G Goude

I Dori

V S Sparacello
email: vitale.sparacello@u-bordeaux.fr

E Starnini

A Varalli

Multi-proxy stable isotope analyses of dentine microsections reveal diachronic changes in life history adaptations, mobility, and tuberculosisinduced wasting in prehistoric Liguria (Finale Ligure, Italy, northwestern Mediterranean)

Keywords:

Multi-proxy stable isotope analyses of dentine microsections reveal diachronic changes in life history adaptations, mobility, and tuberculosis-induced wasting in prehistoric Liguria (Finale Ligure, Italy, northwestern

Mediterranean)

Introduction

The study of dietary intake and its changes during early life has important repercussions in the analysis of prehistoric health, paleodemography, and human life history strategies. In fact, infant breastfeeding and weaning practices (BWPs) -involving exclusive breastfeeding (EBF) duration, termination of breast-feeding (TBF), and timing, and variability of prehistoric BWPs is therefore an important field of research in evolutionary anthropology and bioarchaeology, given the number of information on past life experience, subsistence, and adaptative strategies that it can provide (review in [START_REF] Tsutaya | Reconstruction of breastfeeding and weaning practices using stable isotope and trace element analyses: a review[END_REF].

Isotopic methods investigating these issues aim at identifying the timing of the infant's trophic level shift from a higher trophic position than the mother during EBF (δ 15 N values are about 2 ‰ higher; [START_REF] Herrscher | Longitudinal study of stable isotope compositions of maternal milk and implications for the palaeo-diet of infants[END_REF] towards a value compatible with the adult population's average δ 15 N at the time of TBF [START_REF] Fogel | Nitrogen isotope tracers of human lactation in modern and archaeological populations[END_REF][START_REF] Fuller | Detection of breastfeeding and weaning in modern human infants with carbon and nitrogen stable isotope ratios[END_REF][START_REF] De Luca | δ 15N and δ 13C in hair from newborn infants and their mothers: A cohort study[END_REF]. Several studies on bioarchaeological samples used isotope ratio measurements of bone collagen δ 13 C and δ 15 N from infants of different ages at death, and compared them with the results obtained from females belonging to the same population (e.g., [START_REF] Jay | Iron age breastfeeding practices in Britain: isotopic evidence from Wetwang slack, East Yorkshire[END_REF], or used intra-long-bone analysis [START_REF] Waters-Rist | Infant and child diet in Neolithic hunter-fisher-gatherers from cis-baikal, Siberia: intra-long bone stable nitrogen and carbon isotope ratios[END_REF]. However, the most reliable and detailed approach involves the use of incremental dentine collagen to reconstruct temporal changes in the isotope ratios, and to therefore assess changing childhood diet in individuals who survived early life (e.g. [START_REF] Beaumont | Childhood diet: a closer examination of the evidence from dental tissues using stable isotope analysis of incremental human dentine[END_REF]Beaumont et al., , 2015[START_REF] Beaumont | Comparing apples and oranges: why infant bone collagen may not reflect dietary intake in the same way as dentine collagen[END_REF][START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF]. In addition, changes in δ 15 N values can be related not only to dietary changes, but also to periods of physiological stress and catabolism generated by starvation, long-term disease, or harsh climatic conditions [START_REF] Armit | Difference in death? A lost Neolithic inhumation cemetery with Britain's earliest case of rickets, at Balevullin, Western Scotland[END_REF]Beaumont et al., 2015;[START_REF] Dewitte | The osteological paradox 20 years later: past perspectives, Future Directions[END_REF][START_REF] Henderson | Early life histories of the London poor using δ13C and δ15N stable isotope incremental dentine sampling[END_REF][START_REF] King | Let's talk about stress, baby! Infant-feeding practices and stress in the ancient Atacama desert, Northern Chile[END_REF][START_REF] Montgomery | Strategic and sporadic marine consumption at the onset of the Neolithic: increasing temporal resolution in the isotope evidence[END_REF]; an approach recently supported by experimental data on animals [START_REF] Doi | Starvation effects on nitrogen and carbon stable isotopes of animals: an insight from meta-analysis of fasting experiments[END_REF]. As a result, the "incremental dentine" method is now commonly used in bioarchaeological studies (e.g. [START_REF] Greenwald | Stable isotope evidence of juvenile foraging in prehistoric Central California[END_REF]Scharlotta et al., 2018a, b).

The Neolithic Transition, which involved the adoption of a production economy based on the domestication of plants and animals resulted in changes in human diet and social organization, which had consequences, depending on the local ecology, on overall well-being [sensu [START_REF] Wood | A theory of preindustrial population dynamics: demography, economy, and well-being in Malthusian systems[END_REF], a combination of life expectancy, nutritional status, and health], and demographic patterns (e.g. [START_REF] Bocquet-Appel | Paleoanthropological traces of a neolithic demographic transition[END_REF], 2009, 2011a, 2011b). It has been proposed that the overall demographic growth observed with the Neolithic transition was mainly a consequence of an increase in female fertility when compared to hunter-gatherers, fully compensating for increased infant mortality [START_REF] Armelagos | The origins of agriculture: population growth during a period of declining health[END_REF][START_REF] Page | Reproductive trade-offs in extant huntergatherers suggest adaptive mechanism for the Neolithic expansion[END_REF] Numerous factors may have positively influenced female fertility: food resources predictability and storage, better nutritional status, decreased foraging time and energy expenditure (also positively impacting child care), earlier age at menarche leading to increased reproductive time span, and earlier weaning allowing for a shortening of interbirth interval [START_REF] Armelagos | The origins of agriculture: population growth during a period of declining health[END_REF]review in Bentley et al., 2001;[START_REF] Buikstra | Fertility and the development of agriculture in the Prehistoric Midwest[END_REF][START_REF] Ellison | Introduction. Ecology, reproduction, and human evolution[END_REF][START_REF] Page | Reproductive trade-offs in extant huntergatherers suggest adaptive mechanism for the Neolithic expansion[END_REF][START_REF] Roth | Sedentism and changing fertility patterns in a Northern Athapascan isolate[END_REF].

