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.     

The survival of biochemical information in archaeological bone

The paper gives a review of the advantages of genetic information potentially available from surviving molecules (especially proteins) in bone. Survival of such information depends on degradative processes during burial. Conventional protein chemical and immunochemical investigations on various organic components remaining in buried archaeological bone are described. The results provide information on the chemistry of collagen degradation, and indicate that polymorphic macromolecules survive at too low a concentration to be easily detectable.     

Fish bone chemistry and ultrastructure: implications for taphonomy and stable isotope analysis

This paper reviews the ultrastructure and chemistry of fish bone, with an emphasis on zooarchaeology and stable isotope analysis. On the basis of the chemical composition of the collagen and the relationships between the collagen and mineral phases, fish bone is more susceptible to biotic and abiotic degradation than mammalian bone and is therefore less likely to be recovered in archaeological deposits. The amino acid composition of fish bone differs from that of mammals, most notably with respect to hydroxyproline content. The C:N ratio of fish collagen is, however, very similar and slightly lower than mammalian collagen, and thus the traditional range of acceptable C:N ratios for archaeological bone collagen (2.9–3.6) should not be shifted or extended for fish on the basis of the amino acid composition of collagen. An extensive survey of published archaeological bone collagen C:N ratios demonstrates that fish collagen from archaeological contexts tends to have significantly higher C:N ratios than mammalian collagen. The elevated C:N ratios in fish bone collagen may be the result of abiotic degradation processes that occur within the bone after death, the presence of exogenous humic contaminants, or endogenous lipid contaminants.     

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.     

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

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

Fish ’n chips: ZooMS peptide mass fingerprinting in a 96 well plate format to identify fish bone fragments

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.     

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.     

Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen

Sulphur isotope measurements of bone collagen from archaeological sites are beginning to be applied more often, yet there are no clear criteria to assess the quality of the collagen and therefore the validity of the sulphur isotope values. We provide elemental data from different methods (DNA sequences, amino acid sequences and mass spectrometric measurements) which are used to establish a reliable system of quality criteria for sulphur isotope analyses of bone collagen. The difference in the amount of sulphur from fish and mammalian collagen type I led to the suggestion to use different criteria to assess the in vivo character of the collagen between these two categories. For establishing quality ranges, the bone collagen of 140 modern animals were analysed. The amount of sulphur in fish and mammalian bone collagen is 0.63 ± 0.08% and 0.28 ± 0.07%, respectively. Based on these results we define for mammalian bone collagen an atomic C:S ratio of 600 ± 300 and an atomic N:S ratio of 200 ± 100, and for fish bone an atomic C:S ratio of 175 ± 50 and an atomic N:S ratio of 60 ± 20. These quality criteria were then applied to 305 specimens from different archaeological contexts.     

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.     

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.     

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

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

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.     

Isotopic Comparison of Hair,Nail and Bone: Modern Analyses

This paper presents a comprison of the isotopic values of eight pairings of hair keratin and bone collagen and 12 pairings of hair keratin and nail keratin taken from living humans resident in the U.K., with the aim of examining whether modern human isotopic data can be directly compared to archaeological isotopic data.Results showed that bone collagen was enriched relative to hair keratin from the same individual by +1·4‰ in δ¹³C and +0·86‰ in δ¹⁵N, with some small degree of variability. Isotopic comparison of hair keratin and nail keratin from the same individual showed that there is no significant difference between hair and nail keratin in δ¹³C, but that nail keratin is on average +0·65‰ more enriched in δ¹⁵N than hair.Differences in amino acid composition between hair keratin and bone collagen may account for the carbon isotopic differences between the two proteins, and there is no significant overall carbon isotopic difference between hair and nail. However there are significant isotopic differences for nitrogen in the two pairings, that differences in amino acid composition and turnover times cannot explain. We suggest that these results indicate that constancy of isotopic values between tissues, even for similar proteins, cannot be taken for granted.     

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.     

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.     

Sorting the butchered from the boiled

Is it possible to identify cooked, rather than burnt, bone? Mild heating (≤100 °C,1 h) – typical of cooking – does not lead to detectable changes in any biochemical parameter of bone yet measured. If it is only possible to detect charred bone, how is it possible to detect cooking in the archaeological record? In a previous paper (Koon et al., 2003, J. Arch. Sci.), we used a Transmission Electron Microscopy (TEM) based approach to investigate changes in the organization of the bone protein, collagen, as it is heated, using bone from heating experiments and short term burials. The work revealed that mineralized collagen, despite requiring aggressive treatment to gelatinise the protein (e.g. 90 °C, 240+ h), readily accumulates minor damage. We believe that the presence of mineral matrix stabilises the collagen enabling the damage to accumulate, but preventing it from causing immediate gelatinisation. Once the mineral is removed, the damage can be observed using appropriate visualization methods.     

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.     

Effects of Sodium Hydroxide Treatment and Ultrafiltration on the Removal of Humic Contaminants from Archaeological Bone

This study compared bone collagen extraction techniques that included treatment with sodium hydroxide and 30 kDa ultrafilters using a set of well‐preserved, humic‐contaminated archaeological marine mammal bones. Treatment with sodium hydroxide was effective at removing humic contaminants from archaeological bone, although yields were significantly decreased. Yields were also significantly decreased by ultrafiltration although this study produced no evidence that 30 kDa ultrafilters were effective at selectively removing humic contaminants from archaeological bone. The combination of sodium hydroxide treatment and ultrafiltration did not produce superior results to the treatment involving only sodium hydroxide. Archaeological samples exhibiting darker colouration indicative of humic contamination should be treated with sodium hydroxide to remove these contaminants. Copyright © 2017 John Wiley & Sons, Ltd.     

Introducing δSr analysis in archaeology: a demonstration of the utility of strontium isotope fractionation in paleodietary studies

Isotopic methods are widely used in archaeology to investigate paleodiet. Here, we present a new method to identify trophic level in archaeological human populations and to investigate paleodiet. We demonstrate that strontium isotope compositions (reported as δ^88/86Sr) vary in a mass-dependent manner with increasing trophic level and can elucidate paleodiet in archaeological human populations. We present new mass-dependent strontium isotope data from tooth enamel and bone from individuals buried during the Late Intermediate Period (c. AD 1000–1300) in the large cemeteries of Chiribaya Alta, Chiribaya Baja, San Gerónimo, and El Yaral in the Ilo and Moquegua Valleys of southern Peru. We compare these data to radiogenic strontium isotope data (⁸⁷Sr/⁸⁶Sr) and light stable isotope data (δ¹⁵N_col and δ¹³C_col) from the same individuals to investigate geologic variability in strontium sources as well as marine food consumption among the Chiribaya. Our results demonstrate the utility of measurements of strontium isotope fractionation as a new tool for archaeological investigation of paleodiet. Importantly, this new technique can be used to generate paleodietary (δ^88/86Sr) and paleomobility (⁸⁷Sr/⁸⁶Sr) data from the same specimen, minimizing destructive analyses of invaluable archaeological material, and provides a new way to examine paleodiet through hydroxyapatite, which is particularly important when collagen is poorly preserved.     

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.