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 We also present unpublished osteocalcin sequences of seven mammalian species in addition to the 19 published sequences to highlight phylogenetic limitations of this protein.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.     

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 Diagenesis and Taphonomic History of the Paso Otero 1 Bone Bed, Pampas of Argentina

Samples of guanaco bone from an archaeological site in the Pampas of Argentina have been analysed to understand the diagenetic profile of the bone assemblages that characterized the taphonomic history of the site. Two archaeological occupations of Paso Otero 1 were investigated, encompassing similar landscape settings, climates, and depositional environments. The time span is a c. 2000 year period from c. 4800 to 2800 years . A total of 30 bone samples taken from both occupations were used to provide a preliminary characterization of the diagenetic pathways at the site. The parameters investigated provide a comprehensive account of how both mineral (hydroxyapatite) and bone protein (collagen) have been altered. In order to compare the two bone assemblages in terms of their diagenetic parameters, multivariate analyses were conducted. Results indicate two different diagenetic profiles in the site, % N being one of the variables that accounts for most of the variation in Paso Otero 1. The diagenetic analyses indicate that protein is less preserved in the bone assemblage from the middle stable landscape. Alternative interpretations of the diagenetic profiles are discussed in light of the taphonomic history of the site, and palaeoenvironmental information of the region. One hypothesis stresses the importance of the role of climate in defining the different diagenetic pathways, and the other the continued action of the combined diagenetic factors along time as the main explanation for the variability in the state of preservation of the bones in Paso Otero 1.     

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.     

The taphonomy of cooked bone: characterizing boiling and its physico–chemical effects

Cooking is perhaps the most common pre–burial taphonomic transformation that occurs to bone, yet it is still one of the least understood. Little progress has been made in determining a method of identifying cooked bone in the archaeological record, despite its import for various branches of archaeology. This paper attempts to describe boiling in terms of its physico–chemical effects on bone, and uses a suite of diagenetic indicators to do this. It is shown that cooking for brief periods of time has little distinguishable effect on bone in the short term, but that increased boiling times can mirror diagenetic effects observed in archaeological bone. The relationship between the loss of collagen and alterations to the bone mineral is explored through heating experiments, and the results compared with archaeological data.The possibility of boiling being used as an analogue for bone diagenesis in future studies is raised, and the key relationship between protein and mineral is once again highlighted as vital to our understanding of bone diagenesis.     

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.     

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.     

Actualistic research into dynamic impact and its implications for understanding differential bone fragmentation and survivorship

The relationship between bone mineral density and archaeological bone survivorship has played a critical role in zooarchaeological and taphonomic studies in recent decades. Numerous studies have suggested that higher-density skeletal element portions survive more frequently than lower-density element portions when archaeological assemblages are affected by some taphonomic processes. Interpretations of density mediated destruction have become commonplace in the archaeological literature, and are often used to explain the absence of certain bone elements and element parts in zooarchaeological assemblages. This study explores the effects of rockfall on bovid elements in varied environmental conditions and the differential survivorship of their element parts, and has implications for understanding the taphonomic processes through which bones are subjected to dynamic loading. Actualistic rockfall experiments conducted on twelve samples of frozen, fresh, and semi-dried bovid bones reveal that the generally low-density epiphyseal ends of bone elements resist fracture and analytical deletion with more frequency than the higher-density diaphyses. This evidence suggests that bone density does not correlate with likelihood of breakage or effective archaeological “destruction” when rockfall and other processes that result in dynamic impact are in action. While this research does not question the relationship between bone mineral density and the likelihood for archaeological survivorship as the result of some taphonomic processes, it presents one specific set of taphonomic processes that result in the differential survivorship of low density bone elements parts and the fragmentation and destruction of higher density element parts. This research presents evidence that shows that dynamic impact is a process capable of fragmenting and sometimes destroying high-density elements while low-density elements survive.     

