Collagen deamidation in archaeological bone as an assessment for relative decay rates

Attempts to extend methods for dating archaeological bones beyond that of radiocarbon dating, such as amino acid racemization, have met with limited success owing to the dependence on multiple environmental factors and controls. Despite facing similar challenges, deamidation of glutamine has recently been investigated as a potential indicator of ‘thermal age' in archaeological bones, as well as a measure of their preservation quality. In this study, we undertook a series of simulated diagenetic experiments to understand the various factors affecting deamidation. Further, we analysed bones from different Middle Palaeolithic layers from Grotte Mandrin (France), with the results suggesting potential use of deamidation for relative dating, but only in case of extremely well‐preserved layers. The results also suggested the possible use of attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy as a screening test for soluble collagen before proteomic analysis.     

Bone collagen preservation in the tropics: a case study from ancient Puerto Rico

Bone collagen is a well-characterized and generally robust protein that plays a key role in both radiometric dating and stable isotopic reconstruction of paleodiet. To be useful for such purposes, however, analyzed collagen must be relatively taphonomically unaltered. To date, little research has been conducted to document the taphonomic fate of bone collagen from archaeological sites in the tropics. In the present work, a large (n = 298) dataset of archaeological bone samples from sites on the island of Puerto Rico is examined by means of radiometric, chemical, and elemental analyses. The ultimate conclusion of this work is that while collagen loss may be accelerated in the tropics versus that seen in samples from higher latitudes, what collagen remains is typically sufficiently well-preserved and taphonomically unaltered as to make radiocarbon dating and/or stable isotopic analysis worthwhile.     

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.     

State of preservation of polymorphic plasma proteins recovered from ancient human bones

Protected by the mineral matrix, bone proteins are capable of surviving inhumation periods of several hundreds or thousands of years in soil. While the preservation of the bone matrix protein, collagen I, is the prerequisite for a variety of archaeometric approaches, such as radiocarbon dating and the reconstruction of palaeodiet by stable isotope analysis, little is known about both the rate and state of preservation of non‐collagenous proteins. We succeeded in the isolation, electrophoretic separation (SDS‐PAGE, IEF) and immunological detection (radial immunodiffusion, IEF immunoblotting and ELISA) of plasma proteins preserved in archaeological human bones. However, sample preparation and electrophoretic methods had to be adapted to the specific demands of these aged proteins, since they are not only degraded and fragmented but also cross‐linked to other organic components, either indigenous to the bone or to contaminants from the burial environment. Complex decomposition phenomena are responsible for the altered mode of migration of aged proteins through a gel. After isoelectric focusing, the ancient proteins mainly concentrate below pH 4.45 in the pH‐gradient. Thus, highly negatively charged protein components have a better chance of preservation in bone after death. Isoelectric focusing with subsequent immunoblotting of ancient protein samples revealed protein patterns which showed marked charge‐modifications in comparison with those of modern human plasma proteins due to protein degradation (e.g. α₂‐HS‐glycoprotein and α₁‐antitrypsin). Nevertheless, in combination with different immunological analyses, previous results concerning the selective enrichment of α₂‐HS‐glycoprotein in bone compared with other plasma glycoproteins could be confirmed. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

‘Old wood’ effect in radiocarbon dating of prehistoric cremated bones?

Numerous reports of successful radiocarbon dating of cremated bones have emerged during the last decade. The success of radiocarbon dating cremated bones depends on the temperature during burning and the degree of recrystallisation of the inorganic bone matrix. During cremation bones undergo major morphological and mineralogical changes which have raised some interesting questions and discussion on the origin of the carbon source in archaeologically cremated bones. Recent laboratory experiments reveal that the properties of the combustion atmosphere play a significant role regarding the source carbon in cremated bones. Thus radiocarbon dating cremated bones is potentially dating the wood used for the cremation fire. Here we compare a high precision radiocarbon dated human bone with an associated dendrochronological age from an oak coffin. We find that the age discrepancy between the dendrochronological age and the cremated bone of 73 ± 26 ¹⁴C yr is best accounted for by the so called ‘old wood’ effect.     

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones.     

