Scholarly article on topic 'Compound-specific amino acid isotopic proxies for detecting freshwater resource consumption'

Compound-specific amino acid isotopic proxies for detecting freshwater resource consumption Academic research paper on "History and archaeology"

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{"Amino acids" / "Carbon isotopes" / "Nitrogen isotopes" / Palaeodiet / Zvejnieki / Latvia}

Abstract of research paper on History and archaeology, author of scientific article — Emily C. Webb, Noah V. Honch, Philip J.H. Dunn, Gunilla Eriksson, Kerstin Lidén, et al.

Abstract Of central importance to palaeodietary reconstruction is a clear understanding of relative contributions of different terrestrial (i.e., C3 vs. C4 plants) and aquatic (i.e., freshwater vs. marine) resources to human diet. There are, however, significant limitations associated with the ability to reconstruct palaeodiet using bulk collagen stable isotope compositions in regions where diverse dietary resources are available. Recent research has determined that carbon-isotope analysis of individual amino acids has considerable potential to elucidate dietary protein source where bulk isotopic compositions cannot. Using δ 13CAA values for human and faunal remains from Zvejnieki, Latvia (8th – 3rd millennia BCE), we test several isotopic proxies focused on distinguishing freshwater protein consumption from both plant-derived and marine protein consumption. We determined that the Δ 13CGly-Phe and Δ 13CVal-Phe proxies can effectively discriminate between terrestrial and aquatic resource consumption, and the relationship between essential δ 13CAA values and the Δ 13CGly-Phe and Δ 13CVal-Phe proxies can differentiate among the four protein consumption groups tested here. Compound-specific amino acid carbon-isotope dietary proxies thus enable an enhanced understanding of diet and resource exploitation in the past, and can elucidate complex dietary behaviour.

Academic research paper on topic "Compound-specific amino acid isotopic proxies for detecting freshwater resource consumption"

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Journal of Archaeological Science

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

Compound-specific amino acid isotopic proxies for detecting freshwater resource consumption

Emily C. Webb a, Noah V. Honch a, Philip J.H. Dunn a, Gunilla Eriksson b, Kerstin Liden Richard P. Evershed a' *

a Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom b Archaeological Research Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden

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ARTICLE INFO

ABSTRACT

Article history: Received 2 October 2014 Received in revised form 1 July 2015

Accepted 3 August 2015 Available online 5 August 2015

Keywords: Amino acids Carbon isotopes Nitrogen isotopes Palaeodiet Zvejnieki Latvia

Of central importance to palaeodietary reconstruction is a clear understanding of relative contributions of different terrestrial (i.e., C3 vs. C4 plants) and aquatic (i.e., freshwater vs. marine) resources to human diet. There are, however, significant limitations associated with the ability to reconstruct palaeodiet using bulk collagen stable isotope compositions in regions where diverse dietary resources are available. Recent research has determined that carbon-isotope analysis of individual amino acids has considerable potential to elucidate dietary protein source where bulk isotopic compositions cannot. Using <513Caa values for human and faunal remains from Zvejnieki, Latvia (8th — 3rd millennia BCE), we test several isotopic proxies focused on distinguishing freshwater protein consumption from both plant-derived and marine protein consumption. We determined that the D13CGly.phe and D13CVal-phe proxies can effectively discriminate between terrestrial and aquatic resource consumption, and the relationship between essential d13CAA values and the D13CGly.Phe and D13CVal-Phe proxies can differentiate among the four protein consumption groups tested here. Compound-specific amino acid carbon-isotope dietary proxies thus enable an enhanced understanding of diet and resource exploitation in the past, and can elucidate complex dietary behaviour.

© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Stable carbon and nitrogen isotopic analyses of archaeological human and faunal remains have been used for over three decades to reconstruct palaeodiet and to investigate palaeoecology, and these studies make valuable contributions to our understanding of subsistence practices in diverse environments, and of changing patterns of resource use temporally and spatially (Katzenberg and Pfeiffer, 2000). In recent years, however, it has become increasingly evident that there are significant limitations in the assessment of palaeodiet using bulk protein stable isotope compositions in ecologically-complex regions where diverse dietary resources are available (e.g., Hedges, 2004; Liden et al., 2004; Milner et al., 2004). Differential resource access and exploitation by individuals or societies can have important implications for socioeconomic behaviour, or reveal the social and ecological impact of

* Corresponding author. E-mail address: r.p.evershed@bristol.ac.uk (R.P. Evershed).

environmental perturbations over time. Thus, of central importance in many archaeological contexts is the ability to determine the relative contributions of different protein sources to diet, such as terrestrial C3 plant-derived protein, freshwater protein, terrestrial C4 plant-derived protein, or marine protein resources. Bulk carbon-isotope compositions, however, may mask dietary variability if the contributions from specific resources (e.g., marine protein) are small (Hedges, 2004), or if the natural variability in carbon-isotope compositions between two classes of resources is low, as is often the case when comparing the isotopic compositions of terrestrial plants and freshwater fauna from adjacent lakes and rivers.

