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Citation: Germano R, Higgins OA, Cristiani E, Galbusera A, Esposito C, Neves D, et al. (2026) Health and life-style within the Iron Age Italian group of Pontecagnano (Campania, Italy, Seventh-Sixth century BCE). PLoS One 21(1):
https://doi.org/10.1371/journal.pone.0338448
Editor: Enrico Greco, University of South Florida, ITALY
Received: August 26, 2025; Accepted: November 21, 2025; Published: January 14, 2026
Copyright: © 2026 Germano et al. This is an open entry article distributed underneath the phrases of the Creative Commons Attribution License, which allows unrestricted use, distribution, and copy in any medium, offered the unique writer and supply are credited.
Data Availability: All related information are throughout the paper and its Supporting Information recordsdata.
Funding: Carmen Esposito (CE) was supported by the European Union’s Horizon Europe programme underneath the Marie Skłodowska Curie Actions Postdoctoral Fellowship, Grant Agreement No. 101065320 (TULAR). Dental calculus analyses had been carried out in a laboratory funded by the European Research Council (ERC) Starting Grant HIDDEN FOODS, Grant Agreement No. 639286 (PI: Emanuela Cristiani, EC). Alessia Nava (AN) acquired funding from the European Research Council (ERC) underneath the European Union’s Horizon Europe programme, Grant Agreement No. 101077348 (MOTHERS; challenge web site: https://www.erc-mothers.eu/). Roberto Germano (RG) acquired assist from the Environmental Biology Doctoral School, Sapienza University of Rome. The funders had no position in research design, information assortment and evaluation, choice to publish, or preparation of the manuscript.
Competing pursuits: Alessia Nava (AN) and Luca Bondioli (LB) function Academic Editors for PLOS ONE. This doesn’t alter our adherence to PLOS ONE insurance policies on sharing information and supplies. The authors declare no different competing pursuits.
The research of enamel has emerged as a vital strategy in bioarchaeology, combining strategies comparable to dental histology and micro-residue evaluation to reconstruct particular person life histories [1,2]. Teeth, as dependable organic archives, not solely report the incremental formation of dental mineralized tissues – providing distinctive insights into childhood development and well being [3,4] – however can even protect dental calculus, which serves as a repository for micro‐inclusions associated to weight-reduction plan and even occupational or para-masticatory actions later in life [5,6]. By integrating dental histomorphometry, with a specific deal with dental enamel, and calculus evaluation, it’s attainable to acquire an in depth image of each organic life histories throughout infancy, childhood, and grownup existence, enhancing our understanding of previous populations adaptive capability and cultural behaviors [5–10].
Teeth are wonderful archives as a result of incremental formation of their tissues and resistance to diagenetic and post-depositional alteration [11–15]. They act as particular person organic archives offering details about responses to residing situations and environmental stresses throughout infancy and childhood [1,3,16]. Dental enamel, as soon as shaped, retains its unique construction with out present process physiological reworking [17]. Its rhythmic formation produces microscopic physiological incremental traces repeatedly repeated within the dental crown, permitting the monitoring of knowledge and the power to “count” the times in a person’s life [3,11,18–20].
Dental histology of deciduous and everlasting enamel is a helpful instrument for acquiring data on the event and well being standing of people from earlier than start to the top of dental improvement [3,4], serving to to partially deal with challenges related to the Osteological Paradox [21,22], significantly regarding selective mortality. Histomorphometric evaluation of dental crowns permits the research of formation occasions, the chronology of non-specific physiological stresses observable as Accentuated Lines (ALs), and particular person life histories [1,3]. Stress markers seem concurrently within the crowns of all enamel, forming on the identical time, in the end enabling a pseudo-longitudinal research of childhood by utilizing a number of enamel to acquire a extra complete view of the person’s life historical past [3,19].
While dental histology primarily supplies insights into infancy, childhood, and adolescence, that are the home windows of dental crown formation, the presence of dental calculus (mineralized plaque) extends the temporal vary of research into maturity. Dental calculus – shaped via the mineralization of plaque – preserves a wide selection of knowledge associated to weight-reduction plan, well being, and different facets of every day life. Oral plaque can calcify already inside two weeks, accreting episodically after tooth eruption. If not mechanically eliminated, this report can span months to years previous to dying [5]. For this purpose, it represents a long-term reservoir of dietary/oral/host biomolecules and different particles ensuing from dietary habits, unintended ingestion/inhalation, and the usage of the mouth as a “third hand” in para-masticatory actions [6,9,23,24].
This analysis explores the timing of dental improvement, infancy and childhood well being standing and grownup weight-reduction plan of the inhumed people from the Pontecagnano necropolises (Campania, Italy, Seventh-Sixth century BCE) [25], one of many largest pre-Roman web site in Southern Italy, utilizing histomorphometry and microscopy evaluation of dental calculus from everlasting enamel. The web site of Pontecagnano gives a singular alternative to unravel the bio-cultural responses to rising social and environmental challenges throughout the Orientalizing interval (720–580 BCE) when borders, political steadiness, and networks all around the peninsula had been totally redesigned [26]. These transformations had been accompanied by intensified commerce and cultural contacts with the Greek world, growing social stratification, and shifts in settlement group [27–29]. Such dynamics might have influenced well being and life-style via attainable publicity to new pathogens by way of mobility and change networks, and unequal entry to sources linked to rising of latest social hierarchies, whereas broader financial and environmental pressures might even have affected residing situations. In this context, learning particular person life histories throughout childhood – when people are most weak and completely depending on the care of others – supplies insights into how a inhabitants responds to those bio-cultural challenges and adjustments [7,8,30], enhancing our understanding of the Iron Age group of Pontecagnano and their environmental and social context.
