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.     

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.     

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.     

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.     

Bone survival: The effects of sedimentary abrasion and trampling on fresh and cooked bone

It is commonly assumed that, at least when considering similar sized animals, the bones from all taxa stand an equal chance of preservation. This paper summarizes one aspect of a larger study undertaken in order to assess whether this assumption is true, based on the results of experiments and observations into the effects of a range of pre-depositional processes. The rates of bone destruction by sedimentary abrasion and by trampling are determined for small mammal, fish and frog bones. Patterns of bone loss and fragmentation are examined both between species and within the skeleton, for fresh and boiled bone. It is shown that there is considerable interspecies variation in the ability of bones to withstand these physical forces. Frog bone proved particularly resistant, while within the fish, bone from the Gadidae was less resilient than might be expected, given its predominance in British Medieval archaeological sites and coastal sites of all periods. Within the skeleton, bone shape appears to be a very important determinant of relative survival. Boiling dramatically reduces bone's resistence to destruction. The physical properties of fresh, boiled and burnt bone are compared mechanically, and the dramatic loss of strength induced by heating is demonstrated. The often voiced assumption that fish bone is more prone to destruction than mammal bone is shown to have some validity. The results add more evidence to support the view that comparison of species abundance by fragment counts may not always be appropriate, and that interpretation of skeletal element frequencies should be approached with caution.     

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.     

Bone surface modifications in zooarchaeology

Cutmarks made by stone tools, conchoidal flake scars from hammerstone percussion, carnivore tooth marks, striations from sedimentary abrasion, and other surface modifications on bones from archaeological sites constitute a crucial body of evidence for investigating the role of human behaviors and of nonhuman taphonomic processes in site formation. This paper describes the various kinds of bone surface modifications produced by humans and by nonhuman processes and assesses the current status of bone surface modification studies with regard to such issues as the need for greater analytical standardization, the selection of instruments for examining bone specimens, tactics for identifying the origins of marks on bones, and strategies for inferring human behaviors.     

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.     

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.     

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.     

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.     

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.     

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.     

Why are cut mark frequencies in archaeofaunal assemblages so variable? A multivariate analysis

Cut mark frequencies in archaeological faunal assemblages are so variable that their use has recently created some skepticism. The present study analyses this variability using multivariate statistics on a set of 14 variables that involve differential skeletal element representation, fragmentation processes, carnivore ravaging impact, carcass size and tool type. All these variables affect the resulting cut mark frequencies reported in archaeological sites. A large sample of archaeofaunal assemblages has been used for this study. It was concluded that the best estimator of cut mark frequency in any given assemblage is the percentage of cut-marked long bone specimens (probably due to its better preservation than other anatomical areas), which is determined by fragmentation and carnivore ravaging. Carcass size and tool type also play a major role in differences in cut mark frequencies. Fragmentation is also a key variable determining the abundance of cut-marked specimens. It is argued that general cut mark percentages are of limited value, given the number of variables that determine them, and that a more heuristic approach involves quantifying cut marks in a qualitative manner.     

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 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.     

Reconsideration of criteria for differentiating faunal assemblages accumulated by hyenas and hominids

Zooarchaeologists have established several criteria for differentiating hominid‐ and hyena‐derived faunal assemblages. In some cases, however, the patterns of skeletal part representation and bone surface modification on which these criteria are ultimately based have been observed in fossil bone assemblages of unknown origin, rather than in modern assemblages of known origin. When the proposed criteria are evaluated within an actualistic framework, only three are able to differentiate between hominid‐ and hyena‐created faunal assemblages. I suggest that only these three criteria—proportions of carnivores to ungulates in the assemblage, the preserved condition of long bone specimens (either as whole cylinders or as splintered shaft fragments), and the types of bone surface modifications—should be retained as important factors in a diagnosis of the ancient bone‐collector. The remaining four criteria—the relative proportion of horn pieces in the assemblage, the relative representation of podial bones, the relative representation of small and large bovid skeletal parts, and bovid mortality profiles—are not relevant or applicable to the problem of differentiating hominid‐ from hyena‐derived faunal assemblages. Copyright © 2002 John Wiley & Sons, Ltd.     

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.     

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.