Nitrogen-isotope compositions reflect the trophic position of consumed protein and are known to increase several per mil per trophic level between producers and consumers, and often help in disentangling complex diets (DeNiro and Epstein, 1981; Schoeninger, 1985). There is considerable variability in the degree of 15N-enrichment between diet and consumer tissues; among humans from European archaeological sites, for example, the tissue — diet nitrogen-isotope offsets ranged from —h2 to +5%o (reviewed

http://dx.doi.org/10.1016/jjas.2015.08.001

0305-4403/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

in Hedges and Reynard, 2007), and a recent controlled dietary study estimated trophic offsets as high as +6%o (O'Connell et al., 2012). Trophic relationships inferred from nitrogen-isotope compositions can, however, be obscured or altered, for example, if manure is applied to terrestrial crops (Bogaard et al., 2007; Fraser et al., 2011), in particularly arid environments (Ambrose and DeNiro, 1986; Craine et al., 2009; Grocke et al., 1997; Heaton et al., 1986), or if nitrogen metabolism is affected by physiological stress (Reitsema, 2013; Williams et al., 2011). The possible influence of these factors on d15N values can, however, be difficult to assess. Focusing on compound-specific carbon-isotope compositions, particularly of essential amino acids, eliminates the potentially confounding effect of tissue-diet fractionation and routing vs. biosynthesis of non-essential amino acids on bulk carbon-isotope compositions, and of the environmental and physiological effects discussed above on nitrogen-isotope compositions.

Controlled feeding studies and the relatively few published papers that have analyzed archaeological material have demonstrated that carbon-isotope analysis of individual amino acids has considerable potential to elucidate palaeodiet where traditional bulk isotopic data fail (Choy et al., 2010; Corr et al., 2005; Fogel and Tuross, 2003; Hare et al., 1991; Honch et al., 2012; Howland et al., 2003; Jim et al., 2006). For example, intra-individual offsets between the amino acids phenylalanine and glycine (D13CGly-Phe; Corr et al., 2005) reliably distinguish among individuals consuming a high marine protein diet and those consuming a terrestrial C4 plant diet, and the feasibility of using the relationships between other paired amino acids to identify dietary protein source has also been investigated (e.g., D13CVal-Phe for assessing freshwater resource consumption; Honch et al., 2012). Nonetheless, identifying instances of freshwater protein consumption isotopically remains challenging. In temperate environments, high freshwater protein consumption is typically inferred based on high nitrogen-isotope compositions, C3 plant-like carbon-isotope compositions, and the presence of fish bones or artefacts used to exploit aquatic resources in the archaeological record (Lillie and Richards, 2000; Minagawa and Wada, 1984; Richards et al., 2001). In the absence of corroborative archaeological evidence, however, it is very difficult to assess the significance of freshwater resource contribution to human diet. The development of an array of isotopic proxies which are sensitive to changes in dietary protein source and are highly discriminatory among different protein resources is thus essential to increasingly refined palaeodiet reconstruction.

Here, the objective is to test new and previously proposed iso-topic proxies and relationships focused on distinguishing freshwater resource consumption from both plant-derived (C3 and C4) and marine protein consumption using compound-specific carbonisotope compositions of collagen. We present new amino acid carbon-isotope data for archaeological humans and fauna from Zvejnieki, Latvia (c. 8th - 3rd millennia BCE). Archaeological and zooarchaeological data, as well as previously published bulk carbon- and nitrogen-isotope compositions, strongly indicate that the inhabitants consumed considerable quantities of freshwater resources. Hence, these hunter-gatherer-fisher settlements offer an ideal opportunity to evaluate and refine compound-specific iso-topic proxies for disentangling palaeodiet, which can then be used in other, less well-understood archaeological contexts where multiple resources from marine, terrestrial and freshwater environments were available for consumption.

2. Theoretical considerations for stable carbon-isotope analysis

The reconstruction of palaeodiet using stable isotope analysis is based on the well-tested assumption that tissue isotopic

composition reflects the isotopic composition of consumed food (Ambrose, 1993). Systematic differences exist in isotopic composition between tissue and diet (Ambrose, 1993; DeNiro and Epstein, 1978), which are a result of isotopic discrimination, the differential partitioning of isotopes between phases in a reaction (e.g., ingested food / consumer tissue) caused by the slight mass differences among isotopes of the same element (e.g., 13C, 12C). When diet is protein-sufficient, the 513C values of proteinaceous consumer tissues (e.g., collagen and dentin) largely reflect the carbon-isotope composition of dietary protein as it is derived from the base of the food web (Ambrose, 1993; Kellner and Schoeninger, 2007). Tissue carbon-isotope composition is a weighted average of the isotopic compositions of all component essential and non-essential amino acids (d13CAA). Essential amino acids (e.g., threonine, valine, methionine, isoleucine, leucine, histidine, lysine, and phenylalanine) cannot be generated by the body and therefore must be ingested in sufficient quantities. In contrast, non-essential amino acids (e.g., asparagine/aspartic acid, hydroxyproline, glutamic acid/glutamate, serine, glycine, alanine, proline and arginine) can be assimilated with minimal modification from a dietary source, or may be synthesised de novo using components drawn from the body's biochemical pools; the latter will result in isotopic fractionation (Ambrose and Norr, 1993; Newsome et al., 2014). Tissue essential amino acid S13C values are thus expected to closely approximate dietary essential amino acid 513C values due to direct routing, i.e., D13C tissue AA _ diet AA z 0%. Non-essential amino acid 513C values, however, may show evidence of both direct routing and biosynthesis, depending on the quality, digestibility and amino acid composition of consumed food.

