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Refining human palaeodietary reconstruction using amino

acid δ15N values of plants, animals and humans

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Impact Factor: 2.2 · DOI: 10.1016/j.jas.2014.11.009

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Refining human palaeodietary reconstruction using amino acid d15N

values of plants, animals and humans
Amy K. Styring a, *, Rebecca A. Fraser b, Rose-Marie Arbogast c, Paul Halstead d,
€fer f, Sevasti Triantaphyllou g,
Valasia Isaakidou b, Jessica A. Pearson e, Marguerita Scha
Soultana Maria Valamoti , Michael Wallace , Amy Bogaard b, Richard P. Evershed a
g c

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 l0 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 €historische und Naturwissenschaftliche Archa
Institut für Pra €ologie, University of Basel, Basel, Switzerland
g
School of History and Archaeology, Aristotle University of Thessaloniki, Thessaloniki, Greece

a r t i c l e i n f o a b s t r a c t

Article history: An established method of estimating the trophic level of an organism is through stable isotope analysis of
Received 9 April 2014 its tissues and those of its diet. This method has been used in archaeology to reconstruct past human diet
Received in revised form from the stable nitrogen isotope (d15N) values of human and herbivore bone collagen. However, this
29 September 2014
approach, using the 15N-enrichment of human bone collagen d15N values over associated herbivore bone
Accepted 6 November 2014
Available online 20 November 2014
collagen d15N values to predict the relative importance of animal protein, relies on the assumptions that:
(i) the d15N 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 d15N values have the
Keywords:
Bone collagen
potential to tackle these uncertainties, as they constrain the factors influencing bone collagen d15N
Cereal grains values. In this study, the d15N values of glutamic acid and phenylalanine in human and herbivore bone
Amino acids collagen isolates from Neolithic sites in Germany, Greece and Turkey were determined by gas
Nitrogen chromatography-combustion-isotope ratio mass spectrometry. The fraction of animal protein in total
d15N values dietary protein consumed by the humans was estimated by: (i) comparing bulk human and herbivore
Palaeodiet collagen d15N values, (ii) comparing bulk human and herbivore collagen and ancient charred cereal grain
d15N values, (iii) comparing human bone collagen d15NGlutamic acid and d15NPhenylalanine values, and (iv)
comparing d15NGlutamic acid values of human and herbivore bone collagen and estimated d15NGlutamic acid
values of ancient charred cereal grains. Where determined cereal grain d15N 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 D15NConsumer-Diet values of glutamic acid and
phenylalanine in terrestrial ecosystems, and (ii) D15NGlutamic 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.
© 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/).

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
* Corresponding author. Present address: School of Archaeology, 36 Beaumont
Street, University of Oxford, Oxford, UK. Tel.: þ44 0 1865 288014.
governed by underlying metabolic processes associated with
E-mail address: [email protected] (A.K. Styring). 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/).
 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515 505

extent to which the d15N of human bone collagen lies above the
d15N values of herbivore bone collagen from the same archaeo-
logical 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). In-
terpretations, however, can be confounded by uncertainty
regarding the 15N trophic enrichment factor between diet and
consumer tissues (D15NConsumer-Diet); studies have found this to vary
between 2 and 6‰ (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 d15N values integrate
the d15N 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 d15N values alone.
Determination of individual amino acid d15N values in bone
collagen has the potential to shed more light on the underlying
metabolic pathways responsible for the 15N-enrichment of con-
sumer tissues over diet and to allow further elucidation of the often
complex biochemical processes contributing to the bulk d15N value Fig. 1. Variation of human bone collagen d15N value with fraction of animal protein in
(Hare et al., 1991; Naito et al., 2010a,b; Styring et al., 2010). Equa- the diet, assuming a D15NCollagen-Diet value of 4‰, for humans eating animals and
unmanured or manured plants. In this case, manuring increases the d15N value of plant
tions have been developed to estimate the trophic level of humans
by 2‰. Following this model, a human bone collagen value of 10‰ could be due either
and fauna in aquatic, C3-plant-based and C4-plant-based ecosys- to: (i) consumption of unmanured plants and 75% animal protein, or (ii) consumption
tems based on the fact that the d15N values of the amino acids of manured plants and 50% animal protein.
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 conducive to producing undistorted cereal grains similar to those
this approach is that the d15N values of Glu (d15NGlu) and Phe found on archaeological sites (less than 250  C for more than 6 h;
(d15NPhe) provide an internal trophic level indicator, precluding the Charles et al., in prep), the d15N values of charred einkorn grains
need to rely upon the bone collagen d15N values of preserved fauna, were found to increase by around 1‰ (Fraser et al., 2013a). This was
whose tissues may not in fact have contributed to the human diet. ascribed to the preferential loss of 14N-containing volatiles, during
The very small 15N-enrichment of d15NPhe with trophic level (DPhe) the thermal conversion of starch and protein in the grains to high
suggests that Phe has undergone very little metabolism within the molecular weight melanoidins (Styring et al., 2013), which are
body and therefore bone collagen d15NPhe values broadly reflect relatively resistant to degradation (Almendros and Dorado, 1999).
those of the diet, whereas the large 15N-enrichment in d15NGlu with Biochemical investigation of ancient charred grains from two
trophic level (DGlu) suggests that bone collagen d15NGlu values result archaeological sites in Europe found them to contain similar pro-
from subsequent N metabolism within the consumer. portions of N as their modern charred counterparts and acid-
The limitation of both of these methods is that they rely on the ebaseeacid pre-treatment, which is commonly used to remove
assumption that the d15N values of plants consumed by humans contaminants in radiocarbon dating (Goh, 1991), resulted in little
and the fauna they consume are identical. d15N value de- change in their %N or d15N values (Fraser et al., 2013a; Styring et al.,
terminations of modern plants suggest that this assumption is 2013). Whilst further work is needed to establish the effect of a
implausible, since plants vary widely in their d15N values (e.g. wider range of burial conditions and durations on the biochemical
Craine et al., 2009). One of the proposed reasons for high d15N composition and d15N values of a wider range of cereal grain taxa,
values of human bone collagen, particularly during the European the charred grains preserved on archaeological sites have the po-
Neolithic, is consumption of manured crops by humans, since tential to provide baseline d15N values of plants to include in
manuring increases the d15N values of plants (Bogaard et al., 2007; palaeodietary predictions.
Dürrwa €chter et al., 2006). Studies have shown that manuring of The amino acid d15N values of these cereal grains could also
crops can increase cereal grain and chaff d15N values by as much as improve trophic level estimates by refining the amino acid trophic
10‰ and that d15N values are correlated to the amount of manure level equations. Although amino acids in archaeobotanical charred
applied (Bogaard et al., 2007; Fraser et al., 2011; Kanstrup et al., cereal grains do not survive in sufficient quantities for isotopic
2011). The effect of this increase on crop d15N values is to raise analysis (they contain 0.4% of the total hydrolysable amino acid
the bone collagen d15N value of humans eating manured crops content of fresh cereal grains; Styring et al., 2013), it may be
above that of herbivores eating unmanured plants, leading to possible to estimate their values based upon differences between
overestimation of the importance of animal protein in the diet bulk d15N values and amino acid d15N values determined in modern
(Fig. 1). plants (Chikaraishi et al., 2010, 2011; Styring et al., 2014). Such
In the past, the stable isotope values of plants consumed by calculations are discussed in more detail in Sections 2.5.3 and 2.5.4.
humans on an archaeological site have rarely been determined In this study, the bulk and amino acid d15N values of human and
directly from those preserved, due to concerns about contamina- faunal bone collagen and the bulk d15N values of ancient charred
tion and the robustness of plant isotope values after years of burial. cereal grains and pulse seeds from the archaeological sites of Vai-
Carbonisation is the most common process by which plant material hingen an der Enz, Germany, Makriyalos, Greece and Çatalho €yük,
can survive in archaeological contexts and various studies have Turkey were determined by elemental analysis-isotope ratio mass
examined the changes in the d15N values of cereal grains with spectrometry (EA-IRMS) and gas chromatography-combustion-
charring (Bogaard et al., 2007; Fiorentino et al., 2012; Fraser et al., IRMS (GC-C-IRMS). We use the d15N values obtained to compare
2013a; Kanstrup et al., 2012). Within the charring conditions four different methods for estimating the proportion of animal
506 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515

