Refining human palaeodietary reconstruction using amino acid d 15 N 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 Sch € afer f , 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 0 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 Pr€ ahistorische und Naturwissenschaftliche Arch€ aologie, University of Basel, Basel, Switzerland g School of History and Archaeology, Aristotle University of Thessaloniki, Thessaloniki, Greece 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 d 15 N values Palaeodiet 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 (d 15 N) values of human and herbivore bone collagen. However, this approach, using the 15 N-enrichment of human bone collagen d 15 N values over associated herbivore bone collagen d 15 N values to predict the relative importance of animal protein, relies on the assumptions that: (i) the d 15 N values of plants consumed by humans and herbivores are identical, and (ii) the 15 N- enrichment between diet and consumer is consistent. Bone collagen amino acid d 15 N values have the potential to tackle these uncertainties, as they constrain the factors influencing bone collagen d 15 N values. In this study, the d 15 N 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 d 15 N values, (ii) comparing bulk human and herbivore collagen and ancient charred cereal grain d 15 N values, (iii) comparing human bone collagen d 15 N Glutamic acid and d 15 N Phenylalanine values, and (iv) comparing d 15 N Glutamic acid values of human and herbivore bone collagen and estimated d 15 N Glutamic acid values of ancient charred cereal grains. Where determined cereal grain d 15 N 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 D 15 N Consumer-Diet values of glutamic acid and phenylalanine in terrestrial ecosystems, and (ii) D 15 N Glutamic 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 governed by underlying metabolic processes associated with nutrient assimilation and tissue biosynthesis. In archaeology, the * Corresponding author. Present address: School of Archaeology, 36 Beaumont Street, University of Oxford, Oxford, UK. Tel.: þ44 0 1865 288014. E-mail address: amy.styring@arch.ox.ac.uk (A.K. Styring). Contents lists available at ScienceDirect Journal of Archaeological Science journal homepage: http://www.elsevier.com/locate/jas 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/). Journal of Archaeological Science 53 (2015) 504e515