Differences in the way that C3 and C4 plants incorporate 13C/12C during photosynthesis allow assessment of relative dietary contributions of these different classes of foods, consumed as both plants and plant-consuming fauna. C3 plants (e.g., grains, rice, tubers, fruits and vegetables) have lower (i.e., more negative) 513C values (global average z -26.5%), whereas C4 plants (e.g., tropical grasses, maize, millet and sorghum) have higher 513C values, with an average of around -12.5% (Ambrose, 1993). Terrestrial plants obtain carbon from atmospheric CO2 (average -8%; Marino and McElroy, 1991). In contrast, marine plants obtain carbon from several inorganic sources (e.g., dissolved bicarbonate, carbonate-containing minerals), which are generally 13C-enriched relative to the terrestrial carbon source. Dissolved inorganic carbon enters the marine food web via photosynthetic uptake by algae and phyto-plankton (Corbisier et al., 2006; Mook and Tan, 1991). The carbonisotope compositions of marine organisms are therefore dependent on several factors, including the 513C values of the dissolved inorganic carbon, isotopic fractionation associated with photosynthetic uptake, and the rate of diffusion of CO2 across photosynthetic cells in plants, which is slow for marine plants compared to terrestrial plants (Mook and Tan, 1991; Rau et al., 1996). As a result, C3 marine plant carbon-isotope compositions are often indistinguishable from the isotopic compositions of terrestrial C4 plants. This comparatively small difference in carbon-isotope compositions makes disentangling consumption of marine protein (-5—17%) from C4 plant-derived protein (z -12.5%) challenging. The isotopic composition of freshwater carbon is contingent on a similarly complex array of environmental sources, including, for example, CO2 and dissolved organic carbon evolved from bacterial decay of terrestrial organic matter, dissolution of inorganic carbonates from rocks and soils in the catchment area, photosynthesis of aquatic flora, and carbonate precipitation (Mook and Tan, 1991). In the predominately C3 plant-based ecosystems of northern Europe, freshwater resource 513C values are ~z -27% (Dufour et al., 1999; Katzenberg and Weber, 1999).

3. Archaeological background and sampling

The Zvejnieki archaeological complex encompasses a large Neolithic and Mesolithic hunter-fisher-gatherer cemetery, and two adjacent settlement sites: the extensively excavated Mesolithic Zvejnieki II and the Neolithic Zvejnieki I, which has been partially excavated (Eriksson et al., 2003; Larsson and Zagorska, 2006; Zagorskis, 2004). The cemetery is located on the eastern side of Lake Burtnieks, approximately 50 km inland from the Gulf of Riga (Fig. 1). During the Mesolithic and Neolithic, water levels in Lake Burtnieks were high, and the Zvejnieki site would have been on a small island rather than part of the mainland during this time. The cemetery, consisting of in excess of300 human burials, is one of the largest hunter-fisher-gatherer cemeteries in Europe. Radiocarbon evidence indicates that the cemetery was in use from the Middle Mesolithic (c. 8th millennium BCE) to the Late Neolithic (c. 5th-3rd millennium BCE). Most human graves are single interments, although some graves contain multiple individuals.

Zooarchaeological and stable isotope analyses suggest that freshwater fish were a vital dietary resource for the individuals buried in the Zvejnieki cemeteries. Numerous fish bones (e.g., pike, perch and various species from the carp family), as well as eel and salmon, have been recovered from the habitation layers of Zvejnieki I and II, together with a wide variety of fishing implements. A diverse range of mammalian species, the most prevalent of which are moose, wild boar and beaver, were also found at the settlements. In the burials, in contrast, tooth pendants made of teeth from hunted game, such as moose, wild boar and red deer, predominate, and badger, marten, wild horse, brown bear, fox, wolf, aurochs, seal, otter, beaver and dog also occur (Eriksson et al., 2003). Bulk stable carbon- and nitrogen-isotope analysis has determined that freshwater fish were consumed throughout the period of use of the cemetery, although dependency on this resource appears to have declined by the Late Neolithic (Eriksson, 2006; Eriksson et al., 2003). Terrestrial protein resources also contributed to diet. The 513C values of many of the humans and fish are low (—24—21%) relative to local terrestrial herbivores, but are similar to isotopic compositions from freshwater fish in shallow lakes elsewhere in central Europe (Dufour et al., 1999; Eriksson et al., 2003).

Here, a subset of human skeletal remains from Zvejnieki were analyzed for their compound-specific amino acid carbon-isotope compositions (n = 8; Fig. 2 and Table 1). Individuals with low 513C and high d15N values, which are characteristic of high freshwater protein consumption, were selected for further compound-

specific isotopic analysis. Several archaeological faunal specimens were also selected for amino acid carbon-isotope analysis, representing a variety of dietary ecologies (n = 16; Fig. 2 and Table 1). The archaeological material spans the Middle Mesolithic to Middle Neolithic, after which time freshwater resources were not as widely consumed.

4. Laboratory procedures

Collagen was extracted from bone or tooth dentin powder at the Archaeological Research Laboratory, Stockholm University, using the method described in Brown et al. (1988). Collagen samples of ~0.5 mg were weighed into tin capsules and isotopic analysis was performed using a Carlo Erba NC2500 elemental analyzer coupled to a Finnigan MAT DeltaPlus mass spectrometer via a split interface. Bulk collagen isotopic analyses were performed at the Stable Isotope Laboratory (SIL), Department of Geological Sciences, Stockholm University. The 513C values were calibrated to VPDB using NBS-18, NBS-19, IAEA-CO-1 and IAEA-CO-8, and the d15N values were calibrated to AIR using IAEA-N1, IAEA-N2, IAEA-NO-3 and USGS25. Precision was monitored throughout using in-house standards (acetanilide and pepton), and was ±0.1% for both carbon- and nitrogen-isotope analyses (Eriksson, 2006).