The direct reconstruction of diet and changing dietary composition during early life in Neolithic children is therefore fundamental to our understanding of the adaptive strategies developed concomitantly with the new management of food resources in various local ecologies. In fact, the advantages or disadvantages of the adoption of the Neolithic lifestyle, as well as their long-term evolution, need to be explored at a regional level, due to the mosaic patterns of expansion and crisis that characterized this process throughout the European continent (e.g. [START_REF] Bocquet-Appel | Detection of diffusion and contact zones of early farming in Europe from the space-time distribution of 14C dates[END_REF][START_REF] Cohen | Ancient Health: Skeletal Indicators of Agricultural and Economic Intensification[END_REF][START_REF] Porčić | Demography of the early Neolithic population in Central Balkans: population dynamics reconstruction using summed radiocarbon probability distributions[END_REF][START_REF] Zvelebil | The agricultural transition and the origins of Neolithic society in Europe[END_REF]. This study employs the incremental dentine method (δ 13 C, δ 15 N, and δ 34 S isotopes) in a sample of directly dated children spanning the early Neolithic through the Metal Ages in the region of Finalese in Liguria (northwestern Italy). The purpose of this study is to reconstruct weaning patterns, episodes of stress, and residential mobility, in order to infer changing environmental conditions, reproductive strategies, and fertility, alongside important changes in subsistence and societal organization through time (Bogucki and Crabtree, 2004;[START_REF] Cunliffe | The Oxford Illustrated Prehistory of Europe[END_REF][START_REF] Cunliffe | Europe between the oceans. 9000 BC -AD 1000[END_REF][START_REF] Guidi | Preistoria della complessità sociale[END_REF].

Bioarchaeological background and expectations

The western diffusion of the Neolithic production economy from the Near East reached the northwestern Mediterranean around 5800-5600 BCE, when people belonging to the Impresso-Cardial chrono-cultural complex (ICC) settled in the Liguro-Provençal Arc [START_REF] Binder | In pursuit of a missing transition: the Mesolithic and Neolithic radiocarbon chronology at La Font-aux-Pigeons rockshelter[END_REF]. The Square Mouthed Pottery culture (SMP) developed later and spread throughout Liguria and Northern Italy during the fifth millennium BCE (c. 5000-4300 BCE; [START_REF] Binder | La séquence de l'Impresso-Cardial de l'abri Pendimoun et l'évolution des assemblages céramiques en Provence[END_REF][START_REF] Del Lucchese | Aggiornamenti sulla fase antica della Cultura dei Vasi a Bocca Quadrata in Liguria da una revisione dei materiali ceramici in corso[END_REF]. Later, Liguria was the access road for the spread of the Chassean in northern Italy from France (c. 4300-3700 BCE;[START_REF] Crepaldi | Le Chasséen en Ligurie[END_REF]Maggi, 1997a). The Metal Ages in Liguria are subdivided into the Copper Age (c. 3700-2200 BCE), Bronze Age (Early: 2200-1600 BCE; Middle: 1600-1350 BCE; Late and Final: 1350-900 BCE), and Iron Age (c. 900 BCE to Roman conquest in 180 BCE) [START_REF] Bagolini | The radiocarbon chronology of the Neolithic and Copper Age of northern Italy[END_REF][START_REF] De Marinis | L'età del Rame. La pianura padana e le Alpi al tempo di Ötzi. Brescia: Compagnia della Stampa Massetti Rodella Editori.L'età del Rame. La pianura padana e le Alpi al tempo di Ötzi[END_REF][START_REF] De Marinis | L'età del Rame. La pianura padana e le Alpi al tempo di Ötzi. Brescia: Compagnia della Stampa Massetti Rodella Editori.L'età del Rame. La pianura padana e le Alpi al tempo di Ötzi[END_REF]Spadea, 2004, 2007;Del Lucchese and Maggi, 1998;Maggi et al., 1991-92).

Evidence of prehistoric human occupation in western Liguria comes predominantly from several caves and rock shelters opening within a radius of a few kilometers in the Finalese area, where important sites such as Arene Candide have yielded detailed stratigraphic successions spanning from the Upper Paleolithic to the Roman Age [START_REF] Arobba | Cereal remains, plant impressions and 14C direct dating from the Neolithic pottery of Arene Candide Cave (Finale Ligure, NW Italy)[END_REF]Maggi, 1997a;[START_REF] Tinè | Transizione tra Neolitico antico e Neolitico medio. Le ceramiche dello stile Pollera. Strato 13[END_REF]. Several bioarchaeological studies provide a picture of life during the Neolithic in this area. Based on archaeobotanical and zooarchaeological studies, subsistence was based on a variety of domesticated plants [START_REF] Arobba | Cereal remains, plant impressions and 14C direct dating from the Neolithic pottery of Arene Candide Cave (Finale Ligure, NW Italy)[END_REF][START_REF] Nisbet | Environment and agriculture in the early Neolithic of the Arene Candide (Liguria)[END_REF] and on livestock breeding, especially of sheep [START_REF] Macphail | The soil micromorphological evidence of domestic occupation and stabling activities[END_REF][START_REF] Rowley-Conwy | Arene Candide: a Small Part of a Larger Pastoral System?[END_REF], 1997). Isotopic analyses (δ 13 C/ δ 15 N ratios) suggest a diet based on C 3 cereals, with a significant component of proteins derived from terrestrial animals (Le Bras- [START_REF] Kuzawa | Stratégies de subsistance et analyse culturelle de populations Néolithiques de Ligurie: approche par l'étude isotopique (δ13C et δ15N) des restes osseux[END_REF][START_REF] Goude | Alimentation et modes de vie néolithiques en Ligurie[END_REF]. No significant consumption of fish protein during the SMP period was proposed in light of the collagen isotopic composition (review in [START_REF] Goude | Alimentation et modes de vie néolithiques en Ligurie[END_REF]. The same pattern was confirmed by analyses performed on individuals from the earlier ICC from the same region [START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF]. These results contrast, to a degree, with archaeological (e.g. fishing hooks, shell blow horns made of Charonia sp.), archaeozoological (e.g. shells), and dental calculus findings, which highlight the continuous use of marine resources throughout the Neolithic [START_REF] Desse-Berset | Les poissons[END_REF].

