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

Abstract

Forager focus on wild cereal plants has been documented in the core zone of domestication in southwestern Asia, while evidence for forager use of wild grass grains remains sporadic elsewhere. In this paper, we present starch grain and phytolith analyses of dental calculus from 60 Mesolithic and Early Neolithic individuals from five sites in the Danube Gorges of the central Balkans. This zone was inhabited by likely complex Holocene foragers for several millennia before the appearance of the first farmers ~6200 cal BC. We also analyzed forager ground stone tools (GSTs) for evidence of plant processing. Our results based on the study of dental calculus show that certain species of Poaceae (species of the genus Aegilops) were used since the Early Mesolithic, while GSTs exhibit traces of a developed grass grain processing technology. The adoption of domesticated plants in this region after ~6500 cal BC might have been eased by the existing familiarity with wild cereals.

Editor's evaluation

By combining starch grains analysis from dental calculus and grinding implements, the authors demonstrate the consumption of a large variety of plants by Mesolithic foragers and Neolithic farmers in the Danube Gorges of the Balkans. The data and analyses advance debates on the intensification of plant selection prior to their strict domestication.

https://doi.org/10.7554/eLife.72976.sa0

eLife digest

Before humans invented agriculture and the first farmers appeared in southwestern Asia, other ancient foragers (also known as hunter-gatherers) in southeastern Europe had already developed a taste for consuming wild plants. There is evidence to suggest that these foragers were intensely gathering wild cereal grains before the arrival of agriculture. However, until now, the only place outside southwestern Asia this has been shown to have occurred is in Greece, and is dated around 20,000 years ago.

In the past, researchers proposed that forager societies in the Balkans also consumed wild cereals before transitioning to agriculture. But this has been difficult to prove because plant foods are less likely to preserve than animal bones and teeth, making them harder to detect in prehistoric contexts.

To overcome this, Cristiani et al. studied teeth from 60 individuals found in archaeological sites between Serbia and Romania, which are attributed to the Mesolithic and Early Neolithic periods. Food particles extracted from crusty deposits on the teeth (called the dental calculus) were found to contain structures typically found in plants. In addition, Cristiani et al. discovered similar plant food residues on ground stone tools which also contained traces of wear associated with the processing of wild cereals.

These findings suggest that foragers in the central Balkans were already consuming certain species of wild cereal grains 11,500 years ago, before agriculture arrived in Europe. It is possible that sharing knowledge about plant resources may have helped introduce domesticated plant species in to this region as early as 6500 BC.

This work challenges the deep-rooted idea that the diet of hunter-gatherers during the Palaeolithic and Mesolithic periods primarily consisted of animal proteins. In addition, it highlights the active role the eating habits of foragers might have played in introducing certain domesticated plant species that have become primary staples of our diet today.

Introduction

Forager knowledge and consistent use of wild cereals are still debated and poorly documented outside of the assumed centers of domestication in southwestern Asia (Kotsakis, 2003). For some time, it has been claimed that in the Balkans some forms of intense gathering or incipient human management of local plant and animal species might have occurred before the full-blown transition to the Neolithic (Clarke, 1978; Halstead, 1996; Kotsakis, 2003; Kotzamani and Livarda, 2014; Srejović, 1988; ; y’Edynak and Fleisch, 1983), partly due to the region’s geographical proximity to the Near East. However, the hypothesis of a systematic use of wild grasses of the Poaceae family (e.g., Aegilops spp.; Hordeum spp.) during the Mesolithic remains to be verified in this region.

In southeastern Europe, specifically in its Mediterranean zone, where one would expect a greater spectrum of small seeded grasses, fruits, and nuts, forager consumption of wild cereals is well documented only at Franchthi Cave in Greece. Here, wild barley (Hordeum sp.) appears in the archaeobotanical record starting in the Late Upper Palaeolithic and throughout the Mesolithic, along with oat (Avena sp.), pulses (Lens sp. Mill.), bitter vetch (Vicia ervilia (L.) Willd.), almond (Prunus amygdalus Batscht), and terebinth (Pistacia cf. lentiscus L.) (Hansen, 1991; Van Andel et al., 1987). More recently, at Vlakno Cave in Croatia, starch granules of a wild species of barley (Hordeum spp.), along with those of oat (Avena spp.), were found in the dental calculus of a Mesolithic forager individual, dating to the late eight millennium cal BC burial (Cristiani et al., 2018).

Besides this type of evidence, data about the increase of cereal-type pollen in the Late Mesolithic (LM) come from palynological spectra from across Europe. Although the exclusive reliance on pollen evidence for inferring cultivation can be problematic, consistent evidence for interventions in the forest canopy, marked as disturbances in pollen spectra, might suggest anthropogenic activity. Due to low dispersal rates of cereal-type pollen grains as well as Cerealia-type pollens, their very presence in pollen spectra could be highly indicative of anthropic origin of disturbance phases (Edwards, 1989), and could be interpreted as forest clearances.

Recent methodological advances in our ability to analyse microresidues in the form of microremains along with surface modifications and microresidues on ground stone tools (henceforth GSTs) (Barton et al., 2018; Dubreuil and Nadel, 2015; Radini et al., 2017) have the potential to contribute to this old debate about Balkan and other prehistoric foragers’ familiarity with plant species. Moreover, far from seeing foragers as passive recipients of novelties arriving from Neolithic groups at the time of agricultural transitions, there is now growing evidence of the active role of hunter–gatherers in shaping their landscape ecologies, including plant management, and manipulation of ecosystems through niche constructing (Lombardo et al., 2020; Rowley-Conwy and Layton, 2011; Smith, 2012).

We examine these pertinent issues in hunter–gatherer research by studying dental remains and GSTs found at Mesolithic and Neolithic sites in the Danube Gorges area of the north-central Balkans between present-day Serbia and Romania (Figures 1 and 2; Figure 3). This is one of the best researched areas of Europe regarding the Mesolithic–Neolithic transition period with more than 20 sites spanning the duration of the Epipalaeolithic through to the Mesolithic and EN (~13,000–5500 cal BC) (Bonsall, 2008; Borić, 2011; Borić, 2016; Radovanović, 1996; Srejović, 1972). Open-air sites began appearing in the archaeological record with the start of the Holocene warming on river terrace promontories in the vicinity of strong whirlpools, narrows, and rapids of the Danube, which facilitated intense fishing operations (Borić, 2011). The Early and Middle Mesolithic (~9600–7300 cal BC) deposits at many sites are damaged by later Mesolithic and Neolithic intrusions, but a number of burials have directly been dated by Accelerator Mass Spectrometry to these early phases. From the Early Mesolithic (EM) onwards, these sites became places for a continuous interment of the dead (Borić, 2016; Borić et al., 2014; Radovanović, 1996), thus creating a substantial mortuary record, which is in the excess of 500 individuals. Osteological collections allowed for a host of bioarchaeological analyses to be applied on this material (Bonsall et al., 2013; Borić et al., 2004; Borić and Price, 2013; Mathieson et al., 2018). Fishing seems to have remained one of the main subsistence foci throughout the Holocene, with a possible intensification during the LM (~7300–6200 cal BC), the period that saw an intense inhabitation of the area, with recognizable features in the archaeological record, such as stone-lined rectangular hearths and abundant primary burials placed as extended inhumations parallel with the Danube River. Between ~6200 and 5900 cal BC, there are clearest indications based on both material culture associations and isotope and genomic data (Borić and Price, 2013; Mathieson et al., 2018) that the local Mesolithic foragers came into contact with the first Neolithic groups appearing in this region, and who likely founded several new sites in this area, especially in the downstream part of the region. These documented encounters of two different cultural groups are most clearly observed at the site of Lepenski Vir (Borić, 2016; Borić et al., 2018). After ~5900 cal BC, it seems that the forager cultural specificity was lost and that various sites remained to be used as typical EN Starčevo culture villages up until ~5500 cal BC, when most of the previously used locales were abandoned.

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.

While sources of animal protein in the diet of Mesolithic–Neolithic inhabitants of the area are well understood by now, the significance of plant foods in this region has remained less well known. Nonflaked tools such as pestles, grinders, crushers, and anvils have recently been associated with fruit, seed, and nut processing in early prehistoric and ethnographic contexts (de Beaune, 2004; Dubreuil and Nadel, 2015; Hamon et al., 2021; Pardoe et al., 2019; Wright, 2017). However, this category of artifacts is primarily documented from the LM onwards and only sporadically associated with earlier periods in the region of the Danube Gorges (Antonović, 2006; Borić et al., 2014; Srejović and Letica, 1978).

Such a lack of evidence about the role of plant foods in Mesolithic stems from very limited attempts to recover macrobotanical remains through intense sediment flotation, which has only been applied at two more recently excavated sites in this region—Schela Cladovei (Mason et al., 1996) and Vlasac (Borić et al., 2014). Despite a generally poor preservation of plant remains due to taphonomic issues, recent carpological analyses indicated a relatively wide spectrum of wild resources available to the local Mesolithic foragers. These included drupes, fruits, and berries (Marinova et al., 2013), among which cornelian cherry (Cornus mas L.), hazelnut (Corylus avellana L.), and elderberry (Sambucus nigra L.) were the most frequent taxa (Filipović et al., 2010; Marinova et al., 2013). Molecular record of C. avellana and S. nigra was also found preserved in the dental calculus of two LM individuals from Vlasac, which underwent metagenomic analysis (Ottoni et al., 2021). Moreover, in the study region of the Danube Gorges, at the site of Vlasac, presumed human palaeofeces contained pollen of Amaranthaceae and Cerealia (Cârciumaru, 1978). Evidence from the Mesolithic levels at the site of Icoana, located in the same region, has suggested local cereal cultivation (Cârciumaru, 1973). Pollen provides only indirect evidence for consumption and cultivation and, unfortunately, the 1960–1970s excavations of the sites in the Danube Gorges did not involve any flotation of contextual units associated with burning in domestic contexts. While the extensive program of flotation at the site of Vlasac in the course of more recent work (2006–2009) has not led to the discovery of macrobotanical remains of wild or domesticated cereal grains, it should also be emphasized that the new excavations at this site have taken place in a marginal, upslope part of the site with little or no evidence of domestic features associated with burning that might have preserved macrobotanical remains (Borić et al., 2014). More recently, starch granules identified in dental calculus within a sample of 12 individuals provided evidence for the consumption of domesticated cereals at the site of Vlasac during the LM (Cristiani et al., 2016).

Hence, plant debris recovered in human dental calculus constitute the most reliable line of evidence to unveil the role of plants in the local forager diets. Our previous pilot study has provided the first evidence of domesticated cereal grains and plant food consumption in the LM from the analyses of dental calculus (Cristiani et al., 2016). Based on a more robust sample of human dental calculus, which now also involves numerous EM individuals not included in our previous study, and complementary functional evidence from the most conspicuous assemblage of Mesolithic GSTs from the Danube Gorges area, the present study details forager use of certain species of the Triticeae tribe and other plant foods in the region already since ~9500 cal BC.

Results

Dental calculus

Starch granules were almost ubiquitous in the analyzed individuals and many of them were found still in part associated with dental calculus remains. Six morphotypes have been retrieved in this study (Table 1, Table 2, Table 3). We have not attempted the identification of starch granules less than 5 μm to avoid misinterpretation of transitory and small storage starch granules (Haslam, 2004).

