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.

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.

Figure 1

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Figure 2

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Figure 3

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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.

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.

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.

Figure 10

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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).

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

1. Book

   1. Adams JL
   2. Delgado S
   3. Dubreuil L
   4. Hamon C
   5. Plisson H
   6. Risch R

   (2006)

   Functional analysis of macro-lithic artefacts: A focus on working surface

   In: Sternke F, Eigeland L, Costa LJ, editors. Non-Flint Raw Material Use in Prehistory. Old Prejudices and New Directions. BAR International Series. pp. 4–9.

   • Google Scholar
2. Book

   1. Antonović D

   (2006)

   Stone Tools from Lepenski Vir

   Belgrade: Institute of Archaeology.

   • Google Scholar
3. 1. Asouti E
   2. Ntinou M
   3. Kabukcu C

   (2018) The impact of environmental change on Palaeolithic and Mesolithic plant use and the transition to agriculture at Franchthi Cave, Greece

   PLOS ONE 13:e0207805.

   https://doi.org/10.1371/journal.pone.0207805

   • PubMed
   • Google Scholar
4. 1. Barton H
   2. Mutri G
   3. Hill E
   4. Farr L
   5. Barker G

   (2018) Use of grass seed resources c.31 ka by modern humans at the Haua Fteah cave, northeast Libya

   Journal of Archaeological Science 99:99–111.

   https://doi.org/10.1016/j.jas.2018.08.013

   • Google Scholar
5. 1. Blumenthal C
   2. Wrigley CW
   3. Batey IL
   4. Barlow EWR

   (1994) The heat-shock response relevant to molecular and structural changes in wheat yield and quality

   Functional Plant Biology 21:901–909.

   https://doi.org/10.1071/PP9940901

   • Google Scholar
6. 1. Blumenthal C
   2. Bekes F
   3. Gras PW
   4. Barlow EWR
   5. Wrigley CW

   (1995)

   Identification of wheat genotypes tolerant to the effects of heat stress on grain quality

   Cereal Chem 72:539–544.

   • Google Scholar
7. 1. Bonsall C

   (2008)

   The Mesolithic of the Iron Gates

   Mesolithic Europe 2008:238–279.

   • Google Scholar
8. 1. Bonsall C
   2. Lennon R
   3. McSweeney K
   4. Stewart C
   5. Harkness D
   6. Boronean V
   7. Bartosiewicz L
   8. Payton R
   9. Chapman J

   (2013) Mesolithic and Early Neolithic in the Iron Gates: A Palaeodietary Perspective

   Journal of European Archaeology 5:50–92.

   https://doi.org/10.1179/096576697800703575

   • Google Scholar
9. 1. Borić D
   2. Grupe G
   3. Peters J
   4. Mikić Ž

   (2004) Is the Mesolithic–Neolithic subsistence dichotomy real? New stable isotope evidence from the Danube Gorges

   European Journal of Archaeology 7:221–248.

   https://doi.org/10.1177/1461957104056500

   • Google Scholar
10. Conference

    1. Borić D

    (2011)

    Presented at the Beginnings - New research in the appearance of the Neolithic between Northwest Anatolia and the Carpathian Basin: Papers of the International Workshop

    Adaptations and transformations of the Danube Gorges foragers (c. 13,000–5500 cal. BC): an overview.

    • Google Scholar
11. 1. Borić D
    2. Price TD

    (2013) Strontium isotopes document greater human mobility at the start of the Balkan Neolithic

    PNAS 110:3298–3303.

    https://doi.org/10.1073/pnas.1211474110

    • PubMed
    • Google Scholar
12. 1. Borić D
    2. French CAI
    3. Stefanović S
    4. Dimitrijević V
    5. Cristiani E
    6. Gurova M
    7. Antonović D
    8. Allué E
    9. Filipović D

    (2014) Late Mesolithic lifeways and deathways at Vlasac (Serbia)

    Journal of Field Archaeology 39:4–31.

    https://doi.org/10.1179/0093469013Z.00000000070

    • Google Scholar
13. Book

    1. Borić D

    (2016)

    Deathways at Lepenski Vir: Patterns in Mortuary Practice

    Belgrade: Serbian Archaeological Society.

