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.     

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 long–term survival of bone: the role of bioerosion

Fossil bones (N = 350) spanning more than 350 million years, and covering a wide range of depositional environments, were studied to compare the distribution of microbial destruction features in fossil bones with previously published data sets of bones of archaeological age. The distribution of bioerosion in fossil bones is very different from that found in bone from archaeological sites. Fossil bones typically show little or no bioerosion. Under normal conditions, if a bone is to survive into the fossil record, then rapid bioerosion must be prevented (or halted). This conclusion suggests that early post mortem processes,such as the mode of death, influence the potential of any bone to survive into deep time.     

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.     

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.     

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.     

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.     

Sub‐micron spongiform porosity is the major ultra‐structural alteration occurring in archaeological bone

Total pore volume and pore size distribution are indicators of the degree of post‐mortem modification of bone. Direct measurements of pore size distribution in archaeological bones using mercury intrusion porosimetry (HgIP) and back scattered scanning electron microscopy (BSE‐SEM) reveal a common pattern in the changes seen in degraded bone as compared to modern samples. The estimates of pore size distribution from HgIP and direct measurement from the BSE‐SEM images show remarkable correspondence. The coupling of these two independent approaches has allowed the diagenetic porosity changes in human archaeological bone in the >0.01 µm range to be directly imaged, and their relationship to pre‐existing physiological pores to be explored. The increase in porosity in the archaeological bones is restricted to two discrete pore ranges. The smaller of these two ranges (0.007–0.1 µm) lies in the range of the collagen fibril (0.1 µm diameter) and is presumably formed by the loss of collagen, whereas the larger pore size distribution is evidence of direct microbial alteration of the bone. HgIP has great potential for the characterization of microbial and chemical alteration of bone. Copyright © 2002 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.     

Preservation of fungi in archaeological charcoal

During the analysis of wood charcoal remains from archaeological sites, it is common to find different microorganisms and different forms of degradation present in the plant tissue. However, one may encounter difficulties when attempting to identify these microorganisms and determine when their attack occurred. This paper focuses on preservation aspects related to the microorganisms in wood and demonstrates the structural changes that take place in different types of decayed wood after it was converted into charcoal. The study seeks to determine whether the microbial attack found in archaeological woods took place before the burning of the wood or after. Burning experiments were conducted using wood that had been decayed by various types of fungi including white-rot, brown-rot, and soft-rot. The laboratory burnt wood samples showed decay patterns that were comparable to those observed in archaeological charcoal samples, indicating that signs of fungal infestation and features of decay can be preserved after burning with micromorphological details of mycelium and cell wall attack evident. This indication may provide important information related to the gathering of deadwood as fuelwood. In addition, examples of decayed wood preserved in archaeological charcoal assemblages are described. Their relationship to the archaeological context and environmental conditions may suggest different interpretative models concerning wood management strategies applied by past societies.     

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.     

Confocal laser scanning microscopy: a flexible tool for simultaneous polarization and three-dimensional fluorescence imaging of archaeological compact bone

Wide-field polarized light and epifluorescence microscopy have been used to enhance analysis of archaeological bone tissue, providing information on bone formation, modeling, pathology, preservation, age estimation, and biomechanics. Though valuable, these techniques are limited by their inability to remove out-of-focus light and view multiple levels of a sample, restricting our understanding of the three-dimensional (3-D) microarchitecture of compact bone. Modern technological advances, such as microscopic computerized tomography, allow increasing resolution in 3-D bone imaging, but do not allow fluorescence labeling or polarized-light analysis. Confocal laser scanning microscopy (CLSM) is a valuable tool for 3-D histology. However, its application to the study of compact bone is lacking, especially in archaeological and forensic sciences. The current study investigated CLSM as a tool for fluorescence and polarized-light microscopy of archaeological compact bone in order to demonstrate its advantages. Standard techniques and CLSM are compared in their suitability for imaging well preserved archaeological bones from the Dakhleh Oasis, Egypt. CLSM's high resolution, multi-channel, two- and three-dimensional capabilities augment the flexibility and creativity of compact bone imaging and have the potential to increase the accuracy of quantitative medical and anthropological histomorphometric techniques. CLSM is specifically suggested as a useful tool for the investigation of ancient bone fluorescence caused by the presence of tetracycline and/or other fluorochromes.     

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.     

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

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

Destruction of microstructure in archaeological bone: a case study from Portugal

Sampling of archaeological human bone may not be justified, contrary to former high expectations regarding adult age assessment based on histomorphometry. The alterations in buried bone as a result of bacterial action are readily visible in the scanning electron microscope (SEM). An understanding of the chemical and structural changes to cortical bone requires work at the level of a few microns. This paper reports on problems encountered during analyses of samples of human bone from Mesolithic (ca. 8000 calbp) shell midden sites at Muge in central Portugal, and the methods used to try and overcome these problems. We believe we have shown that these Mesolithic bones are partly comprised of bacterially reprecipitated mineral, which has had collagen removed, with consequent obliteration of bone microstructure. We conclude that microbial destruction of the structure of archaeological bone can be a serious impediment to analysis of the characteristics of the population represented by those skeletal remains. Copyright © 2001 John Wiley & Sons, Ltd.     

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.     

Tracking changes in bone fracture morphology over time: environment, taphonomy, and the archaeological record

Zooarchaeologists have often employed studies of bone fracture morphology as a means of understanding past human cultural activity, and various methodological approaches have been developed for analyzing archaeological broken bone assemblages. It is widely understood that bones degrade over time, however, few studies have attempted to define and quantify the rate at which bones degrade and fracture morphologies change. This study examines degradation in frozen bones (−20 °C) and bones exposed to hot (40 °C) dry conditions. These two simulated environmental conditions represent extreme real-world climates, and allow for an actualistic understanding of the rates of degradation that bones experience in nature. When frozen, bones degrade slowly but significantly, and demonstrate measurable differences in samples frozen for 1, 10, 20, 40, and 60 weeks. In hot, dry conditions, bones degrade very quickly, and demonstrate measurable differences after 1, 3, 7, 14, and 21 days. These data allow for a more detailed understanding of the relationship between the cultural and natural processes that result in bone fracture, and the time period during which bones can be expected to maintain fresh fracture characteristics. This research also has implications for understanding human subsistence and survival strategies and for interpreting the archaeological record.     

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.     

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.     

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.