Scholarly article on topic 'Refining human palaeodietary reconstruction using amino acid δ15N values of plants, animals and humans'

Refining human palaeodietary reconstruction using amino acid δ15N values of plants, animals and humans Academic research paper on "History and archaeology"

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{"Bone collagen" / "Cereal grains" / "Amino acids" / Nitrogen / "δ15N values" / Palaeodiet}

Abstract of research paper on History and archaeology, author of scientific article — Amy K. Styring, Rebecca A. Fraser, Rose-Marie Arbogast, Paul Halstead, Valasia Isaakidou, et al.

Abstract An established method of estimating the trophic level of an organism is through stable isotope analysis of its tissues and those of its diet. This method has been used in archaeology to reconstruct past human diet from the stable nitrogen isotope (δ15N) values of human and herbivore bone collagen. However, this approach, using the 15N-enrichment of human bone collagen δ15N values over associated herbivore bone collagen δ15N values to predict the relative importance of animal protein, relies on the assumptions that: (i) the δ15N values of plants consumed by humans and herbivores are identical, and (ii) the 15N-enrichment between diet and consumer is consistent. Bone collagen amino acid δ15N values have the potential to tackle these uncertainties, as they constrain the factors influencing bone collagen δ15N values. In this study, the δ15N values of glutamic acid and phenylalanine in human and herbivore bone collagen isolates from Neolithic sites in Germany, Greece and Turkey were determined by gas chromatography-combustion-isotope ratio mass spectrometry. The fraction of animal protein in total dietary protein consumed by the humans was estimated by: (i) comparing bulk human and herbivore collagen δ15N values, (ii) comparing bulk human and herbivore collagen and ancient charred cereal grain δ15N values, (iii) comparing human bone collagen δ15NGlutamic acid and δ15NPhenylalanine values, and (iv) comparing δ15NGlutamic acid values of human and herbivore bone collagen and estimated δ15NGlutamic acid values of ancient charred cereal grains. Where determined cereal grain δ15N values are higher than estimated herbivore forage values, estimates of animal protein consumption are significantly lower, emphasising the importance of the plant nitrogen contribution to human bone collagen. This study also highlights the need for further investigation into: (i) the Δ15NConsumer-Diet values of glutamic acid and phenylalanine in terrestrial ecosystems, and (ii) Δ15NGlutamic acid-Phenylalanine values of common plant foods in order to improve the accuracy and more widespread applicability of amino acid-based methods for palaeodietary reconstruction.

Academic research paper on topic "Refining human palaeodietary reconstruction using amino acid δ15N values of plants, animals and humans"

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Journal of Archaeological Science

journal homepage: http://www.elsevier.com/locate/jas

Refining human palaeodietary reconstruction using amino acid ô15N values of plants, animals and humans

Amy K. Styring a *, Rebecca A. Fraser b, Rose-Marie Arbogast c, Paul Halstead d, Valasia Isaakidou b, Jessica A. Pearson e, Marguerita Schäferf, Sevasti Triantaphyllou g, Soultana Maria Valamoti g, Michael Wallace c, Amy Bogaard b, Richard P. Evershed a

a Organic Geochemistry Unit, Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Bristol, UK b School of Archaeology, 36 Beaumont Street, University of Oxford, Oxford, UK c CNRS/UMR 7044 Maison Interuniversitaire des Sciences de l'Homme-Alsace, Strasbourg, France d Department of Archaeology, University of Sheffield, Sheffield, UK

e Department of Archaeology, Classics and Egyptology, University of Liverpool, Liverpool, UK f Institut für Prahistorische und Naturwissenschaftliche Archäologie, University of Basel, Basel, Switzerland g School of History and Archaeology, Aristotle University of Thessaloniki, Thessaloniki, Greece

ABSTRACT

An established method of estimating the trophic level of an organism is through stable isotope analysis of its tissues and those of its diet. This method has been used in archaeology to reconstruct past human diet from the stable nitrogen isotope (515N) values of human and herbivore bone collagen. However, this approach, using the 15N-enrichment of human bone collagen 515N values over associated herbivore bone collagen 515N values to predict the relative importance of animal protein, relies on the assumptions that: (i) the 515N values of plants consumed by humans and herbivores are identical, and (ii) the 15N-enrichment between diet and consumer is consistent. Bone collagen amino acid 515N values have the potential to tackle these uncertainties, as they constrain the factors influencing bone collagen 515N values. In this study, the 515N values of glutamic acid and phenylalanine in human and herbivore bone collagen isolates from Neolithic sites in Germany, Greece and Turkey were determined by gas chromatography-combustion-isotope ratio mass spectrometry. The fraction of animal protein in total dietary protein consumed by the humans was estimated by: (i) comparing bulk human and herbivore collagen 515N values, (ii) comparing bulk human and herbivore collagen and ancient charred cereal grain 515N values, (iii) comparing human bone collagen 515NGlutamic add and 515NPhenylalanine values, and (iv) comparing 515Nautamic acid values of human and herbivore bone collagen and estimated d15NGlutamic acid values of ancient charred cereal grains. Where determined cereal grain 515N values are higher than estimated herbivore forage values, estimates of animal protein consumption are significantly lower, emphasising the importance of the plant nitrogen contribution to human bone collagen. This study also highlights the need for further investigation into: (i) the A15Nc onsumer-Diet values of glutamic acid and phenylalanine in terrestrial ecosystems, and (ii) A15NGlutamic add-Phenylalanine values of common plant foods in order to improve the accuracy and more widespread applicability of amino acid-based methods for palaeodietary reconstruction.

© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/3.0/).

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ARTICLE INFO

Article history:

Received 9 April 2014

Received in revised form

29 September 2014

Accepted 6 November 2014

Available online 20 November 2014

Keywords: Bone collagen Cereal grains Amino acids Nitrogen S15N values Palaeodiet

* Corresponding author. Present address: School of Archaeology, 36 Beaumont Street, University of Oxford, Oxford, UK. Tel.: +44 0 1865 288014. E-mail address: amy.styring@arch.ox.ac.uk (A.K. Styring).

1. Introduction

Stable isotope analysis is routinely used to estimate the trophic position of an organism within a food web, based on the premise that the isotopic composition of a consumer's tissues originates from its diet, but is offset by trophic enrichment factors that are governed by underlying metabolic processes associated with nutrient assimilation and tissue biosynthesis. In archaeology, the

http://dx.doi.org/10.1016/j.jas.2014.11.009

0305-4403/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

extent to which the S15N of human bone collagen lies above the S15N values of herbivore bone collagen from the same archaeological site is an established method of estimating human trophic position, or rather the proportion of animal protein in the human diet (Hedges and Reynard, 2007; Koch, 2007; Lee-Thorp, 2008; Minagawa and Wada, 1984; Schoeninger et al., 1983). Interpretations, however, can be confounded by uncertainty regarding the 15N trophic enrichment factor between diet and consumer tissues (A15NConsumer-Djet); studies have found this to vary between 2 and 6%o (DeNiro and Epstein, 1981; Hare et al., 1991; Minagawa and Wada, 1984; O'Connell et al., 2012; Schoeninger and DeNiro, 1984). Since bulk bone collagen S15N values integrate the S15N values of their constituent amino acids, representing the net effect of dietary protein sources and metabolic cycling within the body, such variation in consumer-diet offsets is not easy to study using bulk collagen S15N values alone.