    • Google Scholar
14. 1. Borić D
    2. Higham T
    3. Cristiani E
    4. Dimitrijević V
    5. Nehlich O
    6. Griffiths S
    7. Alexander C
    8. Mihailović B
    9. Filipović D
    10. Allué E
    11. Buckley M

    (2018) High-resolution AMS dating of architecture, boulder artworks and the transition to farming at Lepenski Vir

    Scientific Reports 8:14221.

    https://doi.org/10.1038/s41598-018-31884-7

    • PubMed
    • Google Scholar
15. 1. Buckley S
    2. Usai D
    3. Jakob T
    4. Radini A
    5. Hardy K

    (2014) Dental calculus reveals unique insights into food items, cooking and plant processing in prehistoric central Sudan

    PLOS ONE 9:e100808.

    https://doi.org/10.1371/journal.pone.0100808

    • PubMed
    • Google Scholar
16. 1. Cârciumaru M

    (1973)

    Compte rendu de l’analyse pollinique des coprolithes d’Icoana--Portes de Fier

    Actes Du VIIIe Congrès International Des Sciences Préhistoriques et Protohostoriques, Beograd 1971:172–173.

    • Google Scholar
17. Book

    1. Cârciumaru M

    (1978) L’analyse pollinique des coprolithes de la station archéologique de Vlasac

    In: Garasanin M, editors. Vlasac-A Mesolithic Settlement in the Iron Gates - Vol. II: Geology-Biology-Anthropology. Beograd. pp. 31–34.

    https://doi.org/10.13140/2.1.1394.4646

    • Google Scholar
18. Book

    1. Clarke DL

    (1978)

    Mesolithic Europe: The Economic Basis

    Duckworth.

    • Google Scholar
19. Book

    1. Costantini L

    (1989)

    Plant exploitation at Grotta dell’Uzzo, Sicily: new evidence for the transition from Mesolithic to Neolithic subsistence in southern Europe

    In: Harris D, Hillman G, editors. Foraging and Farming: The Evolution of Plant Exploitation. Unwin Hyman Ltd. pp. 762–766.

    • Google Scholar
20. 1. Cristiani E
    2. Radini A
    3. Edinborough M
    4. Borić D

    (2016) Dental calculus reveals Mesolithic foragers in the Balkans consumed domesticated plant foods

    PNAS 113:10298–10303.

    https://doi.org/10.1073/pnas.1603477113

    • PubMed
    • Google Scholar
21. 1. Cristiani E
    2. Radini A
    3. Borić D
    4. Robson HK
    5. Caricola I
    6. Carra M
    7. Mutri G
    8. Oxilia G
    9. Zupancich A
    10. Šlaus M
    11. Vujević D

    (2018) Dental calculus and isotopes provide direct evidence of fish and plant consumption in Mesolithic Mediterranean

    Scientific Reports 8:8147.

    https://doi.org/10.1038/s41598-018-26045-9

    • PubMed
    • Google Scholar
22. 1. Cristiani E
    2. Zupancich A

    (2020) Sandstone ground stone technology: a multi-level use wear and residue approach to investigate the function of pounding and grinding tools

    Journal of Archaeological Method and Theory 28:704–735.

    https://doi.org/10.1007/s10816-020-09488-1

    • Google Scholar
23. 1. Cummings LS
    2. Yost C
    3. Sołtysiak A

    (2016) Plant microfossils in human dental calculus from Nemrik 9, a Pre-Pottery Neolithic site in Northern Iraq

    Archaeological and Anthropological Sciences 10:883–891.

    https://doi.org/10.1007/s12520-016-0411-3

    • Google Scholar
24. 1. de Beaune SA

    (2004) The Invention of Technology: Prehistory and Cognition

    Current Anthropology 45:139–162.

    https://doi.org/10.1086/381045

    • Google Scholar
25. 1. Dietrich L
    2. Meister J
    3. Dietrich O
    4. Notroff J
    5. Kiep J
    6. Heeb J
    7. Beuger A
    8. Schütt B

    (2019) Cereal processing at Early Neolithic Göbekli Tepe, southeastern Turkey

    PLOS ONE 14:e0215214.