protein consumed by humans at each of the sites: (i) the ‘Standard 30e60 min, or until any effervescing ceased, and then rinsing in
method’ comparing human and faunal collagen d15N values; (ii) the distilled water three times, 2) treatment with 10 mL of 0.1 M so-
‘Standard method plus plants’, comparing human, faunal and dium hydroxide (NaOH) at 70  C for 60 min, followed by rinsing in
ancient charred cereal grain d15N values; (iii) the ‘Bone collagen distilled water until the solution was clear and the pH neutral, using
d15NGlu and d15NPhe values method’, comparing the difference be- a minimum of three rinses, 3) treatment with 10 ml of 0.5 M HCl at
tween d15NGlu and d15NPhe values in human bone collagen with the 70  C for 30e60 min, followed by three rinses in distilled water and
difference between d15NGlu and d15NPhe values in modern plants, final freeze drying. Grains/seeds were ground to fine homogeneous
and (iv) the ‘Bone collagen and plant d15NGlu values method’, powder using a mortar and pestle and weighed into tin capsules
comparing the d15NGlu value of human and faunal bone collagen ready for bulk d13C and d15N determinations.
and the estimated d15NGlu value of ancient charred cereal grains. The C:N molar ratios of modern grains charred at 230  C for 24 h
Each of these methods is described in more detail in Section 2.5. under a reducing atmosphere have been found to range from 17.9 to
The archaeological sites were chosen because they all date to the 33.4, with an average C:N molar ratio of 22.7 (n ¼ 17; Fraser et al.,
Neolithic, between c. 8000 and 4500 BC, and display evidence of 2013a). Of the ancient charred grain samples from Vaihingen, 13 out
both herding and cultivation, but are located in very different of 13 had C:N molar ratios within this range (18.0e28.6); 3 out of 3
environmental zones. Further details of the archaeological contexts grain samples from Makriyalos had C:N molar ratios within this
can be found in Supplementary Text 1. Inline Supplementary Fig. S1 range (25.3e26.1) and 6 out of 15 grain samples from Çatalho €yük
shows the site locations. Discussion of the relative merits and had C:N molar ratios within a similar range (13.2e21.1). The grains
limitations of each method for estimating the proportion of animal from Çatalho €yük tended to have higher %N contents than those
protein consumed by humans will help to refine future in- from the other sites, which could be due to post-depositional
terpretations of human diet from bone collagen d15N values. In contamination, loss of non-N containing organics during burial or
particular, discussion of current amino acid-based stable isotope natural variation in %N caused by the environment in which they
methodologies is hoped to inform priorities for future work needed were grown. Work by Vaiglova et al. (in press) found no indication
to ground truth this approach. of post-depositional contamination in lentils or peas from Çata-
Inline Supplementary Fig. S1 can be found online at http://dx. €yük, which suggests that the high %N is a factor of the envi-
lho
doi.org/10.1016/j.jas.2014.11.009. ronment in which they were grown. This needs to be investigated
further by N isotope analysis of modern plants from around the site.
2. Materials and methods 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
2.1. Bone collagen extraction and analysis 13.1, with an average C:N molar ratio of 10.8 (n ¼ 15; Fraser et al.,
2013a). Of the ancient charred pulse seed samples from Vaihin-
Human bone was sampled from the compact mid-shaft of the gen, 3 out of 3 had C:N molar ratios within a similar range
femur (preferred), another long bone, or the rib. For faunal sam- (10.0e13.7); 0 out of 1 pulse seed samples from Makriyalos had C:N
pling, the distal humerus was generally sampled, taking sidedness molar ratios within this range (8.0) and 4 out of 8 pulse seed
into account to avoid sampling the same individual more than once. samples from Çatalho €yük had C:N molar ratios within a similar
Collagen was extracted from approximately 1 g of compact bone range (8.7e11.1). The seed samples from Makriyalos and Çata-
using a modified Longin method (Longin, 1971): bone samples were €yük have higher %N contents than those determined in modern
lho
crushed and immersed in 0.5 M HCl until demineralised and then charred pulse seeds, which could also be a feature of the crop
washed three times in Milli Q water. Samples were then gelatinised growing conditions and/or burial environment. Given that the
by adding pH 3 water and heating to 70  C for 48 h. The gelatinous cereal grains from Makriyalos did not have unusually high %N, the
solution was then filtered through an 80 mm Ezee-filter and high %N in the pulse seeds is probably due to a difference in
transferred to clean test tubes and freeze-dried. Bone collagen microscale growing conditions rather than a depositional or envi-
samples of approximately 0.75 mg were weighed into tin capsules ronmental effect that would have increased the %N of both cereal
for determination of d13C and d15N values. Collagen yields ranged grains and pulse seeds. More modern and archaeological plant
from 0.9 to 17.0% and the molar C:N ratios between 2.9 and 3.6, samples need to be analysed to resolve this. To account for the
which is within the accepted range for well-preserved collagen observed effects of experimental charring (at 230  C for 24 h under
(DeNiro, 1985). a reducing atmosphere) on cereal grain and pulse seed d15N values,
a generous offset of 1‰ is subtracted from the measured d15N
2.2. Archaeobotanical sampling and analysis values (cf. Fraser et al., 2013a).