Enamel, composed of roughly 95–97% inorganic supplies (primarily hydroxyapatite) and three–5% natural supplies and water [31,32], develops in incremental layers ranging from the enamel-dentine junction (EDJ) in the direction of the outer floor of the tooth. This rhythmic course of leads to the formation of microscopically seen development markers. These embrace cross-striations, representing every day development increments, and Retzius traces, which type at longer intervals, normally on a near-weekly foundation [12,33,34].
Cross-striations are seen alongside the enamel prisms when noticed underneath transmitted gentle microscopy. They seem as alternating gentle and darkish bands, representing the every day secretion price of ameloblasts [12,31]. These striations end result from the common variation within the price of enamel matrix secretion, reflecting the circadian rhythm of ameloblast exercise [34,35]. The cross-striations function elementary items for establishing a chronological timeline of enamel formation [19,36].
Retzius traces (or striae of Retzius) are incremental traces that, in longitudinal cross sections, seem as sub-parallel traces representing the depositional fronts of the ameloblasts at completely different occasions of improvement [12]. Accentuated Lines (ALs) are stress-affected Retzius traces that, because of non permanent disruptions of the traditional ameloblast exercise, seem darker and extra pronounced in comparison with common Retzius traces [37,38]. It isn’t attainable to affiliate an AL with a selected stress, as ALs are non-specific physiological stress markers that may type in response to varied components, together with malnutrition, infectious ailments, and psychological stress [12,38]. The first notable AL noticed in enamel is normally the Neonatal Line (NL), which marks the start occasion and separates prenatal from postnatal enamel [39–41]. However, a number of research point out it’s attainable to search out ALs in prenatal enamel as effectively [30,42–48]. The presence of the NL and different ALs supplies important data for registering life histories throughout enamel of the identical particular person and figuring out intervals of stress throughout enamel formation [12].
By registering ALs throughout a number of enamel, it’s attainable to assemble a cohesive developmental timeline, providing a longitudinal perspective of childhood development and stress occasions [3,49]. This technique permits to beat limitations related to mortality samples [21,22], by specializing in developmental intervals lengthy earlier than a person’s dying. Overall, the research of dental enamel microstructures permits for the reconstruction of the timing and length of enamel formation, the identification of intervals of physiological stress, and a deeper understanding of previous human experiences [12,33,38,50].
The formation of dental calculus, or tartar, outcomes from the interplay between salivary elements and oral micro organism, resulting in a calcified biofilm on tooth surfaces if not repeatedly eliminated [5,51,52]. The price of calculus accumulation varies amongst people and is influenced by a number of components comparable to weight-reduction plan, oral pH and hygiene, amongst others [53]. During the mineralization, dental calculus can entrap all kinds of microscopic particles and compounds current within the oral cavity and the residing atmosphere, starting from plant micro-remains (e.g., starch granules, phytoliths, wooden fragments, bast fibers), to animal-derived residues (e.g., feather fragments, fish scales, collagen fibers), in addition to fungi, diatoms, mineral particles, and microorganisms. Optical analyses of dental calculus underneath cross-polarized transmitted gentle permit the identification of micro-remains related to meals consumption and every day habits, in addition to different non-dietary practices [6,9,54,55]. In addition to morphologically identifiable particles, dental calculus might protect chemical compounds and biomolecules, comparable to secondary metabolites, bacterial and host DNA, which might be analyzed via biochemical approaches, together with proteomics and metagenomics approaches [56]. Its paleopathological relevance has been more and more acknowledged, as dental calculus can protect microbial signatures and biochemical markers indicative of oral and systemic well being situations [57,58]. Thanks to its heterogeneous composition, dental calculus represents a strong and long-term reservoir for reconstructing not solely historic diets, but additionally well being, occupational habits, and biocultural practices in each historic and fashionable human populations and extinct hominins species comparable to Neanderthals [5,6,10,59].
Although calculus evaluation requires warning because of complicated formation processes, when mixed with isotopic, archaeobotanical and practical proof, it supplies helpful insights into particular person and population-level lifeways [5,60]. More not too long ago, dental calculus has additionally been explored throughout the framework of historic medication. Micro-remains and biochemical markers embedded on this materials have revealed the usage of crops with potential therapeutic properties, suggesting that previous societies possessed empirical data of native botanicals and will have engaged in intentional well being care practices [59]. These findings underscore the potential of dental calculus to disclose beforehand inaccessible facets of historic medicinal data and cultural approaches to well being.