Except for evidence of the utilization of caves for domestic activities, stabling, and funerary purposes (e.g. [START_REF] Arobba | Cereal remains, plant impressions and 14C direct dating from the Neolithic pottery of Arene Candide Cave (Finale Ligure, NW Italy)[END_REF][START_REF] Del Lucchese | The Neolithic burials from Arene Candide: the Bernabò Brea-Cardini excavation[END_REF][START_REF] Maggi | Alberi da foraggio e scalvatura neolitica: nuovi dati dalle Arene Candide[END_REF][START_REF] Rowley-Conwy | Arene Candide: a Small Part of a Larger Pastoral System?[END_REF], 1997), the impact on the people in the territory during Ligurian Neolithic was minimal. No trace of large settlements is present, and only few artifacts are attributed to open-air sites [START_REF] Starnini | Segnalazione di un sito neolitico in località S. Sebastiano di Perti (Finale Ligure-SV)[END_REF]. Humans had little impact on forest coverage until the Late Neolithic [START_REF] Nisbet | Arene Candide: charcoal remains and prehistoric woodland use[END_REF], and land clearing through the extensive use of fire was introduced in Liguria only at the end of the Neolithic (Maggi, 1997b). The archaeological evidence of increasing socioeconomic complexity, population growth, and enlarged exchange networks observed in Italy and Europe during the Metal Ages (reviews in Bogucki and Crabtree, 2004;[START_REF] Cunliffe | The Oxford Illustrated Prehistory of Europe[END_REF][START_REF] Cunliffe | Europe between the oceans. 9000 BC -AD 1000[END_REF][START_REF] Guidi | Preistoria della complessità sociale[END_REF] take the form of quarries and mines, and impacts forest coverage in the Copper Age [START_REF] Maggi | Mid fourth-millennium copper mining in Liguria, north-west Italy: the earliest known copper mines in Western Europe[END_REF][START_REF] De Pascale | Pollen, herds, jasper and copper mines: economic and environmental changes during the 4th and 3rd millennia BC in Liguria (NW Italy)[END_REF], the diffusion of the Bell Beaker ceramics at the beginning of the Bronze Age [START_REF] Chiarenza | Distribution et aspects du Campaniforme en Ligurie (Italie). Congrés Internacional Xarxes al Neolític -Neolithic Networks Rubricatum[END_REF], and the increasingly structured fortified settlements during the Bronze and Iron Age (e.g. [START_REF] Giuggiola | Il villaggio delle Anime: un abitato fortificato dell'età del Ferro nel Finalese[END_REF][START_REF] Maggi | Archeologia dell'Appennino ligure: gli scavi del Castellaro di Uscio : un insediamento di crinale occupato dal Neolitico alla conquista romana[END_REF] in Liguria. However, diet does not seem to vary significantly in western Liguria from the Neolithic throughout the Copper and Bronze Age, at least according to initial C and N isotopic analyses performed on various sites [START_REF] Varalli | Dal Neolitico all'Età del Ferro: indagine paleonutrizionale su alc:une popolazioni della Liguria attraverso un approccio isotopico[END_REF]. It is only in the Iron Age that a dietary shift, i.e. a significant consumption of C 4 plants, can be detected [START_REF] Varalli | Dal Neolitico all'Età del Ferro: indagine paleonutrizionale su alc:une popolazioni della Liguria attraverso un approccio isotopico[END_REF]; an occurrence in northeastern Italy that can be traced earlier to the transition between the Early and Middle Bronze Age [START_REF] Tafuri | Stable isotope evidence for the consumption of millet and other plants in Bronze Age Italy[END_REF]Varalli et al., 2016a).

Expectations

It is generally expected that agriculturalist populations will display an earlier TBF than hunter-gatherers (e.g. [START_REF] Bentley | Fertility changes with the prehistoric transition to agriculture. Perspectives for reproductive ecology and paleodemography[END_REF]. Ethnographic accounts generally agree by showing, on average, a later TBF (between 3-5 years of age) in hunter-gatherers, than in agriculturalists (between 2-4 years), albeit with significant variation (e.g. [START_REF] Dettwyler | When to wean: biological versus cultural perspectives[END_REF][START_REF] Kennedy | From the ape's dilemma to the weanling's dilemma: early weaning and its evolutionary context[END_REF][START_REF] Marlowe | Hunter-gatherers and human evolution[END_REF]Sellen, 2001a[START_REF] Sellen | Lactation, complementary feeding, and human life history[END_REF][START_REF] Sellen | Evolution of infant and young child feeding: implications for contemporary public health[END_REF][START_REF] Sellen | Evolution of human lactation and complementary feeding: implications for understanding contemporary cross-cultural variation[END_REF][START_REF] Sellen | Relationship between subsistence and age at weaning in "preindustrial'' societies[END_REF]. Similar variation, but without consistent association with subsistence mode, is present in the introduction of supplementary foods immediately after birth to 1-1.5 years (Sellen, 2001a;[START_REF] Sellen | Relationship between subsistence and age at weaning in "preindustrial'' societies[END_REF]. It should be noted that the optimal duration of EBF is 6 months (see nutritional recommendations in [START_REF] Who | Global Strategy on Infant and Young Child Feeding[END_REF][START_REF] Butte | Nutrient Adequacy of Exclusive Breastfeeding for the Term Infant during the First Six Months of Life[END_REF], as an earlier introduction of supplementary foods exposes the infant to increased morbidity and gastrointestinal problems [START_REF] Kramer | The optimal duration of exclusive breastfeeding[END_REF]. Conversely, after around 6 months, EBF cannot provide enough essential nutrients to sustain optimal growth, and the reserve of fat deposited in the first six months to buffer nutritional disruptions is rapidly depleted [START_REF] Kuzawa | Stratégies de subsistance et analyse culturelle de populations Néolithiques de Ligurie: approche par l'étude isotopique (δ13C et δ15N) des restes osseux[END_REF]. Cross-cultural surveys indicate that most non-industrial groups adopt attitudes and values about child feeding that are in broad concordance with the optimal practice [START_REF] Sellen | Evolution of infant and young child feeding: implications for contemporary public health[END_REF]. However, suboptimal strategies are common and can be due to contingent factors (health of the mother and/or child), local ecologies and seasonality, and cultural practices (Sellen, 2001a(Sellen, , 2001b[START_REF] Sellen | Evolution of infant and young child feeding: implications for contemporary public health[END_REF].

