Minimum numbers: a consideration

This paper evaluates the various methods used to determine the numbers of individual animals from the numbers of bone fragments found in archaeological sites. It concludes that there is no single “best” method, since the one that is chosen depends upon the result that is desired, and on the particular features of the site and excavation.     

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.     

Recent carbon isotope research and its implications for African archaeology

A number of new developments in the carbon isotope field are reviewed, some of these having immediate and others potential application to African archaeology. Mass spectrometric analysis of¹³C/¹²C ratios in human and animal bone collagen makes it possible to reconstruct prehistoric foodwebs and to draw conclusions about dietary adaptations. Alternatively, where dietary behaviour is known, analysis of the bone of certain animal species allows for reconstruction of past environments. Ratio mass spectrometers are widely available in research institutions, and provided that the proper techniques for sample preparation are followed, this method of analysis should be of immediate use to most Africanists. It is already making an impact on African archaeology. The development of accelerator-based or high energy mass spectrometry (HEMS) radiocarbon dating provides the potential for extending the method back to 100,000 years, particularly in the case of bone samples. Practical applications, however, are still a few years off. In the immediate future HEMS dating is likely to be applied to small samples of ages less than 50,000 years. In competition with HEMS is the use of mini-counters for conventional radiocarbon dating of small samples. This procedure is available in a few laboratories with more under development. Dating of small samples has potential applications in many areas, specifically in the direct age measurement of critical specimens as opposed to the dating of associated materials.     

Biological mineral content in Iberian skeletal cremains for control of diagenetic factors employing multivariate statistics

The aim of this study was to define a strategy for a correct selection of bone samples by employing inductively coupled plasma optical emission spectroscopy (ICP-OES) for reconstructing the biological mineral content in bones through the determination of major elements, trace elements and Rare Earth Elements (REE, lanthanides) in skeletal cremains of ancient Iberians (III–II B.C), discovered in the Necropolis of Corral de Saus (Moixent, Valencia) between 1972 and 1979. The biological mineral content was determined taking into account diagenetic factors. A control method for a better reading of results was applied. To explore large geochemical datasets and to reduce the number of variables, Principal Component Analysis (PCA) was used, thus, providing a deeper insight into the structure of the variance of the dataset. PCA shows that the elemental profiles of bone and soil samples are clearly different. Bone samples obtained from the outer bone layer were shown to have a different elemental composition; more similar to soil samples than samples of the inner bone layer. PCA scores and loadings plots were preferred to dendrograms obtained using Cluster Analysis, due to the limits of the latter one to appreciate the spatial ordering of samples. Partial least squares discriminant analysis (PLS-DA), a frequently used supervised classification method, was applied to differentiate between degradation states of bone samples. PLS-DA results obtained in this study confirmed that changes derived from different burning conditions were associated with transformations in the mineral part of the bones. Accordingly, carbonized bones can be differentiated from cremated bones. Class assignment of bone samples with uncertain thermal conditions in dependence on their elemental composition has shown to be feasible. Consequently, for biochemical-archaeological studies the analysis and statistical classification of carbonized and cremated archaeological bones, as well as those exposed to unknown thermal conditions together with experiments in modern bones, are recommended.     

Carnivore competition, bone destruction, and bone density

In carnivore-modified archaeofaunal assemblages it is important to evaluate the degree to which carnivores have overprinted hominin behavioral signals. To examine the signals of increased competition for discarded bone, we present controlled experimental data on 33 simulated archaeological sites subjected to secondary consumption by spotted hyenas. We examine the relationship between competition, as measured by controlled numbers of hyenas and limb bones, and resultant levels of destruction and correlations between long-bone portion survivorship and bone density. Our results indicate that levels of destruction are equivalent regardless of the numbers of hyenas or long-bones included in the experimental assemblages. Correlations between long-bone epiphyseal and near-epiphyseal portions and bone density, however, do provide an indication of the level of competition. Results from the experimental study are used to highlight divergent levels of carnivore competition for hominin-discarded bone at the Plio-Pleistocene localities FLKN-Zinjanthropus and FLKN levels 1–2 from Olduvai Gorge, Tanzania.     

Characterisation and blind testing of radiocarbon dating of cremated bone

The success of radiocarbon dating of burned or cremated bones depends on the exposed temperature during burning and the degree of re-crystallisation of the inorganic bone matrix. We present a method for characterisation of likely cremated bones by employing visual inspection, infrared spectrometry and carbon stable isotope analysis on the bio-apatite fraction. The method of radiocarbon dating of cremated bones was tested by dating paired samples of bone and associated context materials such as pitch, charcoal and a dendrochronologically dated oak coffin. The dating of these paired test samples were largely performed as blind tests and showed excellent agreement between pitch and bone. The weighted mean age difference of all test samples is observed to −9 ± 60 ¹⁴C yr. To test the indicators and the effects of the degree of burning, a Late-Neolithic human individual has been studied, as this individual exhibits the full spectrum from low temperature burning (charred) to high temperature (“cremated”) from one end of a single bone to the other. This is reflected as a marked step in numerous parameters as well as in a significant difference in ¹⁴C age between the charred and the cremated bone samples.     