Ancient charred cereal grain and pulse seed samples consisted 2.3. Bulk d13C and d15N determinations
of at least ten whole grains/seeds from the same stratigraphic unit,
derived mostly from visible concentrations such as ‘storage de- Bulk 12C/13C analysis was performed by sample combustion in a
posits’. The grains/seeds selected were virtually undistorted Costech 4010 on-line to a VG TripleTrap and Optima dual-inlet mass
morphologically (displaying slight puffing only), resembling mod- spectrometer. Isotope ratios were calculated relative to the VPDB
ern material charred experimentally at around 230  C for a pro- reference by comparison with co-run laboratory standards (of plant
longed period (up to 24 h; Fraser et al., 2013a). Grains/seeds were material) calibrated against NBS-19 and NBS-22. Bulk 15N/14N
examined at x7-45 magnification for visible surface contaminants, analysis was performed by sample combustion on a ThermoFinni-
such as adhering sediment or plant roots; these were removed by gan system comprising an elemental analyser linked under
gentle scraping. Grains/seeds were weighed and placed in glass test continuous flow with a Delta þ XL mass spectrometer (Thermo-
tubes in preparation for an acid-base-acid (ABA) pre-treatment, a Finnigan, Bremen). Isotope ratios were calculated as d15N versus
procedure commonly applied to charcoal and charred plant re- atmospheric N2 by comparison with standards calibrated against
mains prior to radiocarbon and stable isotope analysis (Goh, 1991) IAEA-N-1 and N-2. The relative analytical errors (1 standard devi-
and considered appropriate for use on archaeobotanical remains ation) for replicate analytical standards were ±0.2‰ for d13C and
(Fraser et al., 2013a). This three-step procedure consisted of: 1) ±0.4‰ for d15N. Replicate analyses of a bone collagen sample
treatment with 10 mL of 0.5 M hydrochloric acid (HCl) at 70  C for VAH35 measured in eleven separate mass spectrometry runs had a
 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515 507

standard deviation (1SD) of ±0.3‰ for d13C, ±0.2‰ for d15N and international isotope standard, introduced directly into the ion
±0.2 for the C:N molar ratio (mean C:N of 3.3). For plant material, source in four pulses at the beginning and end of each run. Each
the precision (1SD) among replicates of a well-homogenized reported value is a mean of triplicate d15N determinations. An
modern uncharred barley sample was ±0.2 for %N and ±0.4‰ for amino acid standard mixture, comprising amino acids whose d15N
d15N analysed in 29 separate runs and ±3.5 for %C and ±0.1‰ for values were individually determined by EA-IRMS, was run every 3
d13C, analysed in 21 separate runs. runs in order to monitor instrument performance. The d15N values
of the amino acids in the standard mixture were within 0.8% of
2.4. Amino acid d15N value determinations their d15N 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-
2.4.1. Hydrolysis of bone collagen IRMS chromatogram displaying the separation of amino acids in
Approximately 2 mg of collagen was hydrolysed in culture tubes archaeological human bone collagen.
(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
2.5. Methods of estimating the contribution of animal protein to
before being stored at 18  C until required for analysis. Fractions
human diet
of the protein hydrolysates (250 mL) were transferred to culture
tubes and dried under N2 before undergoing derivatisation. A
The following four methods were used to estimate the propor-
known quantity of norleucine was added to each sample as an in-
tion of animal protein consumed by humans at each of the
ternal standard.
archaeological sites in this study. See Table 1 for a description of
each of the terms used in the equations.
2.4.2. Preparation of amino acid derivatives (N-acetyl-i-propyl
esters)
Amino acids were converted to their i-propyl esters by addition 2.5.1. ‘Standard method’
of 1 mL of a 4:1 v/v mixture of isopropanol and acetyl chloride The ‘Standard method’ assumes that humans eating only plant
(acetyl chloride added dropwise in an ice bath). Culture tubes were protein will have the same bone collagen d15N value as the local
then sealed and heated at 100  C for 1 h. Reagents were evaporated herbivores, whereas humans eating only animal protein will have a
under a gentle stream of N2 at room temperature. Dichloromethane bone collagen d15N value a trophic level higher than the local her-
(DCM) was added (2  0.5 mL) and evaporated in an ice bath to bivores (Hedges and Reynard, 2007). This approach also assumes
remove excess reagents. Amino acid i-propyl esters were then that the 15N trophic enrichment factor between diet and consumer
treated with 1 mL of a mixture of acetic anhydride, triethylamine tissues (D15NConsumer-Diet) is the same for herbivores and carnivores
and acetone (1:2:5, v/v/v; 10 min, 60  C). Reagents were evaporated and for diets of differing protein content, both of which are as-
under a gentle stream of N2 in an ice bath. The samples were dis- sumptions that are still under debate (e.g. Hussey et al., 2014). The
solved in 2 mL ethyl acetate and 1 mL saturated sodium chloride equation for estimating the proportion of animal protein consumed
solution was added. After phase separation, the organic phase was is:
collected and the extraction repeated with an additional 1 mL of h i h i
ethyl acetate. The combined organic phases were evaporated under d15 N hum  d15 N herb
N2 in an ice bath and the residual water removed with successive f ¼ (1)
D15 NConsumerdiet
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 See Supplementary Text 2 for an explanation of how this
stored at 18  C until required for analysis. equation was derived.

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 d15N
values were determined by GC-C-IRMS. A ThermoFinnigan DeltaPlus
XP system (Thermo Electron Corporation) was used to determine
the d15N 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 min1 was used as the carrier gas and the mass spectrom-
eter source pressure was maintained at 9  104 Pa. The separation
of the amino acids was accomplished using a DB-35 capillary col-
umn (30 m  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 min1 to
120  C, then 3  C min1 to 180  C, then 1.5  C min1 to 210  C and
finally 5  C min1 to 270  C and held for 1 min. A Nafion membrane
removed water and a cryogenic trap was employed in order to
Fig. 2. A GC-C-IRMS chromatogram displaying the separation of amino acids in
remove CO2 from the oxidised and reduced sample. archaeological human bone collagen. Amino acids: Ala alanine, Gly glycine, Val valine,
All d15N values are reported relative to reference N2 of known Leu leucine, Nle norleucine, Thr threonine, Ser serine, Pro proline, Asx aspartic acid,
nitrogen isotopic composition, previously calibrated against the AIR Glx glutamic acid, Hyp hydroxyproline, Phe phenylalanine, Lys lysine.
508 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515

Table 1 Table 2
Summary of the terms in equations used to estimate the proportion of Summary of d15NGlu, d15NPhe and D15NGlu-Phe* values determined for different plant
animal protein in human diet. types in previous studies.