Determination of individual amino acid S15N values in bone collagen has the potential to shed more light on the underlying metabolic pathways responsible for the 15N-enrichment of consumer tissues over diet and to allow further elucidation of the often complex biochemical processes contributing to the bulk S15N value (Hare et al., 1991; Naito et al., 2010a,b; Styring et al., 2010). Equations have been developed to estimate the trophic level of humans and fauna in aquatic, C3-plant-based and C4-plant-based ecosystems based on the fact that the S15N values of the amino acids glutamic acid (Glu) and phenylalanine (Phe) increase to different extents with trophic level (8 and 0.4% respectively; Chikaraishi et al., 2011, 2010, 2009, 2007; Steffan et al., 2013). The benefit of this approach is that the S15N values of Glu (515NGlu) and Phe (S15Nphe) provide an internal trophic level indicator, precluding the need to rely upon the bone collagen S15N values of preserved fauna, whose tissues may not in fact have contributed to the human diet. The very small 15N-enrichment of S15NPhe with trophic level (DPhe) suggests that Phe has undergone very little metabolism within the body and therefore bone collagen S15NPhe values broadly reflect those of the diet, whereas the large 15N-enrichment in 515NGlu with trophic level (DGlu) suggests that bone collagen S15NGlu values result from subsequent N metabolism within the consumer.

The limitation of both of these methods is that they rely on the assumption that the S15N values of plants consumed by humans and the fauna they consume are identical. S15N value determinations of modern plants suggest that this assumption is implausible, since plants vary widely in their S15N values (e.g. Craine et al., 2009). One of the proposed reasons for high S15N values of human bone collagen, particularly during the European Neolithic, is consumption of manured crops by humans, since manuring increases the S15N values of plants (Bogaard et al., 2007; Durrwachter et al., 2006). Studies have shown that manuring of crops can increase cereal grain and chaff S15N values by as much as 10% and that S15N values are correlated to the amount of manure applied (Bogaard et al., 2007; Fraser et al., 2011; Kanstrup et al., 2011). The effect of this increase on crop S15N values is to raise the bone collagen S15N value of humans eating manured crops above that of herbivores eating unmanured plants, leading to overestimation of the importance of animal protein in the diet (Fig. 1).

In the past, the stable isotope values of plants consumed by humans on an archaeological site have rarely been determined directly from those preserved, due to concerns about contamination and the robustness of plant isotope values after years of burial. Carbonisation is the most common process by which plant material can survive in archaeological contexts and various studies have examined the changes in the S15N values of cereal grains with charring (Bogaard et al., 2007; Fiorentino et al., 2012; Fraser et al., 2013a; Kanstrup et al., 2012). Within the charring conditions

Fig. 1. Variation of human bone collagen S N value with fraction of animal protein in the diet, assuming a A15NCoiiagen-Diet value of 4%%, for humans eating animals and unmanured or manured plants. In this case, manuring increases the S15N value of plant by 2%. Following this model, a human bone collagen value of 10% could be due either to: (i) consumption of unmanured plants and 75% animal protein, or (ii) consumption of manured plants and 50% animal protein.

conducive to producing undistorted cereal grains similar to those found on archaeological sites (less than 250 °C for more than 6 h; Charles et al., in prep), the S15N values of charred einkorn grains were found to increase by around 1% (Fraseret al., 2013a). This was ascribed to the preferential loss of 14N-containing volatiles, during the thermal conversion of starch and protein in the grains to high molecular weight melanoidins (Styring et al., 2013), which are relatively resistant to degradation (Almendros and Dorado, 1999). Biochemical investigation of ancient charred grains from two archaeological sites in Europe found them to contain similar proportions of N as their modern charred counterparts and acid—base—acid pre-treatment, which is commonly used to remove contaminants in radiocarbon dating (Goh, 1991), resulted in little change in their %N or S15N values (Fraser et al., 2013a; Styring et al., 2013). Whilst further work is needed to establish the effect of a wider range of burial conditions and durations on the biochemical composition and S15N values of a wider range of cereal grain taxa, the charred grains preserved on archaeological sites have the potential to provide baseline S15N values of plants to include in palaeodietary predictions.

The amino acid S15N values of these cereal grains could also improve trophic level estimates by refining the amino acid trophic level equations. Although amino acids in archaeobotanical charred cereal grains do not survive in sufficient quantities for isotopic analysis (they contain 0.4% of the total hydrolysable amino acid content of fresh cereal grains; Styring et al., 2013), it may be possible to estimate their values based upon differences between bulk S15N values and amino acid S15N values determined in modern plants (Chikaraishi et al., 2010, 2011; Styring et al., 2014). Such calculations are discussed in more detail in Sections 2.5.3 and 2.5.4.

In this study, the bulk and amino acid S15N values of human and faunal bone collagen and the bulk S15N values of ancient charred cereal grains and pulse seeds from the archaeological sites of Vaihingen an der Enz, Germany, Makriyalos, Greece and ^atalhoyuk, Turkey were determined by elemental analysis-isotope ratio mass spectrometry (EA-IRMS) and gas chromatography-combustion-IRMS (GC-C-IRMS). We use the S15N values obtained to compare four different methods for estimating the proportion of animal

protein consumed by humans at each of the sites: (i) the 'Standard method' comparing human and faunal collagen 515N values; (ii) the 'Standard method plus plants', comparing human, faunal and ancient charred cereal grain d15N values; (iii) the 'Bone collagen S15NGlu and 515NPhe values method', comparing the difference between 515NGlu and 515NPhe values in human bone collagen with the difference between d15NGlu and d15NPhe values in modern plants, and (iv) the 'Bone collagen and plant d15NGlu values method', comparing the d15NGlu value of human and faunal bone collagen and the estimated d15NGlu value of ancient charred cereal grains. Each of these methods is described in more detail in Section 2.5. The archaeological sites were chosen because they all date to the Neolithic, between c. 8000 and 4500 BC, and display evidence of both herding and cultivation, but are located in very different environmental zones. Further details of the archaeological contexts can be found in Supplementary Text 1. Inline Supplementary Fig. S1 shows the site locations. Discussion of the relative merits and limitations of each method for estimating the proportion of animal protein consumed by humans will help to refine future interpretations of human diet from bone collagen d15N values. In particular, discussion of current amino acid-based stable isotope methodologies is hoped to inform priorities for future work needed to ground truth this approach.

Inline Supplementary Fig. S1 can be found online at http://dx. doi.org/10.1016/jjas.2014.11.009.

2. Materials and methods

2.1. Bone collagen extraction and analysis

Human bone was sampled from the compact mid-shaft of the femur (preferred), another long bone, or the rib. For faunal sampling, the distal humerus was generally sampled, taking sidedness into account to avoid sampling the same individual more than once. Collagen was extracted from approximately 1 g of compact bone using a modified Longin method (Longin, 1971): bone samples were crushed and immersed in 0.5 M HCl until demineralised and then washed three times in Milli Q water. Samples were then gelatinised by adding pH 3 water and heating to 70 °C for 48 h. The gelatinous solution was then filtered through an 80 mm Ezee-filter and transferred to clean test tubes and freeze-dried. Bone collagen samples of approximately 0.75 mg were weighed into tin capsules for determination of 513C and 515N values. Collagen yields ranged from 0.9 to 17.0% and the molar C:N ratios between 2.9 and 3.6, which is within the accepted range for well-preserved collagen (DeNiro, 1985).

2.2. Archaeobotanical sampling and analysis

Ancient charred cereal grain and pulse seed samples consisted of at least ten whole grains/seeds from the same stratigraphic unit, derived mostly from visible concentrations such as 'storage deposits'. The grains/seeds selected were virtually undistorted morphologically (displaying slight puffing only), resembling modern material charred experimentally at around 230 °C for a prolonged period (up to 24 h; Fraser et al., 2013a). Grains/seeds were examined at x7-45 magnification for visible surface contaminants, such as adhering sediment or plant roots; these were removed by gentle scraping. Grains/seeds were weighed and placed in glass test tubes in preparation for an acid-base-acid (ABA) pre-treatment, a procedure commonly applied to charcoal and charred plant remains prior to radiocarbon and stable isotope analysis (Goh, 1991) and considered appropriate for use on archaeobotanical remains (Fraser et al., 2013a). This three-step procedure consisted of: 1) treatment with 10 mL of 0.5 M hydrochloric acid (HCl) at 70 °C for

30-60 min, or until any effervescing ceased, and then rinsing in distilled water three times, 2) treatment with 10 mL of 0.1 M sodium hydroxide (NaOH) at 70 ° C for 60 min, followed by rinsing in distilled water until the solution was clear and the pH neutral, using a minimum of three rinses, 3) treatment with 10 ml of 0.5 M HCl at 70 ° C for 30-60 min, followed by three rinses in distilled water and final freeze drying. Grains/seeds were ground to fine homogeneous powder using a mortar and pestle and weighed into tin capsules ready for bulk d13C and d15N determinations.

