fernandes et al-2015-american journal of physical anthropology

Quantitative Diet Reconstruction of a NeolithicPopulation Using a Bayesian Mixing Model (FRUITS):The Case Study of Ostorf (Germany)

Ricardo Fernandes,1,2,3* Pieter Grootes,1 Marie-Josee Nadeau,4 and Olaf Nehlich5,6

1Institute for Ecosystem Research, University of Kiel, Kiel, Germany2Leibniz-Laboratory for Radiometric Dating and Isotope Research, University of Kiel, Kiel, Germany3McDonald Institute for Archaeological Research, University of Cambridge, Cambridge CB2 1TN, United Kingdom4National Laboratory for Age Determination, Museum of Natural History and Archaeology, Norwegian Universityof Science and Technology, Trondheim, Norway5Department of Anthropology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada6Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

KEY WORDS Neolithic; Ostorf; FRUITS; diet; isotopes; collagen; bioapatite

ABSTRACT OBJECTIVES: The island cemetery siteof Ostorf (Germany) consists of individual human gravescontaining Funnel Beaker ceramics dating to the Earlyor Middle Neolithic. However, previous isotope andradiocarbon analysis demonstrated that the Ostorf indi-viduals had a diet rich in freshwater fish. The presentstudy was undertaken to quantitatively reconstruct thediet of the Ostorf population and establish if dietaryhabits are consistent with the traditional characteriza-tion of a Neolithic diet.METHODS: Quantitative diet reconstruction wasachieved through a novel approach consisting of the useof the Bayesian mixing model Food ReconstructionUsing Isotopic Transferred Signals (FRUITS) to modelisotope measurements from multiple dietary proxies(d13Ccollagen, d

15Ncollagen, d13Cbioapatite, d

34Smethione,14Ccol-

lagen). The accuracy of model estimates was verified bycomparing the agreement between observed and esti-mated human dietary radiocarbon reservoir effects.

RESULTS: Quantitative diet reconstruction estimatesconfirm that the Ostorf individuals had a high proteinintake due to the consumption of fish and terrestrial ani-mal products. However, FRUITS estimates also showthat plant foods represented a significant source of calo-ries. Observed and estimated human dietary radiocarbonreservoir effects are in good agreement provided thatthe aquatic reservoir effect at Lake Ostorf is taken asreference.CONCLUSIONS: The Ostorf population apparentlyadopted elements associated with a Neolithic culture butadapted to available local food resources and imple-mented a subsistence strategy that involved a large pro-portion of fish and terrestrial meat consumption. Thiscase study exemplifies the diversity of subsistence strat-egies followed during the Neolithic. Am J Phys Anthro-pol 000:000000, 2015. VC 2015 Wiley Periodicals, Inc.

The island cemetery site of Ostorf-Tannenwerder,hereafter referred to as Ostorf, in northern Germanyconsists of individual adult graves containing lithics,pottery, and tooth pendants of wild animals as gravegoods (Lubke et al., 2009). Farming was practiced in theWestern Baltic region of Northern Europe from at leastca. 4,000 cal BC (Craig et al., 2011) and pottery found atOstorf includes Funnel Beaker types assigned to theEarly or Middle Neolithic. The majority of calibratedradiocarbon dates obtained on collagen extracted frombone artefacts recovered from the site fall between ca.3,400 and 2,900 cal BC. However, human bone-collagenradiocarbon dates in most instances appear to be severalhundreds of years older than associated animal dates(Olsen et al., 2010). This constitutes an example of die-tary radiocarbon reservoir effect (RRE) caused by theconsumption of aquatic resources that are depleted in14C compared to the contemporary atmosphere (Lantingand van der Plicht, 1998; Cook et al., 2001; Fernandeset al., 2014). A high intake of aquatic protein was previ-ously confirmed by isotope analysis (d15Ncollagen andd13Ccollagen) of human bone collagen. Human bone colla-

Additional Supporting Information may be found in the onlineversion of this article.

Abbreviations: aDNA, Ancient DNA; FRUITS, Food ReconstructionUsing Isotopic Transferred Signals; FTIR, Fourier transform infra-red spectroscopy; RRE, Radiocarbon reservoir effect; SF, Splittingfactor

Grant sponsor: German Research Foundation within the frame ofthe Priority Program SPP 1400; Grant number: DFG project NA776/2; Grant sponsor: Graduate School Human Development inLandscapes of the German Excellence Initiative.

*Correspondence to: Ricardo Fernandes, Leibniz-Laboratory forRadiometric Dating and Isotope Research, University of Kiel, Kiel,Germany. E-mail: [email protected]

Received 14 August 2014; revised 23 May 2015; accepted 26 May2015

DOI: 10.1002/ajpa.22788Published online 00 Month 2015 in Wiley Online Library

(wileyonlinelibrary.com).

2015 WILEY PERIODICALS, INC.

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 00:0000 (2015)

gen d15Ncollagen values were elevated compared withother Neolithic populations (Olsen et al., 2010) as maybe expected from the consumption of fish enriched in15N. Human bone collagen d13Ccollagen values were simi-lar to other inland Neolithic populations, suggestingthat marine species enriched in 13C were not significantdietary components. In addition, a human tooth wearstudy revealed tooth abrasion patterns consistent withthe intake of protein-rich foods (Patolla and Henke,2007). However, similar to the site of Blatterhohle, alsoin Germany, ancient DNA (aDNA) analysis has shownthat the Ostorf individuals share mitochondrial DNAsignatures associated with early Neolithic Europeanfarmers and that these differ from those observed forlate Mesolithic European hunter-gatherers (Bramantiet al., 2009; Deguilloux et al., 2012; Bollongino et al.,2013). In light of all collected evidence, Ostorf has beenboth described as an enclave of a late hunter-gathererpopulation surrounded by Neolithic farmers (Bramantiet al., 2009) or as former farmers that shifted to a modeof subsistence relying preferentially on aquatic resources(Lubke et al., 2009).Much of the debate concerning the Ostorf individuals

derives from the interpretation of human isotope data.However, the methods of interpretation employed weresemi-quantitative and based on the comparison of humanisotope values with pre-defined isotope ranges associatedwith a certain type of diet. Furthermore, the radiocarbonage offsets between associated human and animal arte-facts show some clear outliers (Olsen et al., 2010). Thepresent study builds upon previous work at Ostorf byrepeating radiocarbon measurements on humans andassociated animal remains, including two additional iso-topic dietary proxies (d13Cbioapatite and d

34Smethionine), andquantitatively reconstructing the diet of the Ostorf popu-lation using the Bayesian mixing model FRUITS (Fer-nandes et al., 2012c; Fernandes et al., 2014b). Estimatesgenerated by FRUITS include the relative calorie contri-bution from available food groups, levels of protein intake,and the relative protein contribution from each foodgroup. These estimates can be compared with data frommodern populations or ethnographic case studies to char-acterize the diet of the Ostorf population (Lands et al.,1990; Cordain et al., 2000).The application of this approach to the Ostorf case

study provides an opportunity to gauge the extent towhich a population within a spatially and temporallyNeolithic context that had adopted some Neolithic cul-tural elements also shows aspects which are typicallyassociated with a hunter-gatherer subsistence strategy.

MATERIAL AND SITE DESCRIPTION

Lake Ostorf is part of the Mecklenburg Lake District,and is located ca. 1.5 km southwest of the larger LakeSchwerin (Fig. 1). Within Lake Ostorf, the island of Tan-nenwerder contains a prehistoric cemetery with individ-ual burials. These graves contain a variety of grave goods,including pendants from wild animal teeth (Lubke et al.,2009). Previous isotope and radiocarbon analyses weremade on animal and human remains (Lubke et al., 2009;Olsen et al., 2010). New long-bone samples were takenfrom ten previously radiocarbon dated human adults fornew isotope and radiocarbon analysis (Table 1). Eight ani-mal tooth pendants from each burial were also sampled(Table 1). Collagen extracted from animal tooth dentinwas analyzed isotopically and where possible also by

radiocarbon dating. Since no fish bones were recoveredfrom archaeological excavations, modern fish and musselspecimens were collected from lakes Schwerin and Ostorffor isotope and radiocarbon analysis of the flesh. Part ofthese results was published previously in a separate studythat investigated the ecological variability of RREs (Fer-nandes et al., 2013).

ANALYTICAL METHODS

The following represents a brief description ofemployed analytical methods. A more detailed descrip-tion is provided in Supporting Information File 1.

Bone and teeth pre-treatment

Bone and tooth dentin collagen was extracted at the MaxPlanck Institute for Evolutionary Anthropology (Leipzig,Germany) using an acid-base-acid protocol plus a filtrationstep (Hublin et al., 2012) based on the method developed byLongin (Longin, 1971). Unbleached bioapatite powder(grain size

products, fish) having similar isotopic and nutrientcompositions.

Use of a sufficient number of dietary proxies. To pre-cisely estimate the contributions of defined foodgroups, it is necessary to include a sufficient numberof dietary proxies that, when combined, offer unambig-uous estimates.

Quantification of diet-to-tissue isotopic offsets and die-tary routing. This relies primarily on data obtainedfrom controlled feeding experiments done either onhumans or on omnivorous mammals.