Term Description Plant type n d15NGlu d15NPhe D15NGlu-Phe* Reference

d15N[gr] Mean of ancient charred cereal grain d15N values C3 tree and grass leaves 16 2.0 ± 4.9 10.4 ± 5.0 8.4 ± 1.6 Chikaraishi
d15N[herb] Mean of domestic herbivore collagen d15N values et al., 2010
d15N[hum] Mean of human collagen d15N values Barley and bread wheat 4 5.4 ± 4.2 12.3 ± 4.2 6.9 ± 0.2 Styring et al.,
d15NGlu[gr] Estimated d15NGlu value of ancient cereal grains grains from 2014
on the site Rothamsted, UK
d15NGlu[herb] Mean of domestic herbivore collagen d15NGlu values Barley and bread wheat 4 3.0 ± 2.2 12.0 ± 1.9 9.0 ± 0.3 Styring et al.,
d15NGlu[hum] Mean of human collagen d15NGlu values grains from Bad 2014
d15NPhe[hum] Mean of human collagen d15NPhe values Lauchstadt, Germany
D15NConsumer-Diet 15
N trophic enrichment factor between consumer Broad beans and peas 4 0.9 ± 0.2 0.8 ± 1.1 0.1 ± 1.0 Styring et al.,
and diet d15N, defined as 4‰ in this study from Bad Lauchstadt, 2014
(the mean of the 2e6‰ 15N-enrichment determined Germany
in previous studies; Hedges and Reynard, 2007; C4 grass leaves 7 4.4 ± 6.1 4.0 ± 6.9 0.4 ± 1.7 Chikaraishi
O'Connell et al., 2012) et al., 2010
D15NGlu-Phe* Difference between the d15NGlu and d15NPhe values
of plants at the base of the food chain
15
DGlu N trophic enrichment factor between consumer and
diet collagen d15NGlu, defined as 8.0 ± 1.2‰ in this
D15NGlu-Phe values of these cereal grains were not affected by spe-
study (Chikaraishi et al., 2009) cies or manure application, but differed significantly between lo-
DPhe 15
N trophic enrichment factor between consumer and cations; cereals grown at Rothamsted had a D15NGlu-Phe value
diet collagen d15NPhe, defined as 0.4 ± 0.5‰ in this of 6.9 ± 0.2‰, whereas those grown at Bad Lauchst€ adt had a
study (Chikaraishi et al., 2009)
D15NGlu-Phe value of 9.0 ± 0.3‰ (t(6) ¼ 10.606, p < 0.001). The
G Proportion of animal protein in the human diet
significant difference between the D15NGlu-Phe values of cereal
grains from different sites cautions against using a standardised
D15NGlu-Phe* value as the basis of a trophic level equation, without
2.5.2. ‘Standard method plus plants’ carrying out further D15NGlu-Phe value determinations in the rele-
The ‘Standard method plus plants’ assumes that humans eating vant geographic region of the site, or without further assessment of
only plant protein will have a bone collagen d15N value a trophic variation in this value. We consider the effect of such variation in
level higher than that of associated ancient charred grains and plant D15NGlu-Phe values on the estimate of animal protein con-
humans eating only animal protein will have a bone collagen d15N sumption by humans using Equation (3) (see Section 3.3).
value a trophic level higher than that of local herbivores. Human Chikaraishi et al. (2010) and Styring et al. (2014) found that the
bone collagen d15N values falling between the two indicate a mixed D15NGlu-Phe values of C4 terrestrial plants (n ¼ 7) and pulses (broad
plant-animal diet of varying proportions. The equation is: beans and peas; n ¼ 4) are much higher than that of C3 terrestrial
  h i plants (Table 2), which would influence predictions of plant protein
d15 N½hum  D15 NConsumerdiet  d15 N gr contribution to human diet if humans were eating significant
f ¼ (2)
d15 N½herb  d15 N½gr quantities of C4 plants and/or pulses. Although there is no evidence
for significant C4 plant consumption by humans at the sites in this
See Supplementary Text 3 for an explanation of how this study, since their d13C values are more negative than 18‰ (Sec-
equation was derived. tion 3.1), there is a possibility that pulses played an important role
in the Neolithic diet (cf. Bogaard, 2012, 2013; Valamoti, 2004).
2.5.3. ‘Bone collagen d15NGlu and d15NPhe values method’ The 15N trophic enrichment factor between consumer and diet
The ‘Bone collagen d15NGlu and d15NPhe values method’ was d15N values of Glu and Phe (DGlu and DPhe) from four controlled
developed by Chikaraishi et al. (2009), who use this equation to feeding experiments using green algae, zooplankton and fish were
calculate the trophic level of a consumer: 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
d15 NGlu  d15 NPhe  D15 NGluPhe * in estimates of trophic position in planktonic ecosystems (Hannides
Trophic level ¼ þ1 et al., 2009; McCarthy et al., 2007; McClelland et al., 2003; Schmidt
ðDGlu  DPhe Þ
et al., 2004), terrestrial ecosystems (Chikaraishi et al., 2011) and
Since we are interested in the fraction of herbivore (animal) ancient human skeletal remains (Naito et al., 2010a,b). Recently,
protein consumed in our study, it is necessary to subtract rather two studies have determined the D15NConsumer-Diet values of amino
than add 1, so a human consuming 100% plant protein has a value of acids in seals fed on herring (Germain et al., 2013) and in four large
G ¼ 0: carnivorous fish species (Hoen et al., 2014). They found that the
 h i h i  difference between DGlu and DPhe values in these high trophic level
d15 NGlu hum  d15 NPhe hum  D15 NGluPhe * consumers was much lower than that determined in lower trophic
f¼ 1 (3) position marine organisms, implying that the difference between
ðDGlu  DPhe Þ
DGlu and DPhe values may be affected by the quantity and/or quality
Table 2 summarises the difference in plant d15NGlu and d15NPhe of protein in the diet (Hoen et al., 2014). Since there have been no
15 feeding studies carried out on terrestrial mammals, we use the DGlu
(D NGlu-Phe*) values determined in previous studies. Chikaraishi
et al. (2010) use a D15NGlu-Phe* value of 8.4‰ as the basis of and DPhe values of 8‰ and 0.4‰, since they have been used in
their C3 ecosystem trophic level equation, established from D15NGlu- previous studies of human palaeodiet. In addition, the DGlu and DPhe
Phe values determined in modern C3 leaves (n ¼ 16; Table 2). Styring values determined for seals in the Germain et al. (2013) study yield
et al. (2014) have determined the amino acid d15N values of a difference between DGlu and DPhe values of 2.9‰ (2.9e0‰), which
manured and unmanured bread wheat (Triticum aestivum) and produces unrealistically high estimates (over 100% in most cases) of
barley grains (Hordeum vulgare; n ¼ 8) from the experimental animal protein consumption by humans. It is clear that D15NCon-
farming sites of Rothamsted, UK and Bad Lauchsta €dt, Germany. The sumer-Diet amino acid values need to be determined for mammals
 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515 509