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
39PadinaPAD4Meso-Neo172PO/1FI/3FE/4W/2Ch/1FU/Gr
40PadinaPAD5Meso-Neo6
41VlasacU24 x.30Meso-Neo101P/2Ch
42VlasacH53Meso-Neo22>20051FE/1W
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
Total>1446324>40943824284
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

Type I

Size, shape, morphology, and bimodal distribution that characterize granules of this type are encountered in Europe only in the members of the plant tribe Triticeae (Poaceae family) and considered diagnostic features for taxonomic identification (Henry and Piperno, 2008; Stoddard, 1999; Yang and Perry, 2013). Such distribution involves the presence of large granules (A-Type), mostly with a clear, round to suboval in 2D shape, ranging between 21.1 and 62.7 μm in maximum dimensions (mean size of 41.9 μm), lenticular 3D shape with equatorial groove always visible, a central hilum and high density of deep lamellae concentrated in the mesial part; and small granules (B-Type) with round/suboval shapes, a central hilum, generally smaller than 10 μm (Geera et al., 2006; Stoddard, 1999; Yang and Perry, 2013). A-Type granules possess diagnostic features while smaller B-Type granules are rarely diagnostic to taxa (Yang and Perry, 2013). However, in our archaeological population, variability in the proportion and dimension of small B-Type granules has been noticed, resulting in a unimodal granule size distribution without a clear distinction between A and B granules in some cases. Several studies (Howard et al., 2011; Stoddard and Sarker, 2000) suggested that this characteristic is common in the species of the genus Aegilops of the Triticeae tribe and can be attributed to both environment (Blumenthal et al., 1995; Blumenthal et al., 1994) and genetics (Stoddard and Sarker, 2000). A unimodal starch granule size distribution characterized by normal A-Type granules and a lack/reduced quantity of B-Type granules was also evident in our modern reference collection of local Aegilops species (Figure 6). Based on these observations, Type I category was further divided into two subtypes (Ia and Ib). In subtype Ia, B-Type granules are small, dimensionally uniform (up to 12 μm) and round in shape (Figure 6). Conversely, subtype Ib is characterized by a high variation in starch granule size not allowing for a distinction between A- and B-Type granule, resulting in a unimodal distribution (Figure 9).

Type I (Ia and Ib) is very common in the analyzed samples (Table 2), as already emphasized in our earlier study albeit in different quantities (Cristiani et al., 2016). These starch granules were documented, often lodged in the amyloplast, in most of the analyzed Mesolithic individuals (5 for EM, 16 for LM, 5 for M/N), and in 5 EN individuals of our population (Table 2). A-Type granules recovered in EM and most of the LM individuals were very large, mostly with a clear, round shape, central hilum, and high density of deep lamellae mainly concentrated in the mesial part of the granules. Based on literature (Henry et al., 2011; Yang and Perry, 2013) and our extensive experimental and statistical results on modern botanical collection (Table 4; Table 5; Figures 6, 7 and 9), we confirm that these characteristics are consistent with A-Type granules of most species of the Triticeae tribe.

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. triuncialis5.6050.128.228.211.245.60–50.0615.18
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. monococcum6.6836.620.119.17.116.68–36.6110.44
Table 4—source data 1

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

https://cdn.elifesciences.org/articles/72976/elife-72976-table4-data1-v3.docx
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.

https://cdn.elifesciences.org/articles/72976/elife-72976-table5-data1-v3.xlsx

Subtype Ia

A few LM and M/N individuals (e.g., HV11 and 16, H53, 64.x11, H327, H232) yielded a combination of oval A-Type granules and uniformly small, round B-Type granules (Figure 4m). Our previous claims that this pattern is a recognizable feature of the domestic species of the tribe Triticeae (e.g., Triticum spp./Hordeum spp.) (Figure 6 and 9; Cristiani et al., 2016) are now further supported by a new morphometric analysis of both domestic and wild Triticeae species (Figure 9). Moreover, in the same individuals, A-Type granules could show cratered appearance (Figure 4). Similarly, in the EN individuals, lenticular and oval/suboval A-Type granules with equatorial groove and, an often visible, cratered surface are always associated with very small and uniformly shaped B-Type granules. Type A granules appear damaged in few EN individuals, which may be linked to enzymatic digestion (salivary amylase) although plant food processing could also result in starch damage based on experimental results (Soto et al., 2019; Zupancich et al., 2019).

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).

Subtype Ib

Significant unevenness in granule dimensions and shape was recorded in the EM and most of the LM individuals (e.g., PAD9 and VL45). Granules in this subtype are dimensionally variable and their shapes can range from round to oval (Figure 4b). The well-known limitations in the inclusion, preservation, and recovery of plant debris in dental calculus (Radini et al., 2017) might be responsible for not recognizing this subtype previously.

Type II

Starch granules attributed to this type consist of large aggregates as well as clustered polyhedral/irregular granules (main axis ranging from 5 to 15 μm). They were retrieved from 10 individuals (2 EM, 11 LM, 2 M/N, and 3 EN) (Table 2; Figure 4g). The identification of archaeological specimens is based on published records (Mariotti Lippi et al., 2015) and our experimental reference (Avena barbata L., A. strigosa Schreb., and A. fatua L.) (Figure 7). Granules of this morphotype were grouped in the tribe Aveneae/Poeae based on the fact that such large aggregates are found mostly in the genus Avena L. (oat), which is very common in the region.

Type III

Starch granules attributed to this type are characterized by a polyhedral to subpolyhedral 3D morphology, a central hilum, and fine cracks. They were recovered in 16 individuals (5 for EM, 16 for LM, 2 for M/N, and 1 for EN) (Table 2; Figure 4c and Figure 5b, c). These features are consistent with starch granules of the tribe Paniceae of the grass family Poaceae and very well known in ancient starch research (Madella et al., 2013). In our sample, starch granules assigned to type III reach 21 μm of maximum width, which falls within the size range found in several species of Setaria spp., Panicum spp., and Echinochloa spp. (Lucarini et al., 2016; Figures 6 and 7). Small granules characterized by a round to subpolyhedral 3D morphology, and a central open hilum have been attributed to the tribe Andropogoneae and are here described under the general group of ‘millets’ as is common practice (Madella et al., 2013).

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.

Type IV

Starch granules of this type are identified in 10 individuals (2 EM, 8 LM, 1 M/N, and 2 EN) and are recovered above 6 granules per specimen, with the exception of the only EN individual (Table 2). Diagnostic morphological characteristics for Type IV granules are known in ancient starch research and include a reniform shape in 3D, a collapsed/sunken hilum forming a deep fissure along almost the entire granule, and a size ranging between 12 and 35 μm (Henry et al., 2011). Small cracks were observed departing from such hilum and were very evident under cross-polarized light. In most cases the extinction cross was very bright and showed several lateral arms diverging from the hilum in correspondence with the cracks (Figure 4h). Moreover, lamellae were visible toward the outer part of the granules. All these features are very peculiar and diagnostic of starch granules included in the species of the plant family Fabaceae (Henry et al., 2011), which is mostly known for its several edible domesticated species of legumes (e.g., Lens culinaris Medikus, Vicia faba L., and Pisum sativum L.), but also has a number of wild edible such as vetches (Vicia spp.). While many edible species of the family Fabaceae grow in the Balkans (e.g., Vicia sativa L., V. cracca L., V. hirsuta, V. ervilia, Lathyrus pratensis L., and L. sylvestris L.), an identification at species or genus was not possible due to overlaps in shape and size of starch granules at tribe level, which were observed in our modern reference collection (Figure 7).

Type V

Few starch granules attributed to this type have been identified in eight individuals (2 EM, 5LM, nd 1MN) (Figures 4d and 5a; Table 2). Starch granules reach 23 μm in length and are mostly triangular with round corners and/or have an irregular oval shape (Figure 5a). Overall, lamellae can rarely be visible. The granules show a linear fissure in the center and sometimes the hilum appears as a wide depression. Under polarized light, the hilum is mostly centric while the extinction cross has bent arms. This type was found to have a very close visual match with the starch found in acorns of oaks (Quercus spp.), a Fagaceae member and well known in ancient starch research (Liu et al., 2015; Figure 7i, j).

Type VI

Granules ascribed to this type have been identified in two individuals (1 LM and 1 M/N) (Table 2). They are characterized by a round 3D morphology and a central hilum, which appears as a wide depression, and no lamellae or facets (Figures 4i and 5n–p). Zarrillo and Kooyman, 2017 consider these morphological features diagnostic of some species of drupes and berries. In our sample, starch granules of this morphotype can reach 12 μm of maximum width, which is beyond species of berries and drupes in the Rosaceae family known in literature (Zarrillo and Kooyman, 2017) and in our modern reference (e.g., Prunus spinosa L.). Based on our experimental record, we assign type VI to species of the family Cornaceae (e.g., C. mas) (Figure 7), the remains of which are documented at Vlasac (Filipović et al., 2010).

In addition to starch granules, 42 phytoliths were retrieved in 24 individuals (EM = 4; LM = 13; MN = 2; N = 5). Mostly, short cells, commonly produced in leaves, stems and inflorescences, were identified and attributed to Pooid grasses. Multicellular structures of long cells were identified in Mesolithic individuals (HV25/26, VL70, VL79, and U222) (Figure 4j and k; Figure 5m). Of particular relevance is the recovery of multicellular phytoliths with dendritic appearance. It was observed at least in one case, still embedded in the dental calculus (Figure 4k). Single or multicellular dendritic structures were also identified in Neolithic individuals (VEL-2D and Gârleşti) (Figure 4w, x). A single polylobate cell was found in one LM individual (U64 x.11). With the exception of dendritic structures, characterizing grass inflorescences, different nondietary reasons may be suggested for the inclusion of phytoliths in dental calculus (i.e., accidental ingestion, inhalation, dust in the environment generated by the use of grasses in a variety of activities and uses, such as flooring and kindling) (Norström et al., 2019).

Ground stone tools

Diagnostic use-wear and residues are identified on 44 GSTs from the site of Vlasac. Analyzed tools included functional categories such as handstones (e.g., grinders and crushers) as well as passive bases (anvils) (Table 3). All of the tools are made of sandstone, characterized grains ranging in size between 0.2 and 1 mm densely distributed within the matrix. The combination of different functional modifications (i.e., flattened surfaces, pitted areas, rounding, etc.) on the single specimens suggests the long and complex life histories of the artifacts, often used in different activities. Within the tools displaying diagnostic use-wear, a total of 17 GSTs have surfaces bearing functional areas positively associated with plant food processing (Table 3; Figure 3). The analysis conducted at low magnification on these tools revealed macrotraces resulting in leveled surface crystal grains, sometimes covered by spots of yellowish organic film (sometimes striated) and white compacted powder (Figure 4Z, Aa). At a high magnification, high and low microtopographies of the GST surfaces are affected by a smooth domed, and sometimes striated, micropolish (Figure 4Bb, Cc). The aforementioned combination of use-wear and macroresidues are commonly associated with GSTs used as handstones for crushing and grinding grass grains and/or fruits, such as hazelnuts and/or acorns in our experimental record (Cristiani and Zupancich, 2020; Figure 8).

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).

A total of 137 starch granules have been retrieved from the surfaces of the GSTs characterized by plant-related functional microscopic features. The optical and morphological properties of the starch granules support their attribution to morphotypes already documented in dental calculus from the Danube Gorges sites: Type I assigned to caryopses of the tribe Triticeae (76) (Figure 4Dd, Ee, Ff); Type IV, assigned to the Fabaceae family (13) (Figure 4Ii); Type III assigned to the tribe Paniceae of the grass family Poaceae (16) (Figure 4Gg); and type VI, assigned to berries of the family Cornaceae (32) (Figure 4Hh, Jj).

In sum, several hundreds of starch granules and phytoliths of grass grains of the Triticeae tribe have been identified in the analyzed dental calculus of the Mesolithic population in the Danube Gorges. In addition, residues and use-wear identified on GSTs from LM Vlasac show the existence of a plant food processing technology during this period aimed at preparing a coarse-grained flour through a combination of pounding and grinding gestures (Table 3; Figure 3 and Figure 9). Grit particles, often retrieved in the analyzed dental deposit (Table 2), further confirm the use of sandstone GSTs in food processing. The conclusion about the consumption of partially processed grains is corroborated by the presence of starch granules still lodged in their amyloplast on Mesolithic GSTs and dental calculus, as suggested in our previous study (Cristiani et al., 2016). Interestingly, A-Type granules in EN dental calculus are generally retrieved singularly and exhibit a damage pattern observed when producing fine-grained flour experimentally only through prolonged bidirectional grinding (Dietrich et al., 2019). The pattern of bidirectional grinding is not documented on the examined LM GST from Vlasac, suggesting the existence of two different grain processing modalities typical of respective Mesolithic and Neolithic cultural traditions.

