit that they provide useful guides for the assessment of whether samples are likely to be contaminated or not.     

Bone Preservation and Ancient DNA: The Application of Screening Methods for Predicting DNA Survival

Given the technical difficulties associated with ancient DNA research, any methods that help to identify samples that will yield amplifiable DNA will be of great value. This study examined the relationships between gross preservation, histological preservation, bone size and the ability to amplify short fragments of mitochondrial DNA in 323 goose humeri from the Anglo-Saxon site at Flixborough. Bone size was not a good predictor of the presence of amplifiable DNA, but there was a significant association between both gross and histological preservation and DNA survival. This suggests that it is worthwhile to preferentially select morphologically well-preserved bones for ancient DNA studies. Our results with ancient avian bone mirror those previously obtained with mammalian archaeological bone, although the relationship between DNA survival and histological preservation was stronger for the latter.     

The use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in ancient skeletons

The polymerase chain reaction has been used to extract ancient DNA from a wide range of different types of material. We have considered the possibility of finding residual bacterial DNA in bone that may have been infected with Mycobacterium tuberculosis using this technique. We propose a method of collecting samples and testing for the presence of degraded genetic material in ancient bone. The steps of the polymerase chain reaction are detailed and discussed, as are those for the preparation of the bone sample. Results obtained would suggest that this technique could have wide application in osteoarchaeological research by giving us new information on diseases of antiquity. Future avenues for the investigation of bacterial DNA in ancient bone are suggested.     

Bone preservation in human remains from the Terme del Sarno at Pompeii using light microscopy and scanning electron microscopy

Archaeological bone can show marked and complex alterations depending on the environment in which it was buried. In this study, the state of preservation of 27 femurs recovered from the archaeological site of Pompeii was evaluated by light microscopy and scanning electron microscopy. Most of the bone samples, prepared by the grinding method, showed good histological preservation, although they were characterized by microfissures (microcracking). Nine bone samples showed different states of histological preservation, including worst preservation (two femurs), due to diagenetic processes. Cryostat bone sections stained with thionin or 4′,6′-diamidino-phenylindole (DAPI) revealed the persistence of DNA within some osteocyte lacunae. Scanning electron microscopy analysis showed that ultrastructural characters, such as lamellae and collagen fibres, are recognizable only in unaltered bone. Our results reveal that most Pompeian samples are well preserved since they have a bone microstructure virtually indistinguishable from that of fresh bone. In methodological terms, although each of the various morphological methods used contributes information, histological and histochemical analyses are the most informative for studying the preservation state of bone and allow for rapid essential screening of archaeological bone.     

Ancient Iberian horses: a method to recover DNA from archaeological samples buried under sub-optimal conditions for preservation

Existing methods to extract, amplify, and sequence ancient DNA (aDNA) from horse bone and teeth were optimized to recover DNA from a depositional environment of highly permeable acidic soil. DNA was successfully retrieved using 0.10g of bone powder from horse (Equus sp.) remains dating to 25 K years utilizing the methods optimized for this archaeological material. The genetic analyses were performed in a facility that is dedicated to ancient DNA research (Paleo-DNA Laboratory, Thunder Bay, ON, Canada) and has not been previously used to analyse modern or ancient horse DNA. Research was replicated to obtain reliable sequencing results for six samples from the Iberian Peninsula that were consistent with published sequences of Equus caballus. The archaeological sequence data obtained support hypotheses that promote the significance that the Iberian Peninsula has had to the multi-focal centres of origin for horse domestication and distribution of modern horse breeds. The data presented may provide evidence of the existence of an Iberian refugium for Equus during the last glacial period, 10 K years BP. Further molecular data analyses will enhance the ideas presented by this data and our understanding of horse domestication and phylogeny. The optimization of molecular techniques to successfully obtain DNA using minimally destructive, cosmetically sensitive techniques from archaeological remains endeavours to foster further cooperation between museums and researchers.     

Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA

A principal problem facing human DNA studies that use old and degraded remains is contamination from other sources of human DNA. In this study we have attempted to contaminate deliberately bones and teeth sampled from a medieval collection excavated in Trondheim, Norway, in order to investigate this poorly understood phenomenon. Five pairs of teeth and bone samples were bathed in water containing various concentrations (from 10⁻⁹ and 10⁻²¹ g/l) of purified ΦX174 DNA. Subsequently the samples were subjected to a routine decontamination protocol involving a bleach bath followed by exposure to λ=254 nm ultraviolet light, prior to DNA extraction and analysis for evidence of the persistence of the contaminant. The results support previous speculation that bone is more susceptible to water‐borne sources of contaminant DNA, although both bone and teeth are readily contaminated and are difficult to decontaminate using the tested protocol. We believe that this is largely due to the porous nature of bone and teeth facilitating the deep penetration of the contaminant DNA. To simulate a more realistic handling situation, 27 further teeth were directly handled and washed, then decontaminated, prior to assaying for the residual presence of the handler's DNA. Surprisingly, although our results suggest that a large proportion of the teeth were contaminated with multiple sources of human DNA prior to our investigation, we were unable to contaminate the samples with further human DNA. One potential explanation may be the deposition of sediment or other structural changes that occur within the samples as they desiccate post‐excavation, which may protect samples from subsequent contamination, but also prevent the efficacy of bleach baths in decontaminating specimens. Copyright © 2006 John Wiley & Sons, Ltd.     

Wild or domesticated: DNA analysis of ancient water buffalo remains from north China

Recent zooarchaeological studies on water buffalo (Bubalus sp.) remains from China and south Asia question the traditional view that water buffalo were first domesticated in Neolithic China over 7000 years ago. The results from several recent population genetic studies of modern domesticated buffalo (Bubalus bubalis) are not consistent with each other, placing the original center of buffalo's domestication in south Asia, southeast Asia, or China. This paper reports a study using an ancient DNA approach to analyze water buffalo remains from Neolithic sites in north China to investigate their affinities with modern domesticated water buffalo, and to shed light on the origin of modern domesticated water buffalo in China.A 169 bp fragment of D-loop mitochondrial DNA was successfully amplified and verified for 13 of 24 bone samples obtained from seven archaeological sites along the Wei River valley in Shaanxi Province, China. The bone samples which yielded positive DNA can be dated to 8000–3600 cal. BP. The phylogenetic analysis of the obtained DNA sequences along with modern water buffalo sequences indicated that the ancient water buffalos were not the direct ancestor of modern domesticated water buffalo. However, the phylogenetic analysis, along with BLAST searches of these ancient DNA sequences, did demonstrate their relatedness to water buffalo more so than to any other bovid species, confirming the existence of indigenous wild (but now extinct) water buffalo species (B. mephistopheles) in ancient China.The DNA analysis of these ancient remains failed to establish direct links between modern domesticated water buffalo (B. bubalis) and indigenous water buffalo (B. mephistopheles) from ancient China. If further DNA studies of more ancient remains from other regions of China confirm the observation of solely indigenous water buffalo species in ancient China, it would suggest modern water buffalo might not have been first domesticated in China.     

Bone preservation and DNA amplification

The use of ancient DNA has increased during the past two decades in several scientific disciplines. However, the underlying mechanisms of DNA degradation in bone tissue are poorly understood. Here we address the importance of hydroxyapatite and collagen for DNA preservation in bone. We used two series of bones and teeth, one set of modern experimentally degraded bovid bones and one set of ancient horse bones/teeth. From these samples, we measured crystallinity, DNA presence and extracted collagen. The mtDNA fragments, parts of cytochrome b and the D–loop were amplified and sequenced. Our results show that presence of DNA was strongly related to the crystallinity in the hydroxyapatite and to the amount of collagen. This suggests that the hypothesis that hydroxyapatite has a crucial role in DNA preservation in calcified tissue is valid; and hydroxyapatite and collagen can be used to indicate whether DNA is present in the material. This is what would be expected if DNA is adsorbed to and stabilized by hydroxyapatite in calcified tissue, and collagen is part of the complex system that preserves DNA in bone tissue. Further, since collagen is the preferred material for radiocarbon dating, such bones may be a starting–point for a DNA analysis.     

Deficiencies and challenges in the study of ancient tuberculosis DNA

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.     

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.     

Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen

Sulphur isotope measurements of bone collagen from archaeological sites are beginning to be applied more often, yet there are no clear criteria to assess the quality of the collagen and therefore the validity of the sulphur isotope values. We provide elemental data from different methods (DNA sequences, amino acid sequences and mass spectrometric measurements) which are used to establish a reliable system of quality criteria for sulphur isotope analyses of bone collagen. The difference in the amount of sulphur from fish and mammalian collagen type I led to the suggestion to use different criteria to assess the in vivo character of the collagen between these two categories. For establishing quality ranges, the bone collagen of 140 modern animals were analysed. The amount of sulphur in fish and mammalian bone collagen is 0.63 ± 0.08% and 0.28 ± 0.07%, respectively. Based on these results we define for mammalian bone collagen an atomic C:S ratio of 600 ± 300 and an atomic N:S ratio of 200 ± 100, and for fish bone an atomic C:S ratio of 175 ± 50 and an atomic N:S ratio of 60 ± 20. These quality criteria were then applied to 305 specimens from different archaeological contexts.     

Histological identification of bone fragments in archaeology: telling humans apart from horses and cattle

In archaeology, it is not always possible to identify bone fragments. A novel approach was chosen to assess the potential of histology as an identification tool in those instances in which macroscopical study (e.g. by means of bone surface texture) has failed. This study concentrated on the diaphyses of long bones in three species of comparable size which are relevant to archaeology. Late juvenile and adult human diaphyseal bone structure was compared with the bone structure of horses and cattle. Since bone structure can differ even within a single bone, the restrictions in terms of bone category, bone part and species were deemed necessary for the development of a useful identification method for archaeological bone. The reference series comprised long bones from several individuals to broaden the insight into variations in diaphyseal bone structure within a single species. A general difference in the primary bone types was found between humans and the two large mammals. Human bone displayed lamellar bone types, whereas horses and cattle showed fibrous bone types. The only exceptions were separated growth layers. A difference in the secondary bone structure was also observed. A large number of connecting, primary (Volkmann's) canals, giving the secondary bone a reticular aspect, were common in horses and cattle. They were not, however, present in the human bones studied. To confirm the validity and applicability of these differences, a blind test was conducted on 15 diaphyseal fragments of identified long bones from archaeological sites. The results were very promising. Although four fragments could not be identified due to severe degradation, all the others were correctly attributed. Copyright © 2006 John Wiley & Sons, Ltd.     

A pilot study into the effects of X-ray and computed tomography exposure on the amplification of DNA from bone

This research investigates the intersection of radiography and aDNA, two commonly used methods in bioarchaeology. The goal of this project was to investigate the effects of radiation on the ability to amplify DNA from bone. Bones (n = 124) from domestic pig (Sus scrofa) feet were randomly sorted into a control group and four treatment groups: (1) single exposure X-ray; (2) single exposure CT; (3) multiple exposures X-ray; and (4) multiple exposures CT. Number of PCR cycles required to amplify DNA in 100 bp, 200 bp and 400 bp segments were used as a proxy for the amount of available DNA. In the 200 bp CT sample, distal phalanges required significantly more amplification cycles than did the other bones and were removed from analysis. Results suggest that in general radiation exposure fragments DNA thereby decreasing the amount that is amplifiable. While these results are suggestive, further research is required to elucidate the degree to which radiation fragments DNA in archaeological specimens.     

Comparing the survival of osteocalcin and mtDNA in archaeological bone from four European sites

The small mineral-binding bone protein, osteocalcin, has been applied in a number of studies on ancient bone due to predictions of its long-term stability. However, the intact protein has not been shown to survive in ancient bone devoid of DNA, which is a much more phylogenetically informative biomolecule. In this investigation, the survival of osteocalcin is directly compared to the amplification of mtDNA in a set of 34 archaeological samples from four sites throughout Europe. We also present unpublished osteocalcin sequences of seven mammalian species in addition to the 19 published sequences to highlight phylogenetic limitations of this protein. The results indicate that the intact osteocalcin molecule survives less in archaeological samples than mtDNA and is more subject to the temperature of the archaeological site. Amino acid analyses show the persistence of the dominant protein collagen in samples that failed both osteocalcin and mtDNA analyses. The implications these findings present for biomolecular species identification in archaeological and palaeontological material are that, although proteins do survive beyond ancient DNA, osteocalcin does not appear to be the most ideal target.     