The direct reconstruction of prehistoric BWPs through isotopic analysis reflects the variation described above and generally shows later TBF in Paleo/Mesolithic hunter-gatherers than in Neolithic agriculturalists (e.g. [START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF][START_REF] Howcroft | Infant feeding practices at the Pitted Ware Culture site of Ajvide, Gotland[END_REF][START_REF] Reynard | The known, the unknown and the unknowable: weaning times from archaeological bones using nitrogen isotope ratios[END_REF][START_REF] Tsutaya | Reconstruction of breastfeeding and weaning practices using stable isotope and trace element analyses: a review[END_REF]. Comparable variation is present through the Metal Ages and protohistory (e.g. [START_REF] Fernández-Crespo | Infant and childhood diet at the passage tomb of Alto de la Huesera (north-central Iberia) from bone collagen and sequential dentine isotope composition[END_REF][START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF]Scharlotta et al., 2018aScharlotta et al., , 2018b)), and only in post-medieval times is there evidence from incremental dentine studies of TBF before 2 years of age [START_REF] King | Linear enamel hypoplasia as indicator of systemic physiological stress: evidence from two known age-at-death and sex populations from postmedieval London[END_REF]. Estimates of the introduction of supplementary foods based on isotopic ratios range from 0.5 to 2 years, according to bone and dentine studies (review in [START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF]. However, [START_REF] Millard | A model for the effect of weaning on nitrogen isotope ratios in humans[END_REF] suggests that studies using bone tissue may overestimate EBF cessation by 6-9 months.

In this study, we expect the Ligurian Neolithic to show an isotopic profile compatible with an earlier age of weaning (TBF) than huntergatherers. The scenario of variation in BWPs previously depicted for the Metal Ages [START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF] does not allow for clear expectations. However, we anticipate societal, technological, and subsistence changes to have affected diachronic dietary changes in early life. In particular, we expect a further shortening in BWPs, which would be compatible with the further demographic increase apparent during the Metal Ages, although with significant fluctuations (e.g. [START_REF] Bevan | Holocene population, food production, and climate[END_REF][START_REF] Capuzzo | Highly diverse Bronze Age population dynamics in Central-Southern Europe and their response to regional climatic patterns[END_REF][START_REF] Müller | Demographic traces of technological innovation, social change and mobility: from 1 to 8 million Europeans (6000-2000 BCE)[END_REF]. Furthermore, increased exchange networks may have favored the mobility of people and, concomitantly, materials, and this should be apparent in δ 34 S profiles (see methods).

In addition to subsistence practices, the "local ecology" (sensu McDade, 2003: 111) of Neolithic people in Liguria, and the consequent BWPs, were probably affected by a high infectious load. In fact, the Neolithic skeletal series in the Finalese area are known for having yielded several cases of osteoarticular tuberculosis (TB; [START_REF] Canci | New evidence of tuberculous spondylitis from Neolithic Liguria (Italy)[END_REF][START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millenium BC from Arene candide cave (Liguria, Italy)[END_REF][START_REF] Sparacello | A 6500-year-old Middle Neolithic child from Pollera Cave (Liguria, Italy) with probable multifocal osteoarticular tuberculosis[END_REF][START_REF] Sparacello | Chapter 9: archaeothanatology and palaeobiology of the burials and "scattered human remains" from Arma dell'Aquila (Finale Ligure, Savona)[END_REF]; see also [START_REF] Dori | Environmental correlates of growth patterns in Neolithic Liguria (northwestern Italy)[END_REF]. Tuberculosis is a highly infectious, debilitating, and growth-impairing disease [START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF]. One of the Neolithic individuals included in this study is Arene Candide V (excavations Bernabò-Brea and Cardini;[START_REF] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide. Parte Prima: gli strati con ceramiche[END_REF], an adolescent who died with Pott's spine, a skeletal lesion that is pathognomonic of osteoarticular tuberculosis [START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millenium BC from Arene candide cave (Liguria, Italy)[END_REF]. Since maternal gestation and milk supports the infant's immune system [START_REF] Mcdade | Life history theory and the immune system: steps toward a human ecological immunology[END_REF]McDade and Worthmann, 1999;[START_REF] Pacheco | The impact of the milk glycobiome on the neonate gut microbiota[END_REF][START_REF] Jeurink | Importance of maternal diet in the training of the infant's immune system during gestation and lactation[END_REF], it is possible that Neolithic mothers delayed BWPs, as observed today especially in marginal contexts with limited resources and high infectious risk (e.g. [START_REF] Cantrelle | Breast feeding, mortality in childhood and fertility in a rural zone of Senegal[END_REF][START_REF] Lindstrom | The effects of breastfeeding and birth spacing on infant and early childhood mortality in Ethiopia[END_REF].

The study of Arene Candide V will also provide information on the course of TB in this individual and will contribute to the debate on TB virulence and host-pathogen co-evolution in the Neolithic [START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF]. Tuberculosis causes severe metabolic disturbances (cytokine activation, abnormal protein metabolism, and hormonal unbalance) causing 'consumption' [START_REF] Macallan | Malnutrition in tuberculosis[END_REF][START_REF] Schwenk | Tuberculosis, malnutrition and wasting[END_REF][START_REF] Del Rey | Endocrine and cytokine responses in humans with pulmonary tuberculosis[END_REF]. During such catabolic states, individuals use amino acids from their own body tissues to synthesize new proteins, resulting in an increase of δ 15 N values [START_REF] Beaumont | Childhood diet: a closer examination of the evidence from dental tissues using stable isotope analysis of incremental human dentine[END_REF][START_REF] Beaumont | The great Irish famine: identifying starvation in the tissues of victims using stable isotope analysis of bone and incremental dentine collagen[END_REF][START_REF] Katzenberg | Stable isotope variation in pathological bone[END_REF]. Tracing the onset of this "pathological trophic shift" will test the previous estimates of active TB duration in this individual, c. 2-3 years, based on skeletal gracilization and enamel hypoplasia [START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millenium BC from Arene candide cave (Liguria, Italy)[END_REF][START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF].

Materials and methods

We analyzed seven teeth from seven individuals unearthed from four nearby caves in the Finalese area (Arene Candide Cave, Arma dell'Aquila, Arma delle Anime, Arma dei Parmorari; Liguria region, northwestern Italy; Table 1 and Fig. 1).

