Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture

  1. Emanuela Cristiani  Is a corresponding author
  2. Anita Radini
  3. Andrea Zupancich
  4. Angelo Gismondi
  5. Alessia D'Agostino
  6. Claudio Ottoni
  7. Marialetizia Carra
  8. Snežana Vukojičić
  9. Mihai Constantinescu
  10. Dragana Antonović
  11. T Douglas Price
  12. Dušan Borić  Is a corresponding author
  1. DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Italy
  2. Department of Archaeology, University of York, United Kingdom
  3. Laboratory of General Botany, Department of Biology, University of Rome "Tor Vergata", Italy
  4. Centre of Molecular Anthropology for Ancient DNA Studies; Department of Biology, University of Rome "Tor Vergata", Italy
  5. University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Serbia
  6. Romanian Academy, Institute for Anthropological Research “Francisc I. Rainer”, Romania
  7. Institute of Archaeology, Serbia
  8. Department of Anthropology, University of Wisconsin, United States
  9. Department of Environmental Biology, Sapienza University of Rome, Italy
  10. The Italian Academy for Advanced Studies in America, Columbia University, United States
  11. Department of Anthropology, New York University, United States
10 figures, 5 tables and 1 additional file


Sites in the central Balkans investigated in the article, which provided dental calculus and ground stone tools.

EM = Early Mesolithic; LM = Late Mesolithic; M/N = Mesolithic-Neolithic; EN = Early Neolithic; BA = Bronze Age; MA = Medieval.

Studied teeth photographed under the microscope before dental calculus sampling.
Late Mesolithic ground stone tools from the site of Vlasac featuring use-wear traces and residues related to plant food processing.
Starch granules from Mesolithic and Neolithic dental calculus.

Early Mesolithic: (a) Type Ib (PAD11); (b) Type Ib (PAD9); (c) Type Ib (PAD11); (d) Type V (PAD12); (e) Type III (PAD11); (f) Type Ib (PAD15); Late Mesolithic: (g) Type II (VL82c); (h) Type IV (VL31); (i) Type VI (VL70); (j, k) multicellular structures of long cells embedded in dental calculus (HV25/26, VL70); (l) Type Ia (HV11); Mesolithic–Neolithic: (m) Type Ia (HV16). Neolithic: (n) Type III (LV32a); (o–v) damaged Type I granules (A-Type granules) (VEL-2D); (w) Type I (A-Type granule) (VEL-2D); (x) single dendritic cell (Gârleşti); (y) Type I (A-Type granule) (VEL-2A).

Other dietary and nondietary debris found in Mesolithic dental calculus from the Danube Gorges.

Early Mesolithic: (a) Type V (PAD11); (b, c) Type III (PAD12); (d) Type I (A-Type granule) (PAD12); (e) Type I (A-Type granule) (PAD12); (f) smoke particle (LV50); (g, h) plant fiber embedded in calculus (PAD16); (i) Type Ib (PAD9); (j) feather barbule embedded in calculus (PAD9); Late Mesolithic: (k) Type II (PAD2); (l) polylobate phytolith (US64 x.11); (m) phytoliths (VL79); (n–p) Type VI (VL70,VL83); (q) feather barbules embedded in calculus (HV25/26); (r) echinate pollen grain in calculus (VL83); (s) plant tissue (LV79a); (t) Type II (VL43); (u) Type I (HV11); (v) Type Ia (HV11); Mesolithic-Neolithic: (w) Type I (HV16); (x) wood particle (PAD4); (y) phytoliths (LV28); (z) feather barbule (PAD4).

Starch granule morphological variability within the species of the genus Aegilops and domestic species of the Triticeae tribe.

(a) Aegilops cylindrica; (b) A. neglecta; (c) A. speltoides tauschii; (d) A. caudata; (e) A. triuncialis; (f) A. comosa; (g) A. uniaristata; (h) A. ventricosa; (i) A. geniculata; (j) A. crassa; (k) A. peregrina; (l) Elymus caninus; (m) Bromus tectorum; (n) Agropyron pungens; (o) A. farctus; (p) Dasypyron villosum; (q) Triticum monococcum; (r) Hordeum vulgare; (s) T. dicoccum; (t) T. aestivum.