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 KILL SITES

Age determinations of kill sites, where charcoal is generally absent, has usually depended on radiocarbon analyses of bone material. However, it has been seen with known-age samples that these dates are often in error due to contamination by plant products or ground water carbonates. The use of soil samples from strata both above and below, as well as the level containing the artifacts, offers important advantages. The soil profiles present sequences of dates, whereas the bones are only individual samples. The distribution of the soil date values can indicate upper and lower limits for the age of the kill site. The Taima-taima paleo-indian site in the state of Falcón, Venezuela is presented as an illustration of the application of radiocarbon dating of soil to archaeological problems. A total of 22 samples were processed. It is seen that soils from two profiles indicate a dating of 13 000 years for the kill. Taking into account reasonable extents of error, uncertainties of ± 2000 years can be estimated. The non-carbonate fraction dates are in agreement with the earth samples, but this is probably due to the organic matter extracted from these materials being mostly soil contamination in the porous bone matrix. Fluorine measurements on the bones confirm the Pleistocene origin.     

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.     

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.     

Experimental chemical degradation compared to natural diagenetic alteration of collagen: implications for collagen quality indicators for stable isotope analysis

Stable isotopic ratios from archaeological bone collagen are valid palaeodietary indicators, but depend on sufficiently well preserved molecules and several collagen quality criteria have to be fulfilled (mostly collagen wt%, C%; N%, C/N molar ratio). For a reassessment of these quality criteria, and a better understanding of the chemical degradation of bone collagen, experimentally degraded modern bones and 54 archaeological human bones were investigated. In the course of the experimental degradation, alterations of isotopic ratios were paralleled by altered collagen quality criteria. The contrary was found in the case of the archaeological specimens. This implies that the commonly used collagen quality criteria may be insufficient and do not guarantee that stable isotopic values of the gelatine extracts will still represent the original biological signal.     

Investigation of palaeodiet in the North Caucasus (South Russia) Bronze Age using stable isotope analysis and AMS dating of human and animal bones

This paper addresses the question of human palaeodietary adaptation in the Eneolithic and Early Bronze Age periods of the North Caucasus (South Russia) using stable isotope analysis. One of the key questions is the presence of fish in the diet. AMS radiocarbon dating of archaeological bone collagen has also been carried out to investigate potential radiocarbon reservoir effects in human skeletal material as a result of an aquatic diet. A fish component has previously been suggested in the diet of Iron Age and Bronze Age humans across Eurasia by isotopic research and radiocarbon dating of contemporaneous human and animal bones. In the North Caucasus however, isotopic data is scant. This study presents a new set of stable isotopic data from several Early Bronze Age sites, mainly belonging to the Maikop culture of the North Caucasus. The results show that the diversity in climate and environment across the northern Caucasus may be a causal factor for the patterns observed in the stable isotope values of terrestrial herbivores. This affects the isotopic values of the humans consuming them. The differences in δ¹⁵N and δ¹³C ranges of both humans and fauna were found to correlate strongly with geography and climate; the most enriched isotopic values are found in the dry steppe areas to the north. Overall, a relatively high enrichment in δ¹⁵N values of humans compared to local terrestrial herbivores and carnivores was observed. This indicates that aquatic resources were probably part of the Bronze Age diet in the region although the extent of this needs further investigation. The dramatic effect of environmental factors on isotopic values in the Early Bronze Age of the North Caucasus illustrates how confident conclusions cannot be drawn on the basis of a small number of samples from widely differing regions and time periods. Radiocarbon dating can provide a useful tool for identifying dietary derived reservoir ages in humans, potentially caused by a fish diet. With two possible exceptions, the nine human–animal bone pair dated as part of this study showed no consistent indication for a consistent reservoir effect.     

Túnel: A Case Study of Avian Zooarchaeology and Taphonomy

Although not often considered, there are many osteological characters unique to the avian skeleton that influence the taphonomy of bird bones. These characters are reviewed and their archaeological significance discussed herein. The presence of marrow in many avian long bones is important to interpretation of avian remains from archaeological sites because the presence of marrow affects bone density and, in turn, preservation. Other structural properties that affect avian bone preservation include cortical wall thickness, length and pneumatic state. Based on an analysis of approximately 10,000 bird bones from the archaeological site of Túnel, Tierra del Fuego, Argentina, I found that specific breakage patterns resulted from natural taphonomic processes acting as a result of the unique avian bone characteristics. This information may allow researchers to distinguish breakage patterns in avian bones resulting from natural taphonomic processes from breakage patterns that are culturally induced.     

