Stable isotope investigations of charred barley (Hordeum vulgare) and wheat (Triticum spelta) grains from Danebury Hillfort: implications for palaeodietary reconstructions

Palaeodietary studies typically focus on the analysis of bone collagen due to the limited availability of plant remains. Isotopic analysis of plant remains, however, allow for a more extensive consideration of the contribution of plants to the human diet and can potentially provide information about the environment in which the crops were grown. This paper reports the results of carbon and nitrogen isotope analyses performed on charred barley and wheat grains recovered from pits within Danebury Iron Age hillfort. To the best of our knowledge, this is the first Iron Age site in Britain from which charred grains have been isotopically analysed. Our results suggest that cereals found at the hillfort were grown in several different environmental contexts. The isotope data demonstrate that the herbivores were not consuming a diet primarily based on grains as the δ¹⁵N values of the grains are very similar to those of the herbivores. Palaeodietary investigations typically assume that humans eating plant protein only would have the same δ¹⁵N value as the local herbivores. This assumption is clearly invalid at Danebury, where the humans and animals appear to have consumed either different parts of the same plants or different plants. Researchers typically interpret high differences between human and animal δ¹⁵N values as indicative of diets high in animal protein, however where major plant resources have δ¹⁵N values similar to those of the herbivores our ability to distinguish between plant and animal sources of protein in the diet is limited. Our research has demonstrated that whenever possible it is desirable to measure the isotopic signatures of potential major plant resources in order to understand past subsistence strategies.     

Manuring and stable nitrogen isotope ratios in cereals and pulses: towards a new archaeobotanical approach to the inference of land use and dietary practices

This paper explores the impact of animal manure application on the δ¹⁵N values of a broad range of crops (cereals and pulses), under a range of manuring levels/regimes and at a series of locations extending from northwest Europe to the eastern Mediterranean. We included both agricultural field experiments and areas where ‘traditional’ farming is practised. Our aim is to ground-truth interpretation of δ¹⁵N values in archaeobotanical crop remains as evidence of past growing conditions and husbandry practices. The results confirm the potentially radical impact of manuring on δ¹⁵N values in cereals, depending on manuring level, but indicate only a slight effect on pulses, which can fix atmospheric nitrogen. The expected geographical trend towards greater δ¹⁵N with increasing climatic aridity is not apparent, probably because the growing conditions for crops are ‘buffered’ through crop management. Each of these observations has fundamental implications for archaeobotanical interpretation of δ¹⁵N values as evidence of land use practices and (together with analysis of bone collagen/tooth enamel in potential consumers) palaeodiet.     

An investigation of diet in early Anglo-Saxon England using carbon and nitrogen stable isotope analysis of human bone collagen

The principal aim of this work is to investigate whether protein sources in human diets in early Anglo-Saxon (5th-7th century AD) England varied with geographic location, or with respect to age or sex. The methodology used was analysis of δ¹⁵N and δ¹³C in human bone collagen from 76 adult skeletons from a total of 18 different cemeteries located in inland, riverine and coastal environments. In order to control for the possible effects of socio-economic status on diet, only high status individuals, as identified by grave goods, were studied. Data were analysed using statistical manipulation of the raw results and using IsoSource (Phillips and Gregg, 2003), a mathematical treatment which models the composition of the protein part of human diets using bone stable isotope ratios when the isotopic values for potential protein sources are known or can reasonably be inferred. The results suggest that protein sources varied little according to geographic location and that terrestrial foods dominated at all locations. However there were some subtle patterns. Slightly elevated δ¹³C in skeletons from coastal locations, and δ¹⁵N in those from riverine sites may indicate that communities in these locations made greater use of marine and freshwater resources respectively. If this is correct then it demonstrates that 5th-7th century communities made some use of wild resources even within a predominantly agrarian economy. In general the analysis of the raw data and the IsoSource results were mutually supportive, and this work demonstrates the value of this twin-pronged approach to analysis of human bone stable isotope data.     