We selected unworn teeth without taphonomic damage or pathological lesions (such as caries). We analyzed either the upper or lower first molar of all individuals, except for Arene Candide V, for which the only tooth suitable for sectioning was the upper canine (Table 1). However, it has been previously shown that results from canines and first molars are in broad agreement [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF]. The inability to use the same type of tooth for all the individuals is due to both the nature of the dental series (e.g. incomplete, damaged, or worn molars), and to administrative permits that rightfully limit the possibility of conducting destructive analyses of human remains from archaeological contexts. The method involved in this study destroys half of each selected tooth (see below), and we therefore minimized the impact on the Ligurian skeletal series by selecting isolated fragmentary maxillae/mandibles and one isolated tooth from disturbed contexts other than burials (Table 1). The partial destruction of the canine from the Arene Candide V burial was authorized given the potential information on TB in the Neolithic that it can provide. To minimize loss of future information, an impression of the crown of all teeth was completed using Coltène President's Jet Light Body Plus (polyvinylsiloxane). Specimens were scanned via micro-CT (using a General Electric vtome x s microtomograph hosted by UMS 3626 PLACAMAT and acquired by the LabEx LaScArBx and the Région Nouvelle-Aquitaine. Scanning conditions were 80 kV and 150 μA (with no filter), and numerous macro-photos were taken.

Direct AMS dating was performed on all remains in this study (except for Arma dell'Aquila RS3, which was dated in [START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF] to assess the broad chrono-cultural context of the remains [START_REF] Sparacello | Dating the funerary use of caves in Liguria (northwestern Italy) from the Neolithic to historic times: results from a large-scale AMS dating campaign on human skeletal series[END_REF]. The AMS dates obtained for the individuals extend through most of the Neolithic and Metal Ages noted in Liguria (Table 1). One individual is chronologically contemporaneous with the ICC Neolithic (Arma dell'Aquila RS3; [START_REF] Sparacello | Chapter 9: archaeothanatology and palaeobiology of the burials and "scattered human remains" from Arma dell'Aquila (Finale Ligure, Savona)[END_REF]Sparacello et al., , 2019a)), two with the SMP Neolithic (Arene Candide V, and one juvenile from the scattered remains found in the zone G of Arene Candide cave), two chronologically overlap with the Copper Age at its tail end, one belongs to the Late Bronze Age, and one to the Iron Age of Liguria.

Each tooth was processed for incremental dentine analysis according Beaumont et al. protocol (Beaumont et al., 2013), and the collagen was extracted according to the laboratory standard protocols (e.g. [START_REF] Goude | A multidisciplinary approach to neolithic life reconstruction[END_REF]. A sagittal section of each tooth was performed with a IsoMet® precision saw in order to obtain two equal parts from the tip of a cusp to the tip of a root. Half of the tooth was preserved, and the other half was abraded with aluminum oxide by a sandblaster in order to remove the external surface of the enamel and the root. The clean sample was then demineralized in HCl (0.05 M) at 4 °C for several days and rinsed with distilled water after demineralization was completed. Transverse sections of dentine were realized with a sterilized scalpel, with a thickness of one millimeter when possible, or more when dentine was too fragile to allow for a thinner section (details are available in Supplementary Information Table S1). Each section was then cleaned in NaOH for 20°h to remove potential remaining contaminants, rinsed and solubilized in HCl (0.01 M) at 100 °C for 17 h. Each solubilized microsection was frozen and freeze-dried for 24 h. The collagen was then analyzed by EA-IRMS (Europa Scientific EA analyzer coupled to Europa Scientific 20-20 IRMS; IsoAnalytical Ltd, Crewe, UK). The quality control of each run was checked with in-house standard calibrated with IAEA standards; the measurement error of standards is 0.1‰ for CN and 0.2‰ for S. The preservation of collagen and reliability of isotopic data were controlled according to international recommendations: C ≥ 30 %, N ≥ 10 % [START_REF] Ambrose | Isotopic analysis of paleodiets: methodological and interpretative considerations[END_REF], C/N between 2.9 and 3.6 [START_REF] Deniro | Post-mortem preservation and alteration of in vivo bone collagen isotope ratios on relation to palaeodietary reconstruction[END_REF], S between 0.15 and 0.35, C/S between 300-900 and N/ S between 100-300 [START_REF] Nehlich | Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen[END_REF].

Age at death was estimated from dental development and eruption, epiphyseal fusion, and diaphyseal length of long bones [START_REF] Alqahtani | Brief communication: the London atlas of human tooth development and eruption[END_REF][START_REF] Boccone | Age estimation in subadult Egyptian remains[END_REF][START_REF] Cardoso | Age estimation from stages of epiphyseal union in the presacral vertebrae[END_REF][START_REF] Schaefer | Juvenile Osteology -a Laboratory and Field Manual[END_REF]. We calculated the approximate ages at which the development of dentine starts and ends in each human permanent tooth using the times of tooth formation described in the Queen Mary University of London (QMUL) London atlas [START_REF] Alqahtani | Brief communication: the London atlas of human tooth development and eruption[END_REF]. This work allows estimates of the age of formation for different teeth even in cases where root formation is incomplete. In particular, when the tooth was still forming (e.g. Rc), the age corresponds to the point of formation reached at death; in cases where root formation was complete, the age corresponds to the age of formation of the apical end of the root [START_REF] Alqahtani | Brief communication: the London atlas of human tooth development and eruption[END_REF].

The method described in Beaumont and collaborators in 2013 (see also Beaumont and Montgomery, 2015) was used to determine the ages of the incremental dentine sections of each tooth. This method is based on the assumption that the dentine grows at a constant rate throughout different regions of a tooth (3-5 μm per day, [START_REF] Dean | A longitudinal study of tooth growth in a single individual based on long-and short-period incremental markings in dentine and enamel[END_REF][START_REF] Dean | Rates of dentine mineralization in permanent human teeth[END_REF][START_REF] Hillson | Dental Anthropology[END_REF][START_REF] Liversidge | Increasing human tooth length between birth and 5.4 years[END_REF], although some studies showed variability in growth rate in the coronal and apical areas of the roots [START_REF] Dean | Extension rates and growth in Tooth Heights of modern human and fossil hominin canines and molars[END_REF][START_REF] Dean | Human life history evolution explains dissociation between the timing of tooth eruption and peak rates of root growth[END_REF].