Use of a Bayesian mixing model for data analysis. TheBayesian mixing model Food Reconstruction UsingIsotopic Transferred Signals (FRUITS) was used toproduce estimates of the intake of different foodgroups based on measured dietary proxies and isotopicvalues of available food (Fernandes et al., 2012c; Fer-nandes et al., 2014b). This model is capable of han-dling uncertainties associated with all the parametersdescribed above. It also allows the user to inputdiverse forms of prior information.

Testing model performance. Model predictions shouldbe tested for robustness. This implies assessing thedegree of sensitivity in generated estimates to smallmodifications in the values of model parameters. Inthe present case study, the accuracy of model predic-

tions was also tested by comparing estimated andobserved human dietary RREs.

Modelling parameters

The following describes the model parameters and val-ues considered in the diet reconstruction of the Ostorfpopulation. The robustness of diet estimates produced bythe model was assessed by comparing the estimates gen-erated for different model instances or dietary scenarios.Four dietary scenarios were considered, corresponding totwo different reference d15N plant values (manuredplants for scenarios 1 & 2 vs. unmanured for scenarios 3& 4) and the inclusion or exclusion of sulfur isotope data(included in scenarios 1 & 3 vs. excluded in scenarios 2& 4) in the modeling.

Dietary proxies. Each of the four dietary proxies(d13Ccollagen, d


34Smethionine)employed in this case study is represented in FRUITSby a unique code: 13Ccoll (d13Ccollagen), 15Ncoll (d

15Ncolla-gen), 13Cbioa (d

13Cbioapatite), 34Smeth (d34Smethionine).

Average isotope values of the Ostorf population are giventogether with associated standard error of the mean(standard deviation divided by the square root of the

Fig. 1. Location of Lake Ostorf and the neighboring Lake Schwerin (Germany). The star symbol marks the location of theIsland of Ostorf (Tannenwerder).

DIET RECONSTRUCTION OF THE OSTORF POPULATION 3

American Journal of Physical Anthropology

TABLE1.Samplelistandisotope,elem

ental,andSFresultsforarchaeological(*)andmodern(**)samplesfrom

OstorfIsland(OI),LakeOstorf(LO),andLakeSchwerin

(LS)

KIA

number

Grave

Species

Sex

Age

Fraction

d13Corganic

(&)

d15Norganic

(&)

d34Smethionine

(&)

C (%)

N (%)

S (%)

Atomic

C/N

Atomic

C/S

Atomic

N/S

d13Cbioapatite

(&)

SF

46904*O

I190401

Human

Male

3060

Radiuscollagen

219.8

12.1

4.0

49.0

17.5

0.246

3.3

532

163

214.6

3.2

46906*O

I196101

Human

Male

Tibia

collagen

220.3

13.3

3.1

44.3

15.7

0.208

3.3

568

172

214.7

3.8

46908*O

I190402

Human

Male

Radiuscollagen

221.1

13.6

6.1

48.2

17.5

0.234

3.2

550

171

214.8

3.2

46909*O

I193503

Human

Male

3569

Fem

urcollagen

220.2

11.8

7.6

49.5

17.8

0.262

3.3

504

155

214.8

3.1

46911*O

I193504

Human

Male

3560

Fem

urcollagen

220.0

11.9

6.5

48.3

17.5

0.232

3.2

556

172

215.6

3.3

46913*O

I196104

Human

Fem

ale

Fem

urcollagen

220.0

13.2

2.3

50.0

17.7

0.243

3.3

550

166

215.2

3.7

46915*O

I196105

Human

Fem

ale

Fem

urcollagen

219.5

12.7

1.2

48.9

17.6

0.253

3.3

516

159

215.2

3.4

46917*O

I196107

Human

Fem

ale

Fem

urcollagen

219.7

11.7

4.2

49.4

17.6

0.25

3.3

528

161

213.9

3.2

46919*O

I196108

Human

Male

1730

Fem

urcollagen

219.8

12.8

3.0

49.3

17.7

0.234

3.2

561

173

215.4

3.7

46921*O

I196109

Human

Fem

ale

Fem

urcollagen

219.0

12.9

2.3

49.7

17.4

0.225

3.3

589

177

214.1

3.5

Isotope

average

219.960.2

12.660.2

4.060.3

46905*O

I190401

Wildboar

Dentincollagen

222.1

6.2

7.7

48.9

16.5

0.232

3.5

562

163

46907*O

I196101

Deer

Dentincollagen

221.3

5.8

7.2

48.8

17.1

0.227

3.3

573

172

46910*O

I193503

Deer

Dentincollagen

222.7

5.0

11.3

48.0

17.1

0.229

3.3

559

170

46912*O

I193504

Red

deer

Dentincollagen

223.0

5.4

10.5

49.9

17.5

0.192

3.3

692

208

46914*O

I196104

Wildboar

Dentincollagen

222.6

5.2

8.7

51.5

18.2

0.227

3.3

605

183

46916*O

I196105

Roe

deer

Dentincollagen

222.1

4.4

7.9

46.7

16.2

0.232

3.4

538

160

46918*O

I196107

Carnivore

Dentincollagen

218.0

11.0

15.3

48.3

17.2

0.22

3.3

584

178

46920*O

I196108

Roe

deer

Dentincollagen

221.7

5.2

9.0

49.1

17.2

0.237

3.3

552

166

Isotope

average

222.760.6a

6.060.7a

9.760.9b

46304**LS

Pike

Flesh

bulk

219.6

13.0

20.5

46.6

17.4

0.63

3.1

197

63

46305**LS

Bream

Flesh

bulk

219.3

11.5

22.3

47.1

8.0

0.55

6.9

228

33

46306**LS

Eel

Flesh

bulk

215.0

12.8

1.2

50.3

17.5

0.509

3.3

264

79

46307**LS

Eel

Flesh

bulk

220.6

17.0

24.1

49.7

18.8

0.516

3.1

257

83

46310**LS

Zebra

mussel

Flesh

bulk

227.1

8.1

54.8

10.5

6.1

46311**LO

Zebra

mussel

Flesh

bulk

227.8

14.6

43.9

10.7

4.8

Isotope

average

(musselsnot

included)

218.661.2a

13.661.2a

21.461.1b

Humansexandagedeterminations(when

available)are

alsoincluded.See

textdescription

foruncertainties

ofisotopemeasurements.Meanisotopevalues

ofhumans(bonecolla-

gen),terrestrialanim

als(tooth

dentincollagen),andfish

(bulk

flesh)are

reportedwithanuncertainty

representingthestandard

errorof

themean.

aListedmeanvalues

are

given

asreference

only.Thesewerenot

usedin

themodellingprocess

since

itwaspossible

forcarbon

andnitrogen

stable

isotopes

toobtain

archaeologi-

calreference

values

from

alarger

dataset(see

sectionIsotopiccompositionof

food

groups).

bGiven

thesm

allnumber

ofsamples,standard

deviation

values

(d34ST.anim

als=9.762.6&,d3

4Sfish=21.462.3&)wereusedin

themodellingprocess

tothedefinetheuncertainty

ofd3

4Smethioninevalues

rather

thanlisted

standard

errorof

themean(see

sectionIsotopiccompositionof

food

groups).

4 R. FERNANDES ET AL.


number of individuals) in Table 1. These values wereused in the modeling process to reconstruct the groupdiet. Individual diets were also reconstructed, however,the uncertainty of the dietary proxies (d13Ccollagen,d15Ncollagen, and d

13Cbioapatite) was set at 0.5& to accountfor isotopic variability within and between bones of thesame individual (Schoeninger et al., 1983; Baker et al.,1998; Balasse et al., 1999; Waters-Rist et al., 2011).A more conservative uncertainty of 1& was taken asreference for the d34Smethionine dietary proxy given thelarger instrumental uncertainty associated with thisdietary proxy (see Supporting Information File 1).

Nutrient composition of food groups. Three mainfood groups were considered: terrestrial plant cereals,meat of terrestrial animals, and meat of freshwater fish.Their nutrient content (protein, carbohydrates, lipids,and methionine) is expressed as dry weight carbon con-tent (wtC %) with carbohydrates and lipids combinedinto a single fraction (carbs/lipids). Carbon content canalso be defined using calorie composition values giventhat calorie vs. carbon content ratios for the differentmacronutrients are broadly similar (Morrison et al.,2000; Otten et al., 2006).The macronutrient composition of foods can vary

(FAOSTAT, 2009), however, recorded patterns of humandietary intakes show that the average nutrient contribu-tion of the food groups listed here is relatively constant(see Supporting Information File 2 Table 1). The fol-lowing lists the macronutrient content, rounded to amultiple of five, taken in the modelling process for eachfood group: plant cereals (protein: 1062.5% wtC %;carbs/lipids: 906 2.5% wtC %), meat of terrestrial ani-mals (306 2.5% wtC %; carbs/lipids: 706 2.5% wtC %),meat of freshwater fish (6565% wtC %; carbs/lipids:356 5% wtC %). These values are similar among regionsand time periods with variations in plant cereals andterrestrial animals typically smaller than 5% and infreshwater fish smaller than 10% (see Supporting Infor-mation File 2 Table 1). In the case of plant foods, nutsalso show a protein content of ca. 10% and the selecteduncertainty for the plant food group is sufficiently largeto encompass within a 2r range (ca. 5%) plant foodswith a lower protein content (e.g. fruits, starchy roots).It was assumed that pulses, with a significantly higherprotein content than cereals, did not represent an impor-

tant food source (Jacomet, 2004). The methionine con-tent was established using reported USDA data (USDA,2012) for plants (0.00260.001 wtC %), the flesh of ter-restrial animals (0.0260.01 wtC %), and fish(0.026 0.01 wtC %) expressed as caloric percentage (seeSupporting Information File 2 Tables (24)).