Experimental reference for starch granules identified in the dental calculus and ground stone tools.

(a) Aegilops triuncialis; (b) A. crassa; (c, d) Avena strigosa; (e, f) Setaria italica; (g) Vicia cracca; (h) V. sylvatica; (i) Quercus pubescens; (j) Q. robur; (k) Q. colurna; (l) Cornus mas.

Experimental macroresidues and micropolish associated with grass grains processing compared to macroresidue and micropolish identified on archaeological ground stone tools from the site of Vlasac.

(a–e) Yellowish organic film covering the crystal grains on experimental GSTs used to process oat (a), downy brome (b), wild grass grains (c), and millet (d); smooth domed and flat micropolish developing over the high and low microtopographies associated with oat (Avena barbata) grinding; (f) smooth flat and domed micropolish developing over the surface high and low microtopographies and characterized by narrow microstriations associated with grinding downy brome (Bromus tectorum L.); (g) smooth flat micropolish developed over the high and low microtopographies characterized by sporadic narrow striations associated with grinding wild grass grains (Aegilops ventricosa Tausch); (h) smooth domed polish developed over the high and low microtopographies associated with the grinding of foxtail millet (Setaria italica (L.) P. Beauvois); (i–l) spots of organic film, yellowish in color covering the crystal grains across the surface of archaeological GSTs; smooth domed micropolish identified on archaeological GSTs developing over the high and low surface microtopographies and associated with microstriations (m-o). Starch granules identified on archaeological GSTs. (q) Type I (GST no. INV.80); (r) Type I (GST no. INV.146); (s) Type III (GST no. INV.28); (t) Type VI (GST no. INV.67); (u) Type VI (GST no. INV.10); (v) Type VI (GST no. INV.146); (w) Type I (GST no. INV.71).

Starch granule length in modern wild and domestic cereal species.

(a) Distribution of starch granule length in wild species; (b) distribution of domesticated species; (c) violin plot of comparing the length of starch granules in wild and domesticated species; (d) interquartile ranges (IQRs) of wild and domestic species. IQR corresponds to the difference in the medians of the lower and upper half of the data.

Figure 9—source data 1

Starch granule length in modern wild and domestic cereal species.

Controls for contamination.

(a–w) Evidence of pollutants retrieved from clean working surfaces and dust traps located in different areas of the DANTE Laboratory at Sapienza University of Rome; (x–Ll) dust recorded in storage boxes where groundstone tools were stored at the Faculty of Philosophy, University of Belgrade.


Table 1
Details of dental calculus sampled for the study (n = 60).

*No stable isotope values are currently available for this individual in order to correct the obtained radiocarbon date for the reservoir effect, and the calibrated range should probably be considered too old for its actual age, likely being 200–500 years younger. All calibrated ranges have end points rounded outwards to 5 years. The dates were individually calibrated using OxCal 4.4 and IntCal 20 (Reimer et al., 2020).