    https://doi.org/10.1371/journal.pone.0215214

    • PubMed
    • Google Scholar
26. 1. Divišová M
    2. Šída P

    (2015) Plant Use in the Mesolithic Period. Archaeobotanical Data from the Czech Republic in a European Context – a Review

    Interdisciplinaria Archaeologica - Natural Sciences in Archaeology 6:95–106.

    https://doi.org/10.24916/iansa.2015.1.7

    • Google Scholar
27. 1. Dubreuil L
    2. Nadel D

    (2015) The development of plant food processing in the Levant: insights from use-wear analysis of Early Epipalaeolithic ground stone tools

    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 370:20140357.

    https://doi.org/10.1098/rstb.2014.0357

    • PubMed
    • Google Scholar
28. Book

    1. Dubreuil L
    2. Savage D
    3. Delgado-Raack S
    4. Plisson H
    5. Stephenson B
    6. de Torre L

    (2015) Current Analytical Frameworks for Studies of Use–Wear on Ground Stone Tools

    In: Marreiros JM, Gibaja Bao JF, Ferreira Bicho N, editors. Use-Wear and Residue Analysis in Archaeology. Cham: Springer International Publishing. pp. 105–158.

    https://doi.org/10.1007/978-3-319-08257-8

    • Google Scholar
29. Book

    1. Edwards KJ

    (1989)

    The Cereal Pollen Record and Early Agriculture

    BAR International Series.

    • Google Scholar
30. 1. Filipović D
    2. Allué EA
    3. Borić D

    (2010)

    Integrated carpological and antracological analysis of plant record from the Mesolithic site of Vlasac, Serbia

    Journal of the Serbian Archaeological Society 26:145–161.

    • Google Scholar
31. 1. Fuller DQ
    2. Willcox G
    3. Allaby RG

    (2011) Cultivation and domestication had multiple origins: arguments against the core area hypothesis for the origins of agriculture in the Near East

    World Archaeology 43:628–652.

    https://doi.org/10.1080/00438243.2011.624747

    • Google Scholar
32. 1. Geera BP
    2. Nelson JE
    3. Souza E
    4. Huber KC

    (2006) Composition and properties of A- and B-type starch granules of wild-type, partial waxy, and waxy soft wheat

    Cereal Chemistry Journal 83:551–557.

    https://doi.org/10.1094/CC-83-0551

    • Google Scholar
33. Book

    1. Halstead P

    (1996)

    The development of agriculture and pastoralism in Greece: When, how, who and what

    In: Harris DR, editors. The Origins and Spread of Agriculture and Pastoralism in Eurasia. Institute of Archaeology. pp. 296–309.

    • Google Scholar
34. Book

    1. Hamon C
    2. Plisson H

    (2009)

    Functional analysis of grinding stones: the blind-test contribution In

    In: Skakun N LL, editors. Prehistoric Technology 40 Years Later: Functional Studies and the Russian Legacy. BAR International Series. pp. 29–38.

    • Google Scholar
35. 1. Hamon C
    2. Cagnato C
    3. Emery-Barbier A
    4. Salavert A

    (2021) Food practices of the first farmers of Europe: Combined use-wear and microbotanical studies of Early Neolithic grinding tools from the Paris Basin

    Journal of Archaeological Science 36:102764.

    https://doi.org/10.1016/j.jasrep.2020.102764

    • Google Scholar
36. Book

    1. Hansen JM

    (1991)

    The Palaeoethnobotany of Franchthi Cave

    Indiana University Press.

    • Google Scholar
37. 1. Haslam M

    (2004) The decomposition of starch grains in soils: implications for archaeological residue analyses

    Journal of Archaeological Science 31:1715–1734.

    https://doi.org/10.1016/j.jas.2004.05.006

    • Google Scholar
38. 1. Hastorf CA
    2. Foxhall L

    (2017) The social and political aspects of food surplus

    World Archaeology 49:26–39.

    https://doi.org/10.1080/00438243.2016.1252280

    • Google Scholar
39. 1. Henry AG
    2. Piperno DR

    (2008) Using plant microfossils from dental calculus to recover human diet: a case study from Tell al-Raqā’i, Syria

    Journal of Archaeological Science 35:1943–1950.