Isotopic composition of food groups. The referenceisotopic composition for protein, carbs/lipids, and methio-nine of the food groups is provided in Table 2. Given thelimited number of archaeological food remains recoveredfrom Ostorf, previously published isotope data were usedto define the carbon and nitrogen isotopic composition ofterrestrial animals and freshwater fish. These data,major outliers removed, originate from Late Mesolithicand Neolithic central and northern European sites(Boric et al., 2004; Ogrinc and Budja, 2005; Bosl et al.,2006; Durrwachter et al., 2006; Bocherens et al., 2007;Hedges et al., 2008; Oelze et al., 2011; Dolphin et al.,2013). The average stable isotope values and standarderror of the mean for bone collagen from terrestrial ani-mals and fish are d13C5221.26 0.1&,d15N55.960.1& (n5 268), for the former, andd13C5222.36 0.3&, d15N5 8.860.3& (n5 27), for thelatter. The standard error of the mean, rather than thestandard deviation, is reported given that human longbones integrate food isotopic signals during multiyearsampling periods (Hedges et al., 2007). Isotope values ofterrestrial animals and fish protein and lipids (carbohy-drate contribution assumed to be comparatively negligible)were estimated, from bone-collagen values, relying on pre-viously reported offsets between macronutrient and colla-gen isotopic values for these organisms (Vogel, 1978; Hareet al., 1991; Sholto-Douglas et al., 1991; Tieszen and Fagre,1993; Pinnegar and Polunin, 1999; Fischer et al., 2007;Post et al., 2007; Logan et al., 2008; Warinner and Tuross,2010; Fernandes et al., 2014a). Chosen offsets representapproximate consensus values (terrestrial animals: D13Cpro-tein-collagen522&, D

13Clipids-collagen528&, D15Nprotein-

collagen512&; fish: D13Cprotein-collagen521&, D

13Clipids-collagen527&, D

15Nprotein-collagen512&). To cover theoffset ranges in previously listed studies, a conservativeuncertainty of 1& was taken as reference for estimatedprotein and lipid isotope values (Table 2). Given thatd13Cbioapatite is a dietary proxy that signals the carbon ofthe dietary mix (see section Diet-to-tissue isotope offsets

TABLE 2. Isotope values of the different food group fractions in relationship to the dietary proxy, represented by their FRUITScode, to which the isotope values are relevant. The asterisks mark the different plant isotope values corresponding to manured (*)

plants (dietary scenarios 1 and 2) and unmanured (**) plants (dietary scenarios 3 and 4)

Food group Fraction13Ccoll(&)

15Ncoll(&)

13Cbioa(&)

34Smeth(&)

Plant Bulk 224.061 Plant Protein 226.06 1 4.56 1*

2.06 1**Plant Carbs/Lipids 223.56 1 Plant Methionine 9.76 2.6T. animals Bulk 227.461 T. animals Protein 223.26 1 7.96 1 T. animals Carbs/Lipids 229.26 1 T. animals Methionine 9.76 2.6Fish Bulk 225.461 Fish Protein 223.36 1 10.861 Fish Carbs/Lipids 229.36 1 Fish Methionine 21.46 2.3



and weight contributions), the relevant d13C signal of eachfood group is that of the bulk carbon. Terrestrial animalsand fish d13C bulk values were estimated as a weightedmean (according to nutrient compositionsee above) oflipid and protein d13C values. Sulfur isotope values wereonly available from a limited number of measurementsmade on archaeological (terrestrial animals) and modern(fish) samples (n=12) at Ostorf (Table 1). Given thissmall dataset, the standard deviation of average values,and not the standard error of the mean, was used asuncertainty for each food group (d34ST. animals59.762.6&,d34Sfish521.46 2.3&).Recent research has provided isotope values for bulk

cereal grains, corrected for charring effects, during theEuropean Neolithic (Bogaard et al., 2013). Bulk cerealisotope values within Germany are ca. 4.5& for d15Nand 224.0& for d13C. The elevated d15N values denotethe influence of manuring and this value was taken asbasis for dietary scenarios 1 and 2. A d15N value of 2&represents a typical value for edible European plantsduring the Holocene (Richards and Trinkaus, 2009), andwas taken as basis for dietary scenarios 3 and 4. For allscenarios, bulk isotope plant values are adjusted for iso-tope offsets between bulk d13C values and protein (ca.22&), and between bulk d13C values and carbohydrates(ca. 10.5&, with lipid contribution assumed to be com-paratively negligible) (Tieszen, 1991). In both scenarios,a conservative uncertainty of 1& was assigned to plantd15N and d13C values. No modern or archaeological plantd34S measurements were available and it was assumedthat plants would have the same isotope value(d34Splant=9.76 2.6&) as local terrestrial animals (seeabove).

Diet-to-tissue isotope offsets and weight contribu-tions. Diet-to-collagen and diet-to-bioapatite d13C iso-tope offsets are based on the statistical model presentedby Fernandes et al., (2012b), equivalent to the graph-based model by Kellner and Schoeninger (2007) andlater updated by Froehle et al., (2010). Isotope offsetswere defined using data from controlled feeding experi-ments on omnivorous mammals (Ambrose and Norr,1993; Tieszen and Fagre, 1993; Howland et al., 2003;Jim et al., 2004; Warinner and Tuross, 2009). Statisticalanalysis of these data provided a diet-to-collagen d13Coffset of 4.860.2&, and a diet-to-bioapatite offset of10.16 0.2& However, given that these estimates arebased on data from small mammals, a more conservativeuncertainty (0.5&) was used to account for the possibleeffect of body size (Passey et al., 2005). Statistical analy-sis of animal data showed that the source of bioapatitecarbon is the dietary carbon mix without a bias towardsa particular macronutrient. It also showed that collagencarbon is routed from 7464% dietary protein carbonand the remaining 26% from carbohydrates and lipids(Fernandes et al., 2012b).The source of collagen nitrogen is essentially dietary

protein, and a value of 5.56 0.5& was used for humand15N diet-to-collagen isotope offset. This represents aconsensus value determined from published data onhuman diet-to-hair d15N enrichment values (Minagawaet al., 1986; Schoeller et al., 1986; Minagawa, 1992;Yoshinaga et al., 1996; Hedges et al., 2009; Huelsemannet al., 2009) to which was added the isotope offset (ca.1&) between human hair and bone collagen (OConnelland Hedges, 1999; OConnell et al., 2001; Richards,

TABLE3.Bone-collagen

radiocarbon

resultsobtained

inthisstudyandpreviouslybyOlsen

etal.(2010)forpre-historicadultindividualsandassociatedanim

alremainsfrom

thesiteofOstorf

Grave

Labcode

(anim

al)

14C

(yrBP)

Labcode

(anim

al)

14C

(yrBP)

Lab

code

(human)

14C

(yrBP)

Lab

code

(human)

14C

(yrBP)

RRE(yr)

(Olsen

etal.2010)

RRE(yr)

(thisstudy)

190401

AAR-10590

4400634

UtC-8173

4568640

KIA

-46904

4786622*

168652

386640

196101

AAR-10589

4435636

KIA

-46907

4504624*

UtC-7447

4699649

KIA

-46906

4793626*

264661

289635

190402

AAR-10591

4449634

UtC-7440

4737641

KIA

-46908

4717622*

288653

268640

193503

AAR-10595

4387635

UtC-7445

4356644

KIA

-46909

4581621*

231656

194641

1935204

AAR-10596

4402631

UtC-7446

4299650

KIA

-46911

4702625*

2103659

300640

196104

AAR-10592

4500665

KIA

-46914

4470637

UtC-7448

4827647

KIA

-46913

5061629*

327680

591647

196105

AAR-10594

4398634

UtC-8180

4833638

KIA

-46915

5018625*

435651

620642

196107

AAR-10593

4429632

KIA

-46918

4496634

AAR-9752

4830647

KIA

-46917

4828621*

401657

332640

196108

AAR-10597

4135641

KIA

-46920

4594631

UtC-7449

4970650

KIA

-46919

4968626*

835665

374640

196109

AAR-10598

4303634

UtC-8179

4855649

KIA

-46921

5085664

552660

782672

RRE

represents

theagedifference

betweenhumanandanim

alradiocarbon

ages.WherenoLeibniz

Labanim

aldate

could

beobtained,RRE

represents

theagedifference

betweenthehumandatedattheLeibniz

Lab(KIA

)andtheanim

aldatedattheAarhusLab(AAR).

HumansamplesmeasuredattheUtrechtLab(U

tC)are

alsolisted.

(*weightedmeanof

twomeasurements,seeSupportingInform

ation

File2).



2001; Lehn et al., 2014). The chosen offset is similar tothe ca. 6& estimated from isotope values measured inhuman red blood cells during a controlled feeding experi-ment (OConnell et al., 2012).A review of human and animal data suggests that

there is little isotopic fractionation between methioninein diet and body tissues, provided that there is anadequate protein intake (McCutchan et al., 2003; Rich-ards et al., 2003a; Tanz and Schmidt, 2010; Nehlich,2014). However, a diet-to-collagen methionine offset of06 0.5& was taken as reference.

Prior information. A purely physiological priorassumption based on acceptable levels of dietary proteinintake (Otten et al., 2006), between 5 and 45% of proteincarbon contribution, was incorporated into all dietaryscenarios. The implementation of this prior is describedin Fernandes et al. (2014b).