SiteBurial no.Period attributionAMS datesCalculus location
Lab code and source14C age (BP)Reservoir effect corrected age (BP)95.4 % confidence, cal BCToothSurfaceWeight (mg)
PadinaPAD20Early Meso17Buccal9.6
PadinaPAD25Early Meso38Buccal9.59
PadinaPAD15Early MesoOxA-17145 (Borić, 2011)9310 ± 448870 ± 638240–777038Lingual9.58
PadinaPAD16aEarly MesoPSU-2407 (Mathieson et al., 2018)9340 ± 358907 ± 668275–781534Buccal9.62
PadinaPAD18bEarly MesoPSU-2376 (Mathieson et al., 2018)9715 ± 409424 ± 559115–855048Lingual9.67
PadinaPAD9Early MesoAA-57771 (Borić, 2011)9920 ± 1009480 ± 1109225–849542, 46Lingual9.59
PadinaPAD11Early MesoOxA-16938 (Borić, 2011)9665 ± 549225 ± 708620–829027Lingual9.57
PadinaPAD12Early MesoBM-1146 (Borić, 2011)9331 ± 588750–835027Lingual9.76
PadinaPAD17Early MesoPSU-2375 (Mathieson et al., 2018)9505 ± 359105 ± 628540–823025Buccal9.64
Lepenski VirLV50Early MesoBA-10651 (Borić et al., 2018)9455 ± 389082 ± 628540–802035Buccal9.99
Lepenski VirLV20Early MesoOxA-39629(this paper)10,268 ± 389928 ± 589740–927048Lingual9.73
PadinaPAD26Early Meso14Buccal9.58
PadinaPAD6Early Meso47Lingual9.65
PadinaPAD2Late MesoBM-1143 (Borić, 2011)7738 ± 516650–646536Lingual9.68
Hajdučka VodenicaHV25/26Late Meso44Buccal9.60
Hajdučka VodenicaHV29Late MesoAA-57774 (Borić, 2011)8151 ± 607711 ± 756690–642548Lingual10.72
Hajdučka VodenicaHV8Late MesoOxA-13613 (Borić, 2011)8456 ± 378016 ± 587075–669548Buccal9.61
Hajdučka VodenicaHV11Late Meso48Buccal9.71
Hajdučka VodenicaHV profil ALate Meso27Buccal9.70
Hajdučka VodenicaHV30Late Meso27Buccal9.56
VlasacVL82cLate MesoBRAMS-2588 (Jovanović et al., 2021a)8035 ± 287595 ± 536590–627042Buccal9.68
VlasacVL2Late Meso14Buccal9.54
VlasacVL80aLate Meso26Lingual9.84
VlasacVL55Late MesoBRAMS-2583 (Jovanović et al., 2021b)8377 ± 297837 ± 637035–650033Lingual9.64
VlasacVL74Late MesoBRAMS-2587 (Jovanović et al., 2021b)8149 ± 28*7315–7055*28Lingual9.70
VlasacVL83Late MesoOxA-5826 (Borić, 2011)8200 ± 907760 ± 1007030–642024Lingual9.62
VlasacVL43Late Meso27Lingual
VlasacVL31Late MesoAA-57777 (Borić, 2011)8196 ± 697756 ± 826900–643026Buccal9.58
VlasacVL45Late MesoAA-57778 (Borić et al., 2004)8117 ± 627677 ± 776655–640038Buccal9.50
VlasacVL70Late Meso17Buccal10.42
VlasacVL79Late MesoBRAMS-2448 (Jovanović et al., 2021b)8005 ± 297565 ± 546565–625016Buccal9.60
VlasacU44Late Meso27Buccal9.96
VlasacH232Late MesoOxA-20702 (Borić, 2011)7725 ± 406640–647028Lingual9.92
VlasacH317Late MesoPSU-2381 (Mathieson et al., 2018)8110 ± 357625 ± 716645–627026, 36Lingual9.73
VlasacU115Late Meso28Buccal9.95
VlasacU326Late MesoPSU-2382 (Mathieson et al., 2018)8045 ± 307728 ± 516645–646517Buccal9.94
VlasacU326Late MesoPSU-2382 (Mathieson et al., 2018)8045 ± 307728 ± 516650–64601, 2Buccal9.1
VlasacU64 x.11/H81Late MesoOxA-20762 (Borić, 2011)8125 ± 457685 ± 646645–643020, 26, 27, 29, 30, 31Lingual9.92
VlasacH341Late Meso1Buccal10.12
VlasacVL48Late Meso34Lingual10.06
VlasacU222 x.18Late Meso2Buccal9.54
Lepenski VirLV28Meso-Neo43Buccal9.58
Lepenski VirLV79aMeso-NeoOxA-25091 (Bonsall, 2008)7605 ± 387119 ± 746220–580533Buccal9.69
Hajdučka VodenicaHV16Meso-Neo36Lingual9.54
Hajdučka VodenicaHV19Meso-Neo37Buccal9.58
Hajdučka VodenicaHV13Meso-NeoAA-57773 (Borić, 2011)7435 ± 706995 ± 836020–572017Lingual9.56
PadinaPAD4Meso-NeoAA-57769 (Ottoni et al., 2021)7518 ± 727078 ± 856080–574548Buccal9.73
PadinaPAD5Meso-NeoAA-57770 (Borić, 2011)7598 ± 727158 ± 856230–584515Buccal8.10
VlasacU24 x.30Meso-Neo32Lingual9.97