Isolation and Isotopic Analysis of Individual Amino Acids from Archaeological Bone Collagen: A New Method Using Rp‐hplc

This paper presents a new method for the isolation and isotopic analysis of some individual amino acids from proteins. The technique and its constituent steps are discussed; then isotopic analyses of amino acids from several samples of bone collagen from the Late Roman site of Poundbury, Dorset, UK are presented. The applications of the method are discussed, as well as some advantages of this technique relative to other methods. Although developed for use with archaeological bone collagen, the technique is equally applicable to other proteinaceous materials. The use of reversed‐phase HPLC avoids problems of isotopic fractionation inherent in using ion‐exchange HPLC. Amino acids are isolated preparatively, allowing both carbon and nitrogen isotopic values to be measured on a single sample using CF‐IRMS. Since amino acids are isotopically analysed in an underivatized form (unlike GC‐C‐IRMS), the method also presents the possibility of collecting the CO₂ generated during CF‐IRMS: this would allow the subsequent dating by ¹⁴C‐AMS of individual amino acids isolated from archaeological samples.     

URANIUM-SERIES DATING OF FOSSIL BONES FROM ALPINE CAVES

During the course of an investigation of fossil cave bear populations the uranium-series method for absolute age determination has been applied to bone material. The applicability of the method to bone samples from alpine caves could be demonstrated by the concordance of U/Th and U/Pa ages and cross-checks with the radiocarbon method. Stratigraphic agreement between bone ages and carbonate speleothetn ages also indicates the potential of the uranium-series method as a suitable tool for the age determination of fossil bones from alpine cave environments.     

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.     

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.     

Radiocarbon dating of bone by accelerator mass spectrometry

A companion article describes the sample preparation method used in the production of graphite targets for the Oxford accelerator mass spectrometer (AMS) system. The chemical pretreatment developed for the dating of bone, ivory and tooth samples is described in this paper.     

A novel and non-destructive approach for ZooMS analysis: ammonium bicarbonate buffer extraction

Bone collagen is found throughout most of the archaeological record. Under experimental conditions, collagen is apparently preserved as an intact molecule, with amino acid compositions and isotopic profiles only changing when almost all of the protein is lost. The ubiquity of collagen in archaeological bone has lead to the development of the use of collagen peptide mass fingerprints for the identification of bone fragments—Zooarchaeology by Mass Spectrometry (ZooMS). We report a novel, but a simple method for the partial extraction of collagen for ZooMS that uses ammonium bicarbonate buffer but avoids demineralisation. We compared conventional acid demineralisation with ammonium bicarbonate buffer extraction to test ZooMS in a range of modern and archaeological bone samples. The sensitivity of the current generation of mass spectrometers is high enough for the non-destructive buffer method to extract sufficient collagen for ZooMS. We envisage that a particular advantage of this method is that it leaves worked bone artefacts effectively undamaged post-treatment, suitable for subsequent analysis or museum storage or display. Furthermore, it may have potential as a screening tool to aid curators in the selection of material for more advanced molecular analysis—such as DNA sequencing.     

Serum Proteins in Archaeological Human Bone

Research on the decomposition of bone collagen offers the key to a wealth of hidden information, for instance, to understanding palaeoclimatic conditions; habitat-specific parameters such as altitude; and for the reconstruction of palaeodiet and subsistence patterns. Radiocarbon dating, one of the most commonly used geochronological techniques is also preferentially carried out on bone collagen. Because negatively charged ions, and especially phosphate groups, are responsible for the tight bonding between organic molecules and bone, soluble serum proteins need not necessarily be leached from buried bone. The authors' hypothesis is that intruding minerals, aided by recrystallization of the bone mineral matrix, and colloid formation, aided by humic substances, form protective layers that preserve serum proteins in bone even after long periods of burial. We have used electrophoresis and Western blotting to recover protein fractions from about 150 archaeological bones from various sites and epochs (up to 5500 BC ) and have succeeded in recovering, purifying all, and identifying some of the proteins. Molecular weight bands corresponding to albumin, transferrin and a-2HS-glycoprotein (A2HS) were frequently recovered. This paper presents the method for separating serum proteins from archaeological bone and for their identification. Twenty-five per cent of samples with the respective molecular weight band still gave a positive immunological reaction with antibodies. We conclude that non-collagenous proteins, especially serum proteins, may be well preserved in bone.     

