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.     

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.     

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.     

The strange case of Apigliano: early ‘fossilization’ of medieval bone in southern Italy

The preservation of the osteological material at the medieval site of Apigliano, in southern Italy, is characterized by bones with highly crystalline, and altered, mineral phases. In addition to this, some material retains perfect histological preservation, with the exception of small microfissures present throughout this structure. Diagenetic porosity is indicative of collagen loss via chemical degradation. The levels of residual collagen in these bones are much lower than is predicted from simple models of gelatinization, and thus a more complex explanation for the state of preservation must be sought. Possible explanations for the rapid loss of bone collagen are considered, including a high–temperature event, the acceleration of hydrolysis due to liming and extreme wetting and drying cycles.     

Are Teeth Better? Histological Characterization of Diagenesis in Archaeological Bone–Tooth Pairs and a Discussion of the Consequences for Archaeometric Sample Selection and Analyses

Teeth are often the preferred source material for isotopic and genetic assay involving ancient biomolecules. The assumption is that dental tissue preserves better due to its anatomically protected location, the enamel cap, and lower porosity compared to bone. However, this assumption has not been widely tested. Some similarities in diagenetic processes are to be expected due to similarities in structure and chemical composition of dentine and bone. This has led to the suggestion that bone can be used as an indicator of dental preservation, as a pre‐screening technique in the selection of suitable samples for biomolecular studies. Thus, direct testing of the correlation between bone and tooth preservation and diagenesis is needed. This paper reports the results of the histological characterization of diagenetic alterations within 25 human femur–tooth pairs, from a Medieval to modern (AD 1850) cemetery in Eindhoven, the Netherlands. The results showed that teeth do indeed preserve better overall, but not always, and that this was dependent on the main diagenetic factor(s) at the burial location. Furthermore, good correlations are found between the microstructural preservation of bone and teeth; similar processes of decay were observed within bone and teeth of the same individual. Overall, the study demonstrated that histological analysis of bone is useful for the identification of degradation processes that affect biomolecular preservation in skeletal material. In this way, sample selection and analytical strategies can be optimized. Copyright © 2013 John Wiley & Sons, Ltd.     

DNA in cremated bones from an early bronze age cemetery cairn

The discovery of polymeric DNA in charred wheat grains and other burnt plant remains prompted us to examine a set of cremated bones for the presence of ancient DNA. Extracts of cremated bones from an early Bronze Age cemetery cairn were analysed by hybridization probing and the polymerase chain reaction. Hybridization with two probes targeting human DNA resulted in strong positive signals. Polymerase chain reactions directed at a multicopy sequence in human nuclear DNA were successful, extracts of five burials giving amplification products of the expected size. Exploitation of ancient DNA in cremated bones could be of great value in archaeology as it may enable information pertaining to sex and kinship to be obtained from fragmented material.     

Well preserved non‐collagenous extracellular matrix proteins in ancient human bone and teeth

Ancient bones in a good preservation state, ascertained by microscopic techniques, conserve extracellular matrix proteins over thousands of years. With new techniques, intact extracellular matrix proteins from ancient bones and teeth are extracted and separated by one‐dimensional and two‐dimensional electrophoresis. Proteins were identified in Western blots by special antibodies against different human extracellular matrix (ECM) molecules of bone. We have confirmed different types of ECM human bone molecules such as osteonectin, osteopontin, and alkaline phosphatase with specific antibodies in human bone samples from different age groups. Additionally, we selected bone samples from different cultural time periods, such as the Middle Ages, the Bronze Age and the Late Pre‐Pottery Neolithic Phase (PPNB), and teeth from individuals from the Early Middle Ages and from the Late PPNB. The survival of intact extracellular matrix proteins in ancient bones and teeth dating from recent times to the Late PPNB, and reliable techniques to identify these proteins, present a great challenge to further research. A Match Set with PD‐Quest 7.2 shows that only 16% of protein spots in the teeth are also found in the bone of the same individual. In combination with the results of macro‐ and microscopic investigation, biochemical techniques will help us in obtaining a better understanding of bone and teeth in health and disease. Copyright © 2006 John Wiley & Sons, Ltd.     