    https://doi.org/10.1016/j.jas.2007.12.005

    • Google Scholar
40. 1. Henry AG
    2. Brooks AS
    3. Piperno DR

    (2011) Microfossils in calculus demonstrate consumption of plants and cooked foods in Neanderthal diets (Shanidar III, Iraq; Spy I and II, Belgium)

    PNAS 108:486–491.

    https://doi.org/10.1073/pnas.1016868108

    • PubMed
    • Google Scholar
41. 1. Holden TG
    2. Hather JG
    3. Watson JPN

    (1995) Mesolithic plant exploitation at the Roc del Migdia, Catalonia

    Journal of Archaeological Science 22:769–778.

    https://doi.org/10.1016/0305-4403(95)90006-3

    • Google Scholar
42. 1. Howard T
    2. Rejab NA
    3. Griffiths S
    4. Leigh F
    5. Leverington-Waite M
    6. Simmonds J
    7. Uauy C
    8. Trafford K

    (2011) Identification of a major QTL controlling the content of B-type starch granules in Aegilops

    Journal of Experimental Botany 62:2217–2228.

    https://doi.org/10.1093/jxb/erq423

    • PubMed
    • Google Scholar
43. 1. Jovanović J
    2. Blagojević T
    3. Marković J
    4. Novak M
    5. Bedić Ž
    6. Naumov G
    7. Stojanova Kanzurova E
    8. Los D
    9. Hutinec M
    10. Fidanoski L
    11. Skelac G
    12. Šlaus M
    13. Stefanović S

    (2021a) New radiocarbon dates, stable isotope, and anthropological analysis of prehistoric human bones from the Balkans and Southwestern Carpathian Basin

    Documenta Praehistorica 48:224–251.

    https://doi.org/10.4312/dp.48.18

    • Google Scholar
44. 1. Jovanović J
    2. Power RC
    3. de Becdelièvre C
    4. Goude G
    5. Stefanović S

    (2021b) Microbotanical evidence for the spread of cereal use during the Mesolithic-Neolithic transition in the Southeastern Europe (Danube Gorges): Data from dental calculus analysis

    Journal of Archaeological Science 125:105288.

    https://doi.org/10.1016/j.jas.2020.105288

    • Google Scholar
45. 1. Kotsakis K

    (2003)

    From the Neolithic side: the Mesolithic/Neolithic interface in Greece

    British School at Athens Studies 10:217–221.

    • Google Scholar
46. Book

    1. Kotzamani G
    2. Livarda A

    (2014)

    Plant resource availability and management in Palaeolithic and Mesolithic Greece

    In: Touchais G, Laffineur R, Rougemont F, editors. Physis: L’environmement Naturel et La Relation Homme-Milieu Dans Le Monde Égéen Protohistorique, Aegaeum. Peeters Pub & Booksellers. pp. 229–236.

    • Google Scholar
47. 1. Kubiak-Martens L

    (1999) The plant food component of the diet at the Late Mesolithic (Ertebolle) settlement at Tybrind Vig, Denmark

    Vegetation History and Archaeobotany 8:117–127.

    https://doi.org/10.1007/BF02042850

    • Google Scholar
48. 1. Langejans GHJ

    (2010) Remains of the day-preservation of organic micro-residues on stone tools

    Journal of Archaeological Science 37:971–985.

    https://doi.org/10.1016/j.jas.2009.11.030

    • Google Scholar
49. Book

    1. Lee RB
    2. DeVore I
    3. Lee RB

    (1968) The first intensive survey of a single, crucial stage of human development— Man’s once universal hunting way of life

    In: Devore I, editors. Man the Hunter. Routledge Taylor and Francis Group. pp. 1–432.

    https://doi.org/10.4324/9780203786567

    • Google Scholar
50. 1. Liu L
    2. Field J
    3. Fullagar R
    4. Bestel S
    5. Chen X
    6. Ma X

    (2015) What did grinding stones grind? New light on Early Neolithic subsistence economy in the Middle Yellow River Valley, China

    Antiquity 84:816–833.

    https://doi.org/10.1017/S0003598X00100249

    • Google Scholar
51. 1. Livarda A

    (2010) Spicing up life in northwestern Europe: exotic food plant imports in the Roman and medieval world