The C:N molar ratios of modern grains charred at 230 °C for 24 h under a reducing atmosphere have been found to range from 17.9 to 33.4, with an average C:N molar ratio of 22.7 (n = 17; Fraser et al., 2013a). Of the ancient charred grain samples from Vaihingen, 13 out of 13 had C:N molar ratios within this range (18.0-28.6); 3 out of 3 grain samples from Makriyalos had C:N molar ratios within this range (25.3-26.1) and 6 out of 15 grain samples from ^atalhoyuk had C:N molar ratios within a similar range (13.2-21.1). The grains from ^atalhoyuk tended to have higher %N contents than those from the other sites, which could be due to post-depositional contamination, loss of non-N containing organics during burial or natural variation in %N caused by the environment in which they were grown. Work by Vaiglova et al. (in press) found no indication of post-depositional contamination in lentils or peas from £ata-lhoyuk, which suggests that the high %N is a factor of the environment in which they were grown. This needs to be investigated further by N isotope analysis of modern plants from around the site.

The C:N molar ratios of modern pulse seeds charred at 230 ° C for 24 h under a reducing atmosphere were found to range from 8.8 to 13.1, with an average C:N molar ratio of 10.8 (n = 15; Fraser et al., 2013a). Of the ancient charred pulse seed samples from Vaihingen, 3 out of 3 had C:N molar ratios within a similar range (10.0-13.7); 0 out of 1 pulse seed samples from Makriyalos had C:N molar ratios within this range (8.0) and 4 out of 8 pulse seed samples from ^atalhoyuk had C:N molar ratios within a similar range (8.7-11.1). The seed samples from Makriyalos and £ata-lhoyuk have higher %N contents than those determined in modern charred pulse seeds, which could also be a feature of the crop growing conditions and/or burial environment. Given that the cereal grains from Makriyalos did not have unusually high %N, the high %N in the pulse seeds is probably due to a difference in microscale growing conditions rather than a depositional or environmental effect that would have increased the %N of both cereal grains and pulse seeds. More modern and archaeological plant samples need to be analysed to resolve this. To account for the observed effects of experimental charring (at 230 °C for 24 h under a reducing atmosphere) on cereal grain and pulse seed 515N values, a generous offset of 1%o is subtracted from the measured 515N values (cf. Fraser et al., 2013a).

2.3. Bulk S13C and S15N determinations

Bulk 12C/13C analysis was performed by sample combustion in a Costech 4010 on-line to a VG TripleTrap and Optima dual-inlet mass spectrometer. Isotope ratios were calculated relative to the VPDB reference by comparison with co-run laboratory standards (of plant material) calibrated against NBS-19 and NBS-22. Bulk 15N/14N analysis was performed by sample combustion on a ThermoFinni-gan system comprising an elemental analyser linked under continuous flow with a Delta + XL mass spectrometer (Thermo-Finnigan, Bremen). Isotope ratios were calculated as 515N versus atmospheric N2 by comparison with standards calibrated against IAEA-N-1 and N-2. The relative analytical errors (1 standard deviation) for replicate analytical standards were ±0.2% for 513C and ±0.4% for 515N. Replicate analyses of a bone collagen sample VAH35 measured in eleven separate mass spectrometry runs had a

standard deviation (1SD) of ±0.3% for S13C, ±0.2% for S15N and ±0.2 for the C:N molar ratio (mean C:N of 3.3). For plant material, the precision (1SD) among replicates of a well-homogenized modern uncharred barley sample was ±0.2 for %N and ±0.4% for S15N analysed in 29 separate runs and ±3.5 for %C and ±0.1% for S13C, analysed in 21 separate runs.

2.4. Amino acid S15N value determinations

2.4.1. Hydrolysis of bone collagen

Approximately 2 mg of collagen was hydrolysed in culture tubes (6 M HCl, 2 mL, 100 °C, 24 h). On cooling, the hydrolysates were evaporated under a stream of N2 and redissolved in 2 mL 0.1 M HCl before being stored at -18 °C until required for analysis. Fractions of the protein hydrolysates (250 mL) were transferred to culture tubes and dried under N2 before undergoing derivatisation. A known quantity of norleucine was added to each sample as an internal standard.

2.4.2. Preparation of amino acid derivatives (N-acetyl-i-propyl esters)

Amino acids were converted to their i-propyl esters by addition of 1 mL of a 4:1 v/v mixture of isopropanol and acetyl chloride (acetyl chloride added dropwise in an ice bath). Culture tubes were then sealed and heated at 100 °C for 1 h. Reagents were evaporated under a gentle stream of N2 at room temperature. Dichloromethane (DCM) was added (2 x 0.5 mL) and evaporated in an ice bath to remove excess reagents. Amino acid i-propyl esters were then treated with 1 mL of a mixture of acetic anhydride, triethylamine and acetone (1:2:5, v/v/v; 10 min, 60 °C). Reagents were evaporated under a gentle stream of N2 in an ice bath. The samples were dissolved in 2 mL ethyl acetate and 1 mL saturated sodium chloride solution was added. After phase separation, the organic phase was collected and the extraction repeated with an additional 1 mL of ethyl acetate. The combined organic phases were evaporated under N2 in an ice bath and the residual water removed with successive 1 mL aliquots of DCM and evaporated under N2 in an ice bath. The N-acetyl-i-propyl (NAIP) esters were dissolved in ethyl acetate and stored at -18 °C until required for analysis.

2.4.3. Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS)

Amino acids were identified by GC by comparison of their retention times with those of amino acid standards and their S15N values were determined by GC-C-IRMS. A ThermoFinnigan DeltaPlus XP system (Thermo Electron Corporation) was used to determine the S15N values of derivatised amino acids. The mass spectrometer (EI, 100 eV, three Faraday cup collectors m/z 28, 29 and 30) was interfaced to a Thermo Electron Trace 2000 gas chromatograph via a ThermoElectron gas chromatograph combustion III interface (CuO/NiO/Pt oxidation reactor maintained at 980 °C and reduction reactor of Cu wire maintained at 650 °C). Samples were introduced using a PTV injector held at 200 °C. Helium at a flow of 1.4 mL min-1 was used as the carrier gas and the mass spectrometer source pressure was maintained at 9 x 10-4 Pa. The separation of the amino acids was accomplished using a DB-35 capillary column (30 m x 0.32 mm internal diameter; 0.5 mm film thickness; Agilent Technologies, UK). The oven temperature of the GC was started at 40 °C and held for 5 min before heating at 15 °C min-1 to 120 °C, then 3 °C min-1 to 180 °C, then 1.5 °C min-1 to 210 °C and finally 5 °C min-1 to 270 °C and held for 1 min. A Nafion membrane removed water and a cryogenic trap was employed in order to remove CO2 from the oxidised and reduced sample.

All S15N values are reported relative to reference N2 of known nitrogen isotopic composition, previously calibrated against the AIR

international isotope standard, introduced directly into the ion source in four pulses at the beginning and end of each run. Each reported value is a mean of triplicate S15N determinations. An amino acid standard mixture, comprising amino acids whose S15N values were individually determined by EA-IRMS, was run every 3 runs in order to monitor instrument performance. The S15N values of the amino acids in the standard mixture were within 0.8% of their S15N values measured separately by EA-IRMS, with a precision of better than 0.8% (cf. Styring et al., 2012). Fig. 2 shows a GC-C-IRMS chromatogram displaying the separation of amino acids in archaeological human bone collagen.