Before sectioning, we measured the dentine length of each tooth in order to scale the growth rate to its actual size. For each tooth, the dentine time development was divided by this length to obtain the growth time corresponding to 1 mm. These results were multiplied by the actual measurements of each single slice obtained from each tooth of the sample. In this way, we were able to take into account the variability in growth rate expected between individuals [START_REF] Dean | A longitudinal study of tooth growth in a single individual based on long-and short-period incremental markings in dentine and enamel[END_REF][START_REF] Dean | Preliminary observations on increasing root length during the eruptive phase of tooth development in modern humans and great apes[END_REF][START_REF] Liversidge | Increasing human tooth length between birth and 5.4 years[END_REF]. Further information about sections, age estimates, and isotopic data of the individuals used for incremental dentine analysis are available in Supplementary Information Table S1.

In order to frame the incremental dentine results within the local population δ 13 C, δ 15 N, δ 34 S baseline, we used a sample of adults from western Liguria, in part analyzed in previous studies (Le [START_REF] Kuzawa | Stratégies de subsistance et analyse culturelle de populations Néolithiques de Ligurie: approche par l'étude isotopique (δ13C et δ15N) des restes osseux[END_REF][START_REF] Goude | Alimentation et modes de vie néolithiques en Ligurie[END_REF][START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF], and in part analyzed for this study. Details on the age, sex, absolute date, and chronocultural attribution of the adult sample are available in Supplementary Information Table S2.

Environmental correlates of bone and teeth stable isotope data

Carbon, nitrogen and sulphur isotope ratios (δ 13 C, δ 15 N, δ 34 S) in bone and tooth collagen (organic matter) of archaeological populations are commonly applied to infer diet (relative protein intake), mobility (local vs. non-local), and more generally human behaviors through time (e.g. [START_REF] Drucker | Aquatic resources in human diet in the Late mesolithic in Northern and luxembourg: insights from carbon, nitrogen and sulphur isotope ratios[END_REF][START_REF] Goude | A multidisciplinary approach to neolithic life reconstruction[END_REF][START_REF] Jovanović | Last hunters-first farmers: new insight into subsistence strategies in the Central Balkans through multi-isotopic analysis[END_REF]. While bone biogeochemical composition provides general information covering the time of its formation and renewal (last years of life, depending on age and physiology; e.g. [START_REF] Szulc | Biochemical measurements of bone turnover in children and adolescents[END_REF][START_REF] Hedges | Collagen turnover in the adult femoral mid-shaft: modeled from anthropogenic radiocarbon tracer measurements[END_REF], teeth, and more particularly sequential analysis of teeth, provide more detailed biogeochemical information regarding the moment of dentine formation (i.e. from prenatal to early adulthood; [START_REF] Alqahtani | Brief communication: the London atlas of human tooth development and eruption[END_REF][START_REF] Beaumont | Childhood diet: a closer examination of the evidence from dental tissues using stable isotope analysis of incremental human dentine[END_REF]. Carbon and nitrogen isotope ratios differ in soil and plants according to different environmental parameters, as well as species and physiology (e.g. [START_REF] Farquhar | Carbon isotope discrimination and photosynthesis[END_REF][START_REF] Amundson | Global patterns of the isotopic composition of soil and plant nitrogen[END_REF]. Their specific composition is transferred through the trophic chain, with an increase of heavy isotopes leading to higher δ 13 C and δ 15 N ratios between consumers and their food resources (e.g. [START_REF] Bocherens | Trophic level isotopic enrichment of carbon and nitrogen in bone collagen: case studies from recent and ancient terrestrial ecosystems[END_REF]. This pattern is also valid between breastfed infants and mother's milk (see modern studies of de [START_REF] De Luca | δ 15N and δ 13C in hair from newborn infants and their mothers: A cohort study[END_REF][START_REF] Herrscher | Longitudinal study of stable isotope compositions of maternal milk and implications for the palaeo-diet of infants[END_REF]. Carbon and nitrogen isotope ratios also discriminate between freshwater, marine, or terrestrial environments (eg. [START_REF] Schoeninger | Nitrogen and carbon isotopic composition of bone collagen from marine and terrestrial animals[END_REF], and allow for palaeodietary and mobility reconstructions under conditions of well-established local isotopic baselines (see review for archaeological populations in [START_REF] Goude | Carbon and nitrogen isotopic variability in bone collagen during the Neolithic period: influence of environmental factors and diet[END_REF]. Carbon isotope ratios also allow differentiation between consumers of C 3 (such as wheat, barley) and C 4 plants (such as millet), as C 4 plants exhibit high δ 13 C. Near the seashore, the identification of C 4 plants versus marine resource consumers can be accomplished by integrating δ 15 N data (e.g. [START_REF] Varalli | Dal Neolitico all'Età del Ferro: indagine paleonutrizionale su alc:une popolazioni della Liguria attraverso un approccio isotopico[END_REF]. Added to carbon and nitrogen, sulphur isotope ratios provide relevant information regarding environment, food resource consumption and provenience of individuals [START_REF] Nehlich | The application of sulphur isotope analyses in archaeological research: a review[END_REF]. Moreover, according to the geological terrain, this proxy can be more efficient in documenting mobility than strontium isotope ratios, for example. Measured in sequential tooth collagen, δ 34 S would reflect fetal physiology, mother's milk composition (including diet; [START_REF] Battaglia | Placental transport and metabolism of amino acids[END_REF][START_REF] Holm | Uptake and release of amino acids in the fetal-placental unit in human pregnancies[END_REF], and environment exploited by the mother) and different food consumption and/or mobility during weaning and childhood [START_REF] Nehlich | The application of sulphur isotope analyses in archaeological research: a review[END_REF].

Results

Except for one section of a tooth (AQ_012; from ca. 2-5 years old), all dentine sections (n = 85; from 3 to 18 sections per tooth) provided good collagen quality and reliable C and N stable isotope ratios. After sampling for C and N stable isotope ratios, the remaining collagen allowed for the S analysis of only 15 sections in 5 teeth (Supplementary Information Table S1).

Considering all dentine sections, the results for nitrogen isotope ratios (δ 15 N) range from 7.2-12.2‰ (Δ 5‰), for carbon isotope ratios (δ 13 C) range from -21.2 to -19.2‰ (Δ 2‰), and for sulphur isotope ratios (δ 34 S) range from -2.8-11.5 ‰ (Δ 8.7‰). Among sections within the same individual, the range of δ 15 N varies from 4.6 to 1.7 ‰, the range of δ 13 C varies from 0.4 to 1.1‰, and the range of δ 34 S varies from 0.5 to 2.1‰. The isotopic variation is not correlated with the number of sections performed for each tooth (Pearson's r not significant), albeit the lowest variations recorded for the three isotopes were found in the ICC Neolithic individual Arma dell'Aquila RS3 (AQ 012; Table 1), for which only three sections could be analyzed (Supplementary Information Table S1).