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.

https://cdn.elifesciences.org/articles/72976/elife-72976-fig9-data1-v3.xlsx

Discussion

For some time now, there has been a recognition of the importance of plant foods in forager diets, based on both archaeological and ethnographic evidence (Clarke, 1978; Lee et al., 1968). Research on mineralized dental plaque has significantly advanced our awareness about ancient preagrarian food choices in Europe, Asia, and Africa, thanks to the dental plaque’s potential to preserve plant microremains (Buckley et al., 2014; Cristiani et al., 2018; Cristiani et al., 2016; Cummings et al., 2016; Nava et al., 2021; Norström et al., 2019). However, in many archaeological case studies dating to early prehistory, preservation or recovery biases render plant food evidence invisible. In exceptional cases, good preservation has allowed for the remains of plant macroremains to be found, such as wild cereals at the Epipaleolithic site of Ohalo II in Israel, dating to 23 kya (Nadel et al., 2015; Piperno et al., 2004), or parenchyma remains at the Gravettian site of Dolní Věstonice in the Czech Republic (Pryor et al., 2013). On the other hand, microremains of oat caryopses have been detected on GST found at the Gravettian site of Paglicci cave in Italy (Mariotti Lippi et al., 2015). In a seminal synthesis about plant foods in the European Mesolithic, Zvelebil, 2014 reviews macro- and microbotanical, palynological, artifactual (antler hoes, mattocks, GST), and human osteological (dental size and presence of caries) evidence for the consumption of nuts and fruits by European Holocene foragers, arguing for a form of niche constructing in temperate woodlands by means of deliberate forest clearance in order ‘to increase the productivity of nut and fruit trees and shrubs, wetland plants, and possibly native grasses’ (Zvelebil, 2014). The emphasis is on the existence of some form of husbandry of wild plant species, which did not necessarily lead to domestication. Furthermore, between 200 and 450 indigenous European edible plants (grass seeds, nuts, fruits, roots, tubers, and pulses) are found concentrated in wetland (coastal, lacustrine, and riparian) habitats (Clarke, 1978). Despite preservation and recovery problems, hundreds of Mesolithic sites across Europe have yielded the remains of hazelnuts, acorns, water-chestnuts, and other remains (Zvelebil, 2014).

In this paper, two complementary lines of evidence that we examined provide the first unambiguous and direct support for the consumption and processing of Poaceae grains among other types of edible plants by the Early Holocene foragers in the Danube Gorges area. The chronological framework of the analyzed sample suggests that this interest in and familiarity with various species of wild grasses of the Triticeae tribe (namely grass grains of the genus Aegilops) dates back to at least ∼9500 cal BC. Macrobotanical remains belonging to this genus have not been recovered in Mesolithic and EN sites in the central Balkans (Cristiani et al., 2016: 10301). However, such absence in the archaeobotanical record could be the result of a host of taphonomic and recovery problems and should not be used to exclude the use of this genus during the Mesolithic (contra Cristiani et al., 2016: 4). Newly obtained evidence from the analysis of 61 individuals, which now also involves several EM individuals, lead us to suggest that Aegilops species were consumed in the region since the beginning of the Holocene. The mentioned difficulties associated with the recovery and preservation of botanical remains in local early prehistoric forager contexts along with some voids in the extant data regarding plant use by Mesolithic groups underline the significance of our findings based on the application of relatively recent advances in dental calculus and GST analyses.

We have previously argued that three LM individuals from the site of Vlasac dated to the mid-seventh millennium cal BC (H53, 64.x11, and H232), as well as two presumed EN individuals from Lepenski Vir (8 and 20) exhibit starches consistent with domesticated cereal species, such as Triticum monococcum L. (einkorn wheat), Triticum dicoccum L. (emmer wheat), and/or Hordeum distichon L. (barley) (Cristiani et al., 2016). This observation was based on the bimodal pattern of starch granules distribution, commonly attributed to domestic species and absent in most of the wild species of the genus Aegilops (Howard et al., 2011; Stoddard, 1999). This bimodal pattern is now further retrieved in two other individuals from two different sites (H326 from Vlasac and HV16 from Hajdučka Vodenica) dating to the LM and thus predating the arrival of full-blown agriculture in the region. However, burial 20 from Lepenski Vir, previously published as dating to the EN, has recently been directly AMS-dated to the EM (Table 1). This new chronological attribution does not correspond with our expectations that domesticated grains were introduced in the Danube Gorges area only in the LM. At the face of the current evidence, we explain this inconsistency in our results by arguing that the admittedly small number of starch granules found in this archaeological specimen might have affected the visibility of the potential variation in A-Type vs. B-Type population. Moreover, a large variability of starch granule distributions among different species of the Triticeae tribe has been acknowledged in the literature (Henry and Piperno, 2008; Yang and Perry, 2013) and supported by our experimental reference (Figure 9). Furthermore, fluctuations in environmental and growing conditions have also been recognised as relevant factors affecting starch granule size distribution (Stoddard, 1999; Stoddard and Sarker, 2000).

In addition to starch granules, other microremains, such as phytoliths and burnt debris, were recovered in the dental calculi of the analyzed Mesolithic population (Figure 5l, m, y). The paucity of archaeological phytoliths calls for caution when interpreting their dietary origin. Yet, the presence of few dendritic phytoliths in local forager dental calculus is likely related to plant consumption, as such microremains have experimentally been associated with mechanical destruction of husks and culms of Pooids by grinding (Portillo and Albert, 2014). In the investigated population, phytoliths could potentially provide means of understanding the use of plants as kindling and exposure to potential respiratory irritants generated during daily life activities, but their pathways are too many to narrow them down, and further work is required to better understand the origin of burnt material in dental calculus (Radini et al., 2019; Radini et al., 2017; Scott and Damblon, 2010). The retrieval of phytoliths from herbaceous plants that appear burnt could suggest that plant materials, potentially harvested, were used in a diverse range of activities, including kindling and roofing. Phytoliths of such plants might have also been naturally present in the environment, water, and soil (Norström et al., 2019). Such burnt debris may have reached the mouth by accidental ingestion through food and/or breathing. Other microdebris, such as nettle and wood fibers, might have also been linked to textile. Pathways to wood inclusion in calculus vary from the use of a toothpick to crafting activities (Radini et al., 2016) and the production and maintenance of weapons, such as arrowheads.

For the moment, it remains unclear to what extent plant foods, and species of the Poaceae family, contributed to the diet of the Danube Gorges foragers, and it is equally difficult to be more specific about the range of activities involved in their acquisition prior to processing and consumption – from sporadic collecting of wild grasses to forest clearances, or some form of indigenous system of plant management. Yet, it seems clear from our data that this specific subsistence practice was passed over generations in this regional context up to the first contacts of these foragers with Neolithic, farming groups in the second half of the seventh millennium BC. Previously, we suggested that some sort of exchange between LM foragers and first Neolithic groups in the southern Balkans might have allowed for the introduction of the domesticated species of Triticeae in the Danube Gorges area ∼6500 cal BC (Cristiani et al., 2016). Now, our extended study seems to suggest that this introduction of domesticated cereals, that is more productive cereal strains, from southwestern Asia was preceded by several millennia of collecting and consumption of native grass grains. Adoption of previously unknown plant foods is often facilitated by those local foods that in shape and taste resemble the new arrivals (Livarda, 2010). Incipient management practices on certain wild plant taxa have been documented among nonagricultural societies for environmental and/or cultural reasons, for protecting or promoting the relative abundance of a species, or for reducing the energy involved in its harvesting (Fuller et al., 2011; Smith, 2012).

Another recent study of dental calculus from the Danube Gorges area has reached a conclusion that domesticated cereals started to be used in this region only with the start of the Neolithic (Jovanović et al., 2021b). In the same study, the individuals dated to the EM exhibit a high occurrence of starch granules, which we have shown to be compatible with a variety of wild species of the Triticeae tribe (e.g., genus Aegilops). Jovanović et al., 2021b disregard the evidence of Aegilops consumption during the EM as an ‘implausible pattern’. However, our results based on the combination of a robust sample of analyzed individuals, GSTs, and experimental data are in opposition to the conclusions of the mentioned study.

Most of prehistoric forager groups might have had regular access to plant nutrients and some sort of dependency on specific plants (Fuller et al., 2011). Accordingly, in our analysis of dental calculus and GSTs, we have shown that besides wild grasses, local foragers consumed oat, legumes, minor millet species of the genus Echinochloa and/or Setaria known as ‘forgotten millets’ (Madella et al., 2013; Weber and Fuller, 2008), acorns, and Cornelian cherries (Figure 4; Table 1 and Table 2). Even if secure identification to species or genus level was not possible in every case (Soto et al., 2019), the morphological characteristics of starch granules systematically encountered in our archaeological samples, and the data available for the analysis of material culture, clearly show a contribution to the diet from these plant taxa.

Several starch granules of the tribe Aveneae have been identified in dental calculus, especially during the LM. Very abundant in temperate ecosystems, oat caryopses have been processed as foodstuff already during the Upper Palaeolithic at Paglicci Cave, in the south of Italy (Mariotti Lippi et al., 2015). They have also been documented in the dental calculus of the aforementioned LM individual from Vlakno Cave in the Eastern Adriatic region (Cristiani et al., 2018). Overall, starch granules of this tribe were remarkably well preserved in our dental calculus samples and large aggregates characterizing Aveneae were abundant. Interestingly, starches of this tribe recorded on archaeological GSTs, and associated with the production of flour, consist of abundant single sparse polyhedral granules (Mariotti Lippi et al., 2015). In our case study, exceptionally well-preserved aggregates of Aveneae have been identified in dental calculus only. The concentration of semiopen aggregates (Figures 4g, 5k and u) sustains the hypothesis of a coarse processing of the seeds during the Mesolithic, already suggested for other grass grain taxa.

Small starch granules from grasses of the Poaceae family have been attributed to the Andropogoneae and Paniceae tribes (Figure 5B, C; Madella et al., 2013). Our results confirm previous conclusions about the use of grasses of the tribe Paniceae by the LM foragers (Cristiani et al., 2016), while extending the consumption of this general types of millets to EM foragers too. However, no evidence of this food has been ascertained on the basis of stable isotope analysis (Borić et al., 2004). This pattern could mean that their consumption was not predominant in dietary practices of these Mesolithic foragers. We stress that a great variety of species of C4 plants belonging to the tribe Paniceae and the tribe Andropogoneae (e.g., species of plants of the genera Setaria Beauv. and Echinochloa P. Beauv.) grow well nearby water environments and slow-flowing waters, and it is very likely that a mixture of species from such genera might have contributed to the Mesolithic diet. Furthermore, they often grow in association with each other. The presence of Paniceae and the Andropogoneae tribes, combined with the evidence of several feather barbule fragments from aquatic birds, clearly point to a familiarity with resources from riverine environments other than fish.

Species of the family Fabaceae are well represented in the EM and LM individuals from the Danube Gorges area. While a secure identification to species or genus was not possible in our samples, we know that wild pulses (Lens sp. Mill.) and bitter vetch (V. ervilia (L.) Willd.) were used as foodstuffs for the Mesolithic inhabitants of Franchthi Cave (Asouti et al., 2018; Van Andel et al., 1987). Further evidence for a pre-Neolithic consumption of the species of the tribe Fabeae comes from Uzzo Cave, Italy, where wild legumes (Lathyrus sp. L., Pisum sp. L.) are well represented along with other arboreal fruits (Arbutus unedo L.), acorns (Quercus sp. L.), and wild grapes (Vitis vinifera subsp. sylvestris L.) (Costantini, 1989), as well as from the site of Barma Abeurador in southern France (Vaquer et al., 1986). In addition to the evidence from dental calculus, indirect evidence for the consumption of species of the family Fabaceae has also been retrieved from one GST (Figure 4Ii).

Species of the Fagaceae family (cfr. Quercus ilex L., Quercus sp. L.) were also a significant food resource for the Danube Gorges foragers (Figure 5A). Fragments of acorns were found at several Mesolithic sites across Europe (Holden et al., 1995; Kubiak-Martens, 1999) and in the investigated region (Mason, 1995).

Evidence for the consumption of Cornelian cherries (C. mas L.) has been retrieved in dental calculus belonging to LM and Mesolithic-Neolithic individuals from Vlasac (Figure 5n–p) and also on eight GSTs from the same site. The results based on the analyses of GSTs not only corroborate the evidence for the use of these fruits derived from the calculus analysis, but also support the role of such plants in Mesolithic daily life. Cornelian cherries are the most recurrent macroremains at Vlasac and are documented across Europe as a Mesolithic source of food and medicine (Divišová and Šída, 2015; Filipović et al., 2010). Ethnographically, various fruits commonly referred to as ‘cherries’ and/or ‘berries’ are particularly well documented among Native American groups (Siegfried, 1994; Zarrillo and Kooyman, 2017). Throughout much of North America several species of the Cornaceae and Rosaceae plant families were processed using unmodified pounding stone tools to add to pemmican or to dry as cake. Interestingly, Woodland Cree people used to mix berries and cherries with fish eggs (Siegfried, 1994) and meat (Lowie, 1922).