For each sample, approximately 2 mg of collagen were hydro-lyzed by heating at 110 °C in 6 M HCl for 24 h. Collagen hydrolysates of free amino acids were dried under N2 at 60 °C and re-dissolved in 1 ml HPLC-grade water for LC/IRMS analysis. Amino acid 513C values were obtained using a Surveyor HPLC connected to a Delta V Plus mass spectrometer via an LC Isolink interface (Thermo Scientific). Amino acids were resolved on a Primesep A column (250 mm x 3.2 mm, 5 mm particle size, 100 A pore size, SiELC Technologies Ltd.) using a linear two-phase mobile phase gradient progressing from 100% water to 100% 0.024 M H2SO4 (Dunn et al., 2011; Honch et al., 2012; McCullagh et al., 2006, 2010). Reference CO2 gas was calibrated to VPDB against IAEA L-Glutamic acid (USGS40, 513C: -26.2%; Qi et al., 2003). Accuracy and reproducibility were monitored throughout using a standard amino acid mix incorporating a wide range of 513C values (d13CAsp: -22.4%; 513CGlu: -26.2%; <513CGly: -32.4%; <513CAla: -26.1%; <513CPro: -10.6%%; Sigma Aldrich). The standard deviations of the 513C values for the known amino acids ranged from ±0.4 to ±0.6%, and compared well with the certified or EA-IRMS determined accepted values (±0.02-±0.5%). All archaeological samples were analyzed at least twice and the average d13CAA value is reported herein. All compound-specific carbon-isotope analyses were performed in

Fig. 1. Location of the Zvejnieki archaeological complex

the interior of Latvia.

Fig. 2. Bulk d C and <5 N values for humans and faunal specimens analysed in this paper from Zvejnieki.

Table 1

Descriptions of archaeological human and faunal specimens.

Sample ID Species Context Element Notes Datea

ZVE15 Human Burial 158 Incisor Adult (M) Early Neolithic

ZVE27 Human Burial 228 Scapula Adult (m) Middle Neolithic; 5090 ± 55

ZVE29 Human Burial 277 Femoral bone Young Adult (M) Middle Neolithic; 5545 ± 65

ZVE32 Human Burial 62 1st incisor Tooth Pendant Late Mesolithic

ZVE45 Human Burial 208 Cranial bone Juvenile Middle Neolithic; 5345 ± 60

ZVE56 Human Burial 121 Cranial bone Juvenile Early Neolithic; 6145 ± 80

ZVE57 Human Burial 201 1st molar Child, 9—11 yrs Middle Neolithic; 4865 ± 75

ZVE59 Human Burial 197 Scapula Adult (M) Early Neolithic; 6410 ± 95

AZV70 Dog Burial 114 Canine Tooth Pendant Early Neolithic

AZV72 Dog Burial 132 Canine Tooth Pendant Early Neolithic

AZV73 Dog Burial 146 Canine Tooth Pendant Early Neolithic

AZV77 Dog Burial 153 Canine Tooth Pendant Early Neolithic

AZV82 Dog Burial 167/8 Canine Tooth Pendant No date

AZV56 Moose Zvejnieki II Settlement Bone Middle Mesolithic

AZV96 Moose Burial 221 Antler Modified Antler Middle Neolithic; 5290 ± 105

AZV18 Aurochs Burial 164 Incisor Tooth Pendant Middle Neolithic

AZV11 Wild Horse Burial 42 Incisor Tooth Pendant Late Mesolithic

AZV12 Wild Horse Burial 86 Incisor Tooth Pendant No date

AZV14 Wild Horse Burial 122 Incisor Tooth Pendant Early Neolithic

AZV88 Otter Burial 190 Canine Tooth Pendant Late Mesolithic

AZV64 Perch Zvejnieki II Settlement Bone Tooth Pendant Middle Mesolithic

AZV26 Wild Boar Burial 146 Incisor Tooth Pendant Early Neolithic

AZV35 Wild Boar Burial 168 Canine Tooth Pendant No date

AZV37 Wild Boar Burial 206 Canine Middle Neolithic

a AMS 14C dates (uncalibrated BP) reported when available.

facilities housed in the Organic Geochemistry Unit, School of Chemistry at the University of Bristol.

5. Results

Bulk protein carbon- and nitrogen-isotope compositions are summarized in Fig. 2 and Table 2, and fully reported in Eriksson (2006). For humans, the average 515N value was +12.5 ± 1.0%o and, for 513C values, -23.5 ± 0.8%. The average 513C and 515N values for categories of fauna are as follows: -24.9 ± 1.4% and +13.4 ± 1.4% for dogs, -22.6 ± 0.4% and +3.8 ± 0.9% for moose, -23.3 ± 1.0% and +6.0 ± 0.9% for terrestrial fauna, -25.5 ± 2.8% and +11.1 ± 2.8% for freshwater fauna. For amino

acid isotopic analyses, collagen preservation was assessed by comparing each sample amino acid distribution to the theoretical distribution for collagen, determined relative to glycine and expressed as a percentage. All amino acids were preserved within 10% of the theoretical value (Dunn, 2011). An example chromato-gram of archaeological dentin collagen is presented in Fig. 3. The 513C values are reported for the following amino acids: aspartic acid/asparagine, hydroxyproline, glutamatic acid/glutamine, thre-onine, serine, methionine, histidine, glycine, alanine, proline, valine, isoleucine/leucine, lysine, phenylalanine and arginine. Isoleucine and leucine co-elute and are thus reported as a single value. Aspartic acid and asparagine and glutamic acid and gluta-mine are reported as single values, because asparagine and

Table 2

Zvejnieki human (ZVE prefix) and faunal (AZV prefix) stable isotopic compositions.