Dentine profiles are displayed for the whole sample, by isotope studied, in Fig. 1 (δ 15 N), 2 (δ 13 C), and 3 (δ 34 S). The profiles, by individual, of both δ 15 N δ 13 C are reported in Supplementary Information Fig. 1.

The first microsections in our sample have an age midpoint spanning 1.2 and 2.6 years of age, depending on the individual (Fig. 234and Supplementary Information Fig. 1). Although for some individuals the first section may have included dentine deposited during the first trimester, the earliest δ 15 N value is probably influenced not only by breast milk, but also by food supplementation in all individuals. Nevertheless, for all individuals, the δ 15 N of this first section is the highest ratio recorded (from 12.2-10.1 ‰), followed by a sharp decrease (except for one SMP individual, ACN 022, where the sharp decrease occurs after the second section, age midpoint 2.7 years: Fig. 2, and Supplementary Information Fig. 1). The high δ 15 N ratio of the first microsection indicates that animal protein, in this case breast milk, still had a significant contribution to infant subsistence.

From values derived from dentine of exclusively breastfed individuals, it is possible to extrapolate the theoretical mothers' δ 15 N ratio during pregnancy by using the most recent reference data of mother milk/nail from Herrscher and collaborators [START_REF] Herrscher | Longitudinal study of stable isotope compositions of maternal milk and implications for the palaeo-diet of infants[END_REF], which is ca. 2‰ lower compared to breastfed infant δ 15 N. If we apply this correction to the first dentine microsection of the two SMP Neolithic individuals (δ 15 N 11.9 ‰ and 11.6 ‰), theoretical mothers' δ 15 N ratio should range between 9.9 and 9.6‰, i.e. above the values obtained from bone for the SMP adult females (n = 8; mean = 8.8‰; range 8.5-9.4 ‰; Supplementary Information Table S2), despite the fact that the first microsection probably does not reflect EBF alone. The ICC individual has the lowest δ 15 N in the first section among Neolithic individuals, 10.4‰, but compatible with the 8.5 ‰ found in the only ICC adult female (Arma dell'Aquila 5; Supplementary Information Table S2).

Except for Parmorari 002 from the Late Bronze Age (with a δ 15 N of 12.2 ‰), all individuals from the Metal Ages display a lower δ 15 N in the first microsection than the SMP Neolithic individuals (between 10.8 and 10.1‰), despite having an earlier age estimate midpoint (Fig. 2, and Supplementary Information Fig. 1). Likewise, for all individuals except for Parmorari 002, the theoretical mothers' bone δ 15 N obtained by subtracting 2‰ from the value of the first microsection falls within the range of adults' bone δ 15 N (due to the commingled nature of the remains, it was not possible to select adult females alone. Copper Age δ 15 N: n = 8; mean = 8.6‰; range = 6.8-9.7‰. Bronze Age δ 15 N: n = 5; mean = 8.7‰; range = 7.9-9.0 ‰; Iron Age δ 15 N: n = 2; mean = 7.6‰; range = 7.0-8.4 ‰; Supplementary Information Table S2). For Parmorari 002, a decrease of 3.2‰ would be necessary to reach the upper boundary of the bone δ 15 N range in our Bronze Age sample.

After the first microsection, δ 15 N profiles show a decrease, at least until the age of ca. 4 years old for all individuals, except for the ICC Neolithic individual, which shows a sharp increase in the third and last microsection. The decrease until the age of ca. 4 years old is steeper in the Metal Ages individuals when compared to the two SMP Neolithic individuals (Fig. 2, and Supplementary Information Fig. 1). After this age, the SMP and Metal Ages δ 15 N profiles differ further: three individuals (ANI 002, Late Copper Age; PARM 002, Late Bronze Age; ANI 001, Iron Age) appear to reach a plateau, while one (PARM 003, Copper Age) shows an increase (Fig. 2, and Supplementary Information Fig. 1). On the other hand, the two SMP Neolithic individuals show an interruption in the δ 15 N decrease until c. 5-6 years of age, followed by a further decrease until the age of ca. 6.5 years old (for ACN 022) and 9.5 years old (for Arene Candide V), when they plateau. Arene Candide V then shows a steady and accelerating increase in δ 15 N in the last three microsections, beginning from c. 11.5 years of age until c. 14 years of age, i.e. close to the time of death estimated between 14 and 16 years of age.

Carbon isotope ratio profiles intersect for most of the individuals with dentine growth, except for the low δ 13 C values of the Late Copper Age ANI 002, and for the final part of the section of the Iron Age ANI 001, which rises towards the high δ 13 C values derived from bone tissue of this individual (Fig. 3, and Supplementary Information Fig. 1). The SMP individual Arene Candide V shows a more "sinusoidal" profile, briefly overlapping the low δ 13 C values of ANI 002 around the age of c. 7 years old.

The δ 34 S dentine profiles delineate three groups, which appear to correspond to temporal trends (Fig. 4). The highest δ 34 S ratios are recorded for the ICC Neolithic (Arma dell'Aquila RS3) and the Copper Age (PARM 003) individuals (range from 10 to 11.5‰). The Late Copper Age sample (ANI 002) provides the lowest δ 34 S ratios (-3.4 to -2.8‰). Finally, the Late Bronze Age (PARM 002) and Iron Age (ANI 001) samples show low ratios from 2-4.9 ‰.

Discussion

Through isotopic analyses of incremental dentine, dietary changes in early childhood in a sample of prehistoric children from Liguria were identified. Results suggest divergent timing of BWPs, as well as dietary and mobility differences between the Neolithic and the later Metal Ages individuals.