    Vegetation History and Archaeobotany 20:143–164.

    https://doi.org/10.1007/s00334-010-0273-z

    • Google Scholar
52. 1. Lombardo U
    2. Iriarte J
    3. Hilbert L
    4. Ruiz-Pérez J
    5. Capriles JM
    6. Veit H

    (2020) Early Holocene crop cultivation and landscape modification in Amazonia

    Nature 581:190–193.

    https://doi.org/10.1038/s41586-020-2162-7

    • PubMed
    • Google Scholar
53. Book

    1. Lowie RH

    (1922)

    Material Culture of the Crow Indians

    Lakota Books.

    • Google Scholar
54. 1. Lucarini G
    2. Radini A
    3. Barton H
    4. Barker G

    (2016) The exploitation of wild plants in Neolithic North Africa. Use-wear and residue analysis on non-knapped stone tools from the Haua Fteah cave, Cyrenaica, Libya

    Quaternary International 2016:109.

    https://doi.org/10.1016/j.quaint.2015.11.109

    • Google Scholar
55. 1. Madella M
    2. Lancelotti C
    3. García-Granero JJ

    (2013) Millet microremains—an alternative approach to understand cultivation and use of critical crops in Prehistory

    Archaeological and Anthropological Sciences 8:0130-y.

    https://doi.org/10.1007/s12520-013-0130-y

    • Google Scholar
56. 1. Marinova E
    2. Filipovic D
    3. Obradović D
    4. Allué E

    (2013)

    Wild plant resources and land use in the Mesolithic and early Neolithic south-east Europe: Archaeobotanical evidence from the Danube catchment of Bulgaria and Serbia

    Offa 69:467–478.

    • PubMed
    • Google Scholar
57. 1. Mariotti Lippi M
    2. Foggi B
    3. Aranguren B
    4. Ronchitelli A
    5. Revedin A

    (2015) Multistep food plant processing at Grotta Paglicci (Southern Italy) around 32,600 cal B.P

    PNAS 112:12075–12080.

    https://doi.org/10.1073/pnas.1505213112

    • PubMed
    • Google Scholar
58. Book

    1. Mason SLR

    (1995)

    Acornutopia? Determining the role of acorns in past human subsistence

    In: Wilkins J, editors. Food in Antiquity. University of Exeter Press. pp. 12–24.

    • Google Scholar
59. 1. Mason S
    2. Boroneanţ V
    3. Bonsall C

    (1996)

    Plant remains from Schela Cladovei, Romania

    Mesolithic Miscellany 17:11–14.

    • Google Scholar
60. 1. Mathieson I
    2. Alpaslan-Roodenberg S
    3. Posth C
    4. Szécsényi-Nagy A
    5. Rohland N
    6. Mallick S
    7. Olalde I
    8. Broomandkhoshbacht N
    9. Candilio F
    10. Cheronet O
    11. Fernandes D
    12. Ferry M
    13. Gamarra B
    14. Fortes GG
    15. Haak W
    16. Harney E
    17. Jones E
    18. Keating D
    19. Krause-Kyora B
    20. Kucukkalipci I
    21. Michel M
    22. Mittnik A
    23. Nägele K
    24. Novak M
    25. Oppenheimer J
    26. Patterson N
    27. Pfrengle S
    28. Sirak K
    29. Stewardson K
    30. Vai S
    31. Alexandrov S
    32. Alt KW
    33. Andreescu R
    34. Antonović D
    35. Ash A
    36. Atanassova N
    37. Bacvarov K
    38. Gusztáv MB
    39. Bocherens H
    40. Bolus M
    41. Boroneanţ A
    42. Boyadzhiev Y
    43. Budnik A
    44. Burmaz J
    45. Chohadzhiev S
    46. Conard NJ
    47. Cottiaux R
    48. Čuka M
    49. Cupillard C
    50. Drucker DG
    51. Elenski N
    52. Francken M
    53. Galabova B
    54. Ganetsovski G
    55. Gély B
    56. Hajdu T
    57. Handzhyiska V
    58. Harvati K
    59. Higham T
    60. Iliev S
    61. Janković I
    62. Karavanić I
    63. Kennett DJ
    64. Komšo D
    65. Kozak A
    66. Labuda D
    67. Lari M
    68. Lazar C
    69. Leppek M
    70. Leshtakov K
    71. Vetro DL
    72. Los D
    73. Lozanov I
    74. Malina M
    75. Martini F
    76. McSweeney K
    77. Meller H
    78. Menđušić M
    79. Mirea P
    80. Moiseyev V
    81. Petrova V
    82. Price TD
    83. Simalcsik A
    84. Sineo L
    85. Šlaus M
    86. Slavchev V
    87. Stanev P
    88. Starović A
    89. Szeniczey T
    90. Talamo S
    91. Teschler-Nicola M
    92. Thevenet C
    93. Valchev I
    94. Valentin F
    95. Vasilyev S
    96. Veljanovska F
    97. Venelinova S
    98. Veselovskaya E
    99. Viola B
    100. Virag C
    101. Zaninović J
    102. Zäuner S
    103. Stockhammer PW
    104. Catalano G
    105. Krauß R
    106. Caramelli D
    107. Zariņa G
    108. Gaydarska B
    109. Lillie M
    110. Nikitin AG
    111. Potekhina I
    112. Papathanasiou A
    113. Borić D
    114. Bonsall C
    115. Krause J
    116. Pinhasi R
    117. Reich D