ANALYTICAL RESULTS

Sample preservation

Collagen preservation for human long bones and ani-mal dentine was generally good. Collagen yield valuesobtained by a protocol involving ultra-filtration are notdirectly comparable to standard reference values, how-ever, all collagen yields were above the cut-off referenceof 1% (van Klinken, 1999). The atomic C/N ratiosbetween 3.2 and 3.5 (Table 1) were also all within theexpected collagen range 2.9 to 3.6 (DeNiro, 1985). Simi-larly, sulfur atomic ratios are within the expected rangefor mammalian bones both for C/S (6006 300) and N/S(2006 300) (Nehlich and Richards, 2009).When measured using FTIR-ATR, bone bioapatite of

fresh bones shows SF values between ca. 2.8 and 3.2;the upper limit is extended to ca. 3.9 for relatively well-preserved bone (Hollund et al., 2012). For the Ostorfhuman bone samples SF values were between 3.1 and3.8 (Table 1). This indicates that the bone bioapatitesamples were comparatively well-preserved. Yet, toaccount for possible external contamination (e.g. ionicexchange), the uncertainty for d13Cbioapatite measure-ments was set at a value (0.5&), higher than the instru-mental uncertainty of 0.05& (Fernandes et al., 2012a).

Radiocarbon results

Radiocarbon results from archaeological samplesobtained at the Leibniz Laboratory in Kiel, Germany(lab code KIA) were compared with radiocarbon datesreported by Olsen et al. (2010). These latter were meas-ured at the Utrecht Laboratory, the Netherlands (labcode UtC) and at the Aarhus Laboratory, Denmark (labcode AAR). From each individual context the samehuman bone material was dated in this and in the studyby Olsen et al. However, different animal teeth wereradiocarbon dated in each study. To check the reproduci-bility of the measurements obtained at the Leibniz Labo-ratory where enough material was available, bonesamples were measured twice on the same collagenextract, but with independent combustion, reduction,and AMS counting. These results are provided in Sup-porting Information File 3. The age differences betweenthe repeats range from 8 years, 0.15r, for KIA-46906 to85 years, 2.0r, for KIA-46909 and 100 years, 1.6r, forKIA-46913 and are within the expected 95% statisticalrange (r5 standard uncertainty). This supports the reli-ability of the results.Human dietary RRE is here defined as the difference

in 14C concentration between human bone collagen andthe contemporary atmosphere. The latter is establishedby measuring the radiocarbon age of animal bones foundin association with each individual human. In this wayradiocarbon is employed as a dietary proxy given thataquatic foods have a 14C concentration lower than ter-restrial foods (Fernandes et al., 2014). It is assumedthat terrestrial animals did not consume aquatic foodsand thus have no RRE, although this cannot be fullyexcluded in the case of omnivores or carnivores (Table1). Furthermore, it is also assumed that the local reser-voir effect during the Neolithic is similar to present day.Local freshwater reservoir effects can vary in time withchanging environmental conditions. Nonetheless, thefew comparisons of past and present day freshwater res-ervoir effects within Germany show similar values (Fer-nandes et al., 2014). Average human and animalradiocarbon dates obtained in this study and the result-ing dietary RREs are listed in Table 3, together withresults previously reported by Olsen et al. (2010). Where

TABLE 4. Radiocarbon results from modern aquatic samples. See text description for estimates of reservoir ages

Lab code Lake location Species 14C (yr BP)Reservoirage (yr)

KIA-46304a Schwerin Pike (Esox lucius) 2706 20 ca. 590KIA-46305a Schwerin Bream (Abramis brama) 1906 30 ca. 510KIA-46306a Schwerin Eel (Anguilla anguilla) 2606 25 ca. 580KIA-46307a Schwerin Eel (Anguilla anguilla) 6256 25 ca. 945KIA-46310a Schwerin Zebra mussel (Dreissena polymorpha) 2306 20 ca. 550KIA-48715b Schwerin Zebra mussel (Dreissena polymorpha) 2406 30 ca. 500KIA-49512b Schwerin Zebra mussel (Dreissena polymorpha) 2906 15 ca. 550KIA-49919b Schwerin Zebra mussel (Dreissena polymorpha) 3006 15 ca. 560KIA-46311a Ostorf Zebra mussel (Dreissena polymorpha) 11206 20 ca. 1440KIA-48717b Ostorf Zebra mussel (Dreissena polymorpha) 10856 35 ca. 1345KIA-48718b Ostorf Unidentified bivalve species 11456 30 ca. 1405KIA-49516b Ostorf Unidentified bivalve species 10756 20 ca. 1335KIA-49912b Ostorf Zebra mussel (Dreissena polymorpha) 12956 25 ca. 1555KIA-49921b Ostorf Zebra mussel (Dreissena polymorpha) 11806 15 ca. 1440KIA-49922b Ostorf Unidentified bivalve species 11906 25 ca. 1450

aRadiocarbon results for samples collected in 2011 and reported in Fernandes et al. (2013).b Radiocarbon results from an ongoing study (samples collected in 2012/2013).



no Leibniz Lab animal dates could be obtained, reportedhuman dietary RREs represent the age differencebetween humans dated at the Leibniz Lab and animalsdated at the Aarhus Lab. Four of the five samples datedboth at the Aarhus Lab and the Leibniz Lab agree. Onlythe animal tooth from grave 196108 (AAR-10597)shows a statistically significant difference in agebetween the labs. The animal sample AAR-10597, previ-ously measured by Olsen et al., gave the youngest dateof all the samples, making it a clear outlier (Table 3).Olsen et al. reported some doubts on its reliability, giventhe poor preservation of the sample material. There isno general agreement between the radiocarbon measure-ments reported by the Utrecht Lab and the Leibniz Lab.Six of the nine human bone results of the Utrecht Lab(graves 190401, 193503, 193504, 196104, 196105,and 196109) are significantly younger (by 2.97.2r)than the Leibniz Lab results for the same individuals.No direct comparisons between the Utrecht and Aarhuslab results are possible. Among the samples showingthe largest disagreements between the Leibniz andUtrecht laboratories were those from graves 193503and 193504. In the study by Olsen et al., the associ-ated pairs from these graves (Table 3) indicated nega-tive human dietary RREs (radiocarbon dates fromhumans younger than those from associated animalremains), which suggests that the Utrecht results weretoo young, possibly due to incomplete removal of ayounger contaminant (e.g., humic acids). Overall theresults from the different laboratories confirm a patternof significant human dietary RREs in the Ostorf popu-lation. Some significant differences between the twostudies, in particular a major outlier (grave 196109)

that showed a reservoir effect of 782672 yr, warrantfuture investigation.Radiocarbon results for the flesh of modern fish and

mussels from lakes Schwerin and Ostorf were reportedpreviously (Fernandes et al., 2013) and are given inTable 4. Included are also new radiocarbon dates froman ongoing study that collected bivalve samples from thesame lakes during the second half of 2012 and the firsthalf of 2013. Atmospheric nuclear weapons tests, carriedout mostly between 1954 and 1963, have produced sig-nificant amounts of 14C. Thus, post 1954 AD atmos-pheric 14C concentrations are higher than 100 pMC(percent modern carbon) and their radiocarbon ageshave negative values. The atmospheric radiocarbonage for 2011 was ca. 2320 yr and ca. 2260 yr duringlate 2012 and early 2013. The RRE of aquatic species isthe difference in 14C concentration between the contem-porary atmosphere and the aquatic sample. Thus, to cal-culate the reservoir age of fish and bivalve samplescollected in 2011 it is necessary to add ca. 320 yr toreported radiocarbon ages and 260 yr to samples col-lected during late 2012 and early 2013 (Table 4). Thereservoir ages for Lake Schwerin are, in most cases ca.550 yr with a single eel specimen as an outlier (KIA-46307). This eel also has a high d15N (Table 1) and theolder radiocarbon age has been linked to probable die-tary specialization in planktivorous fauna; remainingspecies are probably associated with a pelagic food webwith radiocarbon ages similar to those of the aquaticflora (see Fernandes et al., 2013 for further details).This is corroborated by the agreement in 14C agesbetween the fish species and the zebra mussel fromLake Schwerin, given that zebra mussels primarily

TABLE 5. Estimates generated by FRUITS for average isotope values of the Ostorf population and for each dietary scenario

Scenario 1a Scenario 2b Scenario 3c Scenario 4d

Food (%)Plant 65617 616 18 566 15 586 17T. animals 23615 296 17 286 15 296 18Fish 1268 106 8 166 9 136 9

Fraction (%)Bulk 5061 506 0 506 1 506 0Protein 1163 106 3 126 3 116 3Carbs/Lipids 3963 406 3 386 3 396 3Methionine 060 06 0

Proxy (Food) (%)13Ccoll (Plant) 57617 536 18 476 14 496 1613Ccoll (T. animals) 25615 336 18 306 15 326 1913Ccoll (Fish) 18611 146 11 236 11 186 1115Ncoll (Plant) 35615 286 14 256 10 246 1115Ncoll (T. animals) 31618 446 21 346 17 416 2215Ncoll (Fish) 34615 286 18 406 15 366 1913Cbioa (Plant) 65617 616 18 566 15 586 1713Cbioa (T. animals) 23615 296 17 286 15 296 1813Cbioa (Fish) 1268 106 8 166 9 136 934Smeth (Plant) 30617 226 13 34Smeth (T. animals) 42620 456 19 34Smeth (Fish) 28615 326 15

Ostorf - Estimated RRE (yr) 2666155 2046 163 3366 161 2686 167Schwerin - Estimated RRE (yr) 101659 786 62 1286 61 1026 63

aPlant d15Nprotein54.5& and d34S included; b Plant d15Nprotein54.5& and d

34S not includedc Plant d15Nprotein52.0& and d

34S included; d Plant d15Nprotein5 2.0& and d34S not included.