2.5. Methods of estimating the contribution of animal protein to human diet

The following four methods were used to estimate the proportion of animal protein consumed by humans at each of the archaeological sites in this study. See Table 1 for a description of each of the terms used in the equations.

2.5.1. 'Standard method'

The 'Standard method' assumes that humans eating only plant protein will have the same bone collagen S15N value as the local herbivores, whereas humans eating only animal protein will have a bone collagen S15N value a trophic level higher than the local herbivores (Hedges and Reynard, 2007). This approach also assumes that the 15N trophic enrichment factor between diet and consumer tissues (D15NConsumer-Diet) is the same for herbivores and carnivores and for diets of differing protein content, both of which are assumptions that are still under debate (e.g. Hussey et al., 2014). The equation for estimating the proportion of animal protein consumed

S15N[hum] - S15N[herb]

^Consumer-diet

See Supplementary Text 2 for an explanation of how this equation was derived.

Fig. 2. A GC-C-IRMS chromatogram displaying the separation of amino acids in archaeological human bone collagen. Amino acids: Ala alanine, Gly glycine, Val valine, Leu leucine, Nle norleucine, Thr threonine, Ser serine, Pro proline, Asx aspartic acid, Glx glutamic acid, Hyp hydroxyproline, Phe phenylalanine, Lys lysine.

Summary of the terms in equations used to estimate the proportion of animal protein in human diet.

Description

815N[gr] 515N[herb] 515N[hum] 815Nciu[gr]

S15Nciu[herb] 815Nciu[hum] S15Nphe[hum] \ 15n

D 1NConsumer-Diet

A15Nclu-Phe* Aciu

Mean of ancient charred cereal grain 515N values Mean of domestic herbivore collagen 515N values Mean of human collagen 515N values Estimated 515NGlu value of ancient cereal grains on the site

Mean of domestic herbivore collagen 515NGlu values Mean of human collagen 515NGlu values Mean of human collagen 815NPhe values 15N trophic enrichment factor between consumer and diet 515N, defined as 4%o in this study (the mean of the 2—6°%o 15N-enrichment determined in previous studies; Hedges and Reynard, 2007; O'Connell et al., 2012)

Difference between the 515NGlu and 815NPhe values

of plants at the base of the food chain

15N trophic enrichment factor between consumer and

diet collagen 515NGlu, defined as 8.0 ± 1.2% in this

study (Chikaraishi et al., 2009)

15N trophic enrichment factor between consumer and

diet collagen 515NPhe, defined as 0.4 ± 0.5% in this

study (Chikaraishi et al., 2009)

Proportion of animal protein in the human diet

25.2. 'Standard method plus plants'

The 'Standard method plus plants' assumes that humans eating only plant protein will have a bone collagen 515N value a trophic level higher than that of associated ancient charred grains and humans eating only animal protein will have a bone collagen d15N value a trophic level higher than that of local herbivores. Human bone collagen 515N values falling between the two indicate a mixed plant-animal diet of varying proportions. The equation is:

(515N[hum] - A15Nconsumer-diet) ~ 515N[gr] _ 515N[herb] - 515N[gr]

See Supplementary Text 3 for an explanation of how this equation was derived.

2.5.3. 'Bone collagen SlsNau and SlsNPhe values method'

The 'Bone collagen 515NGlu and 515Nphe values method' was developed by Chikaraishi et al. (2009), who use this equation to calculate the trophic level of a consumer:

Trophic level

515Nr,„ - 515Nphe — A15N,

NGlu-Phe

(DGlu - DPhe)

Since we are interested in the fraction of herbivore (animal) protein consumed in our study, it is necessary to subtract rather than add 1, so a human consuming 100% plant protein has a value of

G = 0:

_ (515Ng1u jhumj - 515Nphe[hu^ - A15Nqu-Phe*) -1

(DGlu - DPhe)

Table 2 summarises the difference in plant 515NGlu and 515Nphe (A15Na u-phe*) values determined in previous studies. Chikaraishi et al. (2010) use a A15Na u-phe* value of —8.4% as the basis of their C3 ecosystem trophic level equation, established from A15NGlu-Phe values determined in modern C3 leaves (n = 16; Table 2). Styring et al. (2014) have determined the amino acid 515N values of manured and unmanured bread wheat (Triticum aestivum) and barley grains (Hordeum vulgare; n = 8) from the experimental farming sites of Rothamsted, UK and Bad Lauchstädt, Germany. The

Table 2

Summary of S15NGlu, S15NPhe and D15NGlu -Phe* values determined for different plant types in previous studies.

Plant type

n 515NGlu 515NPhe D15NGlu-Phe* Reference

C3 tree and grass leaves 16 2.0: t 4.9 10.4 : 5.0 -8.4 ± 1.6 Chikaraishi et al., 2010

Barley and bread wheat 4 5.4: 4.2 12.3 4.2 -6.9 ± 0.2 Styring et al.,

grains from 2014

Rothamsted, UK

Barley and bread wheat 4 3.0 : 2.2 12.0 1.9 -9.0 ± 0.3 Styring et al.,

grains from Bad 2014

Lauchstadt, Germany

Broad beans and peas 4 0.9 : 0.2 0.8 - 1.1 0.1 ± 1.0 Styring et al.,

from Bad Lauchstadt, 2014

Germany

C4 grass leaves 7 4.4 6.1 4.0 6.9 0.4 ± 1.7 Chikaraishi et al., 2010

D15NGlu -Phe values of these cereal grains were not affected by species or manure application, but differed significantly between locations; cereals grown at Rothamsted had a D15NGlu-Phe value of -6.9 ± 0.2%, whereas those grown at Bad Lauchstadt had a D15NGlu-Phe value of -9.0 ± 0.3% (t(6) = 10.606, p < 0.001). The significant difference between the D15NGlu-Phe values of cereal grains from different sites cautions against using a standardised D15Ngiu -Phe* value as the basis of a trophic level equation, without carrying out further D15NGlu-Phe value determinations in the relevant geographic region of the site, or without further assessment of variation in this value. We consider the effect of such variation in plant D15NGl u-Phe values on the estimate of animal protein consumption by humans using Equation (3) (see Section 3.3). Chikaraishi et al. (2010) and Styring et al. (2014) found that the D15Ngiu -Phe values of C4 terrestrial plants (n = 7) and pulses (broad beans and peas; n = 4) are much higher than that of C3 terrestrial plants (Table 2), which would influence predictions of plant protein contribution to human diet if humans were eating significant quantities of C4 plants and/or pulses. Although there is no evidence for significant C4 plant consumption by humans at the sites in this study, since their d13C values are more negative than -18% (Section 3.1), there is a possibility that pulses played an important role in the Neolithic diet (cf. Bogaard, 2012, 2013; Valamoti, 2004).

