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.     

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.     

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.     

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.     

The effects of PVAc treatment and organic solvent removal on δ13C, δ15N,and δ18O values of collagen and hydroxyapatite in a modern bone

The stable isotopic analysis of archaeological and paleontological bones has become a common method to examine questions of ecology, climate, and physiology. As researchers addressing such questions incorporate museum collections in their studies, it is necessary to understand the isotopic effects of common preservation techniques utilized in such collections to ensure the preservation of original isotopic values. This study examines the effects of PVAc glue (polyvinyl acetate) applied in acetone solution and the subsequent removal of PVAc using various organic solvents on the δ¹³C and δ¹⁵N values of extracted bone collagen, the δ¹³C and δ¹⁸O values of carbonate in bone hydroxyapatite, and the δ¹⁸O values of phosphate in hydroxyapatite. The data demonstrate that isotopic values in the collagen and phosphate are unaffected by any combination of PVAc treatment and solvent application. The carbonates show little variation in δ¹³C values, but exhibit variable δ¹⁸O values upon exposure to the PVAc solution. It is here suggested that δ¹⁸O values from carbonates in PVAc-treated bones do not retain an original isotopic value and should not be included in future studies.     

Sulphur isotope evidence for freshwater fish consumption: a case study from the Danube Gorges,SE Europe

To explore the use of sulphur isotopes as an indicator of the consumption of freshwater fish, we undertook sulphur isotope analysis on bone collagen extracted from humans and animals from five archaeological sites from the Danube Gorges region dating from the Mesolithic to the middle Neolithic periods. The results show a difference in the sulphur isotope values between freshwater and terrestrial ecosystems of 8.7‰. To reconstruct human diets, bone collagen from 24 individuals was analysed for carbon, nitrogen and sulphur isotopic values. The nitrogen isotope ratios ranged from 10.3 to 16.5‰ and the carbon isotope ratios ranged from −20.8 to −18.3‰. Low nitrogen isotope values were found for individuals with low sulphur isotope ratios reflecting the low sulphur isotopic values of the terrestrial animals. The highest human nitrogen isotope values coincided with higher sulphur isotope ratios, which are related to the higher sulphur isotope values of the freshwater fish. Intermediate human sulphur isotopic ratios between these two extremes showed mixed diets of both terrestrial and freshwater resources.     

Dietary changes and millet consumption in northern France at the end of Prehistory: Evidence from archaeobotanical and stable isotope data

Diachronic changes of dietary human habits between the Neolithic and the Bronze Age are mainly identified through archaeological artefacts and archaeozoological and archaeobotanical studies. This paper aims to demonstrate the importance of a multi-disciplinary approach for palaeodietary studies and to identify the food changes between Neolithic and Bronze Age human groups in northern France. These changes are probably linked to the introduction of new crops, such as millet, and the use of stable isotope analysis on bones and teeth proves to be an effective method for assessing the role of this specific cereal in the diet and the economy. Stable isotope analyses were performed on bone and tooth collagen and apatite from eight humans and five domestic animals from a Late Bronze Age site (LBA; Barbuise; 15th–13th c. BC; Aube). The studied corpus is compared with isotopic data from human and animal bones from a nearby Neolithic site (Gurgy; 5th mill. BC; Yonne) and regional Neolithic to Iron sites located in northern France. Moreover, Barbuise data are supplemented by information from an important archaeobotanical study carried out on 21 LBA and Early Iron Age sites in the region. Neolithic and LBA human collagen isotopic ratios (δ¹³C, δ¹⁵N) differ statistically, as do those of some animals. Carbon isotopic ratios of human apatite corroborate collagen results indicating the consumption of ¹³C enriched food by LBA humans and animals compared to Neolithic samples. The high number of occurrences of plant remains in the Bronze Age settlements near the site points to the consumption of C₄ plants, such as millet, and would account for these results.     