The chosen dental pattern for histological evaluation consists of 30 everlasting enamel (Fig 1) from 10 people from the Proprietà Baldi, Chiancone and Proprietà Gaeta Iron Age necropolises of Pontecagnano (Campania, Italy, Seventh-Sixth century BCE) [25]. The pattern dimension displays each the strict choice standards utilized to make sure optimum preservation of dental tissues for histological evaluation and the moral want to reduce harmful sampling of archaeological human stays. The people had been chosen based mostly on the damage stage of particular dental lessons, guaranteeing that canines didn’t exceed put on stage D and molars didn’t exceed put on stage F, in line with Lovejoy’s dental put on scale [61]. Age at dying and organic intercourse of the people had been independently estimated by two of the authors (AN and AS) following customary anthropological strategies [62–64]. Age at dying for the chosen pattern ranges between 16 and 40 years, and each men and women are represented (Table 1).
Fig 1. Dental sample.
The 30 permanent teeth of the 10 individuals from the Iron Age necropolises of Pontecagnano selected for histological analysis, divided by tombs. All teeth are in buccal view.
For every particular person, three everlasting enamel had been sampled: one canine, one first molar, and one second molar. In one case (PTG T.5980), as canines weren’t obtainable, a fourth premolar was chosen as an alternative. When attainable, enamel had been chosen from the identical dental quadrant. These particular lessons of enamel had been chosen for his or her chronological overlapping throughout crown formation, enabling a pseudo-longitudinal research of the people’ childhood from start to round 7 years of age [3,49,65].
The dental calculus samples include a subset of 4 people from the identical samples used for the histological evaluation, and one further particular person (PTG T.8340, not used for histological evaluation because of extreme put on), for a complete of 5 people (Table 1). Only a subset of the unique pattern was used for these analyses, as not all people within the dental pattern had mineralized calculus on their enamel. Dental calculus was sampled from all three tooth lessons in people with it, which was current in delicate to reasonable quantities [66].
The odontoskeletal assortment is stored on the Museo delle Civiltà di Roma underneath the curation of one of many authors (AS). All permits had been obtained for the described research by the Museo delle Civiltà di Roma, which complied with all related laws.
The web site of Pontecagnano (Fig 2), positioned 8 km southeast of Salerno (Campania, Italy), testifies the Etruscan enlargement inside Southern Italy [67]. Pontecagnano was based within the early ninth century BCE by teams of Villanovan tradition from southern Etruria [67,68], however see additionally Peroni [69] for a unique interpretation. The web site construction and territorial group intently resemble the protourban facilities of Tyrrhenian Etruria. The settlement – which then turned the Etruscan metropolis of historic time – was surrounded by quite a few necropolises which yielded over ten thousand burials spanning from the ninth to the third century BCE [70].
Fig 2. Location of Pontecagnano.
Satellite imagery of Italy highlighting the geographical location of Pontecagnano (from NASA Visible Earth project ‒ credits to Jacques Descloitres, MODIS Rapid Response Team, NASA/GSFC).
As in different Iron Age Italian contexts, funerary proof has been instrumental in reconstructing group developments, owing to the abundance and variety of fabric tradition and grave items. The most historic phases in Pontecagnano are principally represented by the Villanovan cremation ritual [67]. Subsequently, within the eighth century BCE, inhumations elevated [71], however cremations had been nonetheless frequent. During the Orientalizing interval (720−580 BCE) Pontecagnano witnessed a big financial enlargement in addition to a rise in cultural contacts with the Greek world [28]. These financial adjustments are mirrored within the necropolises, the place sturdy elite teams had been established, represented by a unique funerary costume. Inhumation turned the prevalent ritual, with just a few cremations representing distinguished people [29]. The character of the grave items and funerary ritual displays the central place of Pontecagnano in relation to the primary territory of Etruria and long-range maritime visitors [72]. Between the Seventh and Sixth centuries a big phenomenon of urbanisation occurred, which was accompanied by the diffusion of the Etruscan tradition [67]. Starting from the fifth century, Pontecagnano was influenced by the Samnites’ enlargement, witnessed by the institution of an Italic funerary ritual [73]. In the primary half of the third century, the Roman enlargement ended the Etruscan-Samnite metropolis [29].
The tombs right here analysed are primarily from the Orientalizing interval. The important cultural and social adjustments of this era was mirrored in new funerary practices and the abandonment of earlier necropolises [29]. Graves had been organized in small plots, emphasizing generational ties, with extra elaborate funerary items influenced by overseas customs [70]. A particular function in each grownup and non-adult graves was the introduction of funerary vessels associated to the consumption and ritual providing of wine, symbolizing privileged relationships with the Greek world and the East [27,28].
Before sectioning, every tooth underwent thorough customary documentation [62]. High-resolution pictures had been taken from a number of angles to protect detailed data of every tooth. To protect the crown surfaces for future research, high-resolution replicas of the tooth crowns had been produced [9,74]. All histological procedures had been carried out on the BIOANTH-Biological Anthropology and Dental Histology laboratory of Sapienza University of Rome.
For the obtainment of histological skinny sections, the NOWA protocol by Esposito et al. [75] was adopted. Teeth had been first embedded in Crystalbond™ (Aremco Products, Inc.), after which in bicomponent epoxy resin (Buehler EpoxiCure™ 2). After curing, the resin blocks had been sectioned utilizing an IsoMet Low Speed Saw (Buehler). A longitudinal reduce was made alongside the bucco-lingual airplane passing subsequent to the tip of the dentine horn, concentrating on the mesio-buccal cusp in molars [11,32]. The first reduce produced two blocklets; the one with the dentine horn was chosen for additional processing, whereas the opposite half was saved for potential future analyses.