Stable hydrogen isotopes of bone collagen in palaeodietary and palaeoenvironmental reconstruction

The stable hydrogen isotope ratios (δD) of bone collagen in archaeological human and animal samples demonstrate a trophic level effect, with increasing δD from herbivores to omnivores to humans, in steps of 10–30‰. In addition the archaeological sites studied (Yarnton, Eton Rowing Lake, Danebury Environs–Suddern Farm, and Windmill Hill in the UK, Balatonszárszó in Hungary, and Huari in Peru) demonstrate geographical variation in δD. The detection of manuring in prehistory by comparison of δ¹⁵N to δD values in humans and a local herbivore (cattle) is also considered. This work is the first to measure δD in a large number and range of archaeological samples, with several animal species and humans. It demonstrates unequivocally that δD is different between species in ancient material, increasing from herbivores to omnivores to carnivores.     

Stable carbon and nitrogen isotope variations in tooth dentine serial sections from Wharram Percy

Here we report δ¹³C and δ¹⁵N measurements of serial sections of human deciduous and permanent tooth dentine from archaeological samples taken from the medieval village site of Wharram Percy, Yorkshire, UK. We found a pattern of enrichment, for both δ¹³C and δ¹⁵N, where the tooth crown was greater than the cervical part of the root, which in turn was greater than the apical portion of the root and the associated rib collagen values. This pattern reflects a decrease in the consumption of isotopically enriched breast milk and the introduction of less enriched weaning foods in the diet. The (mean±SD) difference between the deciduous second molar crowns and corresponding rib samples from the same individuals after 2 years of age was 1.2±0.4‰ for δ¹³C and 3.2±0.8‰ for δ¹⁵N. The δ¹⁵N values are as predicted, but as there were no C₄plants at Wharram Percy, this 1.2‰ enrichment in δ¹³C represents clear evidence of a carbon trophic level effect in collagen from breastfeeding infants. Carbon and nitrogen results also show that the infant diet among those who died in infancy did not differ from those who survived into childhood. This study demonstrates the promise of using dentine serial sections to study the temporal relationships of breastfeeding, weaning, and dietary patterns of single individuals.     

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.     

Diet in the Iron Age cemetery population at Wetwang Slack,East Yorkshire,UK: carbon and nitrogen stable isotope evidence

This paper reports δ¹³C and δ¹⁵N values for human and animal bone collagen from the middle Iron Age site at Wetwang Slack, East Yorkshire, UK. The data indicate a human diet which was high in animal protein, with no evidence for any significant marine food input. No differences were found between high-status vehicle (or ‘chariot’) burials and the rest of the population and no other status differentiations are visible according to burial rite, age or sex groupings, although the data obtained for the older males display an unusual trend. No dietary variation is seen between two site phases and no evidence for an early immigrant group is present. The range of isotope values for the adult human group as a whole is small, indicating that the diet is likely to have been consistent over time and across the population, although two individuals stand out as unusual amongst the 62 analysed.     

The fish of Lake Titicaca: implications for archaeology and changing ecology through stable isotope analysis

Research on past human diets in the southern Lake Titicaca Basin has directed us to investigate the carbon and nitrogen stable isotopes of an important dietary element, fish. By completing a range of analyses on modern and archaeological fish remains, we contribute to two related issues regarding the application of stable isotope analysis of archaeological fish remains and in turn their place within human diet. The first issue is the potential carbon and nitrogen isotope values of prehistoric fish (and how these would impact human dietary isotopic data), and the second is the observed changes in the fish isotopes through time. Out of this work we provide quantitative isotope relationships between fish tissues with and without lipid extraction, and a qualitative analysis of the isotopic relationships between fish tissues, allowing archaeologists to understand these relationships and how these values can be applied in future research. We test a mathematical lipid normalization equation to examine whether future researchers will need to perform lipid extraction procedures for Lake Titicaca fish. We also analyze a number of aquatic plants to better understand the range of isotopic signatures of the Lake Titicaca ecosystem. We use these data to better understand prehistoric human diet and the role that fish may have played in the past as well as potential changes in local lake ecology through time.     

Subsistence and mobility strategies in the Epipalaeolithic: a stable isotope analysis of human and faunal remains at 'Uyun al-Hammam,northern Jordan

This paper investigates evidence for subsistence and settlement activities in the Levantine Middle Epipalaeolithic through the application of stable isotope analysis to human and non-human remains from the site of 'Uyun al-Hammam, northern Jordan. In general, bone from the site suffers a high degree of diagenesis and collagen could not be extracted for analysis here. Carbon and oxygen isotopic values from human tooth enamel samples were variable, but within expected values for the Middle Epipalaeolithic, whereas animal carbon and oxygen isotopic values varied widely, most likely due to hydrological and climatological factors. Carbon and isotopic values for the human samples indicated a predominantly C₃ plant dietary input, while animal samples appeared to have varying amounts of C₄ inputs into their diet. This is the first isotopic analysis conducted on material from the Middle Epipalaeolithic of the southern Levant and, as such, even results constrained by temporal and climatological variants within the region contribute to the overall knowledge of settlement and subsistence strategies during this cultural period.     

The distinction between freshwater- and terrestrial-based diets: methodological concerns and archaeological applications of sulphur stable isotope analysis

Sulphur isotopes in archaeological bone collagen are not routinely analysed in palaeodietary studies. Here we investigate the potential contribution that sulphur isotope analysis can provide toward the study of ancient human diet and economy, with particular emphasis on the distinction between freshwater and terrestrial consumers. For material from the Late Bronze Age site of Chicha in the south-western Eurasian forest-steppe, sulphur isotopes effectively separate freshwater and terrestrial animal food resources. The sulphur isotope data coupled with nitrogen isotope values from Chicha reflect a dietary reliance upon freshwater animal protein (fish) for the Late Bronze Age inhabitants. In contrast, sulphur isotope values for freshwater and terrestrial potential food species from the Eneolithic site of Bil'shivtsi in western Ukraine were indistinguishable, demonstrating that δ³⁴S values cannot always be relied upon to identify freshwater and terrestrial consumers. The data from this study support the adoption of δ³⁴S analysis as a standard component of palaeodietary studies; apart from its potential to distinguish freshwater from terrestrial consumers, it can provide supplementary dietary information not evident from the carbon and nitrogen isotope data. In addition, certain indices are considered that may be used to assess the validity of sulphur isotope data, as currently exist for carbon and nitrogen. According to the analysis of modern collagen samples, N:S appears to be a broad indicator of collagen sulphur isotope quality. However, more work needs to be done to establish an effective means by which highly-altered sulphur isotope values can be identified and thereby removed from consideration.     

Stable isotope analysis of humans from Xiaojingshan site: implications for understanding the origin of millet agriculture in China

Millet agriculture originated in Northern China in the early Neolithic period (ca. 8000 BP), however, the actual importance of millet in human diets is still not clear. To determine the relative contribution of millet in human diets in this period we undertook stable isotope analysis of humans from Xiaojingshan site and fauna from Yuezhuang site, both of which are attributed to the Houli Culture and date to about 8000 years ago. The carbon isotope values of human bone collagen showed that millet (as a C₄ plant) only contributed approximately 25% of dietary protein, with the rest from C₃ based plant and animal sources, if a simple mixing model is used. We did not observe any statistical dietary difference between males and females at the site, although it has been argued that the Houli Culture was a matriarchal society. Finally, we compared our data with other published isotopic data from the contemporary Jiahu site and Xinglongwa site and a number of sites from the subsequent Yangshao Culture and found that millet only became a significant source of dietary protein approximately 1000 years later, as human carbon isotope values from these later sites indicated that almost all of dietary protein came from C₄ (i.e. millet) sources.     