The 15N trophic enrichment factor between consumer and diet 515N values of Glu and Phe (DGl u and DPhe) from four controlled feeding experiments using green algae, zooplankton and fish were found to be 8.0 ± 1.2% and 0.4 ± 0.5%, respectively (compiled by Chikaraishi et al., 2009). These DGlu and DPhe values have been used in estimates of trophic position in planktonic ecosystems (Hannides et al., 2009; McCarthy et al., 2007; McClelland et al., 2003; Schmidt et al., 2004), terrestrial ecosystems (Chikaraishi et al., 2011) and ancient human skeletal remains (Naito et al., 2010a,b). Recently, two studies have determined the D15NConsumer-Diet values of amino acids in seals fed on herring (Germain et al., 2013) and in four large carnivorous fish species (Hoen et al., 2014). They found that the difference between DGlu and DPhe values in these high trophic level consumers was much lower than that determined in lower trophic position marine organisms, implying that the difference between DGlu and DPhe values may be affected by the quantity and/or quality of protein in the diet (Hoen et al., 2014). Since there have been no feeding studies carried out on terrestrial mammals, we use the DGlu and DPhe values of 8% and 0.4%, since they have been used in previous studies of human palaeodiet. In addition, the DGlu and DPhe values determined for seals in the Germain et al. (2013) study yield a difference between DGlu and DPhe values of 2.9% (2.9—0%), which produces unrealistically high estimates (over 100% in most cases) of animal protein consumption by humans. It is clear that D15NCon-sumer-Diet amino acid values need to be determined for mammals

with similar digestive systems to humans (i.e. pigs) in order to resolve these uncertainties.

2.5.4. 'Bone collagen and cereal d15NGlu values method'

The 'Bone collagen and plant 515NGlu values method' takes into account the d15NGlu values of human and faunal bone collagen and the estimated d15NGl u value of ancient charred cereal grains from the same archaeological sites. This method focuses upon bone collagen and plant d15NGlu values because the 15N trophic enrichment factor between consumer and diet collagen 515NGlu (DGlu) is much larger than that of Phe (8.0% compared to 0.4%), allowing greater distinction between the d15NGlu values of humans eating only plant or only animal protein. The d15NGlu value of ancient charred cereal grains can be estimated from comparison of their determined bulk d15N values and known differences between bulk d15N values and d15NGl u values in modern cereal grains. Using the cereal grain amino acid d15N values determined by Styring et al. (2014), it is found that the 515NGlu value relative to the bulk grain d15N value (D15NGl u-Bulk value) was not affected by site or manure application, but differed significantly between T. aestivum (-0.5 ± 0.5%) and H. vulgare (+1.4 ± 0.5%; t(6) = 5.548,p = 0.001). Since the cereal grains from the archaeological sites in this study were predominantly glume wheats (Triticum monococcum and Triticum dicoccum), whose 515NGlu values have not been determined in modern grains, the D15NGlu-Bulk value determined for T. aestivum (from the same genus as the glume wheats) was used to estimate the d15NGlu value of ancient charred cereal grains. It would be advisable to determine A15NGlu-Buu( values for T. monococcum and T. dicoccum in future studies to test the reliability of this approach. Thus, the 515NGlu value of the ancient charred cereal grains was estimated by subtracting 0.5% from the bulk 515N value of charred cereal grains from the site (also corrected for charring by subtracting 1% from the determined bulk 515N value). It is therefore assumed that humans eating 100% cereal protein will have a bone collagen d15NGl u value 8.0% higher than that of the associated charred cereal grain d15NGlu value and humans eating 100% animal protein will have a bone collagen 515NGlu value 8.0% higher than that of the local herbivore bone collagen 515NGlu value. The equation for this method is the same as Equation (2), but substitutes bulk collagen/grain d15N values with d15NGlu values:

(s15Nqu[hum] - Aglu) - 515Ngiu [gr]

515NGlu[herb]-515NGlu[gr])

2.6. Statistical analysis

Independent t-tests were used to detect differences in crop stable isotope values between site, species and manuring regime for the modern field studies. A Kruskal Wallis test with post-hoc Bonferroni-Dunn test was used to detect differences in bulk collagen and amino acid 515N values between the herbivore species at each archaeological site due to non-normal distribution of the data.

3. Results

3.1. Bulk isotope analyses

3.1.1. Vaihingen an der Enz, Germany

Bulk 513C and 515N values of bone collagen isolates from Vaihingen are plotted in Inline Supplementary Fig. S2. For the full 513C and 515N value dataset of bone collagen and archaeobotanical samples from the site, and for further discussion of land use and

palaeodietary interpretation at Vaihingen using bulk collagen isotope values, see Fraser et al. (2013b). Mean 515N values are plotted in Fig. 3a and those used in the estimates of human diet are given in Table 3. Cereal grains (T. monococcum and T. dicoccum) exhibit mean 513C and 515N values of -24.1 ± 0.5% and 4.5 ± 0.5%, respectively. The pulse seeds (Pisum sativum and Lens culinaris) have relatively high 515N values (2.8 ± 1.5%) compared to atmospheric N2. In modern studies, only pulses grown on artificial 'dung-soil' in Evvia, Greece were found to have such high 515N values (Fraser et al., 2011). The 513C and 515N values of the domestic herbivores are very similar to the average values determined at other Linearbandkeramik sites (cf. Durrwachter et al., 2006; Oelze et al., 2011; Bickle and Whittle, 2013), and reflect a terrestrial herbivore diet in a temperate climate. Wild taxa constitute c.15% of the faunal remains (Schafer, 2011) and the wild herbivores have similar 515N values to the domestic herbivores (average 515N value is 6.4 ± 0.4%). Human bone collagen 515N values vary from 8.0 to 10.2%, with a juvenile individual exhibiting a lower bone collagen 515N value of 5.8%. This individual is discounted from the dietary calculations.

Inline Supplementary Fig. S2 can be found online at http://dx. doi.org/10.1016/jjas.2014.11.009.

3.1.2. Makriyalos, Greece

Bulk 513C and 515N values of bone collagen isolates from early Late Neolithic Phase I of Makriyalos (dating to 5500 to 5000 BC) are plotted in Inline Supplementary Fig. S3. The 513C and 515N values of 5 pigs determined by Triantaphyllou (2001) from late Late Neolithic Phase II of Makriyalos (c.4900 to 4500 BC) are also plotted, since pig bones account for a large proportion of the faunal assemblage (Pappa et al., 2004). Mean 515N values are plotted in Fig. 3b and those used in the estimates of human diet are given in Table 3. Cereal grains (T. dicoccum) exhibit mean 513C and 515N values of -24.4 ± 0.1% and 0.4 ± 0.3%, respectively. More cereal grains and pulse seeds need to be analysed from this site to gain a better idea of the crop baseline 515N signature, but these initial isotope determinations provide a basis for further work. The large animal bone assemblage from Phase I is heavily dominated by domesticates (>99%), among which pigs, cattle and sheep are more strongly represented than goats and dogs. Relative proportions differ somewhat both between context types and according to method of quantification (Pappa et al., 2013, 2004). The cattle are not considered to contribute significantly to the human bone collagen isotope values, since their 513C values (-16.1 ± 2.0%) are significantly higher than those of the humans (-20.4 ± 0.3%; t(16.68) = 8.70, p < 0.001) and indicate a C4 plant component to the cattle diet. The human bone collagen 515N values range from 5.3 to 9.3%, with a mean of 7.6 ± 1.0%. These values are very similar to those determined for the bone collagen isotope values of 18 human bone samples from the same Late Neolithic phase determined in a previous study (between 4.9 and 8.3%; Triantaphyllou, 2001).

Inline Supplementary Fig. S3 can be found online at http://dx. doi.org/10.1016/j.jas.2014.11.009.