We conclude that the long-lasting interactions with edible grains (but also wild pulses), documented in the Balkans since the end of the Pleistocene might have allowed enough time for specific eating habits, tastes, and ‘cultural valuation’ (Hastorf and Foxhall, 2017) to develop. Such a shared knowledge about specific plant resources effectively predates the introduction of agriculture in Europe, and might have eventually eased the introduction of domesticated species starting from the second half of the seventh millennium BC. Our results call for more systematic and interdisciplinary research in order to reconstruct plant food traditions and cultural tastes before the introduction of agriculture.

Materials and methods

The examined collection of the teeth previously studied for strontium isotopes (Borić and Price, 2013) from five sites in the Danube Gorges area and the site of Gârleşti from the region of Oltenia in Romania and additionally collected teeth from the sites of the Velesnica and the 2006–2009 excavations at Vlasac contained a total of 155 specimens. Of these, a total of 60 individuals had sufficiently preserved calculi for further analyses (Table 1): 13 individuals date to the EM, 29 to the LM, 9 to the Mesolithic–Neolithic transition phase (M/N), and 8 to the EN. In addition, two later period burials were also included in the analysis as a methodological comparison (Table 1). In order to corroborate data obtained through dental calculus analysis, we also analyzed 101 sandstone GSTs of the 131 implements found at the site of Vlasac during the 1970–1971 excavations and chronologically attributed to the LM (Borić et al., 2014; Srejović and Letica, 1978). While GSTs are well documented during the Mesolithic–Neolithic transition at the site of Lepenski Vir (Antonović, 2006), earlier evidence for their use is available only from the site of Vlasac. This site yielded the richest Mesolithic assemblage of nonflaked stone tools recovered so far in the region (Antonović, 2006; Borić et al., 2014; Srejović and Letica, 1978). GSTs underwent a functional study aimed at verifying their function and potential involvement in plant food processing (Table 3; Figure 3).

Dental calculus – sampling, extraction, decontamination, and examination procedures

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All the sampling was conducted under the stereoscope, as can be seen in Figure 2 (for more details see below), and following strict protocols systematized by Sabil and Fellow Yates (Sabin and Fellow Yates, 2020) with some variation (disposable blades were changed after each sample extraction). Deposits of dental calculus were judiciously left on the teeth for future research. Whenever possible, sampled dental calculus was further subdivided for metagenomic analysis aimed at reconstructing aDNA of oral bacteria (Ottoni et al., 2021).

Decontamination and the extraction procedures for microdebris were conducted in dedicated clean spaces not connected to modern botanical work and under strict environmental monitoring of the DANTE laboratory of Sapienza University of Rome (IT), the BioArch laboratory at University of York (UK), and the aDNA facility of the University of Vienna (Austria). In all of these facilities, strict contamination rules were followed. Cleaning is carried out daily and no food is allowed in order to prevent any type of modern contamination. Bench space surfaces were cleaned prior to the analysis of each sample, using soap and ethanol, followed by covering of the surfaces by aluminum foil, and using of clean starch-free nitrile gloves at all times. Calculus cleaning was carried out under the stereomicroscope, on a Petri dish previously washed and immersed in hot ultrapure water, with magnifications up to ×100. The removal of the mineralized soil adhering on the surface of the calculus was meticulously carried out using sterile tweezers to hold the sample and a fine acupuncture needle to gently scrape off the soil attached to the external layer of the mineralized plaque. The procedure was performed using drops of 0.05 M hydrochloric (HCl) acid to dissolve the mineralized flecks of soil and ultrapure water to block the demineralization, as well as to wash and remove the contaminants. Once the calculus surface was cleaned, the contaminated soil was checked for possible cross-contamination and the clean samples were washed in ultrapure water up to three times in order to remove any trace of sediment. The clean calculus was then dissolved in a solution of 0.5 M HCl and subsequently mounted on slides using a solution of 50:50 glycerol and ultrapure water. Furthermore, control samples from the clean working tables and dust traps were collected and analyzed for comparative purposes in order to prevent any type of modern contamination in these laboratories – this is a practice routinely done in our laboratories, even at times where no archaeological analysis occur, to allow a better understanding of the flow of contaminations through seasons. Our results based on this procedure show that synthetic and plant fibers and hairs, fungal spores and hyphae, palm and conifer pollens, insects’ debris; maize starch granules were detected twice while unidentified small starch granules were very rare; phytoliths and starch granules belonging to species of the Triticeae tribe were never recovered (Figure 10). We did not retrieve any debris morphologically similar to any of the remains in the environmental control samples. Furthermore, starch granules amounted to a neglectable fraction of the laboratory ‘dust’ – suggesting it is extremely unlikely that an event of contamination of starch granules would occur in the lab, where no other remains from dust, way more common, were not found.

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.

The examination of microdebris embedded in the calculus matrix was performed at Sapienza University of Rome and at the University of York using a Zeiss Imager2 cross and an Olympus polarized microscope with magnifications ranging from ×100 to ×630. A modern reference collection of 300 plants native to the Balkans, the Mediterranean region, and Europe was used as a comparison, along with published literature, for the identification of archaeological starch granules. The experimental reference collection also included species documented in the local archaeological record (Filipović et al., 2010; Marinova et al., 2013).

GSTs functional analysis

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GSTs were sampled and analyzed at the Archaeological Collection of the University of Belgrade. Strict protocols were followed for controlling modern contamination during the residue sampling and functional analysis of GSTs: bench surfaces where the work was conducted were cleaned before the analysis of each tool using ethanol, hot water, and covered by aluminum foil; starch-free gloves were used while handling the GSTs; dust samples from the storage boxes and the working tables were collected and analyzed for comparative purposes; use-wear and residue analyses were performed on the surfaces not affected by severe postdepositional modifications and free from concretions. Furthermore, starch granules were considered reliable only when in combination with use-wear traces associated with plant processing.

Functional study involved the analysis of use-wear traces on the GST surfaces at low magnification (×0.75–×168) using a Zeiss Discovery V20 stereomicroscope and at high magnification (×200–×500) with a Zeiss AxioScope metallographic reflected light microscope(Cristiani and Zupancich, 2020; Dubreuil et al., 2015). At low magnification, GSTs were analyzed using a Zeiss Discovery V20 Stereomicroscope, which allowed us to assess the state of preservation of the materials and identify the residues still adhering to the surfaces. Appearance, morphological features, and spatial patterns of macroresidue distribution were considered (Langejans, 2010). Casts of the used areas were taken by means of a high-resolution polyvinylsiloxane (Provil Novo Light Fast Set), and later analyzed at high magnification (up to ×500) at the DANTE Laboratory at the Sapienza University of Rome. Micropolish, abrasions, and microstriations across the tools’ surfaces were identified using a Zeiss AxioScope metallographic microscope, and described following relevant parameters available in literature (Adams et al., 2006; Dubreuil et al., 2015; Hamon and Plisson, 2009).

Microresidues were sampled before surface casts. Ultrapure water was placed on the crevices of surface and left for 1 min on the artifact in order to soften the residues, then pipetted out and stored in a sterile tube. Once in laboratory, the samples were centrifuged and the natant mounted on microscope slides using a 50% solution of purified water and glycerol. Slides were subsequently analyzed in transmitted light using Zeiss Imager2 microscope (×630) and cross-polarizing filters. For archaeological residues, appearance, morphological features, and spatial patterns of distribution were considered (Cristiani and Zupancich, 2020).

An experimental reference collection of used GSTs and starch granules housed at the DANTE laboratory was consulted along with relevant literature and scientific databases. High-resolution images of the identified use-wear and residue were taken at ×630 using a Zeiss Axiocam 305 high definition color camera. Risk of modern contamination from the storage and sampling environment was minimized following a strict cleaning procedure before and during the sampling/analysis.

Testing morphological differences in experimental starch granules from Aegilops, Hordeum, and Triticum species

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Methodologically, we were able to characterize the morphological variability of starch granules in ancient plant species consumed in the investigated area, hence complementing our previous work and its implications (Cristiani et al., 2016). Differences in the starch granules assigned of the tribe Triticeae in the analyzed individuals were identified on the basis of (1) the specific morphology, dimensions, and appearance of Type A and B granules; and (2) the proportion between A-Type and B granules. In particular, during the EM A-Type granules preserved in calculi are very large and round in shape with deep lamellae visible only in the granules’ mesial part. Additionally, B-Type granules with different sizes and shape have been recorded in all of the EM individuals and most of LM individuals (Figure 4). This feature is absent in the EN individuals analyzed in this work, displaying only identical small round B-Type granules, while A-Type granules are large, round to oval/lenticular, with lamellae less pronounced in the mesial part of the grains and well visible craters on their surfaces (Figure 4y). We could not match these differences in our experimental record, which includes various Aegilops species as well as wild species within the genera Hordeum, Elymus L., Agropyron Gaertn., Dasypyron L. growing locally, and in the literature (see Henry et al., 2011). The abovementioned features have consistently been assigned to modern domestic Triticeae species (Triticum spp. and Hordeum spp.) (Cristiani et al., 2016; Piperno et al., 2004; Yang and Perry, 2013). Given the high variability recorded in the dimensions and distribution of starch granules within the modern, locally available, species of the genus Aegilops (Figure 6a–j), further statistical work was carried out in order to interpret starch granules assigned to the tribe Triticeae in Mesolithic-Neolithic transitional contexts.

Caryopses from 11 Aegilops species (A. triuncialis, A. comosa, A. crassa, A. cylindrica, A. geniculata, A. neglecta, A. speltoides tauschii, A. peregrina, A. triuncialis, A. uniaristata, and A. ventricosa), 1 Hordeum species (Hordeum vulgare distichon), and 2 Triticum species (Triticum monococcum and T. dicoccum) grown in the central Balkans were collected. All plant material was grounded using pestle and mortar. Starch powder (0.5 mg) was resuspended in 100 µl of sterile distilled water and vortexed for 5 min. After that, the sample was observed by an optic transmitted light microscopy (Figure 6). Fifty starch granules were randomly selected (for size and shape), counted, and their length measured. Minimum and maximum lengths, mean, and median values with relative standard deviations and interquartile ranges are reported for each species in Table 4. Length distribution and variation of modern starches are reported in Figure 9. Finally, length distribution of the starches from each species was compared with the other species to investigate the existence of significant differences. This statistical analysis was carried out through a pairwise Wilcoxon test (Table 5). Results were considered significant for p values <0.05 (<0.05; *<0.01; ***<0.001) and not significant (ns) for measurements >0.05.

Triticeae starches are known to possess a bimodal distribution, made up of small (B-Type) and large (A-Type) granules (Figure 6). In the analyzed Triticum and Hordeum genera, B-Type grains are more abundant than A-Type, except for H. secalinum Schreb., which does not exhibit the large granules. Differently, in Aegilops genus, the size distribution of starches is characterized by two different trends. The first one, evidenced in A. caudata L., A. cylindrica Host, A. comosa Sm., and A. speltoides tauschii Coss., appears very similar to that of Triticum and Hordeum samples, although B-Type grains are less abundant than their counterparts in wheat and barley. On the other hand, the second cluster (A. crassa Boiss. ex Hohen., A. geniculata Roth, A. neglecta Req. ex Bertol., A. speltoides tauschii Tausch, A. triuncialis L., A. uniaristata Vis., and A. ventricosa Tausch) exhibits larger starches, determining a significant shift of the mean size toward intermediate lengths. In general, the present experimental analysis revealed that Aegilops sp. starch granules show a larger size distribution than Hordeum sp. and Triticum sp. (Figures 6 and 9). This evidence is also supported by the pairwise Wilcoxon test, which highlights that Aegilops spp. starch measurements are significantly different from those obtained for Hordeum sp. and Triticum sp. counts (Table 4).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

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Decision letter

  1. George H Perry
    Senior and Reviewing Editor; Pennsylvania State University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife. However, all reviewers had positive views about your work and paper. Yet the consultation consensus was that the extent of methodological revision and interpretive adjustments that would be required was more than what we felt could be accomplished with a reasonable revision timeframe and effort. Please see the below reviews. If you were to expansively address the comments, and based on feedback from the reviewers, I would be willing to consider a revised version of the manuscript as a new submission, with a high bar of expectations (and no guarantee of full review).