The chosen blocklets had been glued to microscope slides utilizing a UV-adhesive (Loxeal® Engineering Adhesives UV 30–23) and cured underneath UV gentle. The mounted blocklets had been sectioned utilizing the diamond blade noticed to supply skinny sections of roughly 250–300 μm in thickness. Thin sections had been polished utilizing progressively finer abrasive papers (Carbimet Buehler – P800, P1200, P2500) to cut back thickness to an optimum vary of roughly 80–100 μm for histological evaluation [11]. Then, the sections had been polished with alumina suspension (MicroPolish Alumina 0.3 μm, Buehler) after which mounted utilizing Eukitt® mounting medium (O. Kindler GmbH & Co.) and coated with cowl slips. Lastly, high-resolution photomosaics of the skinny sections had been acquired at completely different magnifications (50x, 100x) with polarized gentle utilizing a transmitted gentle microscope (Zeiss Axio IMAGER.M2, Carl Zeiss Microscopy GmbH) geared up with a digital coloration digicam for microscopy (Zeiss Axiocam 807, Carl Zeiss Microscopy GmbH). The photomosaics had been mechanically obtained by way of the “tiles” instrument in ZEN Core (v3.8, Carl Zeiss Microscopy GmbH) software program.
Chronologies of the dental crowns had been constructed utilizing the zig-zag technique proposed by Dean [76]. This technique includes marking prism segments and Retzius traces ranging from the tip of the dentine horn as much as the cervical space of the tooth. Data segments had been traced and measured utilizing ImageJ v.1.54p [77]. The measurements had been then used to calculate the crown formation time (CFT) and the enamel extension charges (EER) utilizing a normal every day secretion price (DSR) of two.85 µm/day [20]. The CFT represents the full time required for full crown formation, calculated by summing the formation occasions of the measured prism segments alongside the crown [78–80]. The EER represents the speed of enamel formation alongside the EDJ, calculated by measuring the space between prism segments and dividing it by the estimated time for every section [20].
In first molars, chronologies had been anchored to start both via the identification of the Neonatal Line (NL), when current, or by aligning the beginning of crown formation with start as per literature conference [80–82]. For worn first molars, the dentine horn tip was digitally reconstructed utilizing an overlay of a well-preserved tooth. The EDJ path was traced, and extra days of enamel formation had been added based mostly on the size of the reconstructed section, offering a calibrated place to begin for the chronology.
ALs had been recognized in the event that they had been seen for a minimum of three-quarters of their size, as advised by Nava et al. [3], and in the event that they had been noticed, a minimum of partially, on each the labial and buccal sides of the crown [4,37,83]. The dental crowns of every particular person had been aligned by registering sequences of ALs (Fig 3), used as temporal markers. The methodology is predicated on the speculation that AL patterns in enamel (morphology and distance) are maintained throughout the completely different enamel forming on the identical time [3,4].
Fig 3. Photomosaics of URM1 and URC teeth from individual PTG T.6059 with matched Accentuated Lines (ALs).
Colors of the star symbols indicate homologous ALs, matched by morphology and spacing along the enamel, enabling alignment of developmental chronologies between teeth.
The prevalence of stresses was calculated because the variety of people with a minimum of one AL in a selected month from start, divided by the variety of people with enamel in formation throughout that very same month, and expressed as a share [3,4]. Statistical analyses and graphs had been carried out utilizing R model 4.5.1 [84].
Dental calculus sampling was performed following the protocol systematized by Sabin and Fellows Yates [85], with some variations (e.g., disposable blades had been modified after every pattern extraction). Decontamination procedures, demineralization and optical analyses adopted customary printed protocols [5,51,85,86] and had been performed in devoted clear areas not linked to fashionable botanical work and underneath strict environmental monitoring on the DANTE-Diet and Ancient Technology laboratory of Sapienza University of Rome.
Calculus samples had been cleaned with the intention to take away soil particles and different potential environmental contaminants hooked up to their floor. Bench surfaces had been cleaned all through the a number of steps of the pattern preparation, utilizing cleaning soap and ethanol, and starch-free nitrile gloves had been used always. Each pattern was decontaminated underneath a stereomicroscope, on a clear Petri dish, with magnifications of as much as 100x. During this process, the elimination of soil adhering to the floor of the calculus was carried out utilizing sterile tweezers to carry the pattern and a wonderful acupuncture needle to softly scrape off the soil hooked up to the exterior layer of the mineralized plaque. Drops of 0.5 M HCl acid had been used to dissolve the flecks of soil and ultrapure water to halt demineralization, in addition to to clean and take away contaminants. The decontamination water was saved in plastic tubes for monitoring functions. The clear calculi had been then washed in ultrapure water as much as thrice to take away any traces of unfastened sediment, dissolved in an answer of 0.5 M HCl, and subsequently mounted on slides utilizing an answer of fifty:50 glycerol and ultrapure water. Furthermore, environmental samples from dust-traps set within the laboratory had been collected with the intention to rule out any sort of recent contamination.
Glass slides had been analyzed utilizing a Zeiss Axio Imager.M2 with magnifications starting from 100x to 630x. A contemporary reference assortment of 300 crops native to the Mediterranean area and Europe, together with printed literature, was used for the identification of archaeological starch granules. Spores had been recognized by comparability with obtainable literature [87,88].