    (2018) The genomic history of southeastern Europe

    Nature 555:197–203.

    https://doi.org/10.1038/nature25778

    • PubMed
    • Google Scholar
61. 1. Nadel D
    2. Piperno DR
    3. Holst I
    4. Snir A
    5. Weiss E

    (2015) New evidence for the processing of wild cereal grains at Ohalo II, a 23 000-year-old campsite on the shore of the Sea of Galilee, Israel

    Antiquity 86:990–1003.

    https://doi.org/10.1017/S0003598X00048201

    • Google Scholar
62. 1. Nava A
    2. Fiorin E
    3. Zupancich A
    4. Carra M
    5. Ottoni C
    6. Di Carlo G
    7. Vozza I
    8. Brugnoletti O
    9. Alhaique F
    10. Cremonesi RG
    11. Coppa A
    12. Bondioli L
    13. Borić D
    14. Cristiani E

    (2021) Multipronged dental analyses reveal dietary differences in last foragers and first farmers at Grotta Continenza, central Italy (15,500-7000 BP)

    Scientific Reports 11:4261.

    https://doi.org/10.1038/s41598-021-82401-2

    • PubMed
    • Google Scholar
63. 1. Norström E
    2. Gustavsson R
    3. Molin F
    4. Gummesson S

    (2019) Micro-fossil analysis of Mesolithic human dental calculus, Motala, Sweden - Indications of health status and paleo-diet

    Journal of Archaeological Science 26:101866.

    https://doi.org/10.1016/j.jasrep.2019.05.031

    • Google Scholar
64. 1. Ottoni C
    2. Borić D
    3. Cheronet O
    4. Sparacello V
    5. Dori I
    6. Coppa A
    7. Antonović D
    8. Vujević D
    9. Price TD
    10. Pinhasi R
    11. Cristiani E

    (2021) Tracking the transition to agriculture in Southern Europe through ancient DNA analysis of dental calculus

    PNAS 118:e2102116118.

    https://doi.org/10.1073/pnas.2102116118

    • PubMed
    • Google Scholar
65. 1. Pardoe C
    2. Fullagar R
    3. Hayes E

    (2019) Quandong stones: A specialised Australian nut-cracking tool

    PLOS ONE 14:e0222680.

    https://doi.org/10.1371/journal.pone.0222680

    • PubMed
    • Google Scholar
66. 1. Piperno DR
    2. Weiss E
    3. Holst I
    4. Nadel D

    (2004) Processing of wild cereal grains in the Upper Palaeolithic revealed by starch grain analysis

    Nature 430:670–673.

    https://doi.org/10.1038/nature02734

    • PubMed
    • Google Scholar
67. Conference

    1. Portillo M
    2. Albert RM

    (2014)

    Revista d’arqueologia de Ponent

    Microfossil evidence for grinding activities.