with similar digestive systems to humans (i.e. pigs) in order to palaeodietary interpretation at Vaihingen using bulk collagen
resolve these uncertainties. isotope values, see Fraser et al. (2013b). Mean d15N values are
plotted in Fig. 3a and those used in the estimates of human diet are
2.5.4. ‘Bone collagen and cereal d15NGlu values method’ given in Table 3. Cereal grains (T. monococcum and T. dicoccum)
The ‘Bone collagen and plant d15NGlu values method’ takes into exhibit mean d13C and d15N values of 24.1 ± 0.5‰ and 4.5 ± 0.5‰,
account the d15NGlu values of human and faunal bone collagen and respectively. The pulse seeds (Pisum sativum and Lens culinaris)
the estimated d15NGlu value of ancient charred cereal grains from have relatively high d15N values (2.8 ± 1.5‰) compared to atmo-
the same archaeological sites. This method focuses upon bone spheric N2. In modern studies, only pulses grown on artificial
collagen and plant d15NGlu values because the 15N trophic enrich- ‘dung-soil’ in Evvia, Greece were found to have such high d15N
ment factor between consumer and diet collagen d15NGlu (DGlu) is values (Fraser et al., 2011). The d13C and d15N values of the domestic
much larger than that of Phe (8.0‰ compared to 0.4‰), allowing herbivores are very similar to the average values determined at
greater distinction between the d15NGlu values of humans eating other Linearbandkeramik sites (cf. Dürrwa €chter et al., 2006; Oelze
only plant or only animal protein. The d15NGlu value of ancient et al., 2011; Bickle and Whittle, 2013), and reflect a terrestrial
charred cereal grains can be estimated from comparison of their herbivore diet in a temperate climate. Wild taxa constitute c.15% of
determined bulk d15N values and known differences between bulk the faunal remains (Sch€ afer, 2011) and the wild herbivores have
d15N values and d15NGlu values in modern cereal grains. Using the similar d15N values to the domestic herbivores (average d15N value
cereal grain amino acid d15N values determined by Styring et al. is 6.4 ± 0.4‰). Human bone collagen d15N values vary from 8.0 to
(2014), it is found that the d15NGlu value relative to the bulk grain 10.2‰, with a juvenile individual exhibiting a lower bone collagen
d15N value (D15NGlu-Bulk value) was not affected by site or manure d15N value of 5.8‰. This individual is discounted from the dietary
application, but differed significantly between T. aestivum calculations.
(0.5 ± 0.5‰) and H. vulgare (þ1.4 ± 0.5‰; t(6) ¼ 5.548, p ¼ 0.001). Inline Supplementary Fig. S2 can be found online at http://dx.
Since the cereal grains from the archaeological sites in this study doi.org/10.1016/j.jas.2014.11.009.
were predominantly glume wheats (Triticum monococcum and
Triticum dicoccum), whose d15NGlu values have not been determined 3.1.2. Makriyalos, Greece
in modern grains, the D15NGlu-Bulk value determined for T. aestivum Bulk d13C and d15N values of bone collagen isolates from early
(from the same genus as the glume wheats) was used to estimate Late Neolithic Phase I of Makriyalos (dating to 5500 to 5000 BC) are
the d15NGlu value of ancient charred cereal grains. It would be plotted in Inline Supplementary Fig. S3. The d13C and d15N values of
advisable to determine D15NGlu-Bulk values for T. monococcum and 5 pigs determined by Triantaphyllou (2001) from late Late Neolithic
T. dicoccum in future studies to test the reliability of this approach. Phase II of Makriyalos (c.4900 to 4500 BC) are also plotted, since pig
Thus, the d15NGlu value of the ancient charred cereal grains was bones account for a large proportion of the faunal assemblage
estimated by subtracting 0.5‰ from the bulk d15N value of charred (Pappa et al., 2004). Mean d15N values are plotted in Fig. 3b and
cereal grains from the site (also corrected for charring by sub- those used in the estimates of human diet are given in Table 3.
tracting 1‰ from the determined bulk d15N value). It is therefore Cereal grains (T. dicoccum) exhibit mean d13C and d15N values
assumed that humans eating 100% cereal protein will have a bone of 24.4 ± 0.1‰ and 0.4 ± 0.3‰, respectively. More cereal grains
collagen d15NGlu value 8.0‰ higher than that of the associated and pulse seeds need to be analysed from this site to gain a better
charred cereal grain d15NGlu value and humans eating 100% animal idea of the crop baseline d15N signature, but these initial isotope
protein will have a bone collagen d15NGlu value 8.0‰ higher than determinations provide a basis for further work. The large animal
that of the local herbivore bone collagen d15NGlu value. The equa- bone assemblage from Phase I is heavily dominated by domesti-
tion for this method is the same as Equation (2), but substitutes cates (>99%), among which pigs, cattle and sheep are more strongly
bulk collagen/grain d15N values with d15NGlu values: represented than goats and dogs. Relative proportions differ
  h i somewhat both between context types and according to method of
d15 NGlu ½hum  DGlu  d15 NGlu gr quantification (Pappa et al., 2013, 2004). The cattle are not
f ¼  h i (4) considered to contribute significantly to the human bone collagen
d15 NGlu ½herb  d15 NGlu gr isotope values, since their d13C values (16.1 ± 2.0‰) are signifi-
cantly higher than those of the humans (20.4 ± 0.3‰;
t(16.68) ¼ 8.70, p < 0.001) and indicate a C4 plant component to the
2.6. Statistical analysis cattle diet. The human bone collagen d15N values range from 5.3 to
9.3‰, with a mean of 7.6 ± 1.0‰. These values are very similar to
Independent t-tests were used to detect differences in crop those determined for the bone collagen isotope values of 18 human
stable isotope values between site, species and manuring regime bone samples from the same Late Neolithic phase determined in a
for the modern field studies. A Kruskal Wallis test with post-hoc previous study (between 4.9 and 8.3‰; Triantaphyllou, 2001).
BonferronieDunn test was used to detect differences in bulk Inline Supplementary Fig. S3 can be found online at http://dx.
collagen and amino acid d15N values between the herbivore species doi.org/10.1016/j.jas.2014.11.009.
at each archaeological site due to non-normal distribution of the
data. 3.1.3. Çatalho€yük, Turkey
Bulk d13C and d15N values of bone collagen isolates from Çata-
3. Results €yük are plotted in Inline Supplementary Fig. S4. Bulk collagen
lho
isotope values were determined by Jessica Pearson and are dis-
3.1. Bulk isotope analyses cussed in more detail in Pearson (2013). The latter also compares
human bone collagen d15N values from different areas, different
3.1.1. Vaihingen an der Enz, Germany buildings and across different levels of the site to determine po-
Bulk d13C and d15N values of bone collagen isolates from Vai- tential differences in the importance of animal protein in the hu-
hingen are plotted in Inline Supplementary Fig. S2. For the full d13C man diet. For the purposes of this study, we have taken the mean
and d15N value dataset of bone collagen and archaeobotanical d15N value of all human bone collagen at the site. Mean d15N values
samples from the site, and for further discussion of land use and are plotted in Fig. 3c and those used in the estimates of human diet
510 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515