Food (%) reports the estimate of carbon/calorie dietary contribution from each food group. Fraction (%) reports the estimate of car-bon/calorie contribution from each food fraction. Proxy (Food) (%) reports the estimate of calorie/carbon contribution from each foodgroup towards each dietary proxy. The last two rows list estimated group human dietary RRE values (estimated using as referencethe aquatic RREs at Lakes Ostorf and Schwerin).



consume phytoplankton (Baker et al., 1998). Similarly,zebra mussel specimens from Lake Ostorf, with radiocar-bon reservoir ages of ca. 1,450 yr, should provide a refer-ence RRE value for the local pelagic fish species (seeFernandes et al., 2013 for further details).

ESTIMATES GENERATED BY FRUITS

Interpreting FRUITS estimates

The interpretation of the estimates generated for par-ticular FRUITS model instances depends on how thesemodel instances were parametrized. Given that the mac-ronutrient concentrations are expressed as carbon con-tent or broadly equivalently calories, generatedestimates represent carbon or calorie contributions (theterms carbon or calorie contributions can be used inter-changeably). For the case study presented here three dif-ferent types of model estimates are generated: (1) the

calorie contribution of each food grouprepresentedhere as Food (%); (2) the calorie contribution of eachfood fraction (bulk, protein, carbs/energy, methionine)represented here as Fraction (%); (3) the calorie contri-bution of each food group towards a specific isotopicproxy (d13Ccollagen, d


34Smethio-nine)represented here as Proxy (Food) (%) where Proxyis each of the isotopic proxies and Food each of the foodgroups. The different estimates are expressed as relativecontributions (adding to 100%) with an associated1-sigma uncertainty (Table 5). The estimate Food (%)expresses the relative calorie contribution of each foodgroup and is also represented in Figure 2 as probabilitydistributions or credible intervals. Likewise, the esti-mate Fraction (%) represents the relative calorie contri-bution from each food fraction. The fraction bulk wasintroduced given that the signal of the dietary proxyd13Cbioapatite is determined by the bulk d

13C food signal.

Fig. 2. Model estimates of calorie intake under the different dietary scenarios for the isotope mean of the Ostorf individuals(credible intervals on the right and probability distributions on the left). Boxes represent a 68% credible interval (corresponding tothe 16th and 84th percentiles) while the whiskers represent a 95% credible interval (corresponding to the 2.5th and 97.5th percen-tiles). The horizontal continuous line represents the estimated mean while the horizontal discontinuous line represents the esti-mated median (50th percentile).



For each food group, a concentration value of 100% wasassigned to the fraction bulk. The concentrations of thefractions protein, carbs/lipids, and methionine wereassigned independently and sum to ca. 100%. This sumis negligibly larger than 100% given the contributionfrom methionine. Thus the sum of all defined fractions(bulk1protein1 carbs/lipids1methionine) is ca. 200%.In the modeling process only the relative concentrationvalues are taken into account, irrespective of the unitsused to define concentration. However, the generatedestimate Fraction (%) is normalized to 100% and thus,since by definition bulk5protein1 carbs/lipids, theFraction (%) estimate for bulk contribution is necessarily50% and the remaining fraction contribution estimatesadd to 50%. From the Fraction (%) estimates the ratioprotein vs. carbs/lipids can be calculated expressing therelative carbon or calorie dietary contribution of thesemacronutrients. Finally, the estimates Proxy (Food) (%)require a specific interpretation for each of the differentisotopic proxies (in FRUITS notation: 13Ccoll, 15Ncoll,

13Cbioa, 34Smeth). As mentioned previously the carbonisotopic signal of the proxy 13Cbioa is defined by thebulk d13C food signal. Thus, the estimates generated for13Cbioa provide the same values for each food group asthe estimates given by Fraction (%). Given that the onlysource of dietary nitrogen is assumed to be protein theestimate for 15Ncoll indicates the relative calorie proteincontribution of the different food groups. It is importantto emphasize that the modeling process takes intoaccount the relative fraction concentrations irrespectiveof the units in which these are expressed (Fernandeset al., 2014b). Given that nitrogen content is similar forproteins in different foods (Merrill and Watt, 1973), rela-tive nitrogen compositions can be expressed in FRUITSas relative carbon or calorie compositions. Similarly to15Ncoll the estimates for 34Smeth indicate the relativecalorie methionine contribution from each food group.The estimates for 13Ccoll differ from those generated for15Ncoll given that 13Ccoll includes a routed carbon con-tribution from carbs/lipids. Estimated fish carbon

Fig. 2. (Continued).



contribution towards 13Ccoll together with observedlocal reservoir effects are here used to provide an esti-mate of individual dietary RREs.

Robustness of model estimates

The values associated with each modeling parameterplus a physiological prior define specific models forwhich FRUITS generates dietary estimates. These esti-mates quantify the relative dietary intake, expressed ascarbon or calorie contributions, of plant, terrestrial ani-mals, and fish food groups. Estimates were generated forfour models corresponding to different dietary scenarios:scenario 1 (manured plants with plant d15Nprotein54.5&and with d34S proxy included); scenario 2 (manuredplants with plant d15Nprotein54.5& and with d

34S proxyexcluded); scenario 3 (unmanured plants with plantd15Nprotein5 2.0& and with d

34S proxy included); sce-nario 4 (unmanured plants with plant d15Nprotein52.0&and d34S proxy excluded). Under the different dietaryscenarios similar probability distributions are observedfor dietary estimates of Food (%) representing the car-bon/calorie contributions from each food group (Fig. 2).This demonstrates that the estimates generated are rela-

tively robust under the different model parameters.Uncertainties associated with each average of estimateare in most cases above 10% (Table 5). For dietary sce-narios 2 and 4, which do not include the d34S proxy, theloss of information results in an increase in the uncer-tainty of model estimates compared with dietary scenar-ios 1 and 3.

Accuracy of model estimates

Given the different modeling assumptions it becomesuseful to verify the accuracy of generated estimates underthe different dietary scenarios. The dietary scenario withthe most accurate estimates should also provide the bestagreement with observed human dietary RREs. For theOstorf population group, the carbon contribution fromfreshwater fish to human bone collagen is given in Table 5and corresponds to the fish contribution towards the die-tary proxy d13Ccollagen (FRUITS code: 13Ccoll). The fishRRE at Lake Ostorf is ca. 1,450 yr, while for LakeSchwerin, the fish RRE is ca. 550 yr if the eel outlier(KIA-46307) is removed (Table 4). The average humandietary RRE in this study was 3736 144 yr with the majoroutlier removed (grave 196109). Table 5 lists, for theOstorf group, the estimated dietary RREs values relyingon the fish 13Ccoll values listed in the same table and thelocal reservoir effects at lakes Schwerin and Ostorf. Theresults show that the best agreement between observedand estimated group dietary RREs is obtained under die-tary scenario 3 and having Lake Ostorf as the mainsource of freshwater fish (Table 5). Dietary scenario 3 isalso the model providing the most precise estimates. How-ever, given the relatively large uncertainties of group esti-mates, individual estimates of human dietary RREsunder dietary scenario 3 were also made. These are repre-sented in Figure 3 and numeric values are given in Table6. Comparison of observed and estimated individual die-tary RREs shows overall a remarkably good agreementwhen considering as reference the reservoir effect at LakeOstorf (1,450 yr). In this case, the difference for 8 out of10 individuals between observed and estimated RREs issmaller than 115 yr and not statistically significant (Table6). The exceptions to the general good agreement betweenobserved and estimated values are samples KIA-46906with a difference of 2296 119 yr and KIA-46921 with adifference of 2866218 yr. However, as mentioned previ-ously, sample KIA-46921 constitutes a clear radiocarbonoutlier. With the exception of KIA-46906, the agreementis extremely poor between observed and estimated RREvalues when considering as reference the reservoir effectat Lake Schwerin (550 yr). These results support theaccuracy of model estimates provided that the reservoireffect at Lake Ostorf is taken as reference. Thus, theradiocarbon results provide also an insight into the mobil-ity patterns of the Ostorf population suggesting signifi-cant periods of residence in the vicinity of Lake Ostorf.

DISCUSSION

The following discussion is based on the approximateaverage values of model estimates under dietary sce-nario 3 given that this scenario provides the best consis-tency between estimated and observed human RREs.The average calorie contribution of each food group(Food (%)) for the Ostorf population was ca. 55% plants,30% terrestrial animals, and 15% fish (Table 5). Theestimate of macronutrient contribution is given by theprotein to carbs/lipids ratio of the values [Fraction (%)]

Fig. 3. Observed (circle symbols) and estimated human die-tary RREs for the Ostorf individuals (identified by KIA number)taking as reference the reservoir effect at Lake Ostorf (trian-gles) and Lake Schwerin (square symbols). Error bars representone standard deviation.