VlasacH53Meso-NeoOxA-16544 (Borić et al., 2014)7035 ± 406015–58053, 28, 29Lingual10.04
Lepenski VirLV4Early Neo33Buccal9.64
Lepenski VirLV73Early NeoBA-10652 (Borić et al., 2018)7265 ± 306973 ± 485980–573534Buccal9.69
Lepenski VirLV8Early NeoAA-58319OxA-25207 (Borić et al., 2018)6825 ± 517097 ± 366690 ± 546984 ± 395715–55205985–575044Lingual9.69
Lepenski VirLV32AEarly NeoOxA-5828 (Bonsall et al., 2013)7270 ± 907032 ± 956065–573042, 43, 36Buccal9.77
Lepenski VirLV17Early NeoAA-58320 (Borić et al., 2018)7007 ± 486787 ± 535775–556515Lingual9.10
PadinaPAD30Bronze AgePSU-23792140–176547Buccal9.69
Velesnica2AEarly NeoOxA-19191 (Bonsall, 2008)7409 ± 387196 ± 476220–59308Lingual9.74
Velesnica2DEarly NeoOxA-19210 (Bonsall, 2008)7327 ± 387183 ± 426215–59259Lingual7.2
GârleştiEarly Neo2Lingual8.22
Lepenski VirLV30MedievalOxA-25218 (Bonsall, 2008)427 ± 23AD1440–149016Lingual9.67
Table 2
Details of the microdebris (starch granules and other microremains) found in the archaeological dental calculus samples (PO = pollen; W = wood; Ch = microcharcoal/burnt debris; Gr = grit; P = phytoliths; FE = feathers; FI = fibers; FU = fungi; S = smoke) (n = 51).
SiteBurial labelChronocultural attributionType I TriticeaeType IIAveneaeType III PaniceaeType IVFabeaeType V FagaceaeType VI CornaceaeIndet.Other
1PadinaPAD20Early Meso7431P/10FI/2FE/2W/1Ch/Gr
2PadinaPAD25Early Meso11P/1Ch/Gr
3PadinaPAD15Early Meso>100461P/4PO/1W/1Ch/Gr
4PadinaPAD16aEarly Meso2031P/10FI/8Ch/Gr
5PadinaPAD9Early Meso>2002FI/2FE
6PadinaPAD11Early Meso>10081
7PadinaPAD12Early Meso361211
8Lepenski VirLV50Early Meso111FI;S
9Lepenski VirLV20Early Meso41
10PadinaPAD2Late Meso131711PO/1FI/2FE/2W/1Ch/Gr
11Hajdučka VodenicaHV25/26Late Meso51512P/1PO/2FI/3FE/1Ch/3FU/Gr
12Hajdučka VodenicaHV29Late Meso5353P/1PO/1FI/1FE/13W/1FU/Gr
13Hajdučka VodenicaHV8Late Meso1
14Hajdučka VodenicaHV11Late Meso>10081
15Hajdučka VodenicaHV profil ALate Meso1413PO/1FI/2FE/3W/1Ch/1FU/Gr
16Hajdučka VodenicaHV30Late Meso1
17VlasacU222 x.18Late Meso21P
18VlasacU326Late Meso>6011P
19VlasacVL82cLate Meso4237251P/3PO/1FE/1W/1Ch/Gr
20VlasacVL2Late Meso2122P/1PO/2FI/1FE/2FU/Gr
21VlasacVL80aLate Meso3155511P/4FE/1W/1Ch/1FU/Gr
22VlasacVL55Late Meso611P/1PO/1FI/1W/1FU/Gr
23VlasacVL74Late Meso111P/3PO/17FI/1Ch/Gr
24VlasacVL83Late Meso6811P/1PO/1FE/2W/5Ch/Gr
25VlasacVL43Late Meso>200121122FE/1W/2Ch/1FU/Gr
26VlasacVL31Late Meso18334PO/5FE/1Ch/2FU/Gr
27VlasacVL45Late Meso238201141PO/2FE/2Ch
28VlasacVL70Late Meso331144P/1FI/7Ch
29VlasacVL79Late Meso1P/2FI
30VlasacU44Late Meso32
31VlasacH232Late Meso<100411PO/1FE
32VlasacU115Late Meso1
33VlasacU64 x.11Late Meso>200103242P/4FI/2FE/3Ch/1FU
34VlasacH341Late Meso1
35Lepenski VirLV28Meso-Neo43162P/2PO/1FE/2W/4Ch/Gr
36Hajdučka VodenicaHV16Meso-Neo>2001FU
37Hajdučka VodenicaHV19Meso-Neo11FE
38Hajdučka VodenicaHV13Meso-Neo1
41VlasacU24 x.30Meso-Neo101P/2Ch
43Lepenski VirLV4Early Neo131
44Lepenski VirLV73Early Neo12917P/2PO/17FI/1FE/3FU/Gr
45Lepenski VirLV8Early Neo1141W
46Lepenski VirLV32AEarly Neo8>2001P/2FE
47Lepenski VirLV17Early Neo145
48Velesnica2AEarly Neo4FU
49Velesnica2DEarly Neo122P/2FU
50GârleştiEarly Neo11P/1Ch
51Lepenski VirLV30Medieval1241P/1PO/4Ch/1FU/Gr
Table 3
Late Mesolithic ground stone tools from the site of Vlasac.
Inv. no.Archaeological contextShapeTool typeLength (cm)Width (cm)Thickness (cm)Weight (g)Volume (cm3)State of preservationPDMMicropolish descriptionMicropolish locationMicrostriation descriptionMicrostriation orientationCristal grain modificationGesture