Fruit and seed biomineralization and its effect on preservation

Mineralised fruits and seeds are frequently found in archaeological sediments but their chemical nature has not been often examined. The nature and the origin of these archaeobotanical remains have to be investigated to understand their taphonomic history. Fruits or seeds can be mineralised not only by replacement mineralisation but also by biomineralisation during the plant life. The mineral components of three fossil fruits sampled on the Pleistocene site of Dmanisi were analysed and compared with their modern analogues. Analyses were carried out by means of an environmental scanning electron microscope, equipped with an energy dispersive X-ray device and by means of a Fourier transform infrared spectrometer. Biogenic carbonates and/or biogenic silica were identified in the fossil and modern fruits of some taxa. Comparison between fossil and modern specimens has shown that molecular reorganisation occurred in carbonate and in biogenic silica during fossilisation, through diagenetic processes. The resulting stable mineral structures confer an exceptional preservation to fruits in sediments. Taking into account these taphonomic specificities (transformation and differential preservation), the chronological and palaeoenvironmental aspects of the mineralised fruits are discussed.     

Restrictions on fluorine depth profiling for exposure age dating in archaeological bones

Archaeological fragments of bone and teeth that are exposed to a humid environment take up fluorine from the surrounding soil. The fluorine ion replaces the hydroxyl group in the mineral phase of the bone, forming chemically more stable fluorapatite. In cortical parts of the long bone diaphysis a fluorine concentration profile can be observed, which decreases from the outer surface and the marrow cavity towards the inner parts of the bone matrix. Geological time spans are needed for this process to reach equilibrium and for the distribution to become uniform. As the shape of the profile, which can be described by a diffusion model, contains information on the exposure duration of the fossil object, several attempts to use fluorine profiling as a dating method have been undertaken. The distribution of fluorine in an archaeological sample however is strongly influenced by environmentally induced processes of bone diagenesis, i.e. alteration in the structure and composition of the mineral phase and degradation of organic components that may make the time information indistinct. The primary chemical composition of bones can thus be obscured by diagenesis within tens, hundreds or thousands of years. This depends more on the diagenetic environment than on the geological age. To observe the impact of environmental influence on the profile shape, samples from several burial sites featuring various soil conditions have been analyzed for their fluorine distribution and preservation state. This paper provides an overview on the restrictions that have to be considered when attempts are undertaken to relate a fluorine diffusion pattern to the archaeological age of a bone specimen.     

Quantifying histological changes in archaeological bones using BSE–SEM image analysis

Recent years have seen rapid developments in the understanding of diagenetic changes to archaeological bones. In particular, the degradation or preservation of proteins and other biomolecules has been explored using an increasingly sophisticated battery of analytical techniques. Problems remain, however, in correlating these parameters with physical changes to bone that may be observed microscopically. This is due, in part, to the problems in reproducibly quantifying histological changes to archaeological bone. This paper introduces a novel method for the accurate quantification of these changes employing image analysis of SEM images. Self–consistency of results was tested using measurements of total calcium content at different magnifications. The term ‘bioerosion index’ is suggested for the measured parameter.     

In situ preservation of archaeological bone: a histological study within a multidisciplinary approach

To make preservation in situ a serious option for the management of archaeological sites, research has to be done on the factors affecting conservation of different archaeological materials, including bone. A European project has been started which deals with bone degradation in a multidisciplinary way. The goals of the project are to develop techniques to describe the preservation of archaeological bone, to make a classification of soil environments according to their preservation potential and to detect what factors in the environment of the bone affect its conservation. One technique used in this project to determine the state of preservation of archaeological bone is histology. The relevance of this technique for archaeological heritage management research is discussed.     

