Philippa Kaur

Sampling, preparation and treatment of modern reference materialA total of 53 modern wood samples were analyzed. The modern reference samples were collected in 2014 during the annual archaeological field mission, from 36 individuals (Table S1). For some trees, two wood samples of different diameters were collected in order to take into account anatomical variability within individual.The collected individuals showed different conditions of growth described in the introduction section and detailed in the Table 1. With the agreement of the Tifigit inhabitants and local authorities, wood sampling was achieved but samples were not collected from trunks, to avoid injuring trees of major symbolic, ecological and economic importance. Only section samples with perfect axial symmetry were retained to avoid any impact of biomechanical constraints (formation of reaction wood) on wood characters.Once collected, the samples were air-dried during a month at the laboratory. The samples were separately wrapped in tin foil and buried in the sand and then charred without oxygen, at 450 °C for 15 to 20 min depending on the size of the sample. As a result, samples were enriched in carbon (content  > 90%)20,26, reached their maximal shrinkage27, and thus are considered to become morphologically comparable to charcoal produced in medieval fires27,28,29,30,31. The minimum and the maximum diameter of wood samples were measured (mm) using a digital measuring calliper before and after carbonization. The diameter used in the following analyses is the mean of the two measurements carried out before carbonization.Archaeological materialTwenty archaeological charcoal fragments of Argan tree identified during a previous analysis session13 were included in this study (Table S2). All the Argan charcoal fragments were collected in the medieval archaeological deposits of Îgîlîz13. They come from various contexts, for the most part from living units, and belong to the period of highest human activity at the site, between the late 11th and early thirteenth centuries.Quantitative eco-anatomical analysis of wood applied to the Argan treeThe approach consists in measuring constitutive elements of wood and aims to understand variations according to intrinsic (inferred by the branch diameter mainly age of tree18, linked to the existence of growth rings that are often difficult to distinguish) and environmental parameters affecting the cambial activity and thus, rate of growth and wood development20,28,29,30. This high resolution analysis of wood structure, particularly of conductive elements, allows addressing numerous questions that have been successfully solved in the case of the olive tree and the grapevine, such as phenology, ecology, climate, impact of human activities and agricultural practices20,24,25,31,32,33.Argania spinosa wood is diffuse-porous with a dendritic and diagonal arrangement of vessel elements in transversal section34. The axial parenchyma bands are in tangential alignment and composed of multicellular strands. In radial alignment, woody rays are 1–3 cells wide and of heterocellular composition (Fig. 6).Figure 6Wood anatomical features of the Argan tree (in blue) and measured anatomical characters (in red).Full size imageTo apply a quantitative eco-anatomy approach to the Argan tree, both modern charred samples and archaeological charcoal are broken manually in transverse anatomical section. The following wood constitutive elements and anatomical characters related to sap conduction and reserve storage are observed and measured under a reflected-light microscope connected with an image analysis system (DFC300 FX Leica camera and LAS Leica software) (Fig. 6): (1) vessel density (DVS—number of vessels / mm2), (2) vessel surface area (SVS, µm2), (3) ray density (DRA—number of rays / mm2), (4) axial parenchyma density (DPA, number of bands / mm2), (5) Density of wood fenestrated zones bordered on one side by the radial alignment of axial parenchyma cells and on the other side, tangentially, by rays (DWF—number of fenestrated zones / mm2).These anatomical features were measured several times (see ‘Statistical analyses’ section) following radial lines from the cambium inwards the sample and crossing a small number of growth rings (i.e. functional rings from a sap conduction point of view). Moreover, the hydraulic conductivity or vascular conductivity (CD) was assessed using the following formula: CD = (SVS/π)2/DVS (after32,35,36,37). Finally, the ratio ‘Conductive surface / total wood area’ (SC) was calculated.Statistical analysesIn order to determine the number of measurements required for an optimal assessment of anatomical features, a rarefaction method was carried out from the analysis of test wood samples. For each one, repeated measurements of anatomical characters (Surface vessel area (SVS), Density of vessels (DVS), Ray density (DRA), Axial