10a1-IIISubangularHandstone/grinder12.710.57.881542645PreservedLight soil concretionSmooth and domedHigh microtopographiesShort narrow with a matt bottomUnidirectionalYMixed
13a1-VIIIRoundHandstone/grinder118.165.68680287PreservedNoneSmooth and domed with sporadic pitsHigh and low microtopographiesNANANLongitudinal
23BV/C/IV-XSubangularIndeterminable11.78.798.76823367PreservedLight soil concretionRough to smooth with domed and flat spotsHigh and low microtopographiesNANANLongitudinal
28BIII-C/VOvalPassive base13.311.87.181283499FracturedNoneSmoothHigh microtopographiesNANAYLongitudinal
56A/II–XIIIRoundPassive base15.914.87.71038368PreservedHeavy surface concretion on one surfaceSmooth domed and flatHigh microtopographiesNANAYMixed
63b/17-XVRoundHandstone/grinder8.46.64.47403141PreservedLight surface abrasionRough to smooth with reticulated and flat spotsHigh and low microtopographiesNANANLongitudinal
65C/I–VIRoundPasssive base10084.355.5680298BrokenFracturesSmooth domed and reticulatedHigh microtopographiesNarrow with a matt bottomMixedNLongitudinal
66C/I II/VRoundIndeterminable9.58.57.61170437PreservedNoneSmooth domed and crateredHigh and low topographiesNANAYMixed
67C/I-C/II–IIISubangularPassive base10.88.45.91633253BrokenLight soil concretion and surface abrasionSmooth and reticulatedHigh microtopographiesShort and deep with a matt bottomUnidirectionalNLongitudinal
71C/I–VRoundHandstone/grinder72.157.945.9309119PreservedNoneSmooth domed to flatHigh microtopographiesLong and shallow with a polished bottomMixedYLongitudinal
75b/18VSubangularHandstone/grander5.535.293.4513757BrokenFracturesSmooth and domedHigh and low microtopographiesShort narrow with a polished bottomUnidirectionalNLongitudinal
80C/II-II/6OvatePassive base9.858.353.72547225BrokenNoneSmooth domedHigh microtopographiesShort and narrow with a matt bottomUnidirectionalYLongitudinal
134b/V3-XIISubangularIndeterminable12.29.578.131433565PreservedNoneSmooth domedHigh and low microtopographiesShort deep with a matt bottomUnidirectionalYLongitudinal
141B/I 0–8.9RoundHandstone/grinder10.89.929.22370NAPreservedSoil concretionSmooth domed and flatHigh microtopographiesNANAYLongitudinal
146B/I-below hearth 9RoundHandstone/grinder6.655.74.66275106PreservedLight soil concretionSmooth domed and reticulatedHigh microtopographiesShort narrow with a matt bottomUnidirectionalNLongitudinal
162A/16XRoundHandstione/grinder10.69.37.521143424PreservedNoneSmooth domedHigh and low microtopographiesShirt narrow with a matt bottomUnidirectionalYLongitudinal
167a/15-VIIRoundIndeterminable8.948.825.65611241BrokenLight surface abrasionRough granular and domedHigh and low topographiesNANAYOrthogonal
Table 4
Summary statistics of the length (μm) of wild grass grains and domestic cereal starch granules.
SpeciesMin.Max.MeanMedianSt. Dev.RangeIQR
A. caudata5.2959.321.616.715.175.29–59.3326.55
A. comosa7.9534.521.521.79.787.95–34.5420.09
A. crassa13.3853.735.333.711.0913.38–53.6919.08
A. cylindrica8.5254.024.223.713.078.52–54.0521.6
A. geniculata11.6147.026.326.08.3911.61–47.0312.87
A. neglecta recta10.5462.735.036.214.4610.54–62.7126.5
A. peregrina9.8453.627.825.99.899.84–53.6211.34
A. speltoides tauschii13.2540.023.522.25.9313.25–39.978.39
A. triuncialis5.6050.–50.0615.18
A. uniaristata14.3562.438.239.312.8714.35–62.3822.83
A. ventricosa14.1040.026.325.77.4414.10–40.0412.77
H. vulgare distichon5.1929.619.722.28.125.19–29.598.32
T. dicoccum6.1741.516.512.88.666.17–41.5514.07
T. monococcum6.6836.620.–36.6110.44
Table 4—source data 1