3.1.3. fatalhoyuk, Turkey

Bulk 513C and 515N values of bone collagen isolates from ^atalhoyuk are plotted in Inline Supplementary Fig. S4. Bulk collagen isotope values were determined by Jessica Pearson and are discussed in more detail in Pearson (2013). The latter also compares human bone collagen 515N values from different areas, different buildings and across different levels of the site to determine potential differences in the importance of animal protein in the human diet. For the purposes of this study, we have taken the mean 515N value of all human bone collagen at the site. Mean 515N values are plotted in Fig. 3c and those used in the estimates of human diet

Fig. 3. Mean S13C and S15N values (with ±1 SD) of human and faunal bone collagen and cereal grains and pulse seeds from: a) Vaihingen an der Enz, Germany, b) Makriyalos, Greek Macedonia, and c) Çatalhôyuk, Turkey. Charred grain and pulse S15N values are adjusted for charring effect (cf. Fraser et al., 2013a,b).

are given in Table 3. The 515N values of cereals (H. vulgare, T. dicoccum, Triticum durum/aestivum, T. monococcum) and pulses (P. sativum) from the site are relatively high and it is unclear whether this is a result of manuring, or a factor of the environment. High plant 515N values can be caused by aridity (Hartman and Danin, 2010; Heaton, 1987), or waterlogged conditions, which result in denitrification (Finlay and Kendall, 2008). The range of isotope values for domestic sheep and goats from the site is very wide, suggesting that they fed across varied ecological zones (Pearson et al., 2007). Human bone collagen 515N values range from 9.2 to 15.1%, which would seem to indicate varied sources of dietary protein.

Inline Supplementary Fig. S4 can be found online at http://dx. doi.org/10.1016/j.jas.2014.11.009.

3.2. Amino acid nitrogen isotope analyses

The bone collagen 515NGlu and 515NPhe values were determined for a subset of five humans, five domestic herbivores and five wild herbivores (three from Makriyalos) on each site. Individuals with bulk collagen 513C and 515N values closest to the mean for their species were selected from Vaihingen and Makriyalos in order to minimise variation. Since glutamine is deamidated to form

glutamic acid during hydrolysis of bone collagen, the 515NGlu value determined by GC-C-IRMS represents both the nitrogen of glutamic acid and the amino-nitrogen of glutamine. The mean bone collagen 515N, 515NGlu and 515NPhe values of each species from each site are given in Table 3. Estimated 515NGlu, 515NPhe and D15NGlu-Phe values of the cereal grains are given in italics. Bone collagen 515N, 515NGlu and 515NPhe values for all of the individuals are given in Inline Supplementary Table S1.

Inline Supplementary Table S1 can be found online at http://dx. doi.org/10.1016/j.jas.2014.11.009.

3.2.1. Vaihingen an der Enz, Germany

There is no significant difference in the 515NPhe value of humans, domestic cattle and wild deer from Vaihingen (10.7 ± 1.1 %; c2 (2) = 1.808, p = 0.405), which contrasts with the significant difference in their bulk 515N values (c2 (2) = 12.5, p = 0.002). This similarity in 515NPhe values has also been observed for terrestrial mammals in other studies (e.g. Naito et al., 2010a,b; Styring et al., 2010) and indicates that the 515NPhe values of the human, cattle and deer diets are very similar, since the 515N value of Phe increases by only 0.4% between diet and consumer. Phe undergoes very little metabolic routing in the body: it is not biosynthesised and is cat-abolised into tyrosine via a metabolic pathway that involves no

Mean 515N values determined for bone collagen isolates and charred cereal grains and pulse seeds from the sites of Vaihingen, Makriyalos and ^atalhöyük.

Species

n Bulk 5 N 515NGlu (%o) (%o)

5 Nphe A15NG,u-Phe (% ) (% )

Vaihingen All humans 47 9.1 ± 0.7 - - -

Humansa 5 9.1 ± 0.1 12.2 ± 1.2 10.7 ± 1.6 1.5 ± 1.8

All domestic 29 6.8 ± 0.8 - - -herbivores

Domestic 5 6.8 ± 0.1 9.7 ± 0.8 10.8 ± 1.1 -1.1 ± 1.3 cattlea

All wild deer 9 6.2 ± 0.4 - - -

Wild deera 5 6.2 ± 0.1 8.7 ± 1.0 10.4 ± 0.9

Cereal grains 13 4.5 ± 0.5 4.0b 10.8c -6.9, -9.0,

Pulse seeds 3 2.8 ± 1.5 - - -

Makriyalos All humans 18 7.6 ± 1.0 - - -

Humansa 5 7.7 ± 0.5 11.1 ± 1.4 9.8 ± 1.7 1.3 ± 1.5

All domestic 54 5.1 ± 1.0 - - -herbivores

Domestic 5 5.2 ± 0.1 7.8 ± 1.4 9.9 ± 1.7 -2.2 ± 1.5 sheepa

All wild deer 3 4.9 ± 0.6 - - -

Wild deera 3 4.9 ± 0.6 6.8 ± 1.6 8.6 ± 2.0 -1.8 ± 1.8

Cereal grains 3 0.4 ± 0.3 -0.1b 9.9c -6.9, -9.0,

^atalhöyük All humans 67 12.5 ± 1.2 - - -

Humansa 5 13.0 ± 1.0 16.7 ± 2.1 13.5 ± 2.2 3.3 ± 0.9

All domestic 204 10.0 ± 1.7 - - -herbivores

Domestic 5 10.0 ± 1.3 12.5 ± 2.1 13.3 ± 2.2 -0.8 ± 0.9 caprinesa

All wild cattle 70 9.9 ± 1.8 - - -

Wild cattlea 5 9.7 ± 1.4 12.7 ± 1.5 12.1 ± 1.6 0.6 ± 0.6

Cereal grains 6 6.7 ± 0.9 6.2b 13.3c -6.9, -9.0,

Pulse seeds 4 2.7 ± 1.7 - - -

a Individuals chosen for amino acid isotope analysis. b Estimated by subtracting 0.5°%o from the average cereal grain 515N value. c Estimated from the average domestic herbivore 515NPhe value (see Section 3.2.1).

d A15NGiu_Phe values determined in modern T. aestivum and H. vulgare grains grown at (i) Rothamsted, UK, and (ii) Bad Lauchstödt, Germany (Styring et al., 2014) and (iii) in modern C3 leaves (Chikaraishi et al., 2010).

breaking of a N bond (Salway, 1999) and therefore no isotopic fractionation would be expected. Unfortunately, the 515Nphe values of human and faunal bone collagen and plant protein cannot be used to estimate the relative contributions of plant and animal protein to the diet because the differences in 515NPhe values between fauna and plants are smaller than instrumental errors.

The human 515NPhe value derives from both the plant and animal contributions to the diet, however, and since the determined human and domestic herbivore 515NPhe values are very similar, it can be inferred that the 515NPhe value of the plants eaten by the humans is also similar to that of the domestic herbivore 515NPhe value, regardless of the relative contributions of animal and plant protein to the human diet. This similarity need not imply, however, that the bulk 515N values of plants eaten by humans and herbivores are also the same, because offsets between 515NPhe and bulk 515N values vary among plant taxa and the 515N values of the 19 other amino acids in plant protein also contribute to the bulk plant 515N value (Styring et al., 2014).

Using the cereal grain amino acid 515N values determined by Styring et al. (2014), it is found that the 515NPhe value relative to the bulk grain 515N value (A15NPhe-Bun< value) is 8.4 ± 1.5%o. Since this value differs slightly between site and species, we have decided to use an average A15NPhe-Bun< value in our calculations. Subtracting the D15NPhe-Bulk cereal grain value from the 515NPhe value inferred for the plant portion of the human diet (Table 3) gives an estimate

of the bulk plant 5 N value if humans were eating only cereal grains. For Vaihingen, the calculated cereal grain 515N value is 2.4%, which is lower than the 515N values determined for the 13 cereal grains from Vaihingen (4.5 ± 0.5%; Fig. 4a). This suggests that the plants consumed by humans from Vaihingen were not restricted to cereal grains with similar 515N values to those determined in this study, but may have included plants with lower 515N values such as pulses (Inline Supplementary Fig. S2).