TABLE6.Estim

atesgeneratedbyFRUITSforeach

Ostorfindividualunder

dietary

scenario3

Human(KIA

)46904(m

)46906(m

)46908(m

)46909(m

)46911

(m)

46919(m

)46913(f)

46915(f)

46917(f)

46921(f)

Food(%

)Plant

63615

49619

37618

57616

51618

50619

45619

55618

72612

63616

T.anim

al

23615

25616

44618

35616

40618

28617

26617

18614

17612

13611

Fish

1468

26613

19611

866

967

22613

28614

27614

1167

24612

Fraction(%

)Bulk

5061

5061

5061

5061

5061

5061

5061

5061

5061

5061

Protein

1163

1564

1563

1162

1263

1464

1564

1464

1062

1364

Carbs/Lipids

3963

3564

3563

3962

3862

3664

3564

3664

4062

3764

Methionine

060

060

060

060

060

060

060

060

060

060

Proxy(Food)(%

)13Ccoll(Plant)

54615

39617

30615

49615

43617

41617

36617

44617

63613

52616

13Ccoll(T.anim

al)

25615

25616

44618

39616

43618

28617

26616

18614

19613

14611

13Ccoll(Fish)

21611

36615

26614

1268

14610

31615

38616

37615

1869

34614

15Ncoll(Plant)

31612

19611

1469

29612

25612

21611

17610

23612

39612

27611

15Ncoll(T.anim

al)

31618

26616

46619

48618

51619

30618

26617

19615

25616

16613

15Ncoll(Fish)

38616

55616

40618

22614

24615

49617

57617

58616

36615

57615

13Cbioa(Plant)

63615

49619

37618

57616

51618

50619

45619

55618

72612

63616

13Cbioa(T.anim

al)

23615

25616

44618

35616

40618

28617

26617

18614

17612

13611

13Cbioa(Fish)

1468

26613

19611

866

967

22613

28614

27614

1167

24612

34Smeth(Plant)

28615

18613

11610

21614

18613

19613

16612

21613

35616

26614

34Smeth(T.anim

al)

41620

37619

63617

65617

66618

39620

34618

24616

36620

23616

34Smeth(Fish)

31616

46617

25614

13610

16612

42618

50617

55617

29615

50617

Observed

RRE(yr)

386640

289635

268640

194641

300640

374640

591647

620642

332640

782672

Ostorf-Estim

atedRRE(yr)

3016161

5186216

3796202

1736121

1996141

4516214

5546231

5396223

2596134

4966206

Schwerin

-Estim

atedRRE(yr)

114661

197682

144677

66646

76653

171681

210687

204685

98651

188678

Food(%

)reports

theestimate

ofcarbon/caloriedietary

contribution

from

each

food

group.Fraction(%

)reports

theestimate

ofcarbon/caloriecontribution

from

each

food

fraction.

Proxy(Food)(%

)reports

theestimate

ofcalorie/carbon

contribution

from

each

food

grouptowardseach

dietary

proxy.Letters

betweenparentheses

infirstrowidentify

females(f)

andmales(m

).Thelast

threerowslist

observed

andestimatedhumandietary

RREvalues

(estim

atedusingasreference

theaquaticRREsatLakes

OstorfandSchwerin).



reported in Table 5. This provides an approximate esti-mate of the caloric contribution of each macronutrient.The estimated intake of ca. 25% dietary calorie protein

is significantly higher than the intake observed in mod-ern western diets (ca. 15%) (Lands et al., 1990), but sim-ilar to that observed in some ethnographic studies ofhunter-gatherer populations (Cordain et al., 2000). Theresults for 15Ncoll (Food) (%) (Table 5) show that themain sources of protein calories were fish and terrestrialanimals at ca. 40% and 35%, respectively. Plant foodsrepresented ca. 25% of protein calorie contribution.The primary goal of the research presented here was

to analyze the dietary pattrns of the Ostorf individualsas a group. However, useful information is also providedby estimated individual diets (Table 6 and Fig. 4). Theseprovide a potential use in future studies to investigate,for instance, relationships between gender or social sta-tus and dietary preferences, as the estimates show somedifferences in individual diets. For instance, althoughthe number of individuals is small, FRUITS outputs sug-gest, with the exception of KIA-46917, that women hada higher fish intake than men (Fig. 4).The overview of estimates under dietary scenario 3 for

the Ostorf group shows that a significant, and possiblythe main, dietary calorie contribution came from plantswhile their major protein calorie contribution came fromfish and terrestrial animals. A high caloric dependenceon plant foods is unusual in hunter-gatherers at suchhigh latitudes (>508 N) (Cordain et al., 2000). There are,however, historical analogues that show that a diet richin protein is compatible with a caloric intake predomi-nantly from plant foods. For instance, it is well-knownfrom historical records that medieval Europeans oftenhad a high protein intake from meat and fish while foodsproduced from cereals (e.g., bread, ale) were the mainsource of energy (Dyer, 1988; Harvey, 1993; Woolgar,2010). Fish consumption was particularly high duringthe medieval period and isotope analysis of medievalindividuals often shows values similar to those observedin the Ostorf individuals (e.g. Bayliss et al., 2004; Yoder,2012). The best estimate performance under dietary sce-nario 3 would suggest that unmanured plants were con-sumed. However, model estimates under dietaryscenarios 1 and 3 are similar and thus it is not possibleto securely establish if the dietary contributions of plantfoods corresponded to manured or unmanured plants.The latter could also include wild plants, the consump-tion of which has been established for the Mesolithicperiod in northern Europe (Kubiak-Martens, 1996;Kubiak-Martens, 1999). Significant periods of residencein the vicinity of Lake Ostorf are suggested by the over-all agreement between the estimates of the fish contribu-tions towards bone-collagen carbon and the localreservoir effect (Fig. 3). Thus the Ostorf population,while within a spatially and temporally Neolithic con-text, adopted some Neolithic cultural elements, but fol-lowed a subsistence strategy with aspects more oftenassociated with a hunter-gatherer diet.Large isotope studies have shown that there is, in gen-

eral, a significant shift in modes of subsistence followingthe Mesolithic-Neolithic transition with a decrease inthe consumption of aquatic resources (Tauber, 1981;Richards et al., 2003b). However, local adaptations thatdeviate from the general pattern have also beenobserved from human isotope studies and the analysis ofarchaeological material evidence (Liden, 1995; Craiget al., 2011; Bollongino et al., 2013). The Ostorf casestudy represents such an example of an adaptation tolocal natural resources and shows that there was a richdiversity of subsistence strategies during the Neolithic.

Fig. 4. Individual model estimates of calorie intake,expressed as credible intervals, for dietary scenario 3 (plantd15N5 2&). Boxes represent a 68% credible interval (correspond-ing to the 16th and 84th percentiles) while the whiskers repre-sent a 95% credible interval (corresponding to the 2.5th and97.5th percentiles). The horizontal continuous line represents theestimated mean while the horizontal discontinuous line repre-sents the estimated median (50th percentile). Mars symbol indi-cates a male while the Venus symbol indicates a female.



CONCLUSIONS

The diet of the Ostorf pre-historic population was esti-mated using a novel approach that included the use ofthe Bayesian mixing model FRUITS. The dietary esti-mates were tested considering dietary scenarios with dif-ferent model parameters and values. Generatedestimates were not overly sensitive to model modifica-tions, confirming the robustness of the results. Theseresults indicate that, in general, the individuals studiedhad a diet with a high protein intake and relied princi-pally on fish and terrestrial animals as their proteinsources. Model estimates also suggest that plant foodsrepresented a significant, possibly the main, source ofcalories. However, the uncertainties associated with esti-mates do not allow for a definitive statement. Observeddietary RREs are in good agreement with generated esti-mates using the local aquatic reservoir effect at LakeOstorf, and incompatible with model estimates if a muchlower or higher local aquatic reservoir effect is applied.Thus the radiocarbon results provide an insight into themobility patterns of the Ostorf population suggestingthat the prehistoric individuals spent a significantamount of time in the vicinity of Lake Ostorf. Overallthese results describe a population chronologicallywithin the local early and middle Neolithic that adopteda subsistence strategy that involved a major proportionof fishing and hunting.

ACKNOWLEDGMENTS

The authors thank two anonymous reviewers for theirconstructive comments, which helped us to improve themanuscript. They thank Dr. JohnMeadows for his insight-ful feedback and English revision and Dr. Harald Lubkefor providing additional background information on theOstorf individuals. Finally, they thank Dr. Detlef Jantzen,Prof. Dr. Friedrich Luth, and Prof. Dr. Thomas Terbergerfor providing access to the archaeological material.

LITERATURE CITED

Ambrose SH, Norr L. 1993. Experimental evidence for therelationship of the carbon isotope ratios of whole diet anddietary protein to those of bone collagen and carbonate. In:Lambert JB, Norr L, editors. Prehistoric human bonearchaeology at the molecular level. Springer-Verlag, BerlinHeidelberg, p 138.

Baker SM, Levinton JS, Kurdziel JP, Shumway SE. 1998.Selective feeding and biodeposition by zebra mussels andtheir relation to changes in phytoplankton composition andseston load. J Shellfish Res 17:12071213.

Balasse M, Bocherens H, Mariotti A. 1999. Intra-bonevariability of collagen and apatite isotopic composition usedas evidence of a change of diet. J Archaeol Sci 26:593598.