CFTs for every tooth had been estimated via histomorphometric evaluation and are reported in Table 2 and S1 Table, alongside the preliminary cusp formation (Ci) and crown completion (Crc) charges over the person’s lifetime. These charges are expressed in days, months, and years of life. Specifically, canine CFTs vary from roughly 1,560 days (4.3 years) to 2,498 days (6.8 years). In distinction, first molars have a shorter CFT, starting from 860 days (2.4 years) to 1,304 days (3.6 years), whereas second molars have CFTs starting from 896 days (2.5 years) to 1,428 days (3.9 years). Regarding Ci, it is very important word that, when the NL was not seen, the apex of the dentin horn tip was thought of day 0 for first molars. For canines and second molars, nevertheless, overlapping ALs are used for calibration, though a few of these are nonetheless worn. It was not attainable to overlay solely the M2 of particular person PTG T.5980, so three years got as Ci on this case, as indicated within the literature [80].
Table 3 exhibits the imply CFTs, Ci and Crc, and their respective customary deviations for every tooth class (excluding the one P4). On common, canines take about 1,977 days (5.4 years) to type, whereas first molars take about 1,094 days (3.0 years) and second molars take about 1,176 days (3.2 years). These values spotlight the variation in CFT between tooth lessons, with canines exhibiting considerably longer CFTs than the primary and second molars. The common Ci for the primary molars is shifted by a number of days earlier than start (−8 days), because the NL was seen in two everlasting first molars. Canines present a median formation onset of 162 days, whereas second molars present a median of 1,158 days. In distinction, the Crc imply values are 1,086 days for M1s, 2,139 days for Cs, and a pair of,333 days for M2s.
Fig 4A presents the expansion curves of dental crowns, exhibiting cumulative size alongside the EDJ as a operate of crown formation days. M1 and M2 crowns exhibit fast development throughout the first 200 days, adopted by a gradual deceleration towards the cervical area. C crowns endure an accelerated development section throughout the first 400 days earlier than slowing progressively. Fig 4B illustrates the variation in EER values relative to crown formation days. Initial EER values are considerably increased than subsequent ones, indicating a quicker ameloblast recruitment price within the cusp area, which progressively slows because it approaches the cervical space. The deceleration in EER values alongside the EDJ is constant throughout all tooth lessons. As proven within the M2 graphs, the curve of particular person PTG T.8356 is shifted ahead in time, reflecting a delayed onset and completion of crown formation (Ci and Crc) in comparison with the opposite people within the pattern.
Fig 4. Enamel-dentine junction (EDJ) growth and enamel extension rate (EER) variation across crown formation days.
Growth curves of dental crowns along the enamel-dentine junction (EDJ) over crown formation days (A) and enamel extension rate (EER) variation over crown formation days (B). The graphs, divided by tooth class, illustrate crown growth dynamics across different individuals, showing EDJ length progression and changes in EER values over time.
Table 4 and S1 Table report respectively the quantity and the chronology of ALs for every tooth and particular person. The common of stress occasions throughout childhood is 25.7 (SD 8.7), with homologous ALs throughout completely different enamel counted as a single stress occasion. Notably, canines persistently recorded the very best variety of ALs amongst all dental lessons.
The month-to-month prevalence of stress occasions – calculated because the proportion of people presenting a minimum of one AL inside a given month, relative to the variety of people with enamel forming throughout that interval – is proven in Fig 5. The distribution displays an uneven sample: prevalence is null throughout the first two months of life, after which it begins to extend; from the seventh month onwards, prevalence drastically will increase, reaching its peak on the twelfth month, the place 80% of the pattern presents a minimum of one AL. This produces a bell-shaped distribution centered on the primary 12 months of life. Subsequent months present fluctuating values, with a second peak noticed across the forty-fourth month. From the sixtieth month, a progressive decline in each the speed and denominator of prevalence values is noticed, and the graph was subsequently reduce at this month.
Fig 5. Graph showing the monthly prevalence of Accentuated Lines (ALs) observed in the sample.
The x-axis represents age in months, while the y-axis indicates prevalence percentage. Peaks at specific ages suggest potential physiological stress events during childhood.
Differential evaluation of stress incidence throughout dental lessons is offered in Fig 6. Density plots of ALs distribution all through the crown point out that each the cuspal (preliminary) and cervical (terminal) areas exhibit fewer stress markers in comparison with the lateral (central) crown portion, which shows the very best focus of ALs.
Fig 6. Density curves illustrate the distribution of Accentuated Lines (ALs) across different tooth classes.
The x-axis represents age in months, while the y-axis shows frequency (left) and density (right). The curves for first molars (M1), canines (C), second molars (M2), and premolar (P4) highlight variations in stress occurrence throughout childhood.
The dental calculus preserved vegetal micro-remains within the type of starch granules, plant fibers, and fungal spores (Table 5).
Starch granules (Fig 7A) had been recovered in three of the 5 people analyzed. Overall, two morphotypes had been recognized on this research. To keep away from misinterpretation, starch granules smaller than 5 μm (transient starches) had been excluded from the evaluation [89].
Fig 7. Micro-remains extracted from dental calculus, observed under optical microscopy.