Fig. 3. Mean d13C and d15N 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) Çatalho€yük, Turkey. Charred grain and pulse d15N values are adjusted for charring effect (cf. Fraser et al., 2013a,b).

are given in Table 3. The d15N values of cereals (H. vulgare, glutamic acid during hydrolysis of bone collagen, the d15NGlu value
T. dicoccum, Triticum durum/aestivum, T. monococcum) and pulses determined by GC-C-IRMS represents both the nitrogen of glutamic
(P. sativum) from the site are relatively high and it is unclear acid and the amino-nitrogen of glutamine. The mean bone collagen
whether this is a result of manuring, or a factor of the environment. d15N, d15NGlu and d15NPhe values of each species from each site are
High plant d15N values can be caused by aridity (Hartman and given in Table 3. Estimated d15NGlu, d15NPhe and D15NGlu-Phe values of
Danin, 2010; Heaton, 1987), or waterlogged conditions, which the cereal grains are given in italics. Bone collagen d15N, d15NGlu and
result in denitrification (Finlay and Kendall, 2008). The range of d15NPhe values for all of the individuals are given in Inline
isotope values for domestic sheep and goats from the site is very Supplementary Table S1.
wide, suggesting that they fed across varied ecological zones Inline Supplementary Table S1 can be found online at http://dx.
(Pearson et al., 2007). Human bone collagen d15N values range from doi.org/10.1016/j.jas.2014.11.009.
9.2 to 15.1‰, which would seem to indicate varied sources of di-
etary protein. 3.2.1. Vaihingen an der Enz, Germany
Inline Supplementary Fig. S4 can be found online at http://dx. There is no significant difference in the d15NPhe value of humans,
doi.org/10.1016/j.jas.2014.11.009. domestic cattle and wild deer from Vaihingen (10.7 ± 1.1‰; c2
(2) ¼ 1.808, p ¼ 0.405), which contrasts with the significant dif-
3.2. Amino acid nitrogen isotope analyses ference in their bulk d15N values (c2 (2) ¼ 12.5, p ¼ 0.002). This
similarity in d15NPhe values has also been observed for terrestrial
The bone collagen d15NGlu and d15NPhe values were determined mammals in other studies (e.g. Naito et al., 2010a,b; Styring et al.,
for a subset of five humans, five domestic herbivores and five wild 2010) and indicates that the d15NPhe values of the human, cattle
herbivores (three from Makriyalos) on each site. Individuals with and deer diets are very similar, since the d15N value of Phe increases
bulk collagen d13C and d15N values closest to the mean for their by only 0.4‰ between diet and consumer. Phe undergoes very little
species were selected from Vaihingen and Makriyalos in order to metabolic routing in the body: it is not biosynthesised and is cat-
minimise variation. Since glutamine is deamidated to form abolised into tyrosine via a metabolic pathway that involves no
 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515 511

Table 3 of the bulk plant d15N value if humans were eating only cereal
Mean d15N values determined for bone collagen isolates and charred cereal grains grains. For Vaihingen, the calculated cereal grain d15N value is 2.4‰,
€yük.
and pulse seeds from the sites of Vaihingen, Makriyalos and Çatalho
which is lower than the d15N values determined for the 13 cereal
Site Species n Bulk d15N d15NGlu d15NPhe D15NGlu-Phe grains from Vaihingen (4.5 ± 0.5‰; Fig. 4a). This suggests that the
( ‰) (‰) ( ‰) (‰) plants consumed by humans from Vaihingen were not restricted to
Vaihingen All humans 47 9.1 ± 0.7 e e e cereal grains with similar d15N values to those determined in this
Humansa 5 9.1 ± 0.1 12.2 ± 1.2 10.7 ± 1.6 1.5 ± 1.8 study, but may have included plants with lower d15N values such as
All domestic 29 6.8 ± 0.8 e e e
pulses (Inline Supplementary Fig. S2).
herbivores
Domestic 5 6.8 ± 0.1 9.7 ± 0.8 10.8 ± 1.1 1.1 ± 1.3 The bone collagen d15NGlu values are much more variable and
cattlea differ even between individuals of the same species (within-spe-
All wild deer 9 6.2 ± 0.4 e e e cies differences account for 21% of the variation). Nevertheless, the
Wild deera 5 6.2 ± 0.1 8.7 ± 1.0 10.4 ± 0.9
difference between the d15NGlu and bone collagen d15N values
Cereal grains 13 4.5 ± 0.5 4.0b 10.8c 6.9, 9.0,
8.4d
(D15NGlu-Bulk) between species is not significant (2.8 ± 0.8‰; c2
Pulse seeds 3 2.8 ± 1.5 e e e (2) ¼ 0.740, p ¼ 0.691). This is likely due to the central role that
Makriyalos All humans 18 7.6 ± 1.0 e e e Glu plays both in the biosynthesis of other amino acids and in the
Humansa 5 7.7 ± 0.5 11.1 ± 1.4 9.8 ± 1.7 1.3 ± 1.5 excretion of waste N in the form of urea. The amino group of Glu
All domestic 54 5.1 ± 1.0 e e e
is donated to keto acids to form other amino acids (Salway, 1999)
herbivores
Domestic 5 5.2 ± 0.1 7.8 ± 1.4 9.9 ± 1.7 2.2 ± 1.5 and the first step in the formation of urea occurs during the
sheepa deamination of Glu by Glu dehydrogenase (Sick et al., 1997). The
All wild deer 3 4.9 ± 0.6 e e e d15N value of Glu is therefore much more sensitive to differences
Wild deera 3 4.9 ± 0.6 6.8 ± 1.6 8.6 ± 2.0
1.8 ± 1.8 in metabolic function, which can vary between individuals and
Cereal grains 3 0.4 ± 0.3 0.1b 9.9c
6.9, 9.0,
8.4d
also tends to reflect the average d15N value of bone collagen amino
€ yük All humans
Çatalho 67 12.5 ± 1.2 e e e acids.
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 e e e 3.2.2. Makriyalos, Greek Macedonia
herbivores
There is no difference in the d15NPhe value of humans, domestic
Domestic 5 10.0 ± 1.3 12.5 ± 2.1 13.3 ± 2.2 0.8 ± 0.9
caprinesa sheep and wild deer from Makriyalos (9.8 ± 1.7‰; c2 (2) ¼ 0.908,
All wild cattle 70 9.9 ± 1.8 e e e p ¼ 0.635), which contrasts with the significant difference in bulk
Wild cattlea 5 9.7 ± 1.4 12.7 ± 1.5 12.1 ± 1.6 0.6 ± 0.6 d15N values between humans and herbivores (c2 (2) ¼ 8.580,
Cereal grains 6 6.7 ± 0.9 6.2b 13.3c 6.9, 9.0, p ¼ 0.014). It can therefore be inferred that the d15NPhe value of the
8.4d
Pulse seeds 4 2.7 ± 1.7 e e e
plant portion of the human diet is similar to the d15NPhe value of
a
domestic herbivore bone collagen (Table 3), regardless of animal
Individuals chosen for amino acid isotope analysis.
protein consumption. Using a D15NPhe-Bulk value of 8.4‰, the d15N
b
Estimated by subtracting 0.5‰ from the average cereal grain d15N value.
c
Estimated from the average domestic herbivore d15NPhe value (see Section value of plants consumed by humans at Makriyalos is estimated to
3.2.1). be 1.5‰, which is slightly higher than the d15N values determined
for the 3 cereal grains from Makriyalos (0.4 ± 0.3‰; Fig. 4b). This
d
D15NGlu-Phe values determined in modern T. aestivum and H. vulgare grains
grown at (i) Rothamsted, UK, and (ii) Bad Lauchsta €dt, Germany (Styring et al., 2014)
implies that the plants consumed by humans from Makriyalos were
and (iii) in modern C3 leaves (Chikaraishi et al., 2010).
not restricted to cereal grains with similarly low d15N values to
those determined in this study. Analysis of greater numbers of
cereal grains and pulses from Makriyalos would reveal whether
breaking of a N bond (Salway, 1999) and therefore no isotopic crops with these higher d15N values are preserved on the site.
fractionation would be expected. Unfortunately, the d15NPhe values Again, bone collagen d15NGlu values are much more variable and
of human and faunal bone collagen and plant protein cannot be differ between and within species (within-species differences ac-
used to estimate the relative contributions of plant and animal count for 17% of the variation). In contrast to individuals at Vai-
protein to the diet because the differences in d15NPhe values be- hingen, there is a significant difference in the D15NGlu-Bulk values
tween fauna and plants are smaller than instrumental errors. between species (c2 (2) ¼ 8.949, p ¼ 0.011). A post-hoc Bonferro-
The human d15NPhe value derives from both the plant and ani- nieDunn test showed the significant difference to be between red
mal contributions to the diet, however, and since the determined deer and humans (p ¼ 0.013), with D15NGlu-Bulk values of the red
human and domestic herbivore d15NPhe values are very similar, it deer lower than the humans. This seems to indicate that the d15NGlu
can be inferred that the d15NPhe value of the plants eaten by the value does not reflect the average of amino acid d15N values in red
humans is also similar to that of the domestic herbivore d15NPhe deer bone collagen at Makriyalos.
value, regardless of the relative contributions of animal and plant
protein to the human diet. This similarity need not imply, however, 3.2.3. Çatalho€yük, Turkey
that the bulk d15N values of plants eaten by humans and herbivores There is no significant difference in the d15NPhe value of humans,
are also the same, because offsets between d15NPhe and bulk d15N domestic sheep/goats and wild/domestic cattle from Çatalho €yük
values vary among plant taxa and the d15N values of the 19 other (13.0 ± 1.6‰; c2 (2) ¼ 2.660, p ¼ 0.264), which contrasts with the
amino acids in plant protein also contribute to the bulk plant d15N significant difference in bulk d15N values (c2 (2) ¼ 9.420, p ¼ 0.009).
value (Styring et al., 2014). It can therefore be inferred that the d15NPhe value of the plant
Using the cereal grain amino acid d15N values determined by portion of the human diet is similar to that of the domestic her-
Styring et al. (2014), it is found that the d15NPhe value relative to the bivores (Table 3), regardless of animal protein consumption. Using a
bulk grain d15N value (D15NPhe-Bulk value) is 8.4 ± 1.5‰. Since this D15NPhe-Bulk value of 8.4‰, the d15N value of plants consumed by
value differs slightly between site and species, we have decided to humans at Çatalho €yük is estimated to be 4.9‰, which is lower than
use an average D15NPhe-Bulk value in our calculations. Subtracting the d15N values determined for the 6 cereal grain samples from the
the D15NPhe-Bulk cereal grain value from the d15NPhe value inferred site (6.7 ± 0.9‰; Fig. 4c). This suggests that, like at Vaihingen, the
for the plant portion of the human diet (Table 3) gives an estimate plants consumed by humans from Çatalho €yük may have included
512 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515