Reviewer #1:

This manuscript presents the results of microbotanical (starch grains and phytoliths) and chemical analyses in dental calculus from 61 Mesolithic and Early Neolithic individuals from several sites in the Danube Gorges of the central Balkans. Microbotanical evidence from 44 grinding stones from one of the studied sites is presented as additional evidence supporting the consumption of plants (wild grasses, pulses and fruits) before the arrival of southwest Asian domestic crops.

Archaeologists have long acknowledged the use of plants by hunter-gatherer societies worldwide prior to the advent of agriculture. In the Introduction, the authors claim that "forager knowledge and consistent use of wild cereals are still debated and poorly documented outside of the assumed centers of domestication in southwestern Asia" (l. 40-41) and that "the hypothesis of a systematic use of wild cereals (e.g., Aegilops spp.; Hordeum spp.) in this region [the Balkans] during the Mesolithic has not been verified until now" (l. 46-47). These claims seem to justify the novelty of this study. However, the beginning of the Discussion reviews evidence for wild grass use by hunter-gatherers in southeastern Europe, including one of the study sites, in the form of macro and microbotanical remains and pollen grains. The present study, thus, adds to this body of evidence (which should be presented in the Introduction, not the Discussion) in a specific region.

The robustness of this study lies in the high number of samples analysed. However, I do not believe that the analysed datasets are comparable. Dental calculus and grinding stones do provide independent evidence for the consumption of plant resources, but only when analysed from the same chronological periods and the same sites. In this study, dental calculus samples come from six sites dating to the Early Mesolithic, Late Mesolithic, the Mesolithic-Neolithic transition and the Early Neolithic, but samples from grinding stones come from a single site and a single occupational deposit (Late Mesolithic). Moreover, the analyses conducted in each set of samples are not consistent either-microbotanical and chemical analyses in dental calculus vs. microbotanical analyses in grinding stones-further precluding the comparability of the resulting datasets. In my opinion, these samples are not comparable and should not be presented as part of the same study.

Regarding the identification of microscopic plant remains in samples from dental calculus and grinding stones, I am puzzled by the identification of some Type I starch grains (Triticeae tribe) as belonging to the genus Aegilops. In a previous study in which several of the authors of the current paper participated (Cristiani et al. 2016), the identification of Triticeae starch grains as Aegilops was specifically disregarded because "Aegilops spp. are absent from assemblages with analyzed macrobotanical remains found at Mesolithic and Early Neolithic sites in the central Balkans" (Cristiani et al. 2016: 4). However, in the present study the authors seem comfortable assigning part of the encountered Triticeae starch grains to this genus without rectifying their previous claim.

Also in regards to the starch assemblage, there seem to be two categories of Type I starch grains, one identified as Aegilops and another as a domestic species within the Triticeae tribe. If the authors believe that this is the case, I recommend separating them into subtypes and treating them a separate taxa for the morphometric analysis.

Regarding the starch grain morphometric analysis, I am troubled by the fact that the length of Type A and Type B starch grains from modern and archaeological Triticeae spp. seems to have been analysed together, instead of analysing Type A and Type B grains separately. This makes it impossible to compare the results of this study to previously published data (e.g., Henry et al. 2011; Yang and Perry 2013) and potentially biases the morphometric data from archaeological starch grains due to the fact that small starch grains (in this case, Type B) are less likely to survive than large starch grains (Haslam 2004).

The phytolith assemblage is smaller and it is unclear whether the authors consider it as evidence for food (l. 265-275) or as 'non-dietary remains' (l. 294). In any case, Figure 4w is mentioned as evidence for 'multicellular dendritic structures' (l. 270), but I do not believe that the image shown in Figure 4w can be qualified as a phytolith. Evidence for sedges is also mentioned in l. 298 but not presented elsewhere.

Overall, I believe that this study presents evidence for the consumption of wild grasses and other plants during the Mesolithic and the transition to the Neolithic in the Danube Gorges of the Balkans, but I do not think that the evidence is properly presented.

I find several of the terms employed by the authors inaccurate or problematic. Perhaps the most paradigmatic is the term 'cereal', used in the title, the Abstract (l. 27) and throughout the manuscript. The term 'cereal' refers to a cultivated plant within the grass family (Poaceae). I do not believe that the authors aim at implying the existence of 'low level food production' (Smith 2001), often referred to as 'cultivation' (e.g., Bowles 2011), in the Mesolithic Danube Gorges, so I recommend the consistent use of the term 'wild cereal' (e.g., l. 33) or the more accurate 'wild grasses'. Other terms which I find potentially problematic include 'complex foragers' (e.g., l. 31)-what is a 'complex forager' and how does it compare to a 'simple' (?) forager? – and the use of the term 'microfossils' when referring to ancient starch grains, which are not fossilised and should therefore be referred to as 'microremains' or 'microscopic remains'. Finally, the term '"forgotten" millets' (l. 478) is used without explaining what is meant by 'forgotten'. If it refers to the fact that the millet species mentioned in the manuscript are no longer consumed in the Balkans, how is it different from Aegilops?

Reviewer #2:

This paper contributes significantly to the debate on the possible intensification of plants exploitation by late foraging populations prior to strict neolithisation. It focuses on a particular kind of botanical remains, starch grains, from two different kind of sources: dental calculus and ground stone tools. The region understudy, the Balkans, is particularly adapted to discuss this question, while as a series of important Mesolithic and Neolithic sites are well excavated in the considered area. The corpus of individuals is important, and allows solid interpretations. The results presented are convincing, and shows the high diversity of plants consumed by foragers. They also bring solid arguments to demonstrate that Late Mesolithic foragers were engaged in the consumption and probably selection of several of the wild plant species that were cultivated in their domestic form during the Neolithic. It keeps open some questions regarding the exact datation of the domestication, at the heart of intense debates among prehistorians.

A couple of improvements could be proposed to enhance the paper :

– In the introduction, Early and Late Mesolithic as well as Early Neolithic would deserve a small paragraph to summarize their respective datations, characteristics in terms of type of sites and subsistence strategies. Meso-Neo contexts should be explained for the readers (real mixed levels or undetermined)?

– A brief review of the archaeobotanical data already available in these contexts prior to this study will also be useful.

–Throughout the paper, the terms cereals, oat, etc is questioning as they introduce an ambiguity in the wild versus domestic nature of the plant remains found. I would suggest the authors to be clearer either in the introductory part (ex: the term cereals is used independently of the domestic or wild nature of the caryopses found) or by precising throughout the text if these terms are referring to undet./wild/domestic species. The best solution would probably to keep the latin designation: Poacee or Triticum Sp. This will help the reader clarify exactly the level of precision of the starch grains and more broadly archaeobotanical analysis to discuss the question of domestication. Accordingly, the title and abstract should be revised (the term cereal is not adequate here, speak rather of Poacee e.g.)

– Some larger references to recent starch grains analysis on Mesolithic/Neolithic worlwide and in European contexts would enlarge the scope.

– Concerning the experimental referentials, the authors should precise which species were tested, and if they fit with the ones found in the archaeological record, in order to ensure that the referential used is transposable to prehistoric remains.

– Though all precautions have been taken to avoid any modern contamination of the surfaces of GSTs, no tests have been undertaken concerning contamination by the surrounding soils. These should be discussed to precise the origin of the starch grains: resulting from grinding actions or resulting from taphonomic transfers from the sediments. In the same idea, in table 4 PDm is mentioned; were the starch grains preserved by the concretions? Was the content of the concretions tested?

– The GST section need some additional information. We need more information about the type of tool (pestle, grinder, crusher, etc). This would help the reader to link the type of action and of tools to the starch remains, and to evaluate the different processing technics involved in the respective Mesolithic and Neolithic traditions.

Some precisions should be added to consolidate the presentation of the corpus. Also be careful about the number of tools analyzed, there seems to be contradictory information from one part to another of the text.

We need more information about GST: raw material, localization of the active surfaces on the blank. Surprisingly, macrotraces from the stereoscopic observation are not characterized anywhere. This should be completed in the text and the figures.

Figure 1: in the legend, give dates and precise type of occupation.

Figure 3: in the legend, precise the datation of tools and their typology. Photos are very small to distinguish use-wear traces on their surface. Besides, some plate with Early Mesolithic and Neolithic tools would be interesting to see if there is an evolution in the characteristics of the tools used, in parallel to the evolution of the plants transformed.

Figure 4 (z, Aa) + Bb, Cc: some precisions are necessary: which tool' number (to refer to the Figure 3)? Besides, scale are not correct (only a white suare with no metric indications)

Figure 7: experimental reference or natural one? If experimental explanations about the experimental tests are necessary. This is also not clear in the text itself.

Figure 9: Problem of legend; some number are missing. Again for archaeological GST, precise which one (number, site, type etc); presented this way it is too general and not rigorous.

Reviewer #3:

This is an ambitious and interesting study that systematically examines plant microfossils, other debris, and biochemical markers from dental calculus. The central result is that cereal grasses in the tribe Triticeae, as well as a range of other plants, were being consumed prior to the introduction of domesticated species in the study region. This result is sound, and a valuable contribution to our understanding of long-term plant-human interactions in the region, especially outside of what we understand to be the zones of domestication for the main Neolithic crops.

However, I have two main analytical concerns with the manuscript:

1) There is some tendency to associate starch morphotypes with taxonomic groups without clarifying whether diagnostic characters are present to make secure identifications. This is the case with types IV and VI, for Fabaceae and Cornaceae. It is important to clarify whether these groups are being names as examples of the morphotype, or identified as the taxa present, in which case we need more information about the discriminating power of diagnostic characters in these taxa vs others. Table 2 suggests the latter (positive IDs). The interpretation of Aegilops at the genus level is similar, it's lacking the necessary context to assume confidence in the assignment. I think this is particularly important given the role of Aegilops in bread wheat evolution.

2) The chemical analytical methods are not suitably described, only that mass profiles were compared to records at NIST. As a result, it is very difficult to assess whether the interpretations are reasonable in light of analytical specificity. Specificity of chemical biomarkers is an important consideration and has in the past led to archaeological misinterpretations of alkaloids and other compounds due to shared characteristics. Additionally, hordenine is not diagnostic to cereals, and its presence is not robust evidence of cereal caryopsis ingestion. This alkaloid is widespread in plants, and was only first described in Hordeum, not restricted to Hordeum and relatives. The methods refer to analyzing the "pellets left from a metagenomic study" which is as-yet unpublished. We need more detail about the processing leading to the analyzed pellet, i.e. is it an untreated piece of the calculus or the derivative of a DNA isolation procedure, which could have implications for analysis.

These two issues lead to some over-interpretations in my view, which I discuss more in my detailed comments. I think the central contribution is a valuable one, and I would advise the authors to pull back on some of their additional interpretations at this time. I am very appreciative that the authors have undertaken this work in conjunction with meta-genomic analysis for a robust multi-proxy treatment of these important remains, which will allow the most robust possible interpretations to be drawn.

I enjoyed reading this study, as above I feel over-interpretation is the biggest issue. A few detailed comments follow.

47: "verified until now" slightly implies that you are definitely verifying it with this study, changing it "has not yet been verified" would be clearer.

139: Salivary amylase is the most likely interpretation for this damage ("enzymatic digestion"), I would caution against suggesting that this could be interpreted as cultural modification via food preparation.

The numerous starch micrographs of comparative materials (Figure 6 and 7) are not very helpful without indicating diagnostic characters, with citation, and with specific relevance to the text explicated in the figure captions.

242-45: It is not clear if this form is diagnostic to the family Fabaceae, only "typical of" that family. The strong implication is that these can be interpreted in that way, but it is important to make clear whether these structures are found in other taxa, and clarify the confidence level of a Fabaceae interpretation on that basis. Also, Fabaceae contains nearly 20,000 species in all temperate and tropical regions globally, so again I would caution against the implication that these structures in this study are linked with the familiar crop species mentioned.

297: I don't fully understand "daily life crafting activities"

335: Specify "foxtail millet" as the common name for S. italica to avoid confusion with other mentions of millets.