Reconstruction of weaning patterns and TB-induced wasting

Studies estimating the timing of the cessation of breast-feeding employing dentine profiles use the point at which δ 15 N values cease to decline and reach a plateau (e.g. [START_REF] Eerkens | Estimating weaning and early childhood diet from serial micro-samples of dentin collagen[END_REF][START_REF] Beaumont | Childhood diet: a closer examination of the evidence from dental tissues using stable isotope analysis of incremental human dentine[END_REF][START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF]Scharlotta et al., 2018b). Based on this principle, most individuals in our sample, regardless of the chronology, show an interruption in the sharp decline of δ 15 N values between the age of 3 and 4 years old (except for one ICC Neolithic individual, Arma dell'Aquila RS3, discussed below). However, between individuals from the SMP Neolithic and the Metal Ages there are differences in the nitrogen level and pattern within this age group. For the Metal Ages individuals, the inversion in δ 15 N values is at the bottom of a "dip" below the average value appearing in adult bone, and is generally followed by a gradual increase. [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF] note this fairly consistent "dip" in δ 15 N values during early childhood before reaching adult levels (see also [START_REF] Henderson | Early life histories of the London poor using δ13C and δ15N stable isotope incremental dentine sampling[END_REF]. They suggest that a continuous decline in δ 15 N beyond TBF may be related to relatively little fractionation of nitrogen due to dietary intake barely meeting the needs of growth (but not resulting in catabolism, see below; [START_REF] Fuller | Nitrogen balance and d15N: why you're not what you eat during pregnancy[END_REF], possibly due to weaning foods of poor protein quality such as C 3 -based gruels [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF]. The interaction between growth and nitrogen isotope discrimination is not completely understood and may overestimate the timing of completion of weaning. Indeed, δ 13 C values in early microsections, which are also associated with the weaning process (e.g. [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF], show a sharp decline between 0.5-1.2 ‰ between 1 and 2 years of age in two Metal ages individuals, and between 2 3 years of age in one. This signal may be related to protein derived from carbohydrate-rich weaning foods, such as vegetable gruels [START_REF] Craig | Stable isotopic evidence for diet at the imperial roman coastal site of Velia (1st and 2nd centuries AD) in Southern Italy[END_REF].

Conversely, for the two SMP Neolithic individuals, the point at which δ 15 N ceases to decline sharply is above the mean value of adults and is followed by a decline after a brief plateau. Elevated δ 15 N levels followed by a gradual decrease have been attributed to 15 N-enriched weaning foods, such as different types of animal protein [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF][START_REF] Tsutaya | Reconstruction of breastfeeding and weaning practices using stable isotope and trace element analyses: a review[END_REF]. This appears consistent with the 13 C-enriched values shown in one SMP Neolithic child in the first two microsections (up to an age of almost 2 years), followed by a decline between 3 and 4 years. However, some authors suggest that elevated δ 15 N levels could result from catabolism related to malnutrition [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF][START_REF] Beaumont | The great Irish famine: identifying starvation in the tissues of victims using stable isotope analysis of bone and incremental dentine collagen[END_REF]. Thus, in the Ligurian Neolithic sample, it is possible that the weaning process corresponded to a period of intense metabolic stress. This may be related to a sudden and significant investment in immune defense by the child due to a high environmental infectious load, which is noted by the presence of several cases of osteoarticular TB [START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millenium BC from Arene candide cave (Liguria, Italy)[END_REF][START_REF] Canci | New evidence of tuberculous spondylitis from Neolithic Liguria (Italy)[END_REF][START_REF] Sparacello | A 6500-year-old Middle Neolithic child from Pollera Cave (Liguria, Italy) with probable multifocal osteoarticular tuberculosis[END_REF][START_REF] Sparacello | Chapter 9: archaeothanatology and palaeobiology of the burials and "scattered human remains" from Arma dell'Aquila (Finale Ligure, Savona)[END_REF]; see also [START_REF] Dori | Environmental correlates of growth patterns in Neolithic Liguria (northwestern Italy)[END_REF]. This increased stress may have tipped the nitrogen balance towards negative values; a scenario consistent with the presence of linear enamel hypoplasia [START_REF] Orellana González | Insights on patterns of developmental disturbances from analysis of linear enamel hypoplasia in a Neolithic sample from Liguria (northwestern Italy)[END_REF], and faltering of postcranial development [START_REF] Dori | Environmental correlates of growth patterns in Neolithic Liguria (northwestern Italy)[END_REF] after c. 2-3 years in the Ligurian Neolithic series. In addition, if animal milk was used introduced during weaning, it may have resulted in an increase in infant morbidity (gastrointestinal infections) and micronutrient deficiencies (reviews in [START_REF] Oliver | Food safety hazards associated with consumption of raw milk[END_REF][START_REF] Howcroft | The Milky Way: the implications of using animal milk products in infant feeding[END_REF][START_REF] Claeys | Raw or heated cow milk consumption: review of risks and benefits[END_REF]. One or more of these factors may have resulted in the δ 15 N values detected.

The analysis of first molars and canines may provide information regarding the time span during which TBF might have occurred, through identification of peak dentine δ 15 N and δ 13 C values (which may be affected by rapid development during the first year of life; De Luca et al., 2012), but does not allow for a high-resolution investigation of the first year of life, when EBF might theoretically have ceased and weaning began [START_REF] Beaumont | Comparing apples and oranges: why infant bone collagen may not reflect dietary intake in the same way as dentine collagen[END_REF]. However, when considering the decrease in δ 15 N between exclusively breast-fed infants and mother's adult tissues (around 2‰ based on a in vivo analysis of a mother-infant pair; [START_REF] Herrscher | Longitudinal study of stable isotope compositions of maternal milk and implications for the palaeo-diet of infants[END_REF], we can infer that the δ 15 N value in the first microsections in most individuals studied here were close to the peak. The two SMP Neolithic individuals would therefore show a smoother pattern with a later peak, possibly between 1.5-2 years of age, than Metal Age individuals, who mostly present a steeper δ 15 N decline and estimated peaks between < 1 and 1.5 years. This estimate is also supported by the few δ 13 C data from early microsections.

In the scenario described above, the individual with the earliest age at death, between 4-6 years of age, Arma dell'Aquila RS3 from the ICC Neolithic, shows a different pattern. This individual's δ 15 N profile between the first and second microsection resembles most Metal Ages individuals, but is then followed by a sharp increase in δ 15 N coupled with no significant increase in δ 13 C, suggesting intense metabolic stress [START_REF] De Luca | δ 15N and δ 13C in hair from newborn infants and their mothers: A cohort study[END_REF][START_REF] Beaumont | The great Irish famine: identifying starvation in the tissues of victims using stable isotope analysis of bