Evaluating bone collagen extraction methods for stable isotope analysis in dietary studies

The specific purpose of this study was to compare three different collagen extraction methods commonly used in isotope laboratories conducting dietary studies. We evaluated their resultant differences in δ¹³C and δ¹⁵N, collagen quality and collagen yield. Our study was based on well-preserved skeletal material from the medieval period in Denmark. Our study shows that there is a systematic significant difference in the yield and the δ¹³C values between the three methods. Using the method of DeNiro and Epstein [DeNiro, M.J., Epstein, S., 1981. Influence of diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45, 341–351] with NaOH as cleaning agent, will, according to our study, give δ¹³C values that are on average ±0.32‰ more positive than using the ultra-filtration method [Brown, T.A., Nelson, D.E., Vogel, J.S., Southon, J.R., 1988. Improved collagen extraction by modified Longin method. Radiocarbon 30 (2), 171–177, modified in Richards, M.P., Hedges R.E.M., 1999. Stable isotope evidence for similarities in the types of marine foods used by late Mesolithic humans at sites along the Atlantic coast of Europe. Journal of Archaeological Science 26, 717–722]. The third method, which is a modified version of the second method, excluded the ultra-filtration step. This method seems to give δ¹³C values that lie in between the other methods. Our study did not show any significant difference in δ¹⁵N values. Although the differences between the methods are very small, we conclude that the use of stable isotope analysis in food determination studies requires adherence to routine methods for preparing and measuring samples.     

Stable isotopic evidence for diet in a Roman and Late Roman population from Leptiminus,Tunisia

This study investigates the diet of the Roman and Late Roman population of Leptiminus on the Mediterranean coast of Tunisia. Stable carbon and nitrogen isotope analysis of human bone collagen and carbonate samples obtained from individuals buried in four cemeteries at Leptiminus was conducted in order to reconstruct the diet of the population, investigate the relative importance of marine vs. terrestrial resources, explore sex-, age-, and status-based variations in diet, examine temporal changes in the types of foods consumed, and compare the diet at Leptiminus with that of other Roman populations. The results of this study indicate that the residents of Leptiminus consumed a diet that was heavily reliant on terrestrial plant resources with the addition of a significant amount of marine resources. There were no significant sex differences in isotope values. In contrast, distinct dietary differences were seen between the adults and children. Nitrogen isotope values suggest that weaning began before the age of two and was completed by about 3 years of age, a finding consistent with previous isotopic studies of Roman samples. A temporal shift in diet is suggested by the nitrogen isotope values measured in samples from the most recent cemetery. A comparison of the data from Leptiminus with that derived from other Roman sites indicates that regional variability in diet existed within the Empire.     

Stable isotopes and diet at Ancient Kerma,Upper Nubia (Sudan)

Carbon and nitrogen stable isotope values were measured from bone collagen extracted from archaeological Nubian human (n = 54) and faunal (n = 61) populations from the site of Kerma, Sudan. Collagen suitable for isotopic analysis was extracted from 22 faunal and 48 human samples from the Eastern cemetery site, dated to the Middle Kerma (c. 2050–1750 BC) and Classic Kerma (c. 1750–1500 BC) periods respectively. The isotopic data indicate that the human dietary regimen included a mix of C₃ and C₄ plant-derived components, with a larger C₄ component than previously reported in archaeological Egyptian Nile Valley populations. Elevated δ¹⁵N values are attributed to consumption of dietary resources from a ¹⁵N-enriched terrestrial ecosystem. The faunal isotope data also indicate the consumption of C₃ and C₄ plants. The large range of δ¹³C values measured in both the human and faunal samples supports previous work suggesting that a significant portion of the populations buried at Kerma may have originated elsewhere, further confirming the Nile Valley as a major corridor for population movement in the region.     

Nitrogen isotopes and the trophic level of humans in archaeology

Human and domesticate animal bone collagen δ¹⁵N values in prehistory differ generally by 3‰ or more from Neolithic to post- Roman times in Northwest Europe, leading to an assumed dietary animal protein fraction of 60–80% using a standard interpretation of δ¹⁵N values. We examine the assumptions on which this model rests and the limitations of our knowledge in the analysis of δ¹⁵N values in archaeology. We have developed a set of models which, with small changes made in assumptions (on the order of 1‰), can produce substantially lower estimates of the dietary animal protein fraction for given δ¹⁵N values. We consider the implications of various dietary animal protein fractions on agricultural carrying capacities and human population densities in prehistory.     