parenchyma density (DPA) and Density of wood fenestrated zones (DWF)) were performed following the aforementioned method and the cumulative mean value was then calculated for each character20,29. For each test sample and anatomical character, the number of measurements of a character required for an optimal assessment was quantified as the minimum number of measurements required to stabilize the mean value (rarefaction curve or cumulative mean curve).Furthermore, different measurement sessions were carried out with the aim of testing possible errors and reproducibility of measurements taken by one or various observers, respectively. The data sets produced were tested using the PCA performed to evaluate the Argan anatomical variability. The ARG8-2 sample was used as test sample. In addition to the initial measurements. The ARG8-2 sample was analyzed 4 times: twice by one operator (ARG8-2 (1-OP1) and ARG8-2 (2-OP1)) and twice by another (ARG8-2 (3-OP2) and ARG8-2 (3-OP2)) at different times. The additional data were incorporated into the PCA as additional individuals for comparison with initial anatomical features of ARG8-2.After showing that measurement errors have no impact on the validity of results and the measurements are reproducible, quantitative eco-anatomical data were processed using a principal component analysis (PCA) in order to evaluate anatomical plasticity in the reference modern material, to appreciate relationships between characters and wood sample caliber and to confront archaeological data to the reference model. PCA was applied on 53 reference modern samples and 7 quantitative variables (anatomical characters) to: (1) validate the hypothesis that there is a significant relationship between the size of the branch and anatomy, as previously demonstrated by analyses of wood development and structure18,20,38 and dendrochronology39; (2) identify the anatomical characters most affected by the age of the branch and, in that case, model the ‘anatomical characters—caliber of the branch’ relationship; (3) develop predictive model that might estimate the minimum branch caliber from eco-anatomical data of archaeological charcoal.Finally, data from analysis of the 20 archaeological charcoal fragments were included in PCA as additional statistical samples. They do not contribute to the development of the reference model, but are compared to the modern reference samples in order to infer the ecological conditions under which Argan trees grew during the Middle Ages. More

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Study area and parcel selectionThe study was conducted in Castro Verde Special Protection Area (SPA), located in southern Portugal (Fig. 1). The climate is Mediterranean, with hot summers (30–35 °C on average in July) and mild winters (averaging 5–8 °C in January), and over 75% of annual rainfall (500–600 mm) concentrated in October–March. The landscape is flat or gently undulating (100–300 m), mainly dominated by open areas used for rainfed pastures (ca. 60%) and annual crops (ca. 25%), and to a less extent by open woodlands (ca. 7%)15.Figure 1(a) Location of the study area within the Castro Verde Special Protected Area (SPA), southern Portugal. (b) Distribution of the 27 sheep (dark grey polygons) and 23 cattle (light grey polygons) grazing parcels and (c) Sampling scheme applied to each parcel surveyed. Bird counts were done at the centroid of the parcel (white dot) whereas vegetation sampling was performed at the indicated 10 points (black dots). The area covered with pastures and annual crops (derived from CORINE land cover 2018—https://land.copernicus.eu/pan-european/corine-land-cover/clc2018) is shown in yellow. The map was done using the version 3.10.0 of QGIS—https://qgis.org/en/site/index.html.Full size imageSince 1995, part of the study area has benefited from a CAP agri-environment aiming to protect the traditional farming system16. This scheme provides financial support to farmers for agricultural practices considered favourable to conservation, including the traditional rotation of cereals and fallows, the maintenance of low stocking rates (usually related with sheep grazing systems), and sowing of crops benefiting grassland birds16. However, in recent years the traditional farming system has been declining, with many farmers converting to specialized livestock systems, mainly, cattle grazing systems, with an increase of stocking rates7,15.Parcel selection started by identifying grasslands grazed by either sheep or cattle, based on parcel-level statistical information from 2010 provided by the Portuguese Ministry of Agriculture7. To minimize potentially confounding effects of adjacent land uses (edge effects) and other non-crop elements within parcels on bird assemblages, we excluded parcels less than 100 m from shrubland or forested areas, with shrub and tree cover  > 5% and with a minimum size of 10 