Summary statistics of the length of wild grass grains and domestic cereal starch granules.

Table 5
Pairwise Wilcoxon test performed on the length distribution of modern starches from Aegilops, Hordeum, and Triticum species (p value: not significant/ns >0.05; *<0.05; **<0.01; ***<0.001).
A. caudataA. comosaA. crassaA. cylindricaA. geniculataA. neglecta rectaA. peregrinaA. speltoides tauschiiA. triuncialisA. uniaristataA. ventricosaH. vulgare distichonT. dicoccum
A. comosans
A. crassa******
A. cylindricansns***
A. geniculatans****ns
A. neglecta recta******ns******
A. peregrina*****nsns**
A. speltoides tauschiinsns***nsns****
A. triuncialis*****nsns*ns*
A. uniaristata******ns******ns*********
A. ventricosans****nsns***nsnsns***
H. vulgare distichonnsns***ns*******************
T. dicoccumns***************************ns
T. monococcumnsns***ns*******************ns*
Table 5—source data 1

Length of modern starch granules of Aegilops, Hordeum, and Triticum species.


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  1. Emanuela Cristiani
  2. Anita Radini
  3. Andrea Zupancich
  4. Angelo Gismondi
  5. Alessia D'Agostino
  6. Claudio Ottoni
  7. Marialetizia Carra
  8. Snežana Vukojičić
  9. Mihai Constantinescu
  10. Dragana Antonović
  11. T Douglas Price
  12. Dušan Borić
Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture
eLife 10:e72976.