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.     

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.     

Dietary shifting in the Nasca Region as inferred from the carbon- and nitrogen-isotope compositions of archaeological hair and bone

The topography of the Andean Region results in short travelling distances among ecologically distinct production zones. As such, it is possible to infer relative changes in residence based on the spatial variability in the isotopic composition of food resources. Here, we determine long-term diet through carbon- and nitrogen-isotope analysis of bone collagen for twenty-nine individuals from Cahuachi, an important ceremonial centre in the Nasca Region. We also reconstruct the temporal sequence of change in dietary isotopic compositions along hair samples for seventeen individuals buried at Cahuachi and the neighbouring site of Huaca del Loro.     

A palaeodietary investigation of carbon (C/C) and nitrogen (N/N) in human and faunal bones from the Copper Age cemeteries of Varna I and Durankulak,Bulgaria

Stable isotope analyses have been applied to human and faunal bone collagen from the Varna I and Durankulak cemeteries to explore palaeodietary adaptations in the Neolithic and Eneolithic (Copper Age). The results suggest both populations primarily utilised terrestrial, C₃-based diets, despite their proximity to the Black Sea. The wider δ¹⁵N range of the Durankulak humans likely indicates the differential utilisation of terrestrial meat sources, which is probably related to the degree to which primary and/or secondary ovicaprid products were consumed, particularly since ovicaprid δ¹⁵N values differ from other herbivores. The isotopic distribution of Varna I reflects a linear relationship between δ¹⁵N and δ¹³C, suggesting that a minority of individuals enriched in both isotopic parameters supplemented their diets with marine resources. These burials include the well known ‘chieftain’ (burial 43) and show notable material wealth by way of grave goods. At the population level, however, there is no significant correlation between stable isotope values and material wealth at Varna I, a fact with implications for theories regarding emergent social/economic hierarchies in Balkan prehistory. Five burials at Durankulak were found to have relatively enriched δ¹³C and δ¹⁵N values with respect to the rest of the population. These burials reflect a prominently marine-based or mixed terrestrial C₃-based diet that included C₄ inputs, possibly from millet, for which the limitations of stable isotope analysis on bulk collagen are not able to differentiate. AMS dating has shown that these burials belong to a much later period.     

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.     

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.     

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.     

Fish as diet resource in North Spain during the Upper Paleolithic

During the last glacial maximum, the west European modern humans adapted to adverse conditions and their populations subsisted exploiting the natural resources available. Fish were always present as a source of animal protein, although their importance in the human diet and their contribution to the expansion of humans has not been fully explored. Based on the compilation of zooarchaeological evidences from north Spanish sites we have discovered a constant pattern of exploitation of freshwater fish (Salmonids) with high incidence in the human diet during the Upper Paleolithic. Shifts in the exploitation of marine resources can be explained by climate-related geographical changes. The characteristics of salmonids that make them target species for supporting human survival in adverse climate periods are identified and discussed.     

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 isotopic analysis of human and faunal remains from the Incipient Chulmun (Neolithic) shell midden site of Ando Island,Korea

We analyzed stable carbon and nitrogen isotope ratios of human and animal remains from the Ando shell midden, South Korea. The Ando site is a rare Incipient Chulmun (Neolithic) site (ca. 6000–5000 BC), which contains well-preserved human and animal bones in shell mounds. The stable isotope results for humans (average δ¹³C = −13.5 ± 0.5‰ and δ¹⁵N = 15.2 ± 0.5‰) indicate that Ando people in the Incipient Chulmun period strongly depended on marine resources. There were no isotopic differences between humans of different sex and age at this site. We compared our data with other previous published isotopic data from the Chulmun sites and found that the Ando people had similar isotope values to the southern Chulmun people (Tongsamdong and Daepo), but different isotopic ratios than the western Chulmun people (Daejukri and Konamri). These results indicate that marine foods were the main food resources in the southern coastal regions, but not in the western coastal regions in Chulmun Korea.     