Bayliss A, Popescu ES, Beavan-Athfield N, Ramsey CB, CookGT, Locker A. 2004. The potential significance of dietaryoffsets for the interpretation of radiocarbon dates: anarchaeologically significant example from medieval Norwich.J Archaeol Sci 31:563575.

Bocherens H, Polet C, Toussaint M. 2007. Palaeodiet of Mesolithicand Neolithic populations of Meuse Basin (Belgium): evidencefrom stable isotopes. J Archaeol Sci 34:1027.

Bogaard A, Fraser R, Heaton THE, Wallace M, Vaiglova P,Charles M, Jones G, Evershed RP, Styring, AK, AndersenNH, Arbogast R-M, Bartosiewicz L, Gardeisen A, KanstrupM, Maier U, Marinova E, Ninov L, Schafer M, Stephan E.2013. Crop manuring and intensive land management byEuropes first farmers. Proc Natl Acad Sci 110:1258912594.

Bollongino R, Nehlich O, Richards MP, Orschiedt J, ThomasMG, Sell C, Fajkosova Z, Powell A, Burger J. 2013. 2000Years of Parallel Societies in Stone Age Central Europe.Science 342:479481.

Boric D, Grupe G Peters J., Mikic Z 2004. Is the Mesolithic-Neolithic Subsistence Dichotomy Real? New Stable IsotopeEvidence from the Danube Gorges. Eur J Archaeol 7:221248.

Bosl C, Grupe G, Peters J. 2006. A Late Neolithic vertebratefood web based on stable isotope analyses. Int JOsteoarchaeol 16:296315.

Bramanti B, Thomas MG, Haak W, Unterlaender M, Jores P,Tambets K, Antanaitis-Jacobs I, Haidle MN, Jankauskas R,Kind C-J, Lueth F, Terberger T, Hiller J, Matsumura S,Forster P, Burger J. 2009. Genetic discontinuity between localhunter-gatherers and central Europes first farmers. Science326:1372140.

Cook GT, Bonsall C, Hedges REM, McSweeney K, Boronean V,Pettitt PB. 2001. A freshwater diet-derived 14C reservoireffect at the Stone Age sites in the Iron Gates gorge. Radio-carbon 43:453460.

Cordain L, Miller JB, Eaton SB, Mann N, Holt SHA, Speth JD.2000. Plant-animal subsistence ratios and macronutrientenergy estimations in worldwide hunter-gatherer diets. Am JClin Nutr 71:682692.

Craig OE, Steele VJ, Fischer A, Hartz S, Andersen SH,Donohoe P, Glykou A, Saul H, Jones, DM, Koch E, Heron CP.2011. Ancient lipids reveal continuity in culinary practicesacross the transition to agriculture in Northern Europe. ProcNatl Acad Sci 108:1791017915.

Deguilloux M-F, Leahy R, Pemonge M-H, Rottier S. 2012.European neolithization and ancient DNA: an assessment.Evol Anthropol 21:2437.

DeNiro MJ. 1985. Post-mortem preservation and alteration ofin vivo bone collagen isotope ratios in relation topalaeodietary reconstruction. Nature 317:806809.

Dolphin AE, Naftel SJ, Nelson AJ, Martin RR, White CD. 2013.Bromine in teeth and bone as an indicator of marine diet.J Archaeol Sci 40:17781786.

Durrwachter C, Craig OE, Collins MJ, Burger J, Alt KW. 2006.Beyond the grave: variability in Neolithic diets in SouthernGermany? J Archaeol Sci 33:3948.

Dyer C. 1988. Changes in diet in the late middle ages: the caseof harvest workers. Agric Hist Rev 36:2137.

Fernandes R, Dreves A, Nadeau M-J, Grootes PM. 2013. AFreshwater Lake Saga: carbon Routing Within the AquaticFood Web of Lake Schwerin. Radiocarbon 55:11021113.

Fernandes R, Huls M, Nadeau M-J, Grootes PM, Garbe-Schonberg C-D, Hollund HI, Lotnyk A, Kienle L. 2012a.Assessing screening criteria for the radiocarbon dating ofbone mineral. Nucl Instrum Method Phys Res Sect B 294:226232.

Fernandes R, Meadows J, Dreves A, Nadeau M-J, Grootes P.2014a. A preliminary study on the influence of cooking on theC and N isotopic composition of multiple organic fractions offish (mackerel and haddock). J Archaeol Sci 50:153159.

Fernandes R, Millard AR, Brabec M, Nadeau MJ, Grootes PM.2014b. Food Reconstruction Using Isotopic TransferredSignals (FRUITS): a Bayesian model for diet reconstruction.PLoS One 9:e87436.

Fernandes R, Nadeau MJ, Grootes PM. 2012b. Macronutrient-based-model for dietary carbon routing in bone collagen andbioapatite. Archaeol Anthropol Sci 4:291301.

Fernandes R, Nadeau M-J, Grootes PM. 2014c. EDTA basedprotocols for the cleaning of ancient bone bioapatite. In DBraekmans, M Carremans, P Degryse, eds, Proceedings of the39th International Symposium for Archaeometry. pp 7378.

Fernandes R, Rinne C, Grootes PM, Nadeau MJ. 2012c.Revisiting the chronology of Northern Germanmonumentality sites: preliminary results. In: Hinz M, MullerJ, editors. Fruhe Monumentalitat und soziale Differenzierung2. p 87103.

Fernandes R, Rinne C, Nadeau M-J, Grootes P. 2014. Towardsthe use of radiocarbon as a dietary proxy: establishing a firstwide-ranging radiocarbon reservoir effects baseline for



Germany. Environ Archaeol. DOI: 10.1179/1749631414Y.0000000034.

Fischer A, Olsen J, Richards M, Heinemeier J,Sveinbjornsdottir AE, Bennike P. 2007. Coast-inland mobilityand diet in the Danish Mesolithic and Neolithic: evidencefrom stable isotope values of humans and dogs. J ArchaeolSci 34:21252150.

Froehle AW, Kellner CM, Schoeninger MJ. 2010. FOCUS: effectof diet and protein source on carbon stable isotope ratios incollagen: follow up to. J Archaeol Sci 37:26622670.

Hare PE, Fogel ML, Stafford TW Jr, Mitchell AD, Hoering TC.1991. The isotopic composition of carbon and nitrogen inindividual amino acids isolated from modern and fossilproteins. J Archaeol Sci 18:277292.

Harvey B (1993). Living and dying in England 1100-1540 : theMonastic Experience. Oxford: Oxford University Press.

Hedges R, Rush E, Aalbersberg W. 2009. Correspondencebetween human diet, body composition and stable isotopiccomposition of hair and breath in Fijian villagers. IsotopesEnviron Health Stud 45:117.

Hedges R, Saville A, OConnell T. 2008. Characterizing the dietof individuals at the Neolithic chambered tomb of HazletonNorth, Gloucestershire, England, using stable isotopicanalysis. Archaeometry 50:114128.

Hedges REM, Clement JG, Thomas CDL, OConnell TC. 2007.Collagen turnover in the adult femoral mid-shaft: modeledfrom anthropogenic radiocarbon tracer measurements. Am JPhys Anthropol 133:808816.

Hollund HI, Ariese F, Fernandes R, Jans MME, Kars H. 2012.Testing and alternative high-throughput tool for investigatingbone diagenesis: FTIR in attenuated total reflection (ATR)mode*. Archaeometry 55:507532.

Howland MR, Corr LT, Young SMM, Jones V, Jim S, Van DerMerwe NJ, Mitchell AD, Evershed RP. 2003. Expression ofthe dietary isotope signal in the compound-specific d13C val-ues of pig bone lipids and amino acids. Int J Osteoarchaeol13:5465.

Hublin J-J, Talamo S, Julien M, David F, Connet N, Bodu P,Vandermeersch B, Richards MP. 2012. Radiocarbon datesfrom the Grotte du Renne and Saint-Cesaire support a Nean-dertal origin for the Cha^telperronian. Proc Natl Acad Sci 109:1874318748.

Huelsemann F, Flenker U, Koehler K, Schaenzer W. 2009.Effect of a controlled dietary change on carbon and nitrogenstable isotope ratios of human hair. Rapid Commun MassSpectrom 23:24482454.

Jacomet S (2004). Archaeobotany: a vital tool in theinvestigation of lake-dwellings. In Menotti F, editor. Living onthe Lake in Prehistoric Europe. Routledge: 150 years of Lake-Dwelling Research. p 162177.

Jim S, Ambrose SH, Evershed RP. 2004. Stable carbon isotopicevidence for differences in the dietary origin of bonecholesterol, collagen and apatite: implications for their use inpalaeodietary reconstruction. Geochim Cosmochim Acta 68:6172.

Kellner CM, Schoeninger MJ. 2007. A simple carbon isotopemodel for reconstructing prehistoric human diet. Am J PhysAnthropol 133:11121127.

Van Klinken GJ. 1999. Bone collagen quality indicators forpalaeodietary and radiocarbon measurements. J Archaeol Sci26:687695.

Kubiak-Martens L. 1996. Evidence for possible use of plantfoods in Palaeolithic and Mesolithic diet from the site ofCaowanie in the central part of the Polish Plain. Veg HistArchaeobot 5:3338.

Kubiak-Martens L. 1999. The plant food component of the dietat the late Mesolithic (Erteblle) settlement at Tybrind Vig,Denmark. Veg Hist Archaeobot 8:117127.