Assessing the distribution of African Palaeolithic sites: a predictive model of collagen degradation

In order to assess the distribution of African Palaeolithic sites and hominin remains, we need an understanding of the factors that may affect the preservation of archaeological material in the fossil record. One of the main factors that is thought to affect seriously the global distribution of fossil remains is taphonomy, but it would seem that taphonomic processes may themselves be overprinted by collagen degradation.The rate of collagen loss is highly temperature dependent, and it has been shown that bone is more likely to be preserved in areas of lower temperature and so lower collagen loss. Hence, it would be expected that fossil fauna remains would be distributed in areas of low collagen loss, i.e. low temperature. Conversely, lithics will not be subject to a temperature bias in their preservation. We have tested this hypothesis through the use of archaeological material.Our results show that the distributions of both lithic and faunal assemblages are non-random across the African continent, with all archaeological sites being found in areas of relatively low collagen degradation. This implies that bone collagen degradation is not the only factor affecting the distribution of African Palaeolithic sites and hominin remains. We suggest that the site distribution is not a reflection of bias in excavation history, but is a real phenomenon reflecting hominin habitat choice.     

Effect of heating on the stable carbon and nitrogen isotope ratios of bone collagen

The effects of heat on the stable carbon and nitrogen isotopic ratios of collagen in bone were studied. Boiling or roasting did not change the δ¹³C and δ¹³N values by more than 1‰. More extreme heating, such as might occur if bones were burned or a body cremated, shifted δ¹³C and δ¹⁵N values by as much as 5 and 4‰ respectively. These large shifts were accompanied by changes in the atomic carbon-to-nitrogen ratios of the collagen. These results indicate that collagen samples extracted from prehistoric bones which display anomalous atomic carbon-to-nitrogen ratios may have been subjected to heating extreme enough to have altered their ${}^{13}\text{C}{}^{12}\text{C}$ and/or ${}^{15}\text{N}{}^{14}\text{N}$ ratios and therefore should not be used for dietary reconstruction.     

The effect of growth on stable nitrogen isotope ratios in subadult bone collagen

Stable nitrogen isotopes have been used to reconstruct infant feeding practices as nursing infants have elevated δ¹⁵N ratios compared with their mothers. However, infancy is also a time of rapid growth, which may alter nitrogen isotope diet‐to‐tissue spacing. Several studies have documented a decrease in δ¹⁵N during growth in tissues with relatively fast accretion rates. This study investigates the effect that the growth of long bones, via collagen accretion, has on δ¹⁵N ratios. Long bones from individuals aged seven to nineteen years were obtained from a protohistoric ossuary in Ontario, Canada. Analysis of juveniles and adolescents permitted the examination of growth in a group who were not also nursing. It is concluded that a nitrogen isotope growth effect is not detectable in bone collagen from juveniles and adolescents, because: (1) δ¹⁵N ratios are not significantly different among the epiphyses, metaphyses and diaphysis of a growing long bone; (2) δ¹⁵N ratios are not significantly different between faster‐growing versus slower‐growing metaphyses; and (3) δ¹⁵N ratios are not significantly different between bones (or areas of a bone) that are still undergoing growth, versus bones that have ceased growing. The relatively slow speed of collagen accretion may explain why a growth effect is not manifested. Ultimately this research lends support to the use of nitrogen isotopes from bone collagen for infant feeding reconstructions. Copyright © 2009 John Wiley & Sons, Ltd.     

Distinguishing between archaeological sheep and goat bones using a single collagen peptide

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.     

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.     

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.     

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.     

A Simple(R) Model to Predict the Source of Dietary Carbon in Individual Consumers

Quantitative individual human diet reconstruction using isotopic data and a Bayesian approach typically requires the inclusion of several model parameters, such as individual isotopic data, isotopic and macronutrient composition of food groups, diet‐to‐tissue isotopic offsets and dietary routing. In an archaeological context, sparse data may hamper a widespread application of such models. However, simpler models may be proposed to address specific archaeological questions. As a consequence of the intake of marine foods, individuals from the first century ad Roman site of Herculaneum showed well‐defined bone collagen radiocarbon age offsets from the expected terrestrial value. Taking as reference these radiocarbon offsets and using as model input stable isotope data (δ¹³C and δ¹⁵N), the performance of two Bayesian mixing model instances (routed and concentration‐dependent model versus non‐routed and concentration‐independent) was compared to predict the carbon contribution of marine foods to bone collagen. Predictions generated by both models were in good agreement with observed values. The model with higher complexity showed only a slightly better performance in terms of accuracy and precision. This demonstrates that under similar circumstances, a simple Bayesian approach can be applied to quantify the carbon contribution of marine foods to human bone collagen.     