The bone collagen 515NGlu values are much more variable and differ even between individuals of the same species (within-spe-cies differences account for 21% of the variation). Nevertheless, the difference between the 515NGlu and bone collagen 515N values (D15NGlu-Bulk) between species is not significant (2.8 ± 0.8%; c2 (2) = 0.740, p = 0.691). This is likely due to the central role that Glu plays both in the biosynthesis of other amino acids and in the excretion of waste N in the form of urea. The amino group of Glu is donated to keto acids to form other amino acids (Salway, 1999) and the first step in the formation of urea occurs during the deamination of Glu by Glu dehydrogenase (Sick et al., 1997). The 515N value of Glu is therefore much more sensitive to differences in metabolic function, which can vary between individuals and also tends to reflect the average 515N value of bone collagen amino acids.

3.2.2. Makriyalos, Greek Macedonia

There is no difference in the 515NPhe value of humans, domestic sheep and wild deer from Makriyalos (9.8 ± 1.7%; c2 (2) = 0.908, p = 0.635), which contrasts with the significant difference in bulk 515N values between humans and herbivores (c2 (2) = 8.580, p = 0.014). It can therefore be inferred that the 515NPhe value of the plant portion of the human diet is similar to the 515NPhe value of domestic herbivore bone collagen (Table 3), regardless of animal protein consumption. Using a A15NPhe-Bun< value of 8.4%, the 515N value of plants consumed by humans at Makriyalos is estimated to be 1.5%, which is slightly higher than the 515N values determined for the 3 cereal grains from Makriyalos (0.4 ± 0.3%; Fig. 4b). This implies that the plants consumed by humans from Makriyalos were not restricted to cereal grains with similarly low 515N values to those determined in this study. Analysis of greater numbers of cereal grains and pulses from Makriyalos would reveal whether crops with these higher 515N values are preserved on the site.

Again, bone collagen 515NGlu values are much more variable and differ between and within species (within-species differences account for 17% of the variation). In contrast to individuals at Vaihingen, there is a significant difference in the A15NGlu-Buu( values between species (c2 (2) = 8.949, p = 0.011). A post-hoc Bonferro-ni—Dunn test showed the significant difference to be between red deer and humans (p = 0.013), with A15NGlu-Buu( values of the red deer lower than the humans. This seems to indicate that the 515NGlu value does not reflect the average of amino acid 515N values in red deer bone collagen at Makriyalos.

3.2.3. fatalhoyuk, Turkey

There is no significant difference in the 515NPhe value of humans, domestic sheep/goats and wild/domestic cattle from ^atalhoyuk (13.0 ± 1.6%; c2 (2) = 2.660, p = 0.264), which contrasts with the significant difference in bulk 515N values (c2 (2) = 9.420, p = 0.009). It can therefore be inferred that the 515NPhe value of the plant portion of the human diet is similar to that of the domestic herbivores (Table 3), regardless of animal protein consumption. Using a D15NPhe -Bulk value of 8.4%, the 515N value of plants consumed by humans at ^atalhoyuk is estimated to be 4.9%, which is lower than the 515N values determined for the 6 cereal grain samples from the site (6.7 ± 0.9%; Fig. 4c). This suggests that, like at Vaihingen, the plants consumed by humans from ^atalhoyuk may have included

8.4%o x

1 Grain I I Human Herbivore

o i ♦

8.4%o x

Grain Human Herbivore

X 8.4%o 1 X

Human Herbivore

Ô15NGIU

15 -(%o)

ô15NBulk (%.)

Estimated &l3NPhe (%o) * Estimated ô16NBL,ik (%o)

Fig. 4. Determined and estimated bulk plant, collagen and amino acid S15N values of plants, herbivores and humans from a) Vaihingen, b) Makriyalos, and c) ^atalhoyuk. Estimated plant S15NPhe values are inferred from human and herbivore bone collagen S15NPhe values. Cereal grain S15N values were estimated by subtracting 8.4% from estimated plant S15NPhe values (A15NPhe-Buik values determined in modern cereal grains).

plants with lower 515N values such as pulses (Inline Supplementary Fig. S4).

Again, the bone collagen 515Nau values are much more variable and differ between and within species (within-species differences account for 32% of the variation). As observed at Vaihingen, the D15NGlu -Bulk values do not differ significantly between species (3.3 ± 0.7%; c2 (2) = 2.880, p = 0.237) and the D15NGlu-Bulk value is similar to that of the individuals from Vaihingen.

3.3. Estimating the proportion of animal protein consumed by humans

Table 3 gives the average bulk 515N values, 515NGlu and 515NPhe values used to estimate the proportion of animal protein consumed by the humans on each archaeological site, using each of the four methods described in Section 2.5. The estimates obtained from each of the methods are presented in Table 4. Estimates made using the 'Standard method' and the 'Standard model plus plants' were carried out using: (i) all human and domestic herbivore bulk bone collagen d15N values, and (ii) using only the bone collagen d15N values from the humans (n = 5) and domestic herbivores (n = 5) whose amino acid 515N values were determined in this study. The D15NConsumer-Djet value for bulk bone collagen is assumed to be 4%.

3.3.1. Vaihingen an der Enz, Germany

Animal protein consumption among humans at Vaihingen is calculated to be 58% if the 'Standard method' includes the bulk collagen d15N values of all of the individuals. The proportion of animal protein consumption calculated using only the bulk collagen d15N values of the individuals chosen for amino acid analysis is the same as that calculated for all individuals, with a smaller range in the 95% confidence interval due to the smaller variation in bulk collagen d15N values.

The calculated proportion of animal protein consumed is much lower using the 'Standard method plus plants' (26%). This is because the cereal grain 515N values are relatively high compared to the estimated herbivore forage 515N values (515NHerbivore -4% = 2.8% compared to 4.5%). This indicates that the 515N values of plants consumed by humans and herbivores at Vaihingen are different and suggests that cereals consumed by humans were likely to have been manured (cf. Fraser et al., 2013b), accounting for the relatively high d15N values.

Using different D15NGlu-Phe* values in the 'Bone collagen 515NGlu and d15NPhe values method' makes a considerable difference to the calculated animal protein consumption (between 11 and 38%), but regardless of the D15NGlu-Phe* value used, the calculated animal protein consumption is much lower than that calculated using only

Estimated proportions of animal protein consumed by humans from Vaihingen, Makriyalos and ^atalhöyük, calculated using the four methods described in Section 2.5, the 515N values given in Table 1 and assuming a A15Ncouagen_diet value of 4%o.

Proportion of animal protein consumed (%) 95% Confidence interval in bracketsa

Method Vaihingen Makriyalos Çatalhôyûk

1. 'Standard method'

(i) All bulk S15N values 58 (50-65%) 63 (49-76%) 63 (54-71%)

(ii) Bulk 515N values from bone collagen isolates used in AA analyses 58 (54-61%) 63 (47-78%) 75 (38-100%)

2. 'Standard method plus plants'

(i) All bulk S15N values 26(13-39%) 68 (57-79%) 55 (40-69%)

(ii) Bulk 515N values from bone collagen isolates used in AA analyses 26 (l6—37%) 69 (55-82%) 70 (27-100%)

3. 'Bone collagen 515NGta and 515NPhe values'

(i) A15NGlx_phe* = -6.9 ± 0.2% 11 (8-13%) 8 (6-10%) 33 (23-42%)

(ii) D15NGlx-Phe* = -9.0 ± 0.3% 38 (30-46%) 36 (28-43%) 61 (46-75%)

(iii) D15NGlx-Phe* = -8.4% ± 1.6% 30 (24-37%) 28 (21-34%) 53 (39-66%)

4. 'Bone collagen and cereal 515NGta values' 4 (0-30%) 41 (18-63%) 40 (0-82%)

a 95% confidence intervals for animal protein consumption were calculated using IsoError (www.epa.gov/wed/pages/models.htm; accessed 9 September 2014) for methods 1,2 and 4. For method 3,95% confidence intervals for animal protein consumption were calculated by taking into account the propagation of standard errors for 515NGiu[hum], S15Nphe[humj. ^^Glx-Phe*. AGlu and APhe.

bulk bone collagen 515N values. This highlights the need for further studies to constrain the A15Nconsumer-Diet. AGlu and Aphe values for diets of differing protein quality and content. particularly in terrestrial mammals. When taking into account the estimated cereal grain 515NGlu values. the calculated animal protein consumption is considerably lower (4%) due to the relatively high plant 515N values.