ha. In January 2019 we visited 100 pre-selected parcels which were grazed by either sheep or cattle in 2010 in order to confirm the parcel land use in the agricultural year of 2018/2019, aiming to sample a balanced proportion of 50 sheep and cattle grazed parcels. Additional livestock information for the agricultural year of 2018/2019 was obtained during systematic visits to targeted parcels (see “Grazing Regime” section from Methods). We ended up with 23 cattle parcels and 27 sheep parcels (Fig. 1).Bird and vegetation dataBreeding birds were sampled twice in each parcel during 7–16 April and 1–15 May 2019 respectively, always by the same observer (R.F.R). This was done to take into account species-specific breeding phenology in the area (early and late breeders)17 and minimize bias due to other factors (like weather or disturbance). Sampling was conducted using standardized 10 min point counts18 carried out at the central point of the parcel (Fig. 1). As the open terrain allowed for high visibility, a large detection radius was used, and all birds detected within 100 m of the central point were identified and counted. This radius is roughly similar to the one previously used for characterizing bird populations in the region19. All counts were carried out in the first four hours after sunrise and in the last two hours before sunset, with none in heavy or persistent rain, or in strong wind conditions. To estimate bird species richness and occurrences in each parcel, we pooled the data from the two counts. Species-level analyses focused on the six most common species, which occurred in  > 30% of the parcels (see Supplementary Table S1). In addition to presence/absence, we also estimated population densities, using the count which yielded the highest estimate of density for each species (assuming this is the best indicator of population density, given the potential phenology and detectability biases above mentioned). Bird densities were based on the number of males simultaneously detected and expressed as breeding pairs/10 ha or males/10 ha (in the case of Little Bustard Tetrax tetrax and Common Quail Coturnix coturnix). Categorization to the genus level was made for the Crested and Thekla larks (Galerida cristata and G. theklae) due to difficulties in correctly identifying all individuals of these two very similar species in the field.Vegetation height and cover were measured once in each parcel, between April 22 and May 6. Vegetation height was estimated in a set of ten 3 m radius plots defined inside the 100 m buffer (Fig. 1). In each plot, ten measurements of vegetation height were taken at random locations, for a total of 100 measurements per parcel. Vegetation height was measured using a 50 cm ruler and was defined as the highest point of vegetation projection within 3 cm of the ruler20. All values were estimated to the nearest half centimeter. When no vegetation was present (bare soil, soil litter, rocks or animal dung) the height was set to zero (0) but these measurements were not considered to estimate the mean height of the sward. Vegetation cover was measured inside a 50 × 50 cm quadrat placed at each of the ten grid points, by visual estimation to the nearest 5% of the percentage of the quadrat area covered by vegetation21 (Fig. 1). Vegetation height and cover measurements were averaged within each parcel.Grazing regimeThe number and type of livestock in each parcel as well as the extent of the grazing period since the start of the year (2019) were gathered from interviews (Supplementary Information S1) to land managers during 1–15 May 2019. This information was further validated, and corrected in a few cases, through field checks during regular visits (made at two-week intervals) to the parcels (see “Bird and vegetation data” section from Methods). Three grazing regime indicators were estimated for the whole period (January–May 2019): livestock type (either sheep or cattle), animal density, and grazing pressure. The animal density in each parcel was calculated as the average density (animals per hectare) of any species (regardless of being sheep or cattle) that grazed the parcel during the 5-months period. Stocking Rate translated animal density into livestock unit (LU) per hectare (LU/ha), between January and May, according to the following criteria: one adult bovine = 1 LU; bovine aged  More