ZVE15 ZVE27 ZVE29 ZVE32 ZVE45 ZVE56 ZVE57 ZVE59

S13Cbuik (%o, VPDB) -24.3 -24.0 -22.6 -23.8 -22.2 -23.1 -23.7 -24.1

815Nbuik (%, AIR) +14.0 +12.1 +11.7 + 12.6 +13.6 +10.9 +12.2 +13.0

Essential amino acids (%, VPDB)

Threonine -21.9 -18.2 -16.4 -20.6 -16.2 -17.1 -18.3 -18.2

Valine -32.1 -29.0 -27.9 -31.4 -26.7 -28.5 -29.6 -31.1

Methionine -36.3 -30.9 -32.8 -31.8 -29.4 -31.1

Isoleucine/leucine -32.4 -32.4 -30.1 -31.9 -29.6 -30.9 -32.0 -32.0

Histidine -27.6 -24.8 -28.1 -26.7 -22.8 -22.2 -25.2

Lysine -27.6 -27.7 -26.3 -27.5 -25.5 -26.7 -27.0 -28.1

Phenylalanine -32.2 -34.5 -30.7 -33.4 -31.3 -33.8 -31.8 -34.8

Nonessential amino acids (%, VPDB)

Aspartic acid/asparagine -26.5 -25.6 -24.3 -25.7 -24.7 -24.2 -25.1 -25.5

Hydroxyproline -25.6 -22.1 -22.4 -24.9 -27.7 -25.3 -25.6 -25.4

Glutamic acid/glutamate -23.1 -25.6 -23.6 -21.7 -17.1 -19.8 -20.4 -23.4

Serine -5.3 -20.8 -8.6 -19.9 -20.9 -22.5

Glycine -15.7 -17.0 -15.6 -15.6 -15.8 -15.3 -16.7 -17.1

Alanine -27.9 -24.9 -24.9 -27.2 -24.4 -23.2 -26.3 -26.7

Proline -26.1 -24.6 -24.0 -25.7 -23.3 -23.8 -24.6 -27.0

Arginine -25.6 -27.0 -26.2 -28.7 -25.8 -26.4 -26.4 -26.0

D13CGly-Phe (%) +16.5 +17.5 +15.1 + 17.8 +15.5 +18.5 +15.1 +17.7

AZV70 AZV72 AZV73 AZV77 AZV82 AZV56 AZV96 AZV18

S13Cbulk (%, VPDB) -23.3 -25.2 -26.2 -23.4 -26.2 -22.8 -22.3 -22.2

815Nbulk (%, AIR) +12.4 +12.7 +13.0 + 15.8 +12.9 +3.1 +4.4 +6.4

Essential amino acids (%, VPDB)

Threonine -19.3 -22.0 -21.9 -20.8 -24.2 -13.8 -16.6 -15.5

Valine -31.7 -33.4 -34.7 -31.4 -34.8 -30.2 -29.1 -29.6

Methionine -44.6 -24.7 -26.3

Isoleucine/leucine -31.8 -33.7 -34.3 -32.1 -34.8 -31.6 -30.7 -31.6

Histidine -26.7 -28.2 -30.6 -27.0 -30.0 -21.5 -21.3 -21.0

Lysine -25.0 -27.0 -28.4 -27.0 -28.8 -22.5 -22.2 -22.4

Phenylalanine -33.8 -35.7 -36.4 -34.9 -36.1 -28.6 -27 -27.1

Nonessential amino acids (%, VPDB)

Aspartic acid/asparagine -23.9 -26.4 -26.2 -24.2 -27.4 -23.5 -22.2 -19.2

Hydroxyproline -26.4 -27.2 -28.6 -25.1 -28.4 -24.8 -24.2

Glutamic acid/glutamate -19.4 -22.8 -23 -21.5 -23.5 -19.3 -17.8

Serine -9.9 -15.4 -13.3 -10.3 -11.6 -20.6

Glycine -14.8 -16.3 -18.6 -14.3 -18.1 -18.1 -17.4 -17.0

Alanine -28.2 -29.8 -29.8 -28.1 -31.4 -27.4 -26.4 -27.6

Proline -24.0 -26.3 -27.7 -24.6 -27.8 -23.9 -23.4 -22.3

Arginine -25.7 -28.2 -28.1 -27.8 -29.2 -23.4 -25.0 -24.1

D13CGly-Phe (%) +19.0 +19.4 +17.8 +20.6 +18.0 +10.5 +9.6 +10.1

AZV11 AZV12 AZV14 AZV88 AZV64 AZV26 AZV35 AZV37

S13Cbulk (%, VPDB) -24.9 -23.4 -24.3 -23.5 -27.4 -22.7 -22.6 -22.9

815Nbulk (%, AIR) +5.8 +5.6 +5.6 + 13.0 +9.1 +6.1 +7.9 +4.9

Essential amino acids (%, VPDB)