3.3.2. Makriyalos, Greek Macedonia

Animal protein consumption among humans at Makriyalos is calculated to be 63% if the 'Standard method' includes the bulk collagen 515N values of all of the individuals. However. 1 out of the 18 individuals exhibits a 515N value (under 6%) below those of the domestic herbivores. either suggesting that this individual consumed no animal protein. or pulses and plants with low 515N values comprised a significant part of their diet. Two out of the 21 individuals whose bone collagen isotope values were determined by Triantaphyllou (2001) also exhibited very low 515N values. Conversely. one individual exhibits a 515N value (515N = 9.3%) greater than the maximum theoretical bone collagen d15N value predicted from the consumption of pure herbivore protein (i.e. 9.1 %).

The calculated proportion of animal protein consumed is higher using the 'Standard method plus plants' (68%). This is because the cereal grain d15N values are relatively low compared to the estimated herbivore forage 515N values (515NHerbivore — 4% = 1.1 % compared to 0.4%). There is the potential for fish consumption at Makriyalos. considering its proximity to the coast and the findings of abundant seashells. Extensive investigations of organic residues in cooking pottery from the site show no evidence. however. of the processing of aquatic commodities as judged by stable carbon isotope determinations of fatty acids and an absence of aquatic lipid biomarker proxies (Evershed et al.. 2008; Cramp and Evershed. 2014; Whelton et al. unpublished). Further d15N value determinations of cereal grains from Makriyalos are necessary in order to ascertain whether the very low cereal grain d15N values measured in this study reflect those of the majority of crops.

Using the 'Bone collagen 515NGlu and 515NPhe values method'. animal protein consumption estimates are much lower (between 8 and 36%). regardless of the A15NGl u-phe* value used. Again. this highlights the need for better understanding of the factors contributing to amino acid and bulk collagen d15N values. The calculated animal protein consumption is also lower (41%) using the 'Bone collagen and cereal d15NGlu values method' despite the fact that the cereal grain d15NGlu value used in this calculation was estimated from the relatively low bulk cereal grain d15N values.

3.3.3. fatalhoyuk, Turkey

Animal protein consumption among humans at ^atalhoyuk is calculated to be 63% if the 'Standard method' includes the bulk collagen 515N values of all of the individuals. Seven out of the 67 individuals exhibit 515N values (over 14%) greater than the maximum theoretical bone collagen d15N value predicted from the consumption of pure herbivore protein. The proportion of animal protein consumption calculated using only the bulk collagen d15N values of the individuals chosen for amino acid analysis is higher than that calculated for all individuals (75%).

The calculated proportion of animal protein consumed is slightly lower using the 'Standard method plus plants' (55%). This is because the cereal grain d15N values are slightly higher than estimated herbivore forage 515N values (515NHerbivore — 4% = 6.0% compared to 6.7%). Cereals consumed by humans may therefore have been manured. increasing their d15N values above that of the herbivore forage.

Using the 'Bone collagen d15NGlu and d15Nphe values method'. animal protein consumption estimates are slightly lower (between 33 and 61%). but they are strongly influenced by the A15NGlu-Phe* value used. The calculated animal protein consumption is also lower (40%) using the 'Bone collagen and cereal 515NGlu values' method. The uncertainties associated with all dietary calculations are large because of the considerable variation in bulk and amino acid d15N values between individuals.

4. Discussion and conclusion

This is the first study to investigate bone collagen amino acid 515N values of humans believed to have been eating only. or predominantly. terrestrial protein. which simplifies the possible dietary inputs to the bone collagen N isotopic signature. We have used the 515N values of bone collagen. bone collagen amino acids. plant protein and plant protein amino acids in four different palaeodietary models to calculate the proportion of animal protein in human diet at three different archaeological sites. Comparison of the results of these calculations highlights limitations of these models and draws attention to the priorities for future work needed to improve their accuracy and reliability.

Bulk bone collagen 515N values average out the 515N values of their constituent amino acids. representing the net effect of dietary protein sources and metabolic cycling within the body. Calculating human animal protein consumption using bulk d15N values also relies upon the assumption that faunal bone collagen preserved on the site is representative of the d15N values of the animals

consumed. This is not the case with the 'Bone collagen 515Nciu and S15Nphe values method' since human bone collagen 515NGiu and S15Nphe values provide an internal indicator of animal protein consumption, although it remains a limitation of the 'Bone collagen and cereal 515NGlu values method'.

The large discrepancies between the estimates of animal protein consumption made with and without taking into account the 515N values of charred cereal grains and pulses illustrate the importance of plant 515N values in palaeodietary interpretations. The relatively high 515N values of cereal grains from Vaihingen (4.5%) and £at-alhóyük (6.7%) could be responsible for the relatively high human bone collagen 515N values, leading to overestimation of animal protein contribution to the diet if the 'Standard method' is used. In contrast, the relatively low 515N values of the cereal grains from Makriyalos (0.4%) could lead to underestimation of animal protein contribution to the diet if their values are not taken into account. In order to be more certain about the plant 515N contribution to the human bone collagen 515N value, it is clear that more cereal grain 515N value determinations need to be carried out at each site and the variability in 515N values of modern plants relevant to human and animal diet needs to be ascertained.

Bone collagen 515NGlu and 515NPhe values of humans and the animals included in their diets have the potential to improve interpretation of human diet in the past, since they separate the influence of diet 515N (515NPhe) from subsequent N metabolism (515NGlu). However, this method is only of use if amino acid 515N values of plants consumed by herbivores and humans are the same. This is particularly unlikely in circumstances when humans are eating manured cereal grains and herbivores are not. This could be the case at Vaihingen and ^atalhóyük, since the determined cereal grain 515N values at these sites are higher than estimated herbivore forage 515N values (515NHerbivore - 4%).

The 'Bone collagen and cereal 515NGlu values method' provides a means of combining the specificity of amino acid 515N values whilst taking into account the 515N value of cereals consumed by humans. Comparison of determined cereal grain 515N values and those estimated from herbivore 515NPhe values indicates whether humans were likely to have been eating the cereals preserved on the site (Section 3.2 and Fig. 4).

Further studies are needed into: (i) the 15N trophic enrichment factor between consumer and diet 515NGlu (DGlu) and 515NPhe (DPhe) in a terrestrial ecosystem through feeding experiments involving terrestrial mammals, and (ii) D15NGlu-Phe values of common plant foods, particularly glume wheats, pulses and plants consumed by herbivores, in order to improve the accuracy and more widespread applicability of both amino acid methods. With these provisos, analysis of amino acid 515N values offers significant potential to elucidate the factors contributing to bulk 515N values of human and non-human bone collagen and thus to achieve more reliable estimates of the contribution of animal protein to human diet.

Acknowledgements

The work reported here was funded by the Natural Environment Research Council (NERC standard grant NE/E003761/1, PI Bogaard). The use of the NERC Life Sciences Mass Spectrometry facilities at the University of Bristol is gratefully acknowledged. We are grateful to Tim H. E. Heaton and the NERC Isotope Geosciences Laboratory, Keyworth for bulk isotope analysis of material from Vaihingen and Makriyalos and to Keri Rowsell, BioArCh, York for Zoo-MS species identification of six of the ^atalhöyük caprines. We would also like to thank Rüdiger Krause for access to material from Vaihingen, Maria Pappa for access to material from Makriyalos and Katheryn C.

Twiss, Nerissa Russell, Clark S. Larsen for access to bone material from Çatalhoyûk.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jas.2014.11.009.

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