Method-dependent variations in stable isotope results for structural carbonate in bone bioapatite

Stable carbon and oxygen isotope values (δ¹³C, δ¹⁸O) were obtained for structural carbonate in the bioapatite of archaeological bones from Guatemala and Sudan using several common analytical methods. For the Sudan samples, the different methods produced δ¹³C values within ±0.1‰ and δ¹⁸O values within ±0.7‰, on average. The isotopic results for the Guatemala samples were similar in reproducibility to the Sudan samples when obtained using methods that employed lower reaction temperatures and reactions in sealed vessels. However, many Guatemala samples had highly variable and extremely low δ¹⁸O values when reacted at higher temperatures in vessels that remained open to cryogenic traps. The latter arrangement caused reaction products to be removed immediately upon their production. The anomalously low δ¹⁸O values are related to the production of a contaminant gas that causes the m/z 46/44 ratio to be lowered, either by adding to the m/z 44 peak or subtracting from the m/z 46 peak. That said, potential contaminant materials were not detectable in “anomalous” bones using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, or inductively-coupled plasma atomic emission spectroscopy. However, subtle structural and chemical differences between “normal” and “anomalous” samples were observed, most notably in the FTIR ν₂ CO₃ domain. We suggest that these changes promote volatilization of an oxyphosphorus compound and oxygen isotope fractionation between PO⁻ derived from this compound and CO₂ derived from bone carbonate. Production of the contaminant gas and the related “anomalous” δ¹⁸O values is reversible if the reaction occurs within a sealed vessel for a sufficient period of time, which allows a “back-reaction” to occur.     

Stable isotope evidence of human diet at the Nukdo shell midden site,South Korea

Carbon and nitrogen stable isotope analysis was undertaken on bone collagen extracted from archaeological human (n = 48) and animal (n = 45) skeletons from the Nukdo site, Location I C, South Korea. This shell midden and grave site is dated from the late Mumun (550–300 BC) to early Iron Age (300 BC-1 AD) periods. The herbivorous mammals fell within the range of C₃ consumers, with average values of δ¹³C = −21.0 ± 0.5‰ and δ¹⁵N = 3.6 ± 0.5‰ for deer (n = 16) and δ¹³C = −20.6 ± 0.5‰ and δ¹⁵N = 4.5 ± 2.0‰ for wild boar (n = 17). Humans from this site averaged δ¹³C = −18.3 ± 0.4‰ and δ¹⁵N = 11.2 ± 0.7‰ for adults (n = 15) and δ¹³C = −18.7 ± 0.7‰ and δ¹⁵N = 12.5 ± 1.1‰ for juveniles (n = 33). These δ¹³C values indicate that there was no significant input of C₄ plants in the human diets and this may be associated with the spread of rice agriculture in the Mumun period. Human bone collagen δ¹³C and δ¹⁵N values indicate that there was some consumption of marine foods, although the main protein sources were from terrestrial foods. The isotope data demonstrate that the humans at Nukdo had mixed diets that included marine and terrestrial protein, including C₃ plants such as rice. Finally, the isotope results from the juveniles indicate that weaning occurred before the age of 1.5 years in this period.     

Carbon and nitrogen isotope signals in eel bone collagen from Mesolithic and Neolithic sites in northern Europe

Carbon and nitrogen isotope analysis was undertaken on collagen extracted from eel bone from six Mesolithic and Neolithic sites in northern Europe. The results were compared with data obtained on other archaeological fish remains and modern eel caught in fresh and brackish water and from the sea. The possibility of discriminating between freshwater, brackish and marine signals in archaeological eel bone is evaluated and the implications for archaeology discussed. Our data suggest that eel found at coastal Mesolithic and Neolithic sites have carbon isotope signals consistent with a marine origin with no evidence of freshwater residency. The sample of eel bone from one inland site is small but indicates carbon isotope values more consistent with freshwater residency or at least values intermediate between freshwater and marine carbon pools.     

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.     

Palaeoenvironmental and chronological investigations of the Magdalenian sites of Goyet Cave and Trou de Chaleux (Belgium), via stable isotope and radiocarbon analyses of horse skeletal remains