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.     

Palaeoenvironmental and chronological investigations of the Magdalenian sites of Goyet Cave and Trou de Chaleux (Belgium), via stable isotope and radiocarbon analyses of horse skeletal remains

Trou de Chaleux and Goyet are caves situated less than 30 km apart in Belgium that contain stratified Magdalenian artefacts and butchered faunal remains. Published radiocarbon dates suggest that both sites were contemporaneously occupied during to the Late-glacial interstadial. It has previously been suggested that the Trou de Chaleux Late-glacial faunal remains might be slightly older than those at Goyet Cave, and that Trou de Chaleux has two phases of occupation during the Late-glacial interstadial. However, the limited number of radiocarbon dates currently available makes it impossible to determine whether the sites are truly contemporaneous, and the assessment of their absolute chronologies is complicated by a plateau in the calibration curve. In this paper, bone collagen δ¹³C and δ¹⁵N signatures of horse bones from the two sites are used to reconstruct local palaeoenvironments. We hypothesise that if occupations at the two sites were contemporaneous, the horse collagen δ¹³C and δ¹⁵N isotope signatures from both sites will be similar, since comparable carbon and nitrogen isotopic values would reflect similar diets, ecologies and environments. To provide clear dating parameters, new AMS radiocarbon determinations are also presented. Results show that horse collagen δ¹³C and δ¹⁵N isotope signatures at the two sites are different, indicating that the populations were not subject to similar diets, ecologies and environments. The new radiocarbon dates confirm that the horse bones from Trou de Chaleux date to the Late-glacial interstadial, but they also show that in Goyet Cave Horizons 1 and 2, older horse remains dating to ca. 32?000–27?000 ¹⁴C yr BP are mixed with Late-glacial horse remains. The δ¹⁵N values of the Goyet Cave horse bones radiocarbon dated to the Pleniglacial are substantially higher than those that have Late-glacial radiocarbon dates. Therefore, we now hypothesise that the majority of the horse bones isotopically analysed from Goyet Cave Horizon 1 are Pleniglacial in age. Statistical analysis of the radiocarbon dates indicates that the Late-glacial occupation at the two sites was contemporaneous, and that while there may appear to be two phases of occupation at Trou de Chaleux, this may be an artefact of the radiocarbon calibration curve.     

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.     

In situ Preservation Solutions for Deposited Iron Age Human Bones in Alken Enge,Denmark

The rich mass deposition of Iron Age human bone material from the Danish site, Alken Enge, is extraordinary not only from an archaeological perspective but also from a preservation point of view. The main find is situated in a waterlogged, anoxic environment which provides excellent preservation conditions and therefore enables in situ preservation of the human bones. However, major differences in local environmental conditions challenge an in situ preservation of the entire site area as parts of the bone material presently deteriorate. In this paper, a multi-proxy environmental monitoring approach is used to document threats and to suggest the best preservation solution for the archaeological finds, whether in or ex situ.     

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.     

Bone diagenesis in the European Holocene II: taphonomic and environmental considerations

We have applied cluster analysis to mercury intrusion porosimetry data from 219 archaeological bones (121 human and 98 animal) and soil chemistry data from 219 accompanying soil samples (1 per bone sample), to investigate the influence of soil chemistry on bone preservation. The samples chosen for the study were obtained from sites ranging in time from the pre-modern to the Mesolithic and were representative of burial environments across Europe (from the Baltic to the Mediterranean). These results represent the single largest database for archaeological bone preservation in the European Holocene to date and demonstrate the potential for large-scale diagenetic studies to help develop long term preservation strategies for our European heritage. Despite the variety of sites and environments, bones could be categorised into only four main diagenetic types. Furthermore, soil chemistry appears to significantly affect only one type of preservation, the pathway characterised by loss of mineral. In neutral to basic soils, taphonomy and in particular the differences between the treatment of human and animal remains, becomes the dominating factor in determining preservation. Using these results, strategies for heritage management of archaeological sites can be suggested; grouping sites into those requiring immediate excavation and those where in situ preservation is viable.