Threonine -20.1 -16.0 -17.1 -17.4 -22 -16.6 -15.3 -12.2

Valine -31.7 -30.1 -32.2 -28.3 -33.4 -28.0 -27.3 -27.1

Methionine -28.5 -28.4 -25.1 -28.9

Isoleucine/leucine -32.7 -30.8 -32.3 -30.9 -32.7 -29.9 -29.0 -30.5

Histidine -29.5 -23.3 -25.2 -24.9 -22.6 -21.9 -30.6

Lysine -26.2 -25.7 -25.0 -27.4 -28.3 -24.6 -24.5 -23.1

Phenylalanine -29.2 -29.1 -27.1 -32.2 -35.8 -28.0 -28.5 -26.9

Nonessential amino acids (%, VPDB)

Aspartic acid/asparagine -23.6 -22.1 -23.6 -23.8 -29.5 -23.4 -20.4 -22.1

Hydroxyproline -26.3 -26.4 -27.4 -23.3 -26.1 -26.4 -25.7 -24.0

Glutamic acid/glutamate -21.2 -18.7 -18.4 -23.5 -22.5 -18.5 -15.8 -18.6

Serine -23.5 -19.1 -18.9 -20.6

Glycine -20.5 -18.0 -18.4 -16.1 -20.0 -19.2 -18.2 -18.6

Alanine -28.1 -27.3 -27.5 -27.0 -30.1 -24.7 -23.6 -25.6

Proline -24.9 -24.4 -25.1 -24.2 -30.9 -22.9 -22.3 -23.36

Arginine -23.6 -25.2 -26.3 -25.2 -30.0 -25.7 -25.6 -24.7

D"CGly-Phe (%) +8.7 +11.1 +8.7 + 16.1 +15.8 +8.8 +10.3 +8.3

glutamine are deaminated during hydrolysis and are thus indistinguishable from aspartic acid and glutamic acid, respectively. Serine typically elutes in a region where the baseline drops with the introduction of the sulphuric acid component of the mobile phase, and the 513CSer values are less accurate because of the difficulty of integrating peaks in such a dynamic region. Low abundance amino

acids (i.e., methionine and histidine) are likewise not considered interpretively useful because of poor reproducibility compared to the more abundant amino acids. All 513Caa values are reported in Table 2 and shown in Fig. 4.

For archaeological humans, the overall range of the 513C values for non-essential amino acids is 13% (-29—15%), and the overall

Fig. 3. An example of an LC/IRMS chromatogram (human dentin collagen, ZVE15). Note that serine (Ser) elutes where the baseline drops due to the introduction of the mobile phase acid (see text). Ref: reference CO2 gas pulses; Asx: asparatic acid/asparagine; Hyp: hydroxyproline; Glx: glutamic acid/glutatmate; Ser: serine; Thr: threonine; Gly: glycine; Ala: alanine; Pro: proline; Val: valine; Met: methionine; Leu/Ile: leucine and isoleucine; Lys: lysine; His: histidine; Phe: phenylalanine; Arg: arginine.

Fig. 4. Average essential and non-essential amino acid carbon-isotope compositions for Zvejnieki humans and fauna. Error bars represent ±1 a where n > 1. The line connecting the human <513Caa values is to serve as a visual aid only, and has no interpretive meaning. Asx: asparatic acid/asparagine; Hyp: hydroxyproline; Glx: glutamic acid/glutamate; Thr: threonine; Gly: glycine; Ala: alanine; Pro: proline; Val: valine; Met: methionine; Leu/Ile: leucine and isoleucine; Lys: lysine; His: histidine; Phe: phenylalanine; Arg: arginine.

range of 513C values for non-essential amino acids for archaeological fauna is slightly larger 17% (-31—14%). No meaningful trends exist in non-essential amino acid carbon-isotope compositions among the humans and various fauna, although perch have moderately lower d13CAA values for some non-essential amino acids. The narrow range in bulk collagen and dentin 513C values thus appears to be reflected in the low variability of the 513C values for non-essential amino acids relative to the essential amino acids (see below). The narrow range in non-essential amino acid <513C values is likely the underlying cause of the similarly low variation determined for the bulk 513C values of collagen and dentin, because nonessential amino acids constitute a large proportion of the carbon (~80%; Ambrose, 1993) present in type I collagen protein.

The overall range of the 513C values for essential amino acids for humans is 20% (-36—16%) and 32% (-45—12%) for fauna. These ranges are, as expected, larger than those observed for non-essential amino acids, and the faunal range is larger than that determined for humans, which is likely a reflection of diverse dietary strategies. Species that consume terrestrial (i.e., aurochs, wild horse and wild boar) or mixed aquatic-terrestrial (i.e., moose) plant resources tend to have higher essential d13CAA values than species which consume freshwater resources (i.e., otter and perch), and this is most clearly discernable in the 513CPhe values (Fig. 4). The similarity of the essential d13CAA values between humans and freshwater fauna (e.g., otter and perch) indicates significant consumption of freshwater fish by the humans included in this study. Further, it is likely that dogs were consuming freshwater fish as well, either by scavenging or by direct feeding.