Uranium and thorium distributions in fossil bones from Olduvai Gorge,Tanzania and Kanam,Kenya

The micro-distributions of uranium and thorium have been studied in fossil bone cortices in samples from Olduvai Gorge, Tanzania and Kanam, Kenya, and compared with modern bones collected from the surface at Olduvai Gorge. Concentrations in excess of 800 ppm U and 200 ppm Th are recorded in sections of some fossil bones, whereas concentrations do not exceed 20 ppm U or 1 ppm Th in the modern bones. In fossil bones, Th has a larger intra-bone variation than U. The distribution profiles of the two elements across the fossil bone cortices are quite distinct and reflect differences in their geochemical behaviour in the supergene environment. In particular, the two possible oxidation states of U, compared with one for Th, allow U to be more mobile than Th, but also more susceptible to variations in the redox potential of the depositional environment. In some fossil bone samples secondary leaching of U has occurred, whereas Th is retained in the bone cortex. U is incorporated into fossil bone earlier than Th.     

RESEARCH NOTES AND APPLICATION REPORTS ELEMENT DISTRIBUTION MAPS IN FOSSIL BONES

The combined application of fission track analysis and neutron activation-induced β-autoradiography to map the trace element distributions of scandium, cobalt, uranium and rare earth elements in fossil bone samples from East Africa is described. Both uranium and the rare earth elements are incorporated into bone apatite during fossilisation, whereas scandium and cobalt occur additionally in any iron-manganese minerals precipitated in pore spaces within the bone cortex. The distribution of uranium is different from that of the rare earth elements in the fossil bone cortices; it enters fossil bone more rapidly and is sensitive to changes in the redox potential of the palaeogroundwater.     

The estimation of the relative frequencies of mammalian species: Khirokitia 1972

The difficulties in the estimation of relative frequencies of species are discussed and a new approach to the recording of bone is proposed. This approach eliminates various biases and allows minimum numbers of individuals and counts of identified specimens to be derived from the same data. Sieved material from Khirokitia is used to provide a comparison of different methods and to illustrate possible ways of recording the information.     

Understanding the variability in freshwater radiocarbon reservoir offsets: a cautionary tale

Freshwater resources in past diets can lead to inaccuracies when attempts are made to ascertain their radiocarbon ages or those of the consumers. Radiocarbon reservoir effects may lead to significant age offsets when the bones or other tissues of these consumers are radiocarbon dated. A number of recent studies have investigated freshwater reservoir offsets. However no study thus far has satisfactorily obtained a ubiquitous freshwater reservoir correction due to variability in the ecosystems analysed. This study tests the possibility of predicting freshwater reservoir effects from the carbonate alkalinity of the water with measurements on modern fish bone and water samples. A predictive capability would be especially valuable in the absence of well-preserved archaeological fish bone. We surveyed samples from lakes and rivers in varying geological settings in Britain and Ireland. Modern fish bone and water samples were analysed to investigate modern radiocarbon offsets from the atmosphere. Archaeological fish bone was also analysed to examine past reservoir offsets at selected sites. Stable carbon and nitrogen isotope values were measured to aid in interpretation of any variability in the offsets. Large freshwater reservoir offsets were measured in some modern and archaeological samples (maximum offset = 1638 ¹⁴C years). The freshwater reservoir offsets in the fish bone were highly correlated with alkalinity of water in modern lake sites analysed. However, a high amount of variation within and between fish species was also evident in the results, precluding the possibility of providing regional corrections for freshwater reservoir offsets from alkalinity although this still may provide a general guideline. The variability is thought to be due to differences in the diet of individual fish.     

CT and micro‐CT analysis of a case of Paget's disease (osteitis deformans) in the Grant skeletal collection

This paper presents a case study in which Paget's disease of bone is differentially diagnosed in an individual from the Grant skeletal collection using non‐destructive computed tomography (CT) and micro‐computed tomography (micro‐CT) analyses of the pubis, in addition to plain film radiography and macroscopic examination. In archaeological and modern osteological samples diagnosis frequently relies on macroscopic examination, plain film radiography and histological examination of bone samples. CT and micro‐CT modalities provide researchers with a non‐destructive view of the internal structure of bone unhampered by the superimposition that is characteristic of plain film radiographs. Given the importance of the increased cortical and trabecular thickness in the differential diagnosis of Paget's disease, these techniques are ideal means by which to non‐destructively examine culturally‐sensitive and scientifically‐valuable human remains for signs of Paget's disease of bone. Copyright © 2009 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.     

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.