Lands WE, Hamazaki T, Yamazaki K, Okuyama H, Sakai K,Goto Y, Hubbard VS. 1990. Changing dietary patterns. Am JClin Nutr 51:991993.

Lanting JN, van der Plicht J. 1998. Reservoir effects andapparent 14C ages. J Irish Archaeol 9:151165.

Lehn C, Rossmann A, Graw M. 2015. Provenancing ofunidentified corpses by stable isotope techniquespresentation of case studies. Sci Justice 55:7288.

Liden K. 1995. Megaliths, agriculture, and social complexity: adiet study of two Swedish Megalith populations. J AnthropolArchaeol 14:404417.

Logan JM, Jardine TD, Miller TJ, Bunn SE, Cunjak RA,Lutcavage ME. 2008. Lipid corrections in carbon andnitrogen stable isotope analyses: comparison of chemicalextraction and modelling methods. J Anim Ecol 77:838846.

Longin R. 1971. New method of collagen extraction forradiocarbon dating. Nature 230:241242.

Lubke H, Luth F, Terberger T. 2009. Fishers or farmers? Thearchaeology of the Ostorf cemetery and related Neolithicfinds in the light of new data. In: Larsson L, Luth F,Terberger T, editors. Innovation and continuitynon-megalithic mortuary practices in the Baltic. New Methodsand Research into the Development of Stone age Society.Romisch-Germanischen Kommission. p 307338.

McCutchan JH, Lewis WM, Kendall C, McGrath CC. 2003.Variation in trophic shift for stable isotope ratios of carbon,nitrogen, and sulfur. Oikos 102:378390.

Merrill AL, Watt BK. 1973. Energy value of foods: basis andderivation. United States Department of Agriculture (USDA),Handbook No. 74. Washington, USA: United StatesGovernment Printing Office.

Minagawa M. 1992. Reconstruction of human diet from d13Cand d15N in contemporary Japanese hair: a stochastic methodfor estimating multi-source contribution by double isotopictracers. Appl Geochemistry 7:145158.

Minagawa M, Karasawa K, Kabaya Y. 1986. Carbon andnitrogen isotope abundances in human feeding ecosystem.Chikyu-Kagaku 20:7988.

Morrison DJ, Dodson B, Slater C, Preston T. 2000. 13C naturalabundance in the British diet: implications for 13C breathtests. Rapid Commun Mass Spectrom 14:13211324.

Nadeau M-J, Grootes PM, Schleicher M, Hasselberg P, Rieck A,Bitterling M. 1998. Sample throughput and data quality atthe Leibniz-Labor AMS facility. Radiocarbon 40:239246.

Nadeau MJ, Schleicher M, Grootes PM, Erlenkeuser H,Gottdang A, Mous DJW, Sarnthein JM, Willkomm H. 1997.The Leibniz-Labor AMS facility at the Christian-Albrechts-University, Kiel, Germany. Nucl Instruments Methods PhysRes B 123:2230.

Nehlich O. 2015. The application of sulfur isotope analyses inarchaeological research: a review. Earth-Science Rev 142:117.

Nehlich O, Richards M. 2009. Establishing collagen qualitycriteria for sulphur isotope analysis of archaeological bonecollagen. Archaeol Anthropol Sci 1:5975.

OConnell TC, Hedges REM. 1999. Isotopic Comparison of Hairand Bone: archaeological Analyses. J Archaeol Sci 26:661665.

OConnell TC, Hedges REM, Healey MA, Simpson AHRW. 2001.Isotopic comparison of hair, nail and bone: modern analyses.J Archaeol Sci 28:12471255.

OConnell TC, Kneale CJ, Tasevska N, Kuhnle GGC. 2012. Thediet-body offset in human nitrogen isotopic values: a con-trolled dietary study. Am J Phys Anthropol 149:426434.

Oelze VM, Siebert A, Nicklisch N, Meller H, Dresely V, Alt KW.2011. Early Neolithic diet and animal husbandry: stableisotope evidence from three Linearbandkeramik (LBK) sitesin Central Germany. J Archaeol Sci 38:270279.

Ogrinc N, Budja M. 2005. Paleodietary reconstruction of aNeolithic population in Slovenia: a stable isotope approach.Chem Geol 218:103116.

Olsen J, Heinemeier J, Lubke H, Luth F, Terberger T. 2010.Dietary habits and freshwater reservoir effects in bones froma Neolithic NE German cemetery. Radiocarbon 52:635644.

Otten J, Pitzi Helliwig J, Meyers L. 2006. DRI, dietaryreference intakes: the essential guide to nutrientrequirements. Washington DC: National Academies Press.

Passey BH, Robinson TF, Ayliffe LK, Cerling TE, SponheimerM, Dearing MD, Roeder BL, Ehleringer JR. 2005. Carbon



isotope fractionation between diet, breath CO2, and bioapatitein different mammals. J Archaeol Sci 32:14591470.

Patolla M, Henke W (2007). The skeletal population fromOstorf (Mecklenburg) - new evidence for its relationships andlife-style. In: Larsson L, Luth F, Terberger T, editors.Innovation and continuity - non-megalithic mortuary prac-tices in the Baltic. New methods and research into the devel-opment of stone age society. Romisch-GermanischenKommission. p 353383.

Pinnegar JK, Polunin NVC. 1999. Differential fractionation ofd13C and d15N among fish tissues: implications for the studyof trophic interactions. Funct Ecol 13:225231.

Post DM, Layman CA, Arrington DA, Takimoto G, QuattrochiJ, Monta~na CG. 2007. Getting to the fat of the matter:models, methods and assumptions for dealing with lipids instable isotope analyses. Oecologia 152:1792189.

Richards MP. 2001. Paleodietary reconstruction. St Martinsuncovered: investigations in the Churchyard of St. Martins-in-the-bull-ring. Birmingham: Oxbow Books, Oxford. p 147151.

Richards MP, Fuller BT, Sponheimer M, Robinson T, Ayliffe L.2003a. Sulphur isotopes in palaeodietary studies: a reviewand results from a controlled feeding experiment. Int JOsteoarchaeol 13:3745.

Richards MP, Schulting RJ, Hedges REM. 2003b. Archaeology:sharp shift in diet at onset of Neolithic. Nature 425:366366.

Richards MP, Trinkaus E. 2009. Isotopic evidence for the dietsof European Neanderthals and early modern humans. ProcNatl Acad Sci 106:1603416039.

Schoeller DA, Minagawa M, Slater R, Kaplan IR. 1986. Stableisotopes of carbon, nitrogen and hydrogen in thecontemporary north American human food web. Ecol FoodNutr 18:159170.

Schoeninger MJ, DeNiro MJ, Tauber H. 1983. Stable nitrogenisotope ratios of bone collagen reflect marine and terrestrialcomponents of prehistoric human diet. Science 220:13811383.

Sholto-Douglas AD, Field JG, James AG, van Der Merwe NJ.1991. 13C/12C and 15N/14N isotope ratios in the SouthernBenguela Ecosystem: indicators of food web relationshipsamong different size-classes of plankton and pelagic fish: dif-ferences between fish muscle and bone collagen tissue. MarEcol Prog Ser 78:2331.

Tanz N, Schmidt H-L. 2010. d34S-Value measurements in foodorigin assignments and sulfur isotope fractionations in plantsand animals. J Agric Food Chem 58:31393146.

Tauber H. 1981. 13C evidence for dietary habits of prehistoricman in Denmark. Nature 292:332333.

Tieszen LL. 1991. Natural variations in the carbon isotopevalues of plants: implications for archaeology, ecology, andpaleoecology. J Archaeol Sci 18:227248.

Tieszen LL, Fagre T. 1993. Effect of diet quality andcomposition on the isotopic composition of respiratory CO2,bone collagen, bioapatite, and soft tissues. In: Lambert JB,Norr L, editors. Prehistoric human bone archaeology at themolecular level. Springer-Verlag, Berlin Heidelberg, p 121155.

USDA (2012). USDA National Nutrient Database for StandardReference, Release 25. Available at: http://ndb.nal.usda.gov/ndb/search/list

Vogel JC. 1978. Isotopic assessment of the dietary habits ofungulates. S Afr J Sci 74:298301.

Warinner C, Tuross N. 2010. Brief communication: tissueisotopic enrichment associated with growth depression in apig: implications for archaeology and ecology. Am J PhysAnthropol 141:486493.

Warinner C, Tuross N. 2009. Alkaline cooking and stableisotope tissue-diet spacing in swine: archaeological implica-tions. J Archaeol Sci 36:16901697.

Waters-Rist AL, Bazaliiskii VI, Weber AW, Katzenberg MA.2011. Infant and child diet in Neolithic hunter-fisher-gatherers from cis-baikal, Siberia: Intra-long bone stablenitrogen and carbon isotope ratios. Am J Phys Anthropol 146:225241.

Weiner S, Bar-Yosef O. 1990. States of preservation of bonesfrom prehistoric sites in the Near East: a survey. J ArchaeolSci 17:187196.

Woolgar CM. 2010. Food and the middle ages. J Mediev Hist36:119.

Yoder C. 2012. Let them eat cake? Status-based differences indiet in medieval Denmark. J Archaeol Sci 39:11831193.

Yoshinaga J, Minagawa M, Suzuki T, Ohtsuka R, Kawabe T,Inaoka T, Akimichi T. 1996. Stable carbon and nitrogenisotopic composition of diet and hair of Gidra-speaking Pap-uans. Am J Phys Anthropol 100:2334.



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