The images show different types of residues: (A) starch granules, suggesting the consumption of plant-based foods; (B) fungal spores of Saccharomyces, possibly associated with the intake of fermented foods or beverages; (C) plant fibers, which may reflect para-masticatory activities.
Morphotype I: Starch granules of this kind had been discovered within the dental calculus samples of three people (PTG T.8340, PTG T.8428, PTG T.5998), with some nonetheless partially embedded throughout the calculus matrix. The morphotype is often characterised by giant granules (sub-type A), which shows a spherical to sub-oval two-dimensional form, a centric hilum, and well-defined lamellae, with most widths starting from 21.1 and 45.1 μm (imply: 33.1 μm). Under polarized gentle, a centric extinction cross with 4 arms is seen. In each experimental and printed reference collections, starch granules of this morphotype are sometimes present in bimodal distributions, with giant sub-type A granules related to smaller granules (sub-type B), normally < 10 μm, additionally spherical/sub-oval in form and with a central hilum [90–92]. However, in archaeological samples, sub-type B granules are sometimes underrepresented or absent, as on this case, probably because of differential preservation. According to the literature and fashionable reference collections, the noticed morphometric options – together with bimodal distribution when preserved – are in keeping with starch granules from members of the Triticeae tribe (Poaceae household) and are thought of diagnostic for taxonomic identification [54,91,92].
Morphotype II: A single starch granule of this kind was recognized in two people (PTG T.8428, PTG T.5998). These granules exhibit a reniform form, with dimension ranging between 12–35 μm, and a collapsed/sunken hilum forming a deep fissure extending alongside virtually all the granule. Under polarized gentle, a brilliant extinction cross was seen with a number of lateral arms (> 4) diverging from the centric hilum [93]. These options are diagnostic of starch granules within the Fabaceae household, identified for a number of edible domesticated legumes (e.g., Lens culinaris Medikus, Vicia faba L., and Pisum sativum L.) and wild vetches (Vicia spp.). Identification at species or genus degree was not attainable because of overlaps in form and dimension at tribe degree, which had been noticed in our fashionable reference assortment.
Several Saccharomyces fungal spores (Fig 7B) had been recognized in three people (PTG T.8356, PTG T.8360, PTG T.5998). Known as “sugar fungi,” they embrace a number of yeasts [94,95].
Plant fibers and wooden components (Fig 7C) had been recognized within the calculus of all of the analyzed people. The fibers are lengthy and, in some circumstances, had been noticed nonetheless embedded within the mineral matrix. Several show diagnostic morphological options – together with distinct longitudinal striations, thickened cell partitions, and occasional Z-twisting – clearly seen underneath polarized gentle. These traits are in keeping with bast fibers derived from crops comparable to Linum usitatissimum or Cannabis sativa and align with established identification standards [96,97]. The presence of those fibers might replicate publicity to fibrous plant supplies via extra-masticatory or occupational actions, comparable to fiber processing, cordage manufacturing, or the usage of plant implements for oral hygiene.
This research represents an strategy to organic life histories reconstruction utilizing dental histomorphometry and calculus micro-residue evaluation on a pattern of everlasting enamel from the Pontecagnano group (Seventh-Sixth century BCE). This interdisciplinary strategy allowed the reconstruction of facets of particular person life histories, together with development trajectories and well being situations, and supplies site-specific insights into life-style throughout the Iron Age. While total lifespan is usually thought to be a direct proxy for human well being and environmental adaptation, the frequency and timing of non-fatal stress occasions, comparable to sicknesses or dietary deficiencies, all through improvement can present important insights right into a group’s adaptive capability and organic success [98].
The CFTs obtained on this research reveal important variations between tooth lessons (Tables 2 and 3), with canines exhibiting the longest formation occasions (imply = 1,977 days, SD = 295 days, N = 9), adopted by second molars (imply = 1,176 days, SD = 179 days, N = 10) and first molars (imply = 1,094 days, SD = 154 days, N = 10). These outcomes are in step with Reid and Dean [80], who documented comparable traits in enamel formation time in fashionable human enamel, highlighting the extended improvement of anterior enamel in comparison with molars. Regarding Ci, our outcomes present that the mesio-buccal cusp of the primary everlasting molars begins formation shortly earlier than start (imply = −8 days, SD = 20 days). However, this estimate needs to be interpreted with warning: of the ten M1s analyzed, the Neonatal Line (NL) was clearly seen in solely two circumstances – particularly, within the mesio-buccal cusp of the LLM1 of particular person PTG T.8390 and within the mesio-buccal cusp of the ULM1 of particular person PTG T.6036. In all different circumstances, crown initiation was anchored to start in line with customary conventions within the absence of a visual NL [80–82]. Canines provoke crown formation a number of months later (imply = 162 days, SD = 64 days), whereas second molars start significantly later (imply = 1,158 days, SD = 218 days), as detailed in Table 3. Reid and Dean [80] report mounted values for Ci, with M1s at all times forming at start, M2s at 3 years of age, and canines at 274 days (for higher) and 200 days (for decrease). Our Ci values for M1 and M2 are usually in keeping with these estimates, whereas Ci values for C are usually decrease. Regarding Crc, our outcomes point out that crowns of M1s full at a median of 1,086 days (SD = 156 days), these of canines at 2,139 days (SD = 307 days), and people of M2s at 2,333 days (SD = 316 days). Notably, the M2 of particular person T.8356 has excessive Ci and Crc values. In truth, this M2 begins forming round 1.5 years later than the others and consequently finishes forming later. However, these values fall throughout the anticipated vary of human variability in enamel formation timing [65] and certain replicate inter-individual developmental variations. Unlike earlier research that depend on literature-based Ci values – leading to Crc estimates derived from mounted beginning factors – our strategy permits for larger precision. The Ci values for C and M2 had been decided via chronological overlap with M1, guaranteeing extra correct estimations of crown completion. Furthermore, the overall traits noticed in our research are in keeping with information obtained by AlQahtani et al. [65] on enamel formation charges amongst tooth lessons.