Caesium-137 as an indicator of geomorphic processes in a drainage basin system

Caesium-137 from fallout from nuclear weapons tests is adsorbed on fine sediments and becomes an effective tracer. It is hypothesised that within a drainage basin, sites undergoing little or no erosion accumulate ¹³⁷ Cs in their upper layers; cultivated soils will have ¹³⁷ Cs uniformly distributed within the cultivated layer; eroded soils, cultivated or not, will have relatively less ^l37 Cs, depending on the severity of erosion. Accumulated sediments will have characteristic ¹³⁷ Cs profiles reflecting temporal fallout variations and sedimentation history. This hypothetical model is largely confirmed by results from Maluna Creek basin, where erosion and accumulation of sediments has taken place. Soils under viticulture have about one third the ¹³⁷ Cs content of soils with grass cover, indicating more severe erosion under cultivation. Caesium-137 profiles in alluvial fan and flood plain deposits correlate with sediment layers and known cultivation history.     

Rock art as an archaeological and social indicator: The neolithisation of the Iberian Peninsula

Rock art has been regarded as a second class archaeological data source. In this paper we use Levantine rock art as a case study, to show how this situation can be reversed. This rock art, found along the Mediterranean region of the Iberian Peninsula, has been considered to be typically Mesolithic due to its distinctive hunting scenes. A review of certain archaeological indicators provides new arguments about its chronology, its socio-cultural attribution, and its significance. We first deal with the chronological issue, presenting the evidence that led to the recent consideration of the Levantine style as Early Neolithic, in synchrony with two other rock art styles (Schematic and Macroschematic). We will further propose that rock art itself is a central and independent source of information to explore the historical context of the Neolithisation of Mediterranean Iberia. Its wide geographical distribution allows us to study different and complementary territories as a single entity. This contrasts with the limitations posed by the study of settlements, whose differences, at a regional scale, are usually interpreted in terms of cultural variability. If we consider rock art as a key aspect in the constitution of social landscapes, it must not be treated just as an ideological by-product of the Neolithisation, but as a key factor in understanding this historical process.     

Radiocarbon dating of marine shell samples. The marine radiocarbon reservoir effect of coastal waters off Atlantic Iberia during Late Neolithic and Chalcolithic Periods

Radiocarbon dates on marine shells have not been used as extensively as charcoal or bone dates for the setting up of absolute chronologies because interpreting these dates is complicated by the marine radiocarbon reservoir effect. Nevertheless marine shellfish were used widely at least during the Holocene and their shells are abundant and usually well preserved in archaeological deposits located near shorelines. Consequently prior research concerning the oceanographic conditions and the marine radiocarbon reservoir effect of a particular coastal area is needed in order to set up reliable chronologies for that region. Values of regional marine radiocarbon reservoir effect – ΔR – of coastal waters off Atlantic Iberia, some of its variability along the Holocene and its correlation with the upwelling phenomenon were determined during previous research. More data related to the ΔR values for western Portuguese coastal waters during the Late Neolithic and the Chalcolithic, a time interval badly sampled in previous research, were recently obtained. These values can be compared with ΔR values already determined for the Gulf of Cadiz. Besides the importance of these values for a better knowledge of the palaeoceanography and palaeoclimatology of Atlantic Iberia, from now on archaeologists can set up reliable chronologies for Late Neolithic and Chalcolithic contexts using marine shell samples of that origin for radiocarbon dating.     

Reburial of shipwrecks in marine sediments: a long-term study on wood degradation

Historical shipwrecks in marine environments are continuously decomposed by wood-degrading organisms, adapted to this specific environment. To protect the wrecks from degradation and to preserve the unique remains for future generations, reburial of wrecks using various covering materials has been suggested as a long-term preservation method. The following experiment was carried out to study the bio-protective effect of sediment. Sound oak, pine, and birch samples were buried above and within marine sediment in Marstrand harbour, and retrieved for analysis after 6, 12, 24, and 36 months. Macroscopic as well as light-microscopic examinations were carried out on each occasion. Marine borers (shipworm, Limnoria), soft rot and tunnelling bacteria were wood degraders immediately above the seabed; soft rot, tunnelling bacteria, and erosion bacteria were active 10 cm below the seabed; erosion bacteria were the only degraders at a depth of 43 cm below the seabed. The wood species had different durability towards the decay agents. After 3 years, wood samples above the seabed were totally decomposed, whereas wood was highly protected at 43 cm depth in sediment. In the sediment, decay decreased significantly with depth of burial. The results suggest that reburial of shipwrecks in marine sediment can be recommended as a simple and efficient method for long-term preservation of the wooden cultural heritage.