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Morphological analysisFood preparation or processing of plant material involve multiple activities and all of them can potentially leave micro-traces in the tartar, together with the environmental components.Eleven dental calculi showed plant record: starches, pollen grains, one trichome, one sporangium, and tissue fragments (Table 1).Table 1 Plant microdebris recovered from dental calculus samples of Casale del Dolce.Full size tablePlant hairTrichomes are epidermal outgrowths characterized by different structure and function. Although plant hairs are some of the most common findings in the overall particulate matter carried by air (as pollen grains), in literature, only very few examples of trichomes in ancient contexts have been reported28,29,30. Trichome identification is not a common area in dental calculus research since they do not have a diagnostic morphology. For this reason, the identification of such type of microdebris must be based on realistic criteria, also in accordance with the geographical and historical context, providing all possible interpretative scenarios. The detection of trichomes in ancient tartar may disclose other lines of evidence than nutrition, representing a reliable archaeological environmental proof31.One plant hair was identified in CDD1 sample (Table 1). This remain (Fig. 2L) falls into the general class of dendritic trichomes and its peculiar morphology has been more specifically termed a candelabrum or abietiform32. The overall structure corresponded to non-glandular and pluricellular trichomes with a central uniseriate axis and whorls of unicellular rays emerging at the joints of the axis. Usually, 4 radii from each node occurred perpendicular to the central axis. As exhaustively reported in literature, dendritic trichomes are known in ferns, different groups of modern monocots and basal eudicots, such as Scrophulariaceae and Platanaceae. Although dendritic, trichomes of ferns and monocots were excluded. Indeed, the first ones possess single secondary branches that alternatingly arise at an angle of 70°–120° with respect to the main axis along a single plane33, while the second ones show morphological features and appearance different from the ancient debris34,35. Candelabrum-like trichomes have been usually detected in Verbascum L. and Platanus L. species36. For this work, an experimental reference collection of trichomes from these plants was created (Supplementary Information 1). The general aspect of mullein trichomes appears to be capitate, bigger, more elongated, and slenderer than the microremain found in tartar sample. In addition, these trichomes seem to possess a pair of secondary elements per side or single secondary branches, which depart from the nodes, only rarely perpendicular to the central axis37,38,39. Thanks to the well-preserved morphology, the ancient candelabrum hair was interpreted as a Platanus sp. foliar trichome based on literature40 and our experimental reference, although mullein cannot be totally excluded. Dimensions, distance between nodes and the number of tapering secondary branches attached to the central axis of the microremain were like those of all plane species documented in literature40,41,42.Figure 2Plant microremains identified by light microscopy in dental calculus samples. Some of the images captured by optic microscopy were shown. Aggregate of Triticeae starch granules and relative polarized image (A); Fabaceae starch granule and relative polarized image (B); Pinaceae pollen grain (C); aggregate of Triticeae starch granules and relative polarized image (D); Cupressaceae pollen grain (E); Poaceae spontaneous group pollen (F); polyhedral starches of morphotype II (G,H); fragments of plant tissues (I–K); dendritic hair (L). The scale bar indicates 15 µm [45 µm for panel (L)]. Small flecks of calculus still attached to microremains can be observed in some panels.Full size imageThis finding leads to consider some paleoenvironmental implications. Fossil pollen analysis has demonstrated that, during the Plio-Pleistocene, Platanaceae were present in the Upper Valdarno (Italy)43. For the Holocene, likely as a consequence of Pleistocene glaciations, fragmentary and scarce evidence of plane tree have been found in Spain and French Mediterranean coast; no record of Platanus sp. has hitherto been found in Italy44,45. This thermophylous taxon has reappeared later as an ornamental tree, providing shade, during Roman times46. As we applied rigorous decontamination protocols, the evidence of this ancient trichome, probably accidentally inhaled by CDD1, may testify the presence of Platanus sp. and humid environments in central Italy during the Neo-Chalcolithic period.Starch granulesMore than 70 starches were retrieved from calculus samples (Table 1). Some of them were found in an extraordinary state of preservation, likely due to intentional ingestion and/or accidental inhalation during the processing of starchy foods. These grains were clustered in three different morphological types, based on the morphometric parameters (i.e., shape, size, presence of lamellae and hilum, aggregation level, and other secondary features) evidenced by literature. They were described using the International Code for Starch Nomenclature47,48.Morphotype I These starches were consistent with those of Triticeae Dumort. tribe and occurred in almost all samples, as the most copious