Differences in EERs had been noticed throughout tooth lessons (Fig 4). M1 and M2 crowns exhibited a fast preliminary extension throughout the first 200 days, adopted by a gradual deceleration towards the cervical area. C crowns confirmed a chronic section of accelerated development, lasting as much as 400 days earlier than slowing down. These findings align intently with these of Guatelli-Steinberg et al. [20], who reported an exponential decline in EER from the cusp to the cervical area in fashionable human enamel. Both research spotlight that the very best EER values happen within the cuspal area, steadily lowering as enamel formation progresses cervically. These variations emphasize the variability in EERs amongst completely different tooth varieties, which might be attributed to distinct morphological and developmental traits. The noticed development in EER values over time follows a basic lower in enamel formation charges as crown improvement advances.
There is a scarcity of comparative information on histomorphometric parameters from the Iron Age within the literature, which refers to completely different chronological and geographical intervals [11,99]. Both Nava et al. [11] and Aris et al. [99] report a diachronic development of slowing childhood development trajectories, even in comparatively current evolutionary occasions. However, solely Aris et al. [99] think about the everlasting dentition. This research supplies, for the primary time, histological information from an Iron Age Italian group.
In this research, the prevalence of ALs offered a longitudinal image of well being situations from start as much as ca. 6 years of age. The month-to-month distribution of ALs (Fig 5) exhibits an asymmetrical, virtually bimodal, distribution with notable peaks at 12 and 44 months from start. These non-fatal stress occasions are sometimes linked to infectious ailments and dietary deficiencies [12,37,38,100,101]. The lack of stress occasions within the first two months of life aligns with maternal antibody safety [102], whereas later peaks could also be associated to elevated publicity to pathogens and/or dietary adjustments. The preliminary rise in stress prevalence, noticed from the third month, might coincide with infants turning into extra bodily engaged with their environment – sitting up, touching objects, and experiencing elevated publicity to environmental pathogens [103].
Differential evaluation of stress registration throughout the tooth crowns (Fig 6) signifies that each the cuspal and cervical areas report stress much less successfully in contrast with the central portion of the crown. If all the crown had been equally vulnerable, a homogeneous distribution of stress markers can be anticipated; as an alternative, the noticed bell-shaped sample underscores the differential recording susceptibility throughout the crown. Thus, analyzing a number of tooth varieties enhances the probability of capturing stress occasions which may in any other case go undetected in a single dental class. This statement confirms earlier findings by Nava et al. [3], who recognized comparable crown regional traits in each deciduous and everlasting enamel. It is necessary to notice that, throughout the first months of life, solely M1s are forming enamel, and solely of their cuspal areas, that are much less vulnerable to emphasize recording. This might assist clarify the preliminary absence of stress markers noticed within the enamel, and consequently within the prevalence curve, throughout the first two months. Conversely, the second peak round 44 months probably displays stress alerts recorded primarily by M2s, whose enamel at that stage is forming within the central crown areas – areas proven to register stress extra successfully.
The ALs prevalence distribution partly aligns with an analogous research offered by Nava et al. [3] in a Roman Imperial pattern from Isola Sacra (Fig 8). Nava et al. [3] interpreted the noticed ALs prevalence distribution of their pattern as indicative of weaning-related stress, with the very best frequency of stress occasions occurring across the first 12 months of life. In our Pontecagnano pattern, nevertheless, the first peak is barely shifted ahead at roughly 12 months. This distinction could also be defined by the differential recording capability throughout the dental crown. As demonstrated by Nava et al. [3], deciduous enamel can report stress occasions throughout the first months of life – a stage when the crowns of deciduous canines and second molars are halfway via their improvement. At this level, enamel is forming within the central areas, that are significantly delicate to physiological stress and effectively suited to its detection. In distinction, in our pattern, stress within the perinatal interval can solely be registered by M1s, which at that stage are forming enamel solely within the cuspal area – an space much less attentive to stress recording. Consequently, the absence of deciduous enamel in our dataset probably shifts the general ALs prevalence profile ahead, doubtlessly resulting in a slight underestimation of early-life stress occasions.
The graph illustrates variations in stress prevalence over time, with the x-axis representing age in months and the y-axis indicating prevalence share. Color-coded bars and development traces spotlight variations and similarities between the 2 populations.