€yük. Estimated
Fig. 4. Determined and estimated bulk plant, collagen and amino acid d15N values of plants, herbivores and humans from a) Vaihingen, b) Makriyalos, and c) Çatalho
plant d15NPhe values are inferred from human and herbivore bone collagen d15NPhe values. Cereal grain d15N values were estimated by subtracting 8.4‰ from estimated plant d15NPhe
values (D15NPhe-Bulk values determined in modern cereal grains).

plants with lower d15N values such as pulses (Inline Supplementary 3.3.1. Vaihingen an der Enz, Germany
Fig. S4). Animal protein consumption among humans at Vaihingen is
Again, the bone collagen d15NGlu values are much more variable calculated to be 58% if the ‘Standard method’ includes the bulk
and differ between and within species (within-species differences collagen d15N values of all of the individuals. The proportion of
account for 32% of the variation). As observed at Vaihingen, the animal protein consumption calculated using only the bulk
D15NGlu-Bulk values do not differ significantly between species collagen d15N values of the individuals chosen for amino acid
(3.3 ± 0.7‰; c2 (2) ¼ 2.880, p ¼ 0.237) and the D15NGlu-Bulk value is analysis is the same as that calculated for all individuals, with a
similar to that of the individuals from Vaihingen. smaller range in the 95% confidence interval due to the smaller
variation in bulk collagen d15N values.
3.3. Estimating the proportion of animal protein consumed by The calculated proportion of animal protein consumed is much
humans lower using the ‘Standard method plus plants’ (26%). This is
because the cereal grain d15N values are relatively high compared to
Table 3 gives the average bulk d15N values, d15NGlu and d15NPhe the estimated herbivore forage d15N values (d15NHerbivore -
values used to estimate the proportion of animal protein consumed 4‰ ¼ 2.8‰ compared to 4.5‰). This indicates that the d15N values
by the humans on each archaeological site, using each of the four of plants consumed by humans and herbivores at Vaihingen are
methods described in Section 2.5. The estimates obtained from different and suggests that cereals consumed by humans were
each of the methods are presented in Table 4. Estimates made using likely to have been manured (cf. Fraser et al., 2013b), accounting for
the ‘Standard method’ and the ‘Standard model plus plants’ were the relatively high d15N values.
carried out using: (i) all human and domestic herbivore bulk bone Using different D15NGlu-Phe* values in the ‘Bone collagen d15NGlu
collagen d15N values, and (ii) using only the bone collagen d15N and d15NPhe values method’ makes a considerable difference to the
values from the humans (n ¼ 5) and domestic herbivores (n ¼ 5) calculated animal protein consumption (between 11 and 38%), but
whose amino acid d15N values were determined in this study. The regardless of the D15NGlu-Phe* value used, the calculated animal
D15NConsumer-Diet value for bulk bone collagen is assumed to be 4‰. protein consumption is much lower than that calculated using only
 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515 513