Trou de Chaleux and Goyet are caves situated less than 30 km apart in Belgium that contain stratified Magdalenian artefacts and butchered faunal remains. Published radiocarbon dates suggest that both sites were contemporaneously occupied during to the Late-glacial interstadial. It has previously been suggested that the Trou de Chaleux Late-glacial faunal remains might be slightly older than those at Goyet Cave, and that Trou de Chaleux has two phases of occupation during the Late-glacial interstadial. However, the limited number of radiocarbon dates currently available makes it impossible to determine whether the sites are truly contemporaneous, and the assessment of their absolute chronologies is complicated by a plateau in the calibration curve. In this paper, bone collagen δ¹³C and δ¹⁵N signatures of horse bones from the two sites are used to reconstruct local palaeoenvironments. We hypothesise that if occupations at the two sites were contemporaneous, the horse collagen δ¹³C and δ¹⁵N isotope signatures from both sites will be similar, since comparable carbon and nitrogen isotopic values would reflect similar diets, ecologies and environments. To provide clear dating parameters, new AMS radiocarbon determinations are also presented. Results show that horse collagen δ¹³C and δ¹⁵N isotope signatures at the two sites are different, indicating that the populations were not subject to similar diets, ecologies and environments. The new radiocarbon dates confirm that the horse bones from Trou de Chaleux date to the Late-glacial interstadial, but they also show that in Goyet Cave Horizons 1 and 2, older horse remains dating to ca. 32?000–27?000 ¹⁴C yr BP are mixed with Late-glacial horse remains. The δ¹⁵N values of the Goyet Cave horse bones radiocarbon dated to the Pleniglacial are substantially higher than those that have Late-glacial radiocarbon dates. Therefore, we now hypothesise that the majority of the horse bones isotopically analysed from Goyet Cave Horizon 1 are Pleniglacial in age. Statistical analysis of the radiocarbon dates indicates that the Late-glacial occupation at the two sites was contemporaneous, and that while there may appear to be two phases of occupation at Trou de Chaleux, this may be an artefact of the radiocarbon calibration curve.     

FOCUS: effect of diet and protein source on carbon stable isotope ratios in collagen: follow up to

We analyzed carbon stable isotope data from bone collagen of animals consuming varied experimental diets, including recently published data from Warinner and Tuross [Warinner, C., Tuross, N., 2009. Alkaline cooking and stable isotope tissue–diet spacing in swine: archaeological implications. Journal of Archaeological Science 36, 1690–1697; this journal]. Comparing regression lines for the relationship between collagen and diet δ¹³C, we show that protein source, and not physiology, explains the apparent taxonomic difference between swine and rodents reported in that paper. Our results reveal a complex relationship between whole diet and dietary protein in determining collagen δ¹³C values, such that in many cases, collagen alone may not provide reliable reconstructions of paleodiet. We advocate the simultaneous use of both collagen and apatite δ¹³C, whenever possible, to assess the diets of prehistoric peoples.     

Temporal changes in diet: a stable isotope analysis of late Iron Age and Roman Dorset,Britain

This study investigates the relationship between diet and cultural change in late Iron Age and Romano-British populations from Dorset, England (1st century BC to the early 5th century AD). Dorset was the only region in Britain to exhibit continuity in inhumation burial rites through these periods and a wide array of environmental, archaeological and material culture evidence is available there. A sample of N = 77 human adult and N = 17 faunal rib samples were utilized for carbon and nitrogen stable isotope analysis to test the hypothesis that Romanization of the diet would result in greater dietary variation.     

Discovery of colon contents in a skeletonized burial: Soil sampling for dietary remains

Recovering dietary and parasitological data from skeletonized burials is often overlooked or attempted without sufficient consideration of preservation possibilities, quality sampling areas or the confounding influences of post-interment processes. A case study of a skeletonized Anasazi burial is presented to demonstrate three important points. First, that macrobotanical remains can be preserved in addition to the more durable pollen and parasite eggs. Second, that the sacrum can act as a structural container for the contents of the lower digestive tract upon decomposition of the body, thereby providing a productive sampling area. Finally, that reliable interpretation of any biological remains from skeletonized burials depends upon the analysis of several control samples. Since awareness of probable data sources plays a central role in what is actually collected and analysed, it is important to alert archaeologists to the possibilities of colon content analysis from skeletonized burials.     

Carbon isotope measurement techniques for bone collagen: Notes for the archaeologist

Stable carbon isotope analysis of archaeological human bone collagen is becoming increasingly useful in diet reconstructions. This paper describes, for archaeologists, the techniques that we have found useful in carrying out such analyses, and discusses the requirements for measurement accuracy and inter-laboratory calibration.