A tougher interpretation considerations the secondary rise within the AL prevalence curve, with a peak round 44 months. Nava et al. [3] acknowledge that, as youngsters change into extra cellular and work together extra actively with their environment, elevated publicity to pathogens might contribute to additional stress occasions. In our pattern, it is usually necessary to notice that this second peak could be very probably defined by the truth that, at this stage, enamel is forming virtually solely within the central areas of M2s – areas which are extremely attentive to stress recording. Additionally, this peak might coincide with weaning completion and the transition to a weight-reduction plan extra intently resembling that of adults, reflecting the metabolic challenges related to the diversification of meals sources, each of which can pose important physiological stress throughout early childhood. Given the non-specific nature of ALs as stress markers, it’s believable that each elevated environmental publicity and the dietary transition towards an adult-like sample contribute to this extra complicated stress profile.
Overall, the ALs prevalence distribution in our Pontecagnano pattern displays the short-term, non-fatal stresses that people skilled throughout the first 6 years of life. It is necessary to focus on that though the sequence thought of on this evaluation is basically a mortality pattern, the chosen people had been adults who handed away lengthy after the entire formation of their dental crowns. Therefore, all people survived the demanding occasions that characterised their childhood.
During the Orientalizing interval, the Etruscan civilization skilled important socio-economic and cultural adjustments, characterised by demographic development, a rise in agricultural exercise, and the emergence of a stratified society [70]. This interval additionally noticed the enlargement of Etruscan tradition throughout varied areas of central Italy, with a notable enlargement into Campania [104,105]. The Villanovan and Etruscan weight-reduction plan was predominantly plant-based, with a reasonable consumption of animal proteins, as demonstrated by isotope evaluation [106,107] and archaeobotanical and zooarchaeological research [108–110]. Nutrition was based on cereals comparable to emmer spelt, durum wheat, barley, and rye [111], whereas legumes – together with beans, chickpeas, lentils, lupins, and peas – complemented the weight-reduction plan by offering another supply of proteins [112]. Animal meals sources included sheep, goats, cattle, wild boars, and small recreation [113].
Dental calculus evaluation of Pontecagnano people revealed the presence of starch granules, fungal spores, and plant fibers (Fig 7), which offer complementary insights into each grownup weight-reduction plan and non-dietary actions. Starch granules from the Triticeae (Poaceae) and Fabaceae households had been detected in three people, indicating the consumption of cereals and legumes in step with diversified agricultural practices already documented for historic Italian populations [110,114]. The broken look of some starch granules might recommend meals processing actions comparable to cooking and/or seed grinding, in addition to enzymatic degradations ensuing from early levels of starch digestion previous to consumption. In addition, the presence of oral ailments comparable to caries, documented in a number of people from the Pontecagnano pattern, is in keeping with a daily consumption of carbohydrates, a sample already effectively documented in agricultural populations because the Neolithic [57,115,116]. Interestingly, quite a few Saccharomyces spores had been recognized in three people (two males and one among unknown intercourse). Their abundance within the dental calculus pattern analyzed might point out common consumption of fermented meals or drinks – a follow already attested in archaeological contexts via the evaluation of the mineralized matrix, primarily by way of proteomic strategies and, extra hardly ever, via optical microscopy as in our research [55,117]. These findings might be contextualized throughout the broader socio-cultural transformations of the Orientalizing interval at Pontecagnano, when agricultural intensification and growing contacts with the Mediterranean world assisted in shaping useful resource availability and dietary habits. Finally, the presence of plant fibers in all people suggests attainable extra-masticatory use of the enamel. In a number of people, lengthy and fragmented fibers had been nonetheless embedded in dental calculus, some exhibiting traits indicative of cortical fibers. The widespread presence of wooden particles within the pattern suggests inhalation related to extra-masticatory actions – a lot of which can be occupational in nature – or the usage of picket implements associated to meals preparation or oral hygiene throughout the Pontecagnano group. As generally noticed in dental calculus research, such inclusions may additionally derive from different environmental or occupational pollution [5].
All in all, the number of microremains recovered from the Pontecagnano people highlights the benefits of dental calculus evaluation for reconstructing historic weight-reduction plan and lifeways. Thanks to its mineralized construction, dental calculus can protect starches, phytoliths, plant stays, and biomolecules comparable to DNA and proteins, offering distinctive direct proof of plant meals consumption that’s typically inaccessible via extra protein-sensitive strategies like conventional isotopic evaluation. Furthermore, the potential taxonomic identification of starch granules can reveal the photosynthetic pathways of crops (C3 vs. C4), providing additional insights into the relative contribution of various plant sources to historic diets. Given the small variety of people with preserved dental calculus, the outcomes of this evaluation needs to be interpreted with warning and are greatest understood as offering detailed insights into particular person life histories, relatively than as consultant of the broader inhabitants.
Although reconstructing toddler feeding practices in previous populations is inherently difficult as a result of shortage of direct proof, our research provides new information to the talk, significantly concerning early-life dietary stress and the physiological impression of weaning. A current isotopic research on people from Pontecagnano [107] throughout the Orientalizing interval suggests a weight-reduction plan based on C3 crops, with restricted consumption of animal proteins and remoted proof of C4 plant consumption – probably millet [107]. According to the WARN mannequin utilized in that research, weaning timing ranged between 0.7 years (t1, onset of weaning) and a pair of.6 years (t2, finish of weaning) [107]. The reported onset of weaning align with the rise in physiological stress markers noticed in our pattern, and the presence of C3 plant consumption align with our evaluation of dental calculus micro-remains.
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