    • Google Scholar
68. 1. Pryor AJE
    2. Steele M
    3. Jones MK
    4. Svoboda J
    5. Beresford-Jones DG

    (2013) Plant foods in the Upper Palaeolithic at Dolní Věstonice? Parenchyma redux

    Antiquity 87:971–984.

    https://doi.org/10.1017/S0003598X00049802

    • Google Scholar
69. 1. Radini A
    2. Buckley S
    3. Rosas A
    4. Estalrrich A
    5. de la Rasilla M
    6. Hardy K

    (2016) Neanderthals, trees and dental calculus: new evidence from El Sidrón

    Antiquity 90:290–301.

    https://doi.org/10.15184/aqy.2016.21

    • Google Scholar
70. 1. Radini A
    2. Nikita E
    3. Buckley S
    4. Copeland L
    5. Hardy K

    (2017) Beyond food: The multiple pathways for inclusion of materials into ancient dental calculus

    American Journal of Physical Anthropology 162:71–83.

    https://doi.org/10.1002/ajpa.23147

    • PubMed
    • Google Scholar
71. 1. Radini A
    2. Tromp M
    3. Beach A
    4. Tong E
    5. Speller C
    6. McCormick M
    7. Dudgeon JV
    8. Collins MJ
    9. Rühli F
    10. Kröger R
    11. Warinner C

    (2019) Medieval women’s early involvement in manuscript production suggested by lapis lazuli identification in dental calculus

    Science Advances 5:eaau7126.

    https://doi.org/10.1126/sciadv.aau7126

    • PubMed
    • Google Scholar
72. Book

    1. Radovanović I

    (1996)

    The Iron Gates Mesolithic

    International Monographs in Prehistory.

    • Google Scholar
73. 1. Reimer PJ
    2. Austin WEN
    3. Bard E
    4. Bayliss A
    5. Blackwell PG
    6. Bronk Ramsey C
    7. Butzin M
    8. Cheng H
    9. Edwards RL
    10. Friedrich M
    11. Grootes PM
    12. Guilderson TP
    13. Hajdas I
    14. Heaton TJ
    15. Hogg AG
    16. Hughen KA
    17. Kromer B
    18. Manning SW
    19. Muscheler R
    20. Palmer JG
    21. Pearson C
    22. van der Plicht J
    23. Reimer RW
    24. Richards DA
    25. Scott EM
    26. Southon JR
    27. Turney CSM
    28. Wacker L
    29. Adolphi F
    30. Büntgen U
    31. Capano M
    32. Fahrni SM
    33. Fogtmann-Schulz A
    34. Friedrich R
    35. Köhler P
    36. Kudsk S
    37. Miyake F
    38. Olsen J
    39. Reinig F
    40. Sakamoto M
    41. Sookdeo A
    42. Talamo S

    (2020) The IntCal20 Northern Hemisphere radiocarbon age calibration curve (0–55 cal kBP)

    Radiocarbon 62:725–757.

    https://doi.org/10.1017/RDC.2020.41

    • Google Scholar
74. 1. Rowley-Conwy P
    2. Layton R

    (2011) Foraging and farming as niche construction: stable and unstable adaptations

    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 366:849–862.

    https://doi.org/10.1098/rstb.2010.0307

    • PubMed
    • Google Scholar
75. 1. Sabin S
    2. Fellow Yates J

    (2020) Dental calculus field-sampling protocol

    Protocols.Io.

    https://doi.org/10.17504/protocols.io.bqecmtaw

    • Google Scholar
76. 1. Scott AC
    2. Damblon F

    (2010) Charcoal: Taphonomy and significance in geology, botany and archaeology

    Palaeogeography, Palaeoclimatology, Palaeoecology 291:1–10.

    https://doi.org/10.1016/j.palaeo.2010.03.044

    • Google Scholar
77. Book

    1. Siegfried EV

    (1994)

    Ethnobotany of the Northern Cree of Wabasca/Desmarais

    University of Calgary.