Bone diagenesis in the European Holocene I: patterns and mechanisms

Diagenetic changes in archaeological bone are known to influence the data derived from such material and have thus been the subject of numerous studies. A number of general trends have been observed, but many of the processes that occur are still not fully understood. We present here the analysis of 195 bones excavated from 32 sites in five different countries from Eurasia characterized using 10 simple diagenetic parameters. The results reveal that most European Holocene archaeological bone can be categorized by only four main diagenetic states related to three distinct trajectories that describe more than 60% of the variation in these parameters. Given the potential amount of variation in the dataset the small number of diagenetic pathways is surprising, but highlights the large importance of a few key factors that influence bone diagenesis. Mercury intrusion porosimetry is a key technique for identifying modes of degradation.     

Infrared and Isotopic Evidence for Diagenesis of Bone Apatite at Dos Pilas, Guatemala: Palaeodietary Implications

The use of stable carbon isotopic analysis of bone apatite to reconstruct prehistoric diets is hindered by the possibility of diagenetic alteration of carbonate during burial. We examine apatite preservation in Classic Period Maya skeletal remains from Dos Pilas, Guatemala, using Fourier transform infrared spectroscopy (FTIR). We use weight % CO₂evolved from apatite, FTIR carbonate/phosphate absorbance ratios, phosphate peak splitting crystallinity indices, fluoride peaks, and stable oxygen isotopic ratios to identify diagenetic change in apatite chemistry. Isotopically light carbon taken up from burial soil is adequately removed from most Dos Pilas bone by treatment with dilute acetic acid, but more severe alteration cannot be reversed by standard preparation methods. Infrared criteria identify recrystallized apatite in a subset of Dos Pilas burials, that is accompanied by isotopic exchange, and that no longer preserves biogenic δ¹³C. These results illustrate that comparatively recent bone may be diagenetically altered and demonstrate a need for systematic evaluation of mineral integrity in all archaeological bone prior to interpreting paleodiets with apatite δ¹³C.     

An assessment of the value of bone density measurements to archaeoichthyological studies

This paper reviews the meaning of the term ‘density’ and the problems associated with the methods of density determination for animal bones in archaeology. It has often been assumed that density is the intrinsic property of most influence in controlling the rate of a bone's decay. Values for whole bone density have been published only for large mammal bone, however. Fish bone appears to be particularly vulnerable to decay, and usually a restricted range of skeletal elements are recovered from archaeological sites. The object of this study was to examine the relationship between fish bone density and the ability of the bone to survive on occupation sites and in archaeological deposits. A set of ‘density’ measurements was established for the bones of cod (Gadus morhua). The usefulness of these measurements as a predictive tool in archaeoichthyological studies is assessed. It was found that ‘density’ as measured did not explain adequately the relative survival of skeletal elements after mechanical abrasion and weathering, or within archaeological deposits.     

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.     

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.     

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.     

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.     

Uranium and Vanadium Concentrations as a Trace Element Method for Identifying Diagenetically Altered Bone in the Inorganic Phase

Archaeologists have generally avoided analyzing inorganic isotopes in bone because of its high porosity, large crystalline lattice spacing, and small crystallite size, making it particularly susceptible to diagenetic alteration. Because the inorganic isotopes are left unstudied, we lose a significant portion of information pertaining to an individual’s life history, such as migration, health, and ranging behavior. Tooth enamel, which does not have the same susceptibility to diagenesis as bone, can be used to extract this information but this means that taxa lacking teeth, such as birds, some species of fish, and some reptiles, are excluded. Here, we present a method that can be used to identify diagenetic alternation in bone. This is done by focusing on abnormal concentrations of vanadium and uranium. Neither element is readily bio-precipitated into hydroxyapatite due to ionic radius, vibrational frequency, atomic mass, and ionic charge. This makes them an ideal marker for diagenetically altered bone. Vanadium occurs in very low concentrations in modern bone, while archaeological bone shows clear evidence of normal, non-diagenetically altered values alongside high concentrations of vanadium in diagenetically altered bone. Uranium also is a measure of diagenetic alteration, as modern bone has concentrations below detectable limits (0.017 ppb), while some archaeological bone contains uranium above detectable limits. The biogeochemistry of these elements in soil and bone are discussed with implications for enamel studies.