group (Table 1; Fig. 2A,D). Some grains were still lodged together. The morphotype was characterised by a bimodal distribution, or rather co-presence of large and small granules. Occasionally, the morphology was not completely intact, probably due to chewing as well as grinding and/or cooking procedures. These starch grains were similar to those occurring in caryopses of cereals, such as Hordeum sp. L. and Triticum sp. L. In particular, the diagnostic starches were oval to sub-round in 2D shape (15–43 µm in length; 10–35 µm in width). They had a central and distinct hilum and, sometimes, no visible lamellae. The small granules (≤ 10 μm in diameter) were spherical in shape with a central hilum. Knowledge about the Neo-Eneolithic period in central Italy is characterized by discontinuous data. The archaeobotanical dataset available for Latium is still limited49 but information about cultivated and wild-collected plants from Casale del Dolce site exists. In fact, the carpological analysis previously conducted50 has identified several caryopses of barley and wheat, supporting our results. The recovery of these starch grains, in almost all samples, suggested that the use of cereals was common and probably frequent for Casale del Dolce people, even if it is quite difficult to correlate presence of plant remains in calculus and quantity of consumed food26. The hypothesis of cereal consumption for this community has been also proposed by stable isotope data. Isotopic values would suggest a subsistence economy based on a great intake of carbohydrates and a lifestyle characterized by a progressive agricultural exploitation, even more evident than other Eneolithic sites of central Italy6,51. Lastly, Triticeae starches have been also found in dental calculus from Grotta dello Scoglietto (southern Tuscany), for the same pre-historical period52.Morphotype II A low number of starch granules with faceted shape, perpendicular extinction cross and, sometimes, evident central fissures was recovered from dental calculus (Table 1; Fig. 2G,H). The morphology appeared oval to polygon (2D) with centric hilum and fissures radiating from it. The most frequent size distribution length was 14–25 μm in length and 13–17 μm in width. This type of grains exists in seeds of grasses belonging to the Andropogoneae Dumort. and Paniceae R. Br. tribes, as shown in the modern reference material19. Since an overlap in size and shape occurs among starches of species related to these tribes, the identification of these plant remains is arduous at a lower taxonomic level. Sorghum sp. Moench (sorghum), Setaria sp. P. Beauv. (foxtail millet) and Panicum sp. L. (millet) can be considered as potential candidates. Unfortunately, no phytolith, which would have helped us in distinguishing between the species of Paniceae53, was detected. In addition, the lack of an isotopic signal specific for this type of consumption and the absence of relative carpological remains for the archaeological site of Casale del Dolce might be due to a limited usage of these plants. In fact, although several species of these genera were diffused in Italy, little is known about their employment. The archaeobotanical evidence of millets (i.e., Panicum sp. and Setaria sp.) from the Late Neolithic period has been discussed; however, their cultivation is certain during the Bronze and Iron Ages52,54,55. Recently, Accelerator Mass Spectrometry-datings of prehistoric charred broomcorn millet grains has pinpointed the earliest occurrence of Panicum miliaceum L. in Europe at the middle of the 2nd millennium BCE (Middle/Late Bronze Age)56.Morphotype III Only one grain contributed to the third type of starch (Table 1; Fig. 2B). It appeared to be consistent with the Fabaceae family, probably Vicieae (Bronn) DC. tribe (e.g., vetches) for its oval to elongated (irregular) shape and kidney-like. The hilum was obscured and sunken, while the lamellae were not fully visible. The size was 42 μm in length and 30 μm in width. Data about pulses are scarce for this period. In northern Italy, a high variety of pulses was already present in the Neolithic57,58 but this starch grain would seem to be one of the few and unique evidence of consumption in central and southern Italy. As this finding refers to a single individual, certainly, it is not expected to provide an exhaustive image of the use of pulses for the period and region but its presence, together with the carpological remains of Fabaceae49,50, could attest plant protein consumption.A single starch granule was not classified because missing diagnostic and distinguishable characteristics. Probably modification events, such as grinding process, cooking procedure in water and/or chewing, and exposure to alfa-amylase, altered its shape.Pollen grainsFour calculus samples showed the presence of different pollen types (Table 1). In total, 49 grains were found. Three of them were detected