Table 4
€yük, calculated using the four methods described in Section 2.5, the
Estimated proportions of animal protein consumed by humans from Vaihingen, Makriyalos and Çatalho
d15N values given in Table 1 and assuming a D15Ncollagen-diet value of 4‰.
Proportion of animal protein consumed (%)
95% Confidence interval in bracketsa

Method Vaihingen Makriyalos €yük

Çatalho
1. ‘Standard method’
(i) All bulk d15N values 58 (50e65%) 63 (49e76%) 63 (54e71%)
(ii) Bulk d15N values from bone collagen isolates used in AA analyses 58 (54e61%) 63 (47e78%) 75 (38e100%)
2. ‘Standard method plus plants’
(i) All bulk d15N values 26 (13e39%) 68 (57e79%) 55 (40e69%)
(ii) Bulk d15N values from bone collagen isolates used in AA analyses 26 (16e37%) 69 (55e82%) 70 (27e100%)
3. ‘Bone collagen d15NGlu and d15NPhe values’
(i) D15NGlx-Phe* ¼ 6.9 ± 0.2‰ 11 (8e13%) 8 (6e10%) 33 (23e42%)
(ii) D15NGlx-Phe* ¼ 9.0 ± 0.3‰ 38 (30e46%) 36 (28e43%) 61 (46e75%)
(iii) D15NGlx-Phe* ¼ 8.4‰ ± 1.6‰ 30 (24e37%) 28 (21e34%) 53 (39e66%)
4. ‘Bone collagen and cereal d15NGlu values’ 4 (0e30%) 41 (18e63%) 40 (0e82%)
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 d15NGlu[hum],
d15NPhe[hum], D15NGlx-Phe*, DGlu and DPhe.

bulk bone collagen d15N values. This highlights the need for further €yük, Turkey
3.3.3. Çatalho
studies to constrain the D15NConsumer-Diet, DGlu and DPhe values for Animal protein consumption among humans at Çatalho €yük is
diets of differing protein quality and content, particularly in calculated to be 63% if the ‘Standard method’ includes the bulk
terrestrial mammals. When taking into account the estimated cereal collagen d15N values of all of the individuals. Seven out of the 67
grain d15NGlu values, the calculated animal protein consumption is individuals exhibit d15N values (over 14‰) greater than the
considerably lower (4%) due to the relatively high plant d15N values. 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
3.3.2. Makriyalos, Greek Macedonia
than that calculated for all individuals (75%).
Animal protein consumption among humans at Makriyalos is
The calculated proportion of animal protein consumed is
calculated to be 63% if the ‘Standard method’ includes the bulk
slightly lower using the ‘Standard method plus plants’ (55%). This is
collagen d15N values of all of the individuals. However, 1 out of the 18
because the cereal grain d15N values are slightly higher than esti-
individuals exhibits a d15N value (under 6‰) below those of the
mated herbivore forage d15N values (d15NHerbivore e 4‰ ¼ 6.0‰
domestic herbivores, either suggesting that this individual consumed
compared to 6.7‰). Cereals consumed by humans may therefore
no animal protein, or pulses and plants with low d15N values
have been manured, increasing their d15N values above that of the
comprised a significant part of their diet. Two out of the 21 in-
herbivore forage.
dividuals whose bone collagen isotope values were determined by
Using the ‘Bone collagen d15NGlu and d15NPhe values method’,
Triantaphyllou (2001) also exhibited very low d15N values.
animal protein consumption estimates are slightly lower (between
Conversely, one individual exhibits a d15N value (d15N ¼ 9.3‰)
33 and 61%), but they are strongly influenced by the D15NGlu-Phe*
greater than the maximum theoretical bone collagen d15N value
value used. The calculated animal protein consumption is also
predicted from the consumption of pure herbivore protein (i.e. 9.1‰).
lower (40%) using the ‘Bone collagen and cereal d15NGlu values’
The calculated proportion of animal protein consumed is higher
method. The uncertainties associated with all dietary calculations
using the ‘Standard method plus plants’ (68%). This is because the
are large because of the considerable variation in bulk and amino
cereal grain d15N values are relatively low compared to the esti-
acid d15N values between individuals.
mated herbivore forage d15N values (d15NHerbivore e 4‰ ¼ 1.1‰
compared to 0.4‰). There is the potential for fish consumption at
Makriyalos, considering its proximity to the coast and the findings 4. Discussion and conclusion
of abundant seashells. Extensive investigations of organic residues
in cooking pottery from the site show no evidence, however, of the This is the first study to investigate bone collagen amino acid
processing of aquatic commodities as judged by stable carbon d15N values of humans believed to have been eating only, or pre-
isotope determinations of fatty acids and an absence of aquatic lipid dominantly, terrestrial protein, which simplifies the possible di-
biomarker proxies (Evershed et al., 2008; Cramp and Evershed, etary inputs to the bone collagen N isotopic signature. We have
2014; Whelton et al. unpublished). Further d15N value de- used the d15N values of bone collagen, bone collagen amino acids,
terminations of cereal grains from Makriyalos are necessary in or- plant protein and plant protein amino acids in four different
der to ascertain whether the very low cereal grain d15N values palaeodietary models to calculate the proportion of animal protein
measured in this study reflect those of the majority of crops. in human diet at three different archaeological sites. Comparison of
Using the ‘Bone collagen d15NGlu and d15NPhe values method’, the results of these calculations highlights limitations of these
animal protein consumption estimates are much lower (between 8 models and draws attention to the priorities for future work needed
and 36%), regardless of the D15NGlu-Phe* value used. Again, this to improve their accuracy and reliability.
highlights the need for better understanding of the factors Bulk bone collagen d15N values average out the d15N values of
contributing to amino acid and bulk collagen d15N values. The their constituent amino acids, representing the net effect of dietary
calculated animal protein consumption is also lower (41%) using protein sources and metabolic cycling within the body. Calculating
the ‘Bone collagen and cereal d15NGlu values method’ despite the human animal protein consumption using bulk d15N values also
fact that the cereal grain d15NGlu value used in this calculation was relies upon the assumption that faunal bone collagen preserved on
estimated from the relatively low bulk cereal grain d15N values. the site is representative of the d15N values of the animals
514 A.K. Styring et al. / Journal of Archaeological Science 53 (2015) 504e515

consumed. This is not the case with the ‘Bone collagen d15NGlu and Twiss, Nerissa Russell, Clark S. Larsen for access to bone material
d15NPhe values method’ since human bone collagen d15NGlu and from Çatalho€yük.
d15NPhe values provide an internal indicator of animal protein
consumption, although it remains a limitation of the ‘Bone collagen Appendix A. Supplementary data
and cereal d15NGlu values method’.
The large discrepancies between the estimates of animal protein Supplementary data related to this article can be found at http://
consumption made with and without taking into account the d15N dx.doi.org/10.1016/j.jas.2014.11.009.
values of charred cereal grains and pulses illustrate the importance
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