Stable Carbon and Oxygen Isotope Spacing Between Bone and Tooth Collagen and Hydroxyapatite in Human Archaeological Remains

Spacing between stable isotope values in bones and teeth is a valuable tool for examining dietary influences and diagenesis. This study examines carbon and oxygen isotope values from collagen and hydroxyapatite (structural carbonate and phosphate) in archaeological human bones and teeth to derive species‐specific correlation equations and isotope spacing values. The δ¹³C_collagen and δ¹³C_{structural carbonate} in bone and dentin collagen show a strong correlation (R = 0.87, 0.90, respectively) with an average Δ¹³C_carb‐coll spacing of 5.4‰. The consistency of this isotope spacing with other large mammals and in humans with both low and high protein intake (as indicated by enriched δ¹⁵N values) suggests a similar allocation of protein‐derived carbon and whole diet‐derived carbon to collagen and structural carbonates, respectively, as other terrestrial mammals regardless of absolute meat intake. The δ¹⁸O_{structural carbonate} and δ¹⁸O_phosphate show the strongest correlation in enamel (R = 0.65), weaker correlations in dentin (R = 0.59) and bone (R = 0.35), with an average Δ¹⁸O_carb‐phos of 7.8‰. This isotope spacing is slightly lower than previously reported for large mammals and limited available data for humans. The results potentially indicate species‐specific fractionations and differing access to body water and blood‐dissolved inorganic carbonates in the presence of collagen formation. The use of correlation between δ¹⁸O_{structural carbonate} and δ¹⁸O_phosphate to determine diagenetic state is not recommended. The strength of this correlation observed in bones and teeth is variable and alternate indicators of diagenetic state (i.e. C:N ratios of collagen) provide more robust and independent evidence of isotope preservation despite presence/absence of a strong isotope correlation. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

The effects of PVAc treatment and organic solvent removal on δ13C, δ15N,and δ18O values of collagen and hydroxyapatite in a modern bone

The stable isotopic analysis of archaeological and paleontological bones has become a common method to examine questions of ecology, climate, and physiology. As researchers addressing such questions incorporate museum collections in their studies, it is necessary to understand the isotopic effects of common preservation techniques utilized in such collections to ensure the preservation of original isotopic values. This study examines the effects of PVAc glue (polyvinyl acetate) applied in acetone solution and the subsequent removal of PVAc using various organic solvents on the δ¹³C and δ¹⁵N values of extracted bone collagen, the δ¹³C and δ¹⁸O values of carbonate in bone hydroxyapatite, and the δ¹⁸O values of phosphate in hydroxyapatite. The data demonstrate that isotopic values in the collagen and phosphate are unaffected by any combination of PVAc treatment and solvent application. The carbonates show little variation in δ¹³C values, but exhibit variable δ¹⁸O values upon exposure to the PVAc solution. It is here suggested that δ¹⁸O values from carbonates in PVAc-treated bones do not retain an original isotopic value and should not be included in future studies.     

Patterns of Diagenesis in Bone II: Effects of Acetic Acid Treatment and the Removal of Diagenetic CO3

Archaeological bones of varying preservation have been treated with 0·1 M acetic acid in order to investigate the effect on structural and chemical alterations caused by diagenesis. Acetic acid is commonly used as a “cleaning agent” for removing diagenetic carbonate from bone and enamel, in an attempt to recover original, biogenic signals for use in dietary and¹⁴C dating studies.Diagenetic parameters were measured before and after treatment on a range of archaeological bones with good and bad preservation. Histological preservation defined the behaviour of the correlating parameters, where correlation coefficients between carbonate content and crystallinity, microporosity and macroporosity increased significantly after treatment. For histologically well preserved material, acetic acid is effective at returning carbonate content to around that of modern bone. Where bone is extensively damaged by micro-organisms, “loose” diagenetic material can be removed, but a fraction largely composed of hypermineralized bioapatite remains, which, we believe, cannot be reliably used to obtain accurate biological signals.     

An Investigation of Possible Fluorosis in Human Dentition Using Synchrotron Radiation

Macroscopic analysis of collections of archaeological human dentition from sites in the United Arab Emirates varying in chronological age and geographical location revealed the presence of enamel defects on the buccal, lingual and occlusal surfaces of some of the teeth. The morphology of these enamel defects suggested a possible diagnoses of fluorosis. Because not all teeth in the samples studied exhibited the defect, a conclusive diagnosis could not, however, be offered. Given that fluoride can enter the crystal lattice of hydroxyapatite (the major mineral component of calcified tissue including bones and teeth), and that the uptake of higher than optimal levels of fluoride can alter the molecular structure of dentition, it was thought that the application of X-ray diffraction to this problem may aid a more conclusive diagnoses. This paper details the application of synchrotron radiation to this problem. Initially, details of the archaeological problem are outlined. This is followed by a discussion of synchrotron radiation and the advantages it has over conventional X-rays. The materials and methods used in the analysis are provided, followed by a discussion of the results. While further work is required to fully attribute the enamel defects to the effects of fluoride, this paper illustrates that synchrotron radiation, in particular X-ray diffraction, can be applied to archaeological materials as a means of quickly and effectively obtaining a structural analysis.     