    • Google Scholar
78. 1. Smith BD

    (2012) A Cultural Niche Construction Theory of Initial Domestication

    Biological Theory 6:260–271.

    https://doi.org/10.1007/s13752-012-0028-4

    • Google Scholar
79. 1. Soto M
    2. Inwood J
    3. Clarke S
    4. Crowther A
    5. Covelli D
    6. Favreau J
    7. Itambu M
    8. Larter S
    9. Lee P
    10. Lozano M
    11. Maley J
    12. Mwambwiga A
    13. Patalano R
    14. Sammynaiken R
    15. Vergès JM
    16. Zhu J
    17. Mercader J

    (2019) Structural characterization and decontamination of dental calculus for ancient starch research

    Archaeological and Anthropological Sciences 11:4847–4872.

    https://doi.org/10.1007/s12520-019-00830-7

    • Google Scholar
80. Book

    1. Srejović D

    (1972)

    New Discoveries at Lepenski Vir: Europe’s First Monumental Sculpture

    Thames and Hudson.

    • Google Scholar
81. Book

    1. Srejović D
    2. Letica Z

    (1978)

    Vlasac: A Mesolithic Settlement in the Iron Gates

    Serbian Academy of Sciences and Arts.

    • Google Scholar
82. Book

    1. Srejović D

    (1988)

    The Neolithic of Serbia: Archaeological Research 1948–1988

    University of Belgrade.

    • Google Scholar
83. 1. Stoddard FL

    (1999) Survey of Starch Particle-Size Distribution in Wheat and Related Species

    Cereal Chemistry Journal 76:145–149.

    https://doi.org/10.1094/CCHEM.1999.76.1.145

    • Google Scholar
84. 1. Stoddard FL
    2. Sarker R

    (2000) Characterization of starch in Aegilops species

    Cereal Chemistry Journal 77:445–447.

    https://doi.org/10.1094/CCHEM.2000.77.4.445

    • Google Scholar
85. Book

    1. Van Andel TH
    2. Sutton SB
    3. Vitaliano CJ

    (1987)

    Landscape and People of the Franchthi Region

    Indiana University Press.

    • Google Scholar
86. 1. Vaquer J
    2. Geddes D
    3. Barbaza M
    4. Erroux J

    (1986) Mesolithic plant exploitation at the Balma Abeurador (France)

    Journal of Archaeology 5:1–18.

    https://doi.org/10.1111/j.1468-0092.1986.tb00127.x

    • Google Scholar
87. 1. Weber SA
    2. Fuller DQ

    (2008)

    Millets and their role in early agriculture

    Pragdhara 18:e90.

    • Google Scholar
88. 1. Wright KI

    (2017) Ground-stone tools and hunter-gatherer subsistence in Southwest Asia: Implications for the transition to farming

    American Antiquity 59:238–263.

    https://doi.org/10.2307/281929

    • Google Scholar
89. 1. Yang X
    2. Perry L

    (2013) Identification of ancient starch grains from the tribe Triticeae in the North China Plain

    Journal of Archaeological Science 40:3170–3177.

    https://doi.org/10.1016/j.jas.2013.04.004

    • Google Scholar
90. 1. y’Edynak G
    2. Fleisch S

    (1983) Microevolution and biological adaptability in the transition from food-collecting to food-producing in the Iron Gates of Yugoslavia

    Journal of Human Evolution 12:279–296.

    https://doi.org/10.1016/S0047-2484(83)80150-X

    • Google Scholar
91. 1. Zarrillo S
    2. Kooyman B

    (2017) Evidence for berry and maize processing on the Canadian Plains from starch grain analysis

    American Antiquity 71:473–499.

    https://doi.org/10.1017/S0002731600039779

    • Google Scholar
92. 1. Zupancich A
    2. Mutri G
    3. Caricola I
    4. Carra ML
    5. Radini A
    6. Cristiani E

    (2019) The application of 3D modeling and spatial analysis in the study of groundstones used in wild plants processing

    Archaeological and Anthropological Sciences 11:4801–4827.

    https://doi.org/10.1007/s12520-019-00824-5

    • Google Scholar
93. 1. Zvelebil M

    (2014) Plant use in the Mesolithic and its role in the transition to farming

    Proceedings of the Prehistoric Society 60:35–74.

    https://doi.org/10.1017/S0079497X00003388

    • Google Scholar

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.

[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.

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

   "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

   "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

   "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

   "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

   "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

    "This ORCID iD identifies the author of this article:" 0000-0003-0166-627X

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