in CDD2, 4, and 9 (Fig. 2C,EF), while the remaining ones (46), both in single and in aggregate form, were retrieved from only one individual (CDD7) (e.g., in Fig. 3). All palynomorphs were identified according to morphometric parameters described in literature and evidenced in the Palynological Database59 and the names of the pollen types refer to literature60,61,62.Figure 3Plant micro-remains detected by morphological analysis in the dental calculus of CDD7 sample. Representative images obtained by light microscopy analysis were shown. Aggregates of pollen and spores (A,B); Pinaceae and Cupressaceae pollen grains (C); Brassicaceae pollen grain (D); Pinaceae pollen grains (E,F); Cupressaceae pollen (G); Quercus deciduous pollen (H); Alchemilla type pollen (I); sporangium of Monylophyta (J). The scale bar indicates 15 µm. Small flecks of calculus still attached to microparticles can be observed in some panels.Full size imageIn this paragraph we describe the pollen grains found in CDD2, 4, and 9 samples.The ancient microremain embedded in sample CDD2 was apolar and medium in size (63 µm in diameter), showing a morphology which typically occurs in Poaceae63,64. The stenopalynous nature of such type of pollen (that is, uniform monoporate) makes its systematic identification difficult. Although a low taxonomic determination limits paleoecological inferences, the evidence of Poaceae pollen is usually interpreted as indicative of open grasslands65.One ancient palynomorph displayed morphological traits consistent with Pinaceae (sample CDD4). It appeared as a bisaccate monad with an elliptic corpus and medium reticulation on bladders59,66,67. Including sacci, the dimension was 56 µm in equatorial view.A non-saccate Cupressaceae-type pollen, instead, was found in sample CDD9. It appeared spherical (with polar and equatorial axes of 30 µm) and inaperturate at light microscope; the protoplast exhibited itself star-like. Pollen grains produced by several species of Cupressaceae are considered morphologically uniform68. Since prehistoric times, Gymnosperm wood has been widely used as raw material and firewood, while needles, nuts and inner bark represented the edible parts of these trees69. Noteworthy is that the resins of these plants, possessing adhesive qualities and antibacterial properties, might have been also appreciated by Neanderthal14. Cupressaceae pollen grain is generally scarce in ancient sediments and one of the most underrepresented palynomorph in archaeological context. Several archaeobotanical studies have demonstrated the use of Juniperus L. species in the Mediterranean basin since the Holocene. In particular, the use of them as a source of aromatic foliage and resins employed for medicinal purposes, wood as fuel and for construction of dwellings, and fresh or dried berries as food has been proposed70,71,72. Sporadic fossil discoveries of Cupressus sp. L, instead, are rather sparse in the Mediterranean area, although some ancient record has been registered in Italy during the Quaternary73. Thus, the investigated plant microdebris testify the presence of Cupressaceae and provide additional evidence about the possible existence of evergreen Mediterranean forests, during the Neo-Chalcolithic period, in the Sacco River Valley.Pollen grains in CDD7CDD7 specimen (Fig. 1B), an adult male affected by severe malocclusion, preserved an interesting set of microparticles at microscopic analysis; therefore, we decided to report and discuss separately the data obtained from his calculus.Eleven pollen grains out of 46 were not distinguishable due to the lack of diagnostic characteristics. The remaining 35 were found (singly, in pairs, or aggregates; Table 1, Fig. 3) in good or excellent state of conservation. The latter appeared as clusters of Pinaceae pollen (Gymnosperm) and other palynomorphs, including spores. Examples are shown in panels A and B of Fig. 3.Two Cupressaceae, ten Pinaceae and one Poaceae pollen, presenting the same morphological features described in the previous paragraph, were also found in this sample (e.g., see Fig. 3C,E,F,G).In addition, pollen grains from four herbaceous plants, namely Cyperaceae, Urticaceae, Trifolium, and Alchemilla species, and from the arboreal genus Corylus L. were detected and aredescribed below. Although pollen morphological variation within Cyperoideae subfamily is notable, one ancient microremain, possessing a pear-shape and a scabrate sculpture on its surface, appeared belonging to the genus Carex74,75. In equatorial view it was triangular and the polar axis length was 41 µm. A second pollen grain was recognised as Urticaceae-type; it exhibited spheroidal shape (equatorial diameter 23 μm) and scabrate ornamentation. This morphology occurs both in Parietaria sp. and Urtica sp. pollen grains59,62 and it is very difficult to distinguish them by optical microscope, especially if degraded. The shape