358: This is the first mention of the amyloplast context. This should be introduced and quantified in the Results section, and more information should be given here on the reliability of this context for determining the degree of processing.

387-388: I disagree that the common view is that groups of people without agriculture and domesticated plants are merely foraging opportunistically, I think this is a slightly outdated view. The bulk of domestication and early agriculture/pre-agriculture literature have come to recognize a longterm mutualism of humans and plants even in what we would interpret as wild forms. This doesn't affect the interpretation here in any problematic way and I do think it is an important topic to explore, only that I think the authors are setting up the result against a "common view" that isn't all that common.

410: I would advise against interpreting cultivation on this basis without further exploration of the ecological characters of these species. Several cultigen wild relatives are notoriously weedy, and so in many cases there's a strong possibility that these taxa colonize newly opened landscapes with no human intervention. In either case, cultivation here is an over-interpretation.

411: Chenopodiaceae has been subsumed within Amaranthaceae.

461: The current study does not provide direct evidence for management of wild stands, only their consumption. This interpretation should be contextualized as likely management in light of other literature and ecological expectations, but it should be clear that management is not being tested here.

470-474: This section is a bit unclear, I can't tell what evidence is being interpreted by whom as implausible in the context of the cited study.

544-560: I'm afraid I find this paragraph to be over-interpreted. As mentioned above, the methods of the GC-MS approach are underdeveloped, and the specificity of the results is not clear, therefore without that extra information, interpreting specific compounds without regard to their botanical breadth and chemical lookalikes is not useful. Re omega-3 fatty acids, I think it's a bit of a reach to try and interpret them in an archaeobotanical context when the riverine resources would be such a clear contributor of these compounds.

Figure 8 is not called out in the text, and I'm not sure of its purpose.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture" for further consideration by eLife. Your article has been evaluated by George Perry (Senior Editor) and two reviewers.

The reviewers appreciated your efforts in addressing their collective previous comments, and they commented that they now agree that the paper presents compelling evidence for the exploitation of wild grasses prior to the introduction of domestic cereals in the Balkans. However, there are some remaining issues as pointed out by one of the reviewers that need to be addressed, as outlined below:

The identification of Type Ia starch grains as a domestic cereal seems to be based on the absence of a bimodal pattern of starch granules distribution in MOST (but not all) wild Aegilops species. Moreover, other wild Triticeae taxa have not been considered as potential origin for these starch grains. For example, Yang and Perry (2013: Figure 2b in JAS) found a bimodal assemblage in Leymus chinensis. This taxon does not naturally occur in the Balkans, but others within the genus do (e.g., Leymus racemosus). Other potential contributors include the genus Bromus, widely distributed throughout Eurasia. For these reasons, I do not think Type Ia should be assigned to a domestic cereal; at most, it can be suggested that it likely does not belong to the genus Aegilops.

https://doi.org/10.7554/eLife.72976.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

This manuscript presents the results of microbotanical (starch grains and phytoliths) and chemical analyses in dental calculus from 61 Mesolithic and Early Neolithic individuals from several sites in the Danube Gorges of the central Balkans. Microbotanical evidence from 44 grinding stones from one of the studied sites is presented as additional evidence supporting the consumption of plants (wild grasses, pulses and fruits) before the arrival of southwest Asian domestic crops.

Archaeologists have long acknowledged the use of plants by hunter-gatherer societies worldwide prior to the advent of agriculture. In the Introduction, the authors claim that "forager knowledge and consistent use of wild cereals are still debated and poorly documented outside of the assumed centers of domestication in southwestern Asia" (l. 40-41) and that "the hypothesis of a systematic use of wild cereals (e.g., Aegilops spp.; Hordeum spp.) in this region [the Balkans] during the Mesolithic has not been verified until now" (l. 46-47). These claims seem to justify the novelty of this study. However, the beginning of the Discussion reviews evidence for wild grass use by hunter-gatherers in southeastern Europe, including one of the study sites, in the form of macro and microbotanical remains and pollen grains. The present study, thus, adds to this body of evidence (which should be presented in the Introduction, not the Discussion) in a specific region.

We thank the Reviewer for their comments. As suggested by the reviewer, we have restructured the article so that the part of the discussion mentioned by the reviewer is presented in the Introduction. However, we would like to emphasize that all previous studies that mention wild grains among Mesolithic foragers in the Balkans present circumstantial evidence and the novelty of our findings does not simply “add to this body of evidence” but rather provides the first direct evidence for the use of wild grains among Holocene foragers in the Balkans.

The robustness of this study lies in the high number of samples analysed. However, I do not believe that the analysed datasets are comparable. Dental calculus and grinding stones do provide independent evidence for the consumption of plant resources, but only when analysed from the same chronological periods and the same sites. In this study, dental calculus samples come from six sites dating to the Early Mesolithic, Late Mesolithic, the Mesolithic-Neolithic transition and the Early Neolithic, but samples from grinding stones come from a single site and a single occupational deposit (Late Mesolithic). Moreover, the analyses conducted in each set of samples are not consistent either-microbotanical and chemical analyses in dental calculus vs. microbotanical analyses in grinding stones-further precluding the comparability of the resulting datasets. In my opinion, these samples are not comparable and should not be presented as part of the same study.

We agree with the Reviewer that dental calculus and grinding stones provide independent evidence for the consumption of plant foods when analyzed from the same chronological periods and the same sites. However, we still believe that the two lines of evidence are comparable, complementary, and reinforce the conclusions reached about the use of wild cereal grasses. There is no region of the world where one could expect a perfect preservation and match of two different strands of evidence due to a host of reasons, including taphonomic and other preservation biases and the nature of the record. In the new version of the article, we explained better why we primarily find the category of ground stone artifacts from the Late Mesolithic period onwards, which relates to the observed pattern of only sporadic use of grounds stone in earlier periods in the region of the Danube Gorges and the appearance of these types of tools only in the Late Mesolithic (Antonović et al., 2006; Borić et al., 2014; Srejović and Letica, 1978). Likewise, it would be unrealistic to expect us to analyze all sites and their ground stone assemblages for this study and we believe that a sample of over 100 artefacts suffices in order to reach robust and reliable conclusions. We also believe that the evidence from the site of Vlasac, as one the most representative sites in the Danube Gorges area, characterized by a highly developed Late Mesolithic practice of using ground stones for wild grain cereal processing, reflects wider practices of forager ground stone use across the region.

Regarding the identification of microscopic plant remains in samples from dental calculus and grinding stones, I am puzzled by the identification of some Type I starch grains (Triticeae tribe) as belonging to the genus Aegilops. In a previous study in which several of the authors of the current paper participated (Cristiani et al. 2016), the identification of Triticeae starch grains as Aegilops was specifically disregarded because "Aegilops spp. are absent from assemblages with analyzed macrobotanical remains found at Mesolithic and Early Neolithic sites in the central Balkans" (Cristiani et al. 2016: 4). However, in the present study the authors seem comfortable assigning part of the encountered Triticeae starch grains to this genus without rectifying their previous claim.

We thank the Reviewer for their comment. We have now explicitly rectified our previous claims that “it was possible to exclude wild species of the Triticeae […] that could have been eaten at the time in the region […]. Furthermore, Aegilops spp. are absent from assemblages with analyzed macrobotanical remains found at Mesolithic and Early Neolithic sites in the central Balkans.”. We underline that our current conclusion about the consumption of the species of genus Aegilops in the Mesolithic Danube Gorges is not based on the absence of macro remains in the archaeological record but is rather grounded in the analysis of a very large pool of Mesolithic individuals (tot. 61 individuals). In our 2016 study, we included only 9 Mesolithic individuals out of which only 5 yielded Triticeae tribe starch granules. In our current study, we analysed 26 Late Mesolithic individuals out of which 16 yielded Triticeae starch granules; 8 Mesolithic-Neolithic individuals out of which 5 yielded Triticeae tribe starches; and, more importantly, several Early Mesolithic individuals (not included in our previous article) of which 5 yielded Triticeae tribe starches. Hence, we observed a large population of ancient Triticeae starch granules, which made our current conclusions more robust. The analysis of so many individuals also provided a time-depth perspective on the presence of various species. The morphological variability identified in the ancient Triticeae starch population from the Danube Gorges could not be evaluated extensively in 2016 PNAS as only 3 Late Mesolithic individuals yielded starch granules attributed to the Triticeae tribe. Moreover, no Early Mesolithic individuals were analysed at that time. In the dental calculus of the 3 Late Mesolithic individuals discussed in 2016, only one type of ancient Triticeae granules was identified (described in the text as “subtype Ib”), which we attributed to domesticated cereals. Well-known limitations in the inclusion, preservation, and recovery of plant debris in dental calculus could be responsible for the absence of Aegilops starch granules before. However, besides mentioning the absence of Aegilops species in the macrobotanical record of the region, which could also be the result of a host of taphonomic and recovery problems and should not be used to exclude the use of this genus during the Mesolithic (contra Cristiani et al. 2016: 4). In our 2016 paper, we also explicitly claimed that “it was possible to exclude wild species of the Triticeae […] on the basis of their morphology as well as most recent literature on phylogenetic evaluation of Poaceae species”. The morphological criteria used for interpreting ancient Triticeae starch granules haven’t changed in the current work and, indeed, the morphological parameters used in identifying wild (i.e. Aegilops) vs. domesticated species of the Triticeae tribe have only been reinforced, thanks also to newly added statistical analysis. However, we believe that the analysis of a larger subset of individuals, now also including Early Mesolithic individuals, allowed us to observe the morphological variability of the ancient starch population, something that we previously could not fully comprehend given a limited number of individuals analyzed.

Also in regards to the starch assemblage, there seem to be two categories of Type I starch grains, one identified as Aegilops and another as a domestic species within the Triticeae tribe. If the authors believe that this is the case, I recommend separating them into subtypes and treating them a separate taxa for the morphometric analysis.

We thank the Reviewer for this comment. Following this suggestion, we have now separated two subtypes of Type I starch grains. (l.176-223)

Regarding the starch grain morphometric analysis, I am troubled by the fact that the length of Type A and Type B starch grains from modern and archaeological Triticeae spp. seems to have been analysed together, instead of analysing Type A and Type B grains separately. This makes it impossible to compare the results of this study to previously published data (e.g., Henry et al. 2011; Yang and Perry 2013) and potentially biases the morphometric data from archaeological starch grains due to the fact that small starch grains (in this case, Type B) are less likely to survive than large starch grains (Haslam 2004).

We thank the Reviewer for stressing this. We agree to discuss Type A and Type B grains separately so that our results can be compared to already published other data about archaeological Triticeae spp.

Moreover, following the Reviewer’s suggestion, in the “Results” we have made clearer that we have not attempted the identification of starch granules less than 5 μm to avoid misinterpretation of transitory and small storage starch granules (l.171-172) and referred to the work by Haslam 2004.

The phytolith assemblage is smaller and it is unclear whether the authors consider it as evidence for food (l. 265-275) or as 'non-dietary remains' (l. 294). In any case, Figure 4w is mentioned as evidence for 'multicellular dendritic structures' (l. 270), but I do not believe that the image shown in Figure 4w can be qualified as a phytolith. Evidence for sedges is also mentioned in l. 298 but not presented elsewhere.

We have now explained the presence of phytoliths in the dental calculus matrix (l. 501-506). We corrected the mistake about the presence of sedges, which has now been fixed in the text.

Overall, I believe that this study presents evidence for the consumption of wild grasses and other plants during the Mesolithic and the transition to the Neolithic in the Danube Gorges of the Balkans, but I do not think that the evidence is properly presented.

We hope that all the corrections and changes we applied to the text according to the Reviewer’s suggestions helped us presenting the archaeological evidence in a much proper way.

I find several of the terms employed by the authors inaccurate or problematic. Perhaps the most paradigmatic is the term 'cereal', used in the title, the Abstract (l. 27) and throughout the manuscript. The term 'cereal' refers to a cultivated plant within the grass family (Poaceae). I do not believe that the authors aim at implying the existence of 'low level food production' (Smith 2001), often referred to as 'cultivation' (e.g., Bowles 2011), in the Mesolithic Danube Gorges, so I recommend the consistent use of the term 'wild cereal' (e.g., l. 33) or the more accurate 'wild grasses'.

We thank the Reviewer for Their comment. We have now used only ‘wild cereal’, ‘wild grasses,’ or ‘grass grains’.