Fish bone chemistry and ultrastructure: implications for taphonomy and stable isotope analysis

This paper reviews the ultrastructure and chemistry of fish bone, with an emphasis on zooarchaeology and stable isotope analysis. On the basis of the chemical composition of the collagen and the relationships between the collagen and mineral phases, fish bone is more susceptible to biotic and abiotic degradation than mammalian bone and is therefore less likely to be recovered in archaeological deposits. The amino acid composition of fish bone differs from that of mammals, most notably with respect to hydroxyproline content. The C:N ratio of fish collagen is, however, very similar and slightly lower than mammalian collagen, and thus the traditional range of acceptable C:N ratios for archaeological bone collagen (2.9–3.6) should not be shifted or extended for fish on the basis of the amino acid composition of collagen. An extensive survey of published archaeological bone collagen C:N ratios demonstrates that fish collagen from archaeological contexts tends to have significantly higher C:N ratios than mammalian collagen. The elevated C:N ratios in fish bone collagen may be the result of abiotic degradation processes that occur within the bone after death, the presence of exogenous humic contaminants, or endogenous lipid contaminants.     

Sex identification of ancient DNA samples using a microfluidic device

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.     

Molecular identification and characterization of Mycobacterium tuberculosis complex in ancient Egyptian mummies

A considerable number of molecular studies have provided evidence for the presence of Mycobacterium tuberculosis complex (MTB) DNA in ancient skeletal and mummified material. Moreover first studies on the differentiation of sub‐types of the MTB (M. tuberculosis, M. bovis, M. africanum, M. microti, M. canettii) have successfully been performed on ancient tissue samples. In our present study we extend previous analyses and investigate bone and soft tissue samples from 118 ancient Egyptian mummies and skeletons from the Pre‐ to Early Dynastic site of Abydos and different tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period (c. 2050–500 BC). The samples were tested for the presence of MTB DNA and further identified by spoligotyping. Twenty‐six samples provided molecular evidence for the presence of ancient mycobacterial DNA by amplification of a 123 base pair fragment of the repetitive element IS6110. Out of the 26 positive samples, 12 provided a complete spoligotyping signature, which was compared to an international database. Ten further cases showed an incomplete, patchy hybridization pattern, while in four cases no spoligotyping signature could be obtained. Interestingly, they all show either a M. tuberculosis or M. africanum pattern, but none revealed a M. bovis specific pattern. In the material from a Middle Kingdom tomb (used exclusively between c. 2050–1650 BC) several samples revealed a M. africanum type specific spoligotyping signature, while samples from later periods provided patterns typical for M. tuberculosis. This study clearly shows that spoligotyping can be used for the characterization of members of the MTB in historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type. Copyright © 2004 John Wiley & Sons, Ltd.     

Reliability of the Assessment of Periodontal Disease in Historical Populations

Periodontal disease is the destruction of all portions of periodontium. However, only periodontitis, based on whether attachment loss has occurred, can be detected in archaeological material. Therefore, several methods have been used to assess the frequency of periodontitis in past populations. The main scope of this study is to determine whether diagnoses of periodontitis through various methods are consistent with diagnoses obtained using the radiographic method. The samples (35 individuals, 576 teeth) were selected from Radom Cemetery's collection (in use between 1790 and 1811). For each individual, a periodontal status assessment was made using the following parameters: (i) measuring the distance between the cement–enamel junction (CEJ) and the crest of the alveolar bone (AC); (ii) the degree of root exposure; (iii) presence of the inter‐dental septum; (iv) dental calculus; (v) scoring system on the texture of the alveolar bone (Kerr's method) and (vi) radiography. In the studies, a correlation was found between the radiographic images and Kerr's method (r = 0.92) and root furcation (r = 0.96). Increasing degree of change on the alveolar bone is connected with increases in the CEJ–AC distance. A relationship between the frequency of the prevalence of dental calculus and changes on the alveolar bone was found. It means that the radiographic method, which is not always possible to use in archaeological collections, can be replaced by the presented macroscopic methods. Copyright © 2016 John Wiley & Sons, Ltd.