Kruskal-Wallis (KW) non-parametric one-way analysis of variance tests were used to compare the D13CGly-Phe and D13CVal-Phe values, bulk 513C and d15N values, and individual essential and non-essential amino acid isotopic compositions of humans, dogs, terrestrial fauna, moose and freshwater fauna. Both proxies, the d15N values, and the 513C values of lysine, phenylalanine, glycine, alanine, glutamate/glutamic acid, aspartate/aspartic acid, and arginine were statistically significantly different (KW, p < 0.005), whereas the bulk 513C values and 513CPro and 513CVal values were not statistically distinguishable. Note, however, that the sample sizes for each group of specimens was small (n = 2-n = 8), adding uncertainty to these statistical determinations. Further, the nonnormal distribution of the data and small sample sizes preclude additional post-hoc testing (i.e., to assess the extent to which pairs of groups are different from each other). For the two isotopic proxies (D13CGly-Phe and D13CVal-Phe) a visual assessment of Fig. 5a and b indicates that humans, dogs and freshwater fauna have similar D13CGly-Phe values, and D13CVal-Phe values, although there is some overlap between the lowest human proxy values and the highest terrestrial fauna proxy values for the latter.

6. Discussion

Comparative d13CAA values from a variety of ecological and archaeological settings are used to contextualize the Zvejnieki human isotopic results. All data were generated using a similar laboratory methodology and are fully reported in Honch et al. (2012). Briefly, archaeological human isotopic compositions

Fig. 5. The (A) D13CGly - Phe proxy and D13CVal - Phe proxy and (B) the underlying d13CGly, d13CVal and d13CPhe values for Zvejnieki humans and fauna.

representing high marine protein consumption (Japan and Greenland), terrestrial C4-derived protein consumption (Meso-america), high freshwater protein consumption (Iron Gates Region, Europe) and high C3-derived protein consumption (Eastern Europe) are used to assess the efficacy of the proxies tested here in distinguishing among different dietary regimes. Although there is necessarily some degree of uncertainty in all palaeodietary reconstructions, the interpretation of the dietary isotopic compositions for these four groups is well-supported by archaeological and zooarchaeological evidence (Honch et al., 2012). Statistical analysis has determined that there are significant differences among C3 terrestrial protein consumers, C4 terrestrial protein consumers, freshwater protein consumers (including Zvejnieki human samples) and marine protein consumers for both D13CGly-Phe (Corr et al., 2005) and D13CVal-Phe (Honch et al., 2012) proxies (KW, p < 0.005; Fig. 6). Further, for those individual amino acids for which sufficient data were obtained (see Honch et al., 2012), there are significant differences among dietary protein groups (valine, lysine, phenyl-alanine, glycine, alanine, proline, glutamate/glutamic acid, aspar-tate/aspartic acid, and arginine; KW, p < 0.005).

The application of the D13CGly-Phe proxy to detect high marine protein consumption is well-established, and has been tested on archaeological material (Choy et al., 2010; Corr et al., 2005; Honch et al., 2012). It is expected that relatively high D13CGly-Phe values will be observed at high levels of marine protein consumption, and lower D13CGly-Phe values (i.e., less than +10%) will be observed when the dietary protein source is predominately terrestrial. The precise relationship between % contribution of marine protein to

diet and the D13CGly-Phe value for humans is currently unknown, although we hypothesize that more marine protein will lead to increasingly large D13CGly-Phe values. For the Zvejnieki human and faunal data, the human, dog, otter and perch D13CGly-Phe values range from +15 to +21%, whereas the moose, horse, wild boar and aurochs values are generally <+10% (Table 2). On the basis of ecological, archaeological and zooarchaeological evidence, this division is consistent with the presence and absence of substantial freshwater resources in the diet. Corr et al. (2005) determined that, for high marine protein consumers, the high D13CGly-Phe values relative to terrestrial protein consumers were primarily caused by high d13CGly values, as is also the case for the high marine protein consumer values included here. The high freshwater protein consumers from the Iron Gates Region have low 513CPhe values (-30.4 ± 1.1 %) and, although the absolute 513CPhe and d13CGly values differ from those determined for the Zvejnieki humans, the spacing is similar compared to other dietary groups (+16.7 ± 1.3% for Zvejnieki and +19.7 ± 1.5% for Iron Gates; Fig. 6). The high D13CGly -Phe values for freshwater protein consumers are driven primarily by the very low 513CPhe values. We hypothesise that the low 513CPhe values observed here and in other studies for freshwater resources may be influenced by contributions of terrestrial carbon to total carbon present in freshwater lakes and rivers, which is likely to be 13C-depleted relative to marine carbon (see Section 2).