Other terms which I find potentially problematic include 'complex foragers' (e.g., l. 31)-what is a 'complex forager' and how does it compare to a 'simple' (?) forager? –

The term “complex” foragers has been used in archaeological literature for a very long time (e.g. Price, T.D. & J. A. Brown. [ed.] 1985. Prehistoric hunter-gatherers: The emergence of cultural complexity. Orlando, FL: Academic Press, Inc) and it primarily refers to ethnographic and archaeological foragers that are sedentary, focused on particularly abundant resources of the area and characterized by complex traits of social and cultural organization. Ethnographic examples comprise foragers of the NW Coast and the best known archaeological examples are the Natufian foragers in the Levant.

and the use of the term 'microfossils' when referring to ancient starch grains, which are not fossilised and should therefore be referred to as 'microremains' or 'microscopic remains'.

We thank the Reviewer for Their comment. We have changed the term “microfossils” into “micro-remains”.

Finally, the term '"forgotten" millets' (l. 478) is used without explaining what is meant by 'forgotten'. If it refers to the fact that the millet species mentioned in the manuscript are no longer consumed in the Balkans, how is it different from Aegilops?

We thank the Reviewer for raising this point and giving us the chance to better explain what we mean by ‘forgotten millets’ in the text. Small millet species (e.g., Setaria viridis, S. verticillata) are often referred to as ‘forgotten’ millets to differentiate them from the major species of millets that are generally considered of much economic importance (cf. Madella M., Lancelotti C., and García-Granero J.-J. 2013 Millet microremains—an alternative approach to understand cultivation and use of critical crops in Prehistory. Archaeol Anthropol Sci (2016) 8:17–28 DOI 10.1007/s12520-013-0130-y; Weber SA, Fuller DQ (2008) Millets and their role in early agriculture. Pragdhara 18: 69–90).

We agree with the Reviewer that this definition should have been presented in the text, so we have now changed the phrase “Accordingly, in our analysis of dental calculus and GSTs, we have shown that besides cereals, local foragers consumed oat, legumes, “forgotten” millets, acorns, and Cornelian cherries (Figure 4; Tables 1,2)” into “Accordingly, in our analysis of dental calculus and GSTs, we have shown that besides wild grasses, local foragers consumed oat, legumes, minor millet species of the genus Echinochloa and/or Setaria also known as “forgotten millets” (Madella et al., 2016; Weber and Fuller, 2008), acorns, and Cornelian cherries (Figure 4; Tables 1,2).

Reviewer #2:

This paper contributes significantly to the debate on the possible intensification of plants exploitation by late foraging populations prior to strict neolithisation. It focuses on a particular kind of botanical remains, starch grains, from two different kind of sources: dental calculus and ground stone tools. The region understudy, the Balkans, is particularly adapted to discuss this question, while as a series of important Mesolithic and Neolithic sites are well excavated in the considered area. The corpus of individuals is important, and allows solid interpretations. The results presented are convincing, and shows the high diversity of plants consumed by foragers. They also bring solid arguments to demonstrate that Late Mesolithic foragers were engaged in the consumption and probably selection of several of the wild plant species that were cultivated in their domestic form during the Neolithic. It keeps open some questions regarding the exact datation of the domestication, at the heart of intense debates among prehistorians.

A couple of improvements could be proposed to enhance the paper :

– In the introduction, Early and Late Mesolithic as well as Early Neolithic would deserve a small paragraph to summarize their respective datations, characteristics in terms of type of sites and subsistence strategies. Meso-Neo contexts should be explained for the readers (real mixed levels or undetermined)?

We have now added a small paragraph summarizing chronology, characteristics in terms of type of sites, and subsistence strategies in the introduction as suggested by the Reviewer (l.80-107)

– A brief review of the archaeobotanical data already available in these contexts prior to this study will also be useful.

In the introduction (l. 52-69 and l.118-139), we have now provided a review of the extant archaeobotanical data.

–Throughout the paper, the terms cereals, oat, etc is questioning as they introduce an ambiguity in the wild versus domestic nature of the plant remains found. I would suggest the authors to be clearer either in the introductory part (ex: the term cereals is used independently of the domestic or wild nature of the caryopses found) or by precising throughout the text if these terms are referring to undet./wild/domestic species. The best solution would probably to keep the latin designation: Poacee or Triticum Sp. This will help the reader clarify exactly the level of precision of the starch grains and more broadly archaeobotanical analysis to discuss the question of domestication. Accordingly, the title and abstract should be revised (the term cereal is not adequate here, speak rather of Poacee e.g.)

We have now specified when we refer to wild or domestic cereal. Alternatively, we have used “grass grains” referring to wild/domestic cereal or “wild grasses of the Poaceae family”. We have also revised the title and the abstract using “wild cereals”.

– Some larger references to recent starch grains analysis on Mesolithic-Neolithic worlwide and in European contexts would enlarge the scope.

Following the Reviewer’s suggestion, we have now added new references. (l. 434-437)

– Concerning the experimental referentials, the authors should precise which species were tested, and if they fit with the ones found in the archaeological record, in order to ensure that the referential used is transposable to prehistoric remains.

with regards to the different species of the Triticeae tribe used for our archaeological interpretations, we have detailed them in the descriptions of the morphotypes as well as in Figures 6, 7, and 9. For types II, III, and IV, species used as references are mentioned in the description of the morphotype. In figure 8 details the plant species used for experimental activity have also been detailed. We also used already published data. We mentioned this in the description of the morphotypes.

– Though all precautions have been taken to avoid any modern contamination of the surfaces of GSTs, no tests have been undertaken concerning contamination by the surrounding soils. These should be discussed to precise the origin of the starch grains: resulting from grinding actions or resulting from taphonomic transfers from the sediments. In the same idea, in table 4 PDm is mentioned; were the starch grains preserved by the concretions? Was the content of the concretions tested?

The PDM mentioned in Table 4 refers to the alteration observed across the entire surface of GST. The collection of the samples was performed only on the GST surface free of any concretion or with no visible post-depositional alteration so to avoid sampling residues not associated with the actual use of the tool (i.e., contamination, soil concretion).

– The GST section need some additional information. We need more information about the type of tool (pestle, grinder, crusher, etc). This would help the reader to link the type of action and of tools to the starch remains, and to evaluate the different processing technics involved in the respective Mesolithic and Neolithic traditions.

Thank you for highlighting this. Following the Reviewer’s suggestion, we have now added the column “Tool type” in Table 3, indicating the type of tool.

Some precisions should be added to consolidate the presentation of the corpus. Also be careful about the number of tools analyzed, there seems to be contradictory information from one part to another of the text.

We thank the Reviewer for pointing this out. The number of analyzed tools is now consistent throughout the text.

We need more information about GST: raw material, localization of the active surfaces on the blank. Surprisingly, macrotraces from the stereoscopic observation are not characterized anywhere. This should be completed in the text and the figures.

We thank the Reviewer for the comment. Details regarding the GST raw material have now been included in the text (p. 16 lines 341-342). Figure 3 has been edited, indicating the active surface of the tool, its functional area(s), and the activity performed. Figure 8 has been modified by adding microphotographs of the macro traces. Furthermore, a description of the type of surface modification identified at low magnification has been added in the text (p.16 lines 346-349).

Figure 1: in the legend, give dates and precise type of occupation.

As requested, we have now provided information about the sample chronology in the figure 1 legend.

Figure 3: in the legend, precise the datation of tools and their typology. Photos are very small to distinguish use-wear traces on their surface. Besides, some plate with Early Mesolithic and Neolithic tools would be interesting to see if there is an evolution in the characteristics of the tools used, in parallel to the evolution of the plants transformed.

We thank the Reviewer for the comment. Inventory numbers along with the type of tools have now indicated in Figure 3. Also, the functional areas have now been highlighted in the pictures to ease the reader. As for the assemblage chronology, all the GSTs we analyzed refer to the Late Mesolithic occupation of the site of Vlasac, as reported in the caption.

Figure 4 (z, Aa) + Bb, Cc: some precisions are necessary: which tool' number (to refer to the Figure 3)? Besides, scale are not correct (only a white suare with no metric indications)

Following the Reviewer’s comment, in the caption of Figure 4 we have now added the tool inventory number. Also, a metric indication has now been added to the scales.

Figure 7: experimental reference or natural one? If experimental explanations about the experimental tests are necessary. This is also not clear in the text itself.

All of the starch granules presented in Figure 7 are at their natural state (i.e., not processed with a ground stone) from our modern reference collection.

Figure 9: Problem of legend; some number are missing. Again for archaeological GST, precise which one (number, site, type etc); presented this way it is too general and not rigorous.

We thank the Reviewer for spotting this. Figure 9 (now Figure 8) has been edited, and details concerning the inventory number and the type of tool added. We did not add information on the site as all the archaeological GSTs come from Vlasac.

Reviewer #3:

This is an ambitious and interesting study that systematically examines plant microfossils, other debris, and biochemical markers from dental calculus. The central result is that cereal grasses in the tribe Triticeae, as well as a range of other plants, were being consumed prior to the introduction of domesticated species in the study region. This result is sound, and a valuable contribution to our understanding of long-term plant-human interactions in the region, especially outside of what we understand to be the zones of domestication for the main Neolithic crops.

However, I have two main analytical concerns with the manuscript:

1) There is some tendency to associate starch morphotypes with taxonomic groups without clarifying whether diagnostic characters are present to make secure identifications. This is the case with types IV and VI, for Fabaceae and Cornaceae. It is important to clarify whether these groups are being names as examples of the morphotype, or identified as the taxa present, in which case we need more information about the discriminating power of diagnostic characters in these taxa vs others. Table 2 suggests the latter (positive IDs). The interpretation of Aegilops at the genus level is similar, it's lacking the necessary context to assume confidence in the assignment. I think this is particularly important given the role of Aegilops in bread wheat evolution.

We thank the Reviewer for their comments. In the new version of the article, we have changed the description of morphotypes into the following (lines 316-321): “Granules ascribed to this type have been identified in two individuals (1LM, 1M/N) (Table 2). They are characterized by a round 3D morphology and a central hilum, which appears as a wide depression, and no lamellae or facets (Figure 4i, Figure 5n-p). Zarrillo and Kooyman (2006) consider these morphological features diagnostic of some species of drupes and berries. In our sample, starch granules of this morphotype can reach 12 μm of maximum width, which is beyond species of berries and drupes in the Rosaceae family known in the literature (Zarrillo and Kooyman 2006) and in our modern reference (e.g. Prunus spinosa). Based on our experimental record, we assign type VI to species of the family Cornaceae (e.g., Cornus mas L.) (Figure 7), the remains of which are documented at Vlasac (Filipović et al. 2010).”

We have also re-written the descriptions of Type I, better explaining the diagnostic characters used in the interpretation of starch assigned to Aegilops.

2) The chemical analytical methods are not suitably described, only that mass profiles were compared to records at NIST. As a result, it is very difficult to assess whether the interpretations are reasonable in light of analytical specificity. Specificity of chemical biomarkers is an important consideration and has in the past led to archaeological misinterpretations of alkaloids and other compounds due to shared characteristics. Additionally, hordenine is not diagnostic to cereals, and its presence is not robust evidence of cereal caryopsis ingestion. This alkaloid is widespread in plants, and was only first described in Hordeum, not restricted to Hordeum and relatives. The methods refer to analyzing the "pellets left from a metagenomic study" which is as-yet unpublished. We need more detail about the processing leading to the analyzed pellet, i.e. is it an untreated piece of the calculus or the derivative of a DNA isolation procedure, which could have implications for analysis.

We thank the Reviewer for this comment. Upon reflection and considering the Reviewer’s comments, we decided not to present the results of GCMS in the new version of the article as we performed the analysis only on a select number of individuals.

47: "verified until now" slightly implies that you are definitely verifying it with this study, changing it "has not yet been verified" would be clearer.

Thank you for highlighting this. We have now changed this phrase into “However, the hypothesis of a systematic use of wild grasses of the Poaceae family (e.g., Aegilops spp.; Hordeum spp.) during the Mesolithic remains to be verified in this region.”

139: Salivary amylase is the most likely interpretation for this damage ("enzymatic digestion"), I would caution against suggesting that this could be interpreted as cultural modification via food preparation.