The limits of biomolecular palaeopathology: ancient DNA cannot be used to study venereal syphilis

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.     

Pre-screening techniques for identification of samples suitable for radiocarbon dating of poorly preserved bones

Under certain environmental conditions, post-depositional diagenetic loss of bone collagen can severely reduce the number of bones from a particular archaeological site that are suitable for stable isotopic analysis or radiocarbon dating. This study examined nearly 300 bones from 12 archaeological sites across southern England known to yield poor or variable preservation to try to identify one, or more, pre-screening technique(s) that would indicate suitable collagen preservation for radiocarbon dating. The most reliable method was shown to be the percent nitrogen (%N) of whole bone powder, which has an 84% chance of successfully predicting whether or not a bone will yield sufficient (i.e. >1% weight) collagen for dating.     

Implications of heat-induced changes in bone on the interpretation of funerary behaviour and practice

Bones submitted to heat experience structural and chromatic modifications. In particular, heat-induced bone warping and thumbnail fractures have been linked to the burning of fleshed and green bones – where the soft tissues have been removed from the bones soon after death – in contrast to dry bones. Those have been suggested as indicators of the state of the individual before being burned thus allowing inferences about the funerary behaviour of archaeological populations. A large sample of 61 skeletons submitted to cremation has been examined for the presence of both of these heat-induced features. Although uncommon, bone warping and thumbnail fractures were present in some of the skeletons demonstrating that its presence is not restricted to the burning of non-dried bones as generally believed. Rather than being an indicator of the presence of bones with soft tissues, bone warping seems to be more of an indicator of the preservation of collagen–apatite links which can be maintained on dry bones with low collagen deterioration. In addition, our results also do not confirm thumbnail fractures as an exclusive sign of the burning of bones with soft tissues. As a result, these heat-induced changes should be used with caution when trying to infer about the pre-burning state of an individual.     

Distinguishing between archaeological sheep and goat bones using a single collagen peptide

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.     

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.     

Diagenesis and survival of osteocalcin in archaeological bone

It has been demonstrated that the protein osteocalcin can survive in bone in the archaeological record, and postulated that it has the potential to survive over geological time periods. The precise mechanism for this longevity of survival is not yet fully understood, and has not been extensively studied in comparison to other diagenetic aspects of archaeological bone. We report a comparison between osteocalcin survival and the state of preservation of more than 60 bones from 14 archaeological sites. The amount of osteocalcin, assayed immunologically, was compared with diagenetic parameters that measure: the amount of ‘collagen’ in the bone, the mineral changes, the porosity, and the histological preservation of the material. The findings indicate that microbial taphonomy and mineral alteration of bone have a profoundly damaging effect on the preservation of osteocalcin.     

The crystallinity of ancient bone and dentine: new insights by transmission electron microscopy

We studied various archaeological and palaeontological bones and dentines from different burial environments by Fourier transform infrared spectroscopy (FT–IR), X–ray diffraction (XRD) and transmission electron microscopy (TEM), in the framework of a general study of diagenesis. FT–IR and XRD were used to evaluate the global preservation state of the bone and dentine mineral phase by determining a splitting factor (SF) or a crystallinity index (CI), respectively. These data can be combined with studies on the nanometer scale made with TEM. This latter technique,coupled with electron microdiffraction, provides determination of dimensions and shapes of individual bone and dentine apatite nanocrystals as well as of secondary minerals formed during diagenesis. It enables us to distinguish between heat–induced recrystallization processes and crystal growth in solution occurring during diagenesis.     

Experimental effects of bone size and temperature on bone diagenesis

In the ground, bone undergoes chemical and physical changes which affect its preservation. This fact has important implications for dating and other analytical procedures involving bone, as well as faunal analysis where differential preservation of bones of different species may affect conclusions regarding the relative significance of an animal to the economy of a given society. The diagenic processes in bone range from minor changes in the bone protein to complete structural and chemical breakdown.Using fresh cow bone, we conducted laboratory experiments which simulate the effect of temperature and bone size on the rate and nature of bone disintegration in archaeological sites. Temperature influences the rate of chemical change, and bone size and density affect the accessibility of the molecular constituents of bone to extrinsic chemical reactions. These findings clarify the importance of two well-known concepts in bone taphonomy. (1) The rate of chemical breakdown in bone tissues is related to the proximity of a given unit of tissue to the bone surface. This means that, in archaeological bone samples, tissue near the surface may be different chemically from tissue away from the surface and great care is necessary in choosing and preparing bone samples for analytical procedures. (2) In general, small bones are not as well preserved as large bones, therefore small animals are likely to be underrepresented in faunal assemblages.     

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.