Honch et al. (2012) proposed instead that the relationship between 513CVal and 513CPhe values may be useful in distinguishing among marine, freshwater, C4 plant-derived, and C3 plant-derived protein consumers. Controlled feeding studies have confirmed

Fig. 6. The (A) D13CGly - Phe proxy and D13CVal - Phe proxy and (B) the underlying d13CGly, d13CVal and d13CPhe values for Zvejnieki humans and additional human specimens with well-understood diets from Honch et al. (2012).

that the 513C values of essential amino acids do not change substantially from diet to primary and secondary consumer tissues (Hare et al., 1991; Howland et al., 2003; Jim et al., 2006). Thus, the 513C values of phenylalanine and valine are expected to primarily reflect isotopic variability at the base of the food web with minimal isotopic modification. Although there are differences in the absolute 513CVal and 513Cphe values of different freshwater ecosystems, the D13Cval -Phe values of the Zvejnieki humans compared to local terrestrial fauna and the comparative human samples are distinct from terrestrial protein consumers. Terrestrial fauna had D13CVal-Phe values of less than +1.5%, whereas freshwater fauna had values greater than +2.0%. Humans and dogs had a range of D13CVal-Phe values, from as low as +0.1% to +5.5%. Interestingly, some of the Zvejnieki individuals appear to have consumed a mixed C3 terrestrial and freshwater diet, which was not apparent at the bulk iso-topic level and not clearly resolved by the sole use of the D13Cay -Phe proxy. The high marine protein consumers and high freshwater protein consumers had average D13CVal-Phe values of +5.8 ± 1.7% and +6.3 ± 1.1 %, respectively, whereas C3 and C4 terrestrial protein consumers had considerably lower values (+0.8 ± 0.4% and +0.1 ± 0.6%, respectively; Figs. 5 and 6).

Although the D13CGly-Phe and D13CVal-Phe proxies clearly distinguish between aquatic protein consumers and terrestrial C3 and C4 plant-derived protein consumption, they cannot as yet be reliably used to differentiate between marine and freshwater resource

consumption. Thus, although the proxies are valuable in regions where only one kind of aquatic resource is likely to have been available (marine or freshwater), they will be of limited use in more complex socioeconomic or ecological contexts. Instead, bivariate plots of these indices vs. other essential d13CAA values can be used to distinguish among all four dietary protein groups, and may potentially be able to characterise the protein sources consumed in mixed diets (Fig. 7). For example, the relationship between d13CLys and the D13CVal-Phe proxy visually discriminates among all four diet groups, clearly separating terrestrial from aquatic, C3 from C4 and freshwater from marine protein consumers (Fig. 7b). Lysine, an essential amino acid, is a limiting amino acid in cereals and most plants, and is found in somewhat larger quantities in legumes. Meat and other animal products, however, are the most abundant dietary source of lysine, especially aquatic fauna (Young and Pellett, 1994). Here, freshwater consumers tended to have lower d13CLys values and higher D13CVal-Phe values, whereas marine protein consumers had higher d13CLys and higher D13CVal-Phe values. Because there is some variation globally in 513C values of terrestrial and freshwater resources, the measured d13CLys values are not expected to be the same across regions; however, the Zvejnieki human d13CLys values agree well with those for freshwater fauna from the same archaeological site, and the D13CVal-Phe proxy values are, as expected, higher than those of C3-plant consuming terrestrial fauna. Further analyses from well-constrained archaeological human and faunal

Fig. 7. Bivariate comparisons of (A) d13CLys vs. D13CGly _ Phe and (B) d13CLys vs. D13CVal _ Phe values for Zvejnieki humans and fauna (left) and all archaeological human data (right). For the Zvejnieki plots: ♦ Humans; □ Dogs; • Freshwater Fauna; a Moose; Terrestrial Fauna. For archaeological human comparative data plots: C3 Terrestrial Protein Consumers; • C4 Terrestrial Protein Consumers; ▲ Freshwater Protein Consumers; ♦ Zvejnieki Humans; ♦ Marine Protein Consumers.

contexts, as well as modern controlled feeding studies, are needed to improve the certainty with which such relationships can be applied, but this outcome shows considerable promise for ultimately refining palaeodietary reconstruction in complex ecosystems around the world.

7. Conclusion

Compound-specific carbon-isotope analysis of amino acids shows considerable potential as a method for resolving complex palaeodietary reconstructions. As the body of published 513Caa values grows, the isotopic proxies discussed herein and elsewhere (Corr et al., 2005; Honch et al., 2012) will become increasingly valuable to archaeologists in distinguishing among terrestrial (C3 vs. C4), freshwater and marine resource consumption. Here, our goal was to explore how freshwater resource consumption by humans is expressed at the amino acid level compared to other dietary protein sources (C3 terrestrial, C4 terrestrial, and marine protein), in order to develop broadly applicable isotopic proxies and relationships for use in less well-understood archaeological contexts. We determined that (i) the D13CGly-phe and D13CVal-phe proxies can effectively discriminate between terrestrial and aquatic resource consumption, and (ii) the relationship between essential d13CAA values and the D13CGly-Phe and D13CVal-Phe proxies can differentiate among the four protein consumption groups tested here. Further, individuals with 'hidden' mixed diets, such as the few Zvejnieki individuals for whom a mixed C3 and freshwater protein-based diet may be hypothesised, can be distinguished from those with a diet dominated by one type of dietary resources. Crucially, however, the compound-specific amino acid isotopic proxies discussed here must be applied in a context-sensitive manner. For example, the D13CGly-Phe or D13CVal-Phe proxies could be used to identify freshwater protein consumption for inland sites, but not in an estuarine environment, where both freshwater and marine aquatic resources would be readily available for human consumption. Instead, either proxy coupled with an essential amino acid (e.g., lysine) would be more useful. This investigation has demonstrated that amino acid stable isotopic compositions and compound-specific isotopic proxies enhance our understanding of diet and resource exploitation in the past, and can elucidate complex dietary behaviours.

Acknowledgments

This work was conducted as part of a responsive mode grant from the UK Natural Environment Research Council (NERC: NE/ D004535/1). NERC is also thanked for mass spectrometry facilities.

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