We agree that enzymatic digestion is likely the main explanation for damage observed in starch granules entrapped in archaeological dental calculus. However, recent experimental use of ground stone tools for processing various grains has resulted in the production of damaged granules. We have mentioned these results as an alternative scenario for explaining the presence of damaged starch granules in Neolithic individuals. Following the Reviewer’s comment and recent experimental results, we have now changed the phrase into the following: “Type A granules appear damaged in few EN individuals, which may be linked to enzymatic digestion (salivary amylase) although plant food processing could also result in starch damage based on experimental results (Ma et al. 2019; Zupancich et al. 2019).”

The numerous starch micrographs of comparative materials (Figure 6 and 7) are not very helpful without indicating diagnostic characters, with citation, and with specific relevance to the text explicated in the figure captions.

We thank the Reviewer for the comment. Each figure and the relevant panel are now cited correctly in the text. A description of the diagnostic features characterizing each of the granule types identified has been added to the text with reference to figures 6 and 7.

242-45: It is not clear if this form is diagnostic to the family Fabaceae, only "typical of" that family. The strong implication is that these can be interpreted in that way, but it is important to make clear whether these structures are found in other taxa, and clarify the confidence level of a Fabaceae interpretation on that basis. Also, Fabaceae contains nearly 20,000 species in all temperate and tropical regions globally, so again I would caution against the implication that these structures in this study are linked with the familiar crop species mentioned.

Many thanks for this comment. To the best of our knowledge, these characteristics are peculiar and diagnostic of starch granules included in the species of the plant family Fabaceae and not found in other taxa. Following the Reviewer’s suggestions, we have slightly changed the phrasing into the following: “All these features are very peculiar and diagnostic of starch granules included in the species of the plant family Fabaceae (Henry et al., 2011), […]. While many edible species of the family Fabaceae grow in the Balkans (e.g., Vicia sativa, V. hirsuta, V. ervilia, Lathyrus pratensis, L. sylvestris), an identification to species or genus was not possible due to overlaps in shape and size of starch granules at tribe level, which were observed in our modern reference collection” (l.298-305).

297: I don't fully understand "daily life crafting activities"

We have changed this into “daily life activities”.

335: Specify "foxtail millet" as the common name for S. italica to avoid confusion with other mentions of millets.

Thanks for this suggestion. We have now specified this in the revised version of the article text.

358: This is the first mention of the amyloplast context. This should be introduced and quantified in the Results section, and more information should be given here on the reliability of this context for determining the degree of processing.

We thank the Reviewer for this comment. We have now mentioned the recovery of starch granules in the amyloplast in the results.

387-388: I disagree that the common view is that groups of people without agriculture and domesticated plants are merely foraging opportunistically, I think this is a slightly outdated view. The bulk of domestication and early agriculture/pre-agriculture literature have come to recognize a longterm mutualism of humans and plants even in what we would interpret as wild forms. This doesn't affect the interpretation here in any problematic way and I do think it is an important topic to explore, only that I think the authors are setting up the result against a "common view" that isn't all that common.

We thank the Reviewer for this comment. We agree that our statement (“This perspective is different from commonly held views about only opportunistic use of available plant foods by foragers”) was inaccurate, so we decided to delete it.

410: I would advise against interpreting cultivation on this basis without further exploration of the ecological characters of these species. Several cultigen wild relatives are notoriously weedy, and so in many cases there's a strong possibility that these taxa colonize newly opened landscapes with no human intervention. In either case, cultivation here is an over-interpretation.

In the mentioned statement, which is now moved to the introduction (lines 66-69), we were citing an interpretation discussed by Edwards (1989). However, we have deleted the part of the phrase referring to cultivation.

411: Chenopodiaceae has been subsumed within Amaranthaceae.

Many thanks for pointing this out. We have now fixed this in the text.

461: The current study does not provide direct evidence for management of wild stands, only their consumption. This interpretation should be contextualized as likely management in light of other literature and ecological expectations, but it should be clear that management is not being tested here.

We agree with the Reviewer. We have avoided mentioning management in the phrase.

470-474: This section is a bit unclear, I can't tell what evidence is being interpreted by whom as implausible in the context of the cited study.

We have rephrased the section in the following way: “In the same study, the individuals dated to the Early Mesolithic exhibit a high occurrence of starch granules, which we have shown to be compatible with a variety of wild species of the Triticeae tribe (e.g., genus Aegilops). The evidence of Aegilops consumption during the Early Mesolithic was disregard as an “implausible pattern” (lines 542-545).

544-560: I'm afraid I find this paragraph to be over-interpreted. As mentioned above, the methods of the GC-MS approach are underdeveloped, and the specificity of the results is not clear, therefore without that extra information, interpreting specific compounds without regard to their botanical breadth and chemical lookalikes is not useful. Re omega-3 fatty acids, I think it's a bit of a reach to try and interpret them in an archaeobotanical context when the riverine resources would be such a clear contributor of these compounds.

We agree with the Reviewer that in the analyzed context, the presence of omega-3 fatty acids could be more easily explained by referring to the consumption of riverine resources rather than plant foods (although hazelnut DNA was recently recovered in the dental calculus of one Late Mesolithic individual from Vlasac – Ottoni et al. 2021). Upon reflection, we decided not to discuss GCMS results in the article as they were carried out only on a small subset of individuals.

Figure 8 is not called out in the text, and I'm not sure of its purpose.

We thank the Reviewer for this comment. We decided to remove this figure.

[Editors’ note: what follows is the authors’ response to the second round of review.]

The reviewers appreciated your efforts in addressing their collective previous comments, and they commented that they now agree that the paper presents compelling evidence for the exploitation of wild grasses prior to the introduction of domestic cereals in the Balkans. However, there are some remaining issues as pointed out by one of the reviewers that need to be addressed, as outlined below:

The identification of Type Ia starch grains as a domestic cereal seems to be based on the absence of a bimodal pattern of starch granules distribution in MOST (but not all) wild Aegilops species. Moreover, other wild Triticeae taxa have not been considered as potential origin for these starch grains. For example, Yang and Perry (2013: Figure 2b in JAS) found a bimodal assemblage in Leymus chinensis. This taxon does not naturally occur in the Balkans, but others within the genus do (e.g., Leymus racemosus). Other potential contributors include the genus Bromus, widely distributed throughout Eurasia. For these reasons, I do not think Type Ia should be assigned to a domestic cereal; at most, it can be suggested that it likely does not belong to the genus Aegilops.

We thank the reviewer for this comment.

We would like to stress that the possibility that archaeological Type Ia granules could be ascribed to other wild Triticeae taxa has been considered yet excluded based on experimental data, as reported in the discussion.

Species of the genera Elymus (e.g., Elymus caninus) and Agropyrum (e.g., Agropyrum pungens; A. farctus) growing in the Balkans have been analyzed and conclusions were published in our article in 2016 (Cristiani et al. 2016). We excluded the possibility that A-type granules from species of these genera could be compared to the archaeological Type Ia on morphological grounds. In particular, (a) starch granules extracted from these species are overall smaller than the Type Ia starch granules retrieved in the dental calculus of Mesolithic indiivduals in the Danube Gorges, (b) they show a different morphology (generally oval), and (c) starch granules do not show bimodal distribution. We feel these results on the morphology of starch granules from wild Triticeae taxa should be considered reliable as only the plant material from the region under study has been evaluated.

As suggested by the reviewer, we also explored species of the genus Bromus as we are aware they could be recognized as a possible contributor to the Type Ia population of starch granules from the Mesolithic Danube Gorges. To the best of our knowledge, only species of the Bromideae tribe, and in particular Bromus catharticus (Stoddard and Sarker 2000), are known to have a bimodal distribution. However, this species is not native to the Eurasian region. Other species of the genus Bromus (e.g., Bromus tectorium, Bromus secalinus, Bromus alopecuros, Bromus hordeaceus, Bromus arvensis) have also experimentally been analysed for the presence of starch granules. However, they were excluded based on the morphology, distribution, and dimensions of their granules. In particular, in these species, the granules are smaller than those in our archaeological record (around or below 11 μm). We also considered the genus Dasypyron (e.g. Dasypyron villosum), which is not bimodal.

Our conclusions are also supported by previously published data (Stoddard and Sarker 2000). Based on our experimental data, we confidently exclude that species of the genus Aegilops, and other wild species of the Triticeae and Bromideae tribes could be the origin of Type Ia granules. Consequently, we support the possibility that Type Ia granules should be ascribed to domesticated species of the Triticeae tribe available in the Balkans since ca. 6500 BC.

Finally, to show how wild taxa of the Triticeae tribe mentioned above are different in their morphology, dimensions, and distribution from the archaeological Type Ia, we have supplemented Figure 6 to include also some of the wild Triticeae taxa we experimentally analyzed for the presence of starch granules.

https://doi.org/10.7554/eLife.72976.sa2

Article and author information

Author details

  1. Emanuela Cristiani

    DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing – review and editing
    For correspondence
    emanuela.cristiani@uniroma1.it
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2748-9171
  2. Anita Radini

    1. DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
    2. Department of Archaeology, University of York, York, United Kingdom
    Contribution
    Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2099-2639
  3. Andrea Zupancich

    DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
    Contribution
    Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9503-1234
  4. Angelo Gismondi

    Laboratory of General Botany, Department of Biology, University of Rome "Tor Vergata", Rome, Italy
    Contribution
    Formal analysis, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9257-9667
  5. Alessia D'Agostino

    Laboratory of General Botany, Department of Biology, University of Rome "Tor Vergata", Rome, Italy
    Contribution
    Formal analysis, Writing - original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  6. Claudio Ottoni

    1. DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
    2. Centre of Molecular Anthropology for Ancient DNA Studies; Department of Biology, University of Rome "Tor Vergata", Rome, Italy
    Contribution
    Formal analysis, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8870-1589
  7. Marialetizia Carra

    DANTE - Diet and Ancient Technology Laboratory, Department of Oral and Maxillofacial Sciences, Sapienza University of Rome, Rome, Italy
    Contribution
    Writing – review and editing
    Competing interests
    No competing interests declared
  8. Snežana Vukojičić

    University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Belgrade, Serbia
    Contribution
    Resources, Writing – review and editing
    Competing interests
    No competing interests declared
  9. Mihai Constantinescu

    Romanian Academy, Institute for Anthropological Research “Francisc I. Rainer”, Bucharest, Romania
    Contribution
    Resources, Writing – review and editing
    Competing interests
    No competing interests declared
  10. Dragana Antonović

    Institute of Archaeology, Belgrade, Serbia
    Contribution
    Resources, Writing – review and editing
    Competing interests
    No competing interests declared
  11. T Douglas Price

    Department of Anthropology, University of Wisconsin, Madison, United States
    Contribution
    Resources, Writing – review and editing
    Competing interests
    No competing interests declared
  12. Dušan Borić

    1. Department of Environmental Biology, Sapienza University of Rome, Rome, Italy
    2. The Italian Academy for Advanced Studies in America, Columbia University, New York, United States
    3. Department of Anthropology, New York University, New York, United States
    Contribution
    Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing – review and editing
    For correspondence
    dusan.boric@uniroma1.it
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0166-627X

Funding

H2020 European Research Council (639286)

  • Emanuela Cristiani

National Science Foundation (BCS-0235465)

  • T Douglas Price
  • Dušan Borić

NOMIS Stiftung

  • Dušan Borić

Wellcome Trust (209869/Z/17/Z)

  • Anita Radini

British Academy (SG-42170)

  • Dušan Borić

British Academy (LRG-45589)

  • Dušan Borić

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors wish to thank the late Živko Mikić (University of Belgrade), the late Borislav Jovanović (Serbian Academy of Sciences and Arts), Duško Šljivar (National Museum in Belgrade), and Jelena Rankov (National Museum, Belgrade) for permissions to sample the osteological collections from the Danube Gorges. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

Senior and Reviewing Editor

  1. George H Perry, Pennsylvania State University, United States

Publication history

  1. Received: August 11, 2021
  2. Accepted: November 30, 2021
  3. Accepted Manuscript published: December 1, 2021 (version 1)
  4. Version of Record published: January 21, 2022 (version 2)
  5. Version of Record updated: January 25, 2022 (version 3)

Copyright

© 2021, Cristiani et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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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
(2021)
Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture
eLife 10:e72976.
https://doi.org/10.7554/eLife.72976

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