of a third ancient monad, attributed to Trifolium-type (Fabaceae), was subprolate in equatorial view (46 μm) with scabrate ornamentation76. The Alchemilla-type (Rosaceae) microremain (26 μm equatorial view, Fig. 3I) was radially symmetrical, elliptic and prolate in shape77. Finally, another pollen type was found and attributable to Corylus sp. L. (Betulaceae). It was oval in equatorial view (19 μm), smooth, and tripolar with deep oncusis in each pore78.Seven pollen grains were single, prolate, isopolar, and elliptic in equatorial view (polar axis 19–25 µm long). They were tricolpate, with long and narrow colpi. Pores were at times indistinct. Pollen of the different species of Fagaceae shows a high variability in form, size, sculpturing; for this reason, most of them overlap in morphology. The ancient palynomorphs in exam were closely similar to a Quercus-type (examples in Fig. 3A,H)79,80.The last 10 grains showed a morphology (3-colpate, reticulate and subprolate) ascribable to Brassicaceae pollen grains (example in Fig. 3D). This is a stenopalynous family in which pollen varies among the genera but rarely in the species under the same genus81,82.Intriguingly, pollen findings in sample CDD7 were numerous and deriving also from insect-pollinated plants (e.g., Brassicaceae). This evidence appeared like a honey palynospectrum. This type of assemblage has been never registered in dental calculus deposits and, especially for the aggregates, the hypothesis of accidental inhalation seems implausible. Precisely, the presence of aggregates induced us to reflect upon a common origin of the whole pollen record. However, for single granules, to date, the supposition of aspiration cannot be completely excluded, due to the multiple pathways of inclusion of such type of microparticles27. The high pollen variety could be explained by the presence of residues of natural matrices, as well as honey or beehive products (e.g., wax, propolis), in the calculus sample. To support our hypothesis, we prepared a reference collection based on modern multifloral honey samples (Supplementary Information 1, panel E–J).Archaeological finds of bee products are quite rare83,84,85,86,87,88. Since the end of the upper Palaeolithic, honey has been employed as sweetener, while beeswax for technological, ritual, cosmetic and medicinal applications89,90. Regarding the latter, Bernardini et al.91 have found fascinating traces of a filling with beeswax, highlighting Neolithic dentistry procedures. It is important to recall that bees may also visit non-nectariferous plants (e.g., Poaceae, Betulaceae like Corylus sp.) for collecting pollen as protein source. Moreover, Pinaceae (Pinus sp. L. and Abies sp. Mill.) and Fagaceae (Fagus sp. L. and Quercus sp. L.), among others, emit sweet secretions and may be classified as honeydew producers88. Therefore, it is not unlikely to discover pollen grains of pine, hazel, oak, and cereals mixed with melliferous taxa. In fact, similarly, Carboni et al.92 have observed a lump of pollen inside an Eneolithic vessel, suggesting the use of a fermented honey-based drink, the mead, for ritual purposes.According to all this evidence, the pollen record detected in the present ancient calculus could be likely interpreted as direct honey consumption and/or remain of food or beverage including honey as natural sweetener. However, the use of conifer resins as antimicrobial or flavouring agents, mixed to honey or alone, cannot be excluded, together with the hypothesis of inhalation of bisaccate pollen from the immediate environment.Unfortunately, for the investigated site, no evidence supporting the previous hypotheses exists. Nevertheless, it is possible that the individuals from Casale del Dolce practised bee-keeping culture near woodland pastures, although this interpretation cannot be definitive.Currently, pollen spectra from beehive products are used to deduce plant biodiversity of the areas visited by insects for nectar collection93,94. Bearing in mind this indication and the typical habitats of the identified plant taxa, some ecological implications were inferred. A thermophilic broad-leaved forest mainly made up of conifers (such as Pinus) and several deciduous trees (such as Quercus and Corylus), together with wet grasslands (Cyperaceae, Urticaceae, Alchemilla sp.), was outlined by pollen analysis. This hypothesis would seem consistent with Coubray’s work50, who has identified the wood charcoals found in the archaeological site of Casale del Dolce as Carpinus L., Quercus, Maloideae, Cornus L., Corylus, Ulmus L., Fraxinus L., and Acer L. remains. In addition, palynological analyses performed in the same region95,96,97 have detected similar vegetational elements.Other plant microremainsWe detected an unusual range of microparticles, that is, fragments of plant tissues and a sporangium, rarely documented in human dental calculus investigations (Table 