Stable carbon and nitrogen isotope ratios of bone collagen: Variations within individuals,between sexes,and within populations raised on monotonous diets

The stable carbon and nitrogen isotope ratios of collagen of seven bones from each of three rabbits raised on a monotonous diet, and of two bones from each of eight female and seven male mink raised on another monotonous diet, were determined. The ranges of δ¹³C values and δ¹⁵N values were 0·5‰ and 0·6‰ for the rabbit bones and 1·0‰ and 1·4‰ for the mink bones. Uncertainties in the δ¹³C and δ¹⁵N values for prehistoric human diets estimated from the isotopic composition of collagen from the small numbers of bones which are typically available for analysis, and thus likely to be of the order of ±1‰.     

Bone Degradation and Environment: Understanding,Assessing and Conducting Archaeological Experiments Using Modern Animal Bones

Archaeological experiments that use modern bones to replicate past animal bone assemblages have often failed to consider the effects of environment, storage and preparation on modern bones. Often, these experiments make little mention of the conditions to which bones were subject during their storage and preparation for use in experiments. In other instances, these variables are reported but not considered as factors that contribute to the nature of the results obtained. This study considers previously reported data concerning the degradation of frozen bones (−20°C), and bones exposed to hot, dry conditions (40°C), and presents new data for bones exposed to room temperature environments (22°C) and refrigerated environments (2°C), and bones that are frozen (−20°C) and then thawed (22°C). These conditions are all relevant to understanding the nature of bone degradation and the use of bones in modern archaeological experimentation. This article also surveys a range of previously reported experiments that utilise modern bones to create analogies to the past and considers different methodological approaches and their relationship to the condition of bones at the time of their fracture and fragmentation. The longitudinal data presented in this study demonstrate differential rates of bone degradation over time in various environmental conditions. This degradation results in dramatic changes in bone fracture morphology, bone strength and utility for bone tool production. These observations have significant implications for experiments that utilise modern bones, especially when experimental data are used to create analogies to the archaeological past. Copyright © 2012 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.     

Calcium isotopes in archaeological bones and their relationship to dairy consumption

The calcium isotope ratios (δ^44/42Ca) of bones from humans and fauna from three archaeological sites, Taforalt, Abu Hureyra, and Danebury, are evaluated in order to assess whether calcium isotope ratios of bones can be used to detect dairy consumption by adult humans. At each site the fauna δ^44/42Ca is the same regardless of species, while the humans have lower δ^44/42Ca than the local animals by 0.24–0.41‰ (site means). However we cannot ascribe this difference to dairy consumption, given this human–faunal difference also occurs in Epipalaeolithic and Mesolithic adult humans, where dairy consumption is unlikely. Rather, this difference appears to be a result of differences in metabolic processes or other aspects of diet between humans and fauna. Minimal isotopic change in sequential acid leaches of bone powders and consideration of the high calcium concentration in bone suggest that bone calcium isotope ratios are not substantially affected by diagenetic change.     

Kana Tamata or Feasts of Men: An Interdisciplinary Approach for Identifying Cannibalism in Prehistoric Fiji

By integrating osteological, taphonomic, archaeological and stable isotopic data, we test for cannibalism in the Lau Group, Fiji and discuss the potential underlying cause(s) and context(s) of this behaviour. First, we compare taphonomic and element representations of human skeletal material from two contexts in Fiji, examining human bone fragments from archaeological sites, including middens and burials in the Lau Island Group. Fourteen sites produced human remains. Only two of those sites included distinct human burial contexts, but in the remaining 12 sites, the human bone was recovered from middens or contexts where midden was mixed with possible secondary burials. A total of 262 number of identified specimens per species, representing an estimated 15 minimum number of individuals make up the Lau human assemblage. Second, we analysed bones contained in 20 individual human burials from four different sites that are housed at the Fiji Museum for comparative purposes. Third, we examine previously published stable isotopic (δ¹³C, δ¹⁵N) analysis of bone collagen to gauge protein consumption of likely cannibalised humans in midden contexts and potential cannibals from primary burials. We model a cannibalistic diet category within the context of isotopically measured Pacific Islands food groups and apply an isotopic mixing model to gauge plausible dietary contributions from six sources including human flesh. Isotopic mixing models of the Lauan samples illustrate a high diversity in reconstructed diets. The percent contribution of human flesh is low for all individual Lauans. We conclude that mortuary rituals evidenced by sharp‐force trauma may suggest non‐nutritive and non‐violent practices that may have included the consumption of small amounts of human flesh. Copyright © 2012 John Wiley & Sons, Ltd.