1)69,98,99,100.Among the first, one microparticle was made up of plant cells associated to a scalariform xylem vessel (Fig. 2I), while another debris showed wood cells with simple pits (Fig. 2J). A brown-yellowish fragment was also photographed (Fig. 2K). As reported in literature99, no evidence of charring or burning may be attributed to this type of darkening colouring but, if so, it would suggest an involuntary inhalation of ash particles from trees or shrubs used for fire. Thus, this type of microremain could derive from both non-edible and edible plants. In general, all these fragments retrieved from calculus might be the result of some activities, such as chewing of fresh plant organs, food and/or other uses of bark, oral hygiene procedures with woody dental picks, and/or use of teeth as a third hand99,101,102.The second type of uncommon microparticle, found in sample CDD7 (Table 1), appeared morphologically like a sporangium, probably from Monylophyta (Fig. 3J). It was brownish in colour and ovoid in shape. This type of microremain has never been observed in so ancient human dental calculus. A more specific taxonomical identification is very complex and would be risky, since at palaeobotanical and/or archaeological level there is no evidence to support this finding. However, considering that sporangia are typically attached to the abaxial surface of the leaf and that airborne dispersal capability of fern spores into stronger wind currents is rare and improbable100,103, the recovery of the whole sporangium allowed us to hypothesize a voluntary use of fern leaves.Biochemical analysisGC–MS approach revealed the presence of organic compounds derived from the matter ingested and/or inhaled by the individuals. However, the potential of the biomolecular approach on dental calculus is still highly challenging and the capacity to trace the origin of some molecules is still difficult, due to the multifactorial dental calculus’s aetiology31,104.In Supplementary Information 2 (SI2), the molecules detected in each sample were listed and clustered in chemical classes. The chromatographic profiles were dominated by a series of C6 to C30 n-alkenes and n-alkanes, not reported in SI2 because ubiquitous and not taxonomically specific. They could probably come from degradation of oral bacteria or consumed food, representing, for instance, fragments of unsaturated or saturated lipids14,105,106,107.The typology of residues accumulated in dental calculus and their adsorption capacity determine the lipid profile of this matrix, considering that different foods naturally possess variable lipid composition. For this reason, it is difficult to associate fatty acids to specific dietary sources. The presence of fatty acids (e.g., odd, short, and long chains), ubiquitous components of organic matter, could be considered indicator for consumption of animal fats or plant oils (e.g., oil-rich seeds and fruits)104,108,109,110,111,112,113. Long-chained polyunsaturated fatty acid derivatives (PUFAs; e.g., eicosapentaenoic acid, EPA), abundant in dried fruits114, were detected in some samples. Polyunsaturated omega-3 fatty acids have been rarely identified in archaeological contexts115, due to their highly inclination to oxidative alteration116. However, dental calculus has shown itself conservative for this type of molecules31. The consumption of aquatic organisms cannot be excluded, being rich of PUFAs114 and considering the proximity of the ancient settlement to the Sacco River.Monoterpene derivatives, non-specific compounds with volatile nature, retrieved from some samples, such as citronellol, menthol and pinanol (commonly found in leaves, fruit, and bark of a wide range of plant species), could generically indicate the ingestion of plant materials or waxes109.In CDD5 calculus, azulene and coumarin derivatives were also recovered. These secondary metabolites usually occur in species belonging to Apiaceae, Asteraceae, and Rutaceae families, well known medicinal plants possessing a wide range of biological activities117,118. As suggested by Hardy et al.14, the plant species rich in such type of bitter-tasting compounds might have been ingested for self-medication.Two alkaloids were found: trigonelline and hordenine, respectively, in CDD4 and CDD7 specimens. The first one, whose accumulation takes place in various plant species (i.e., Achillea sp. L.) and especially in Fabaceae seeds (e.g., Trigonella sp. L., Trifolium sp. L., and Medicago sp. L.)119,120, might represents a further proof for consumption of pulses.Hordenine, which naturally occurs in certain grasses, like cereals (e.g., barley, millet, and sorghum)121, could demonstrate the ingestion of starchy material, as already testified by the detection of a Triticeae starch granule in the same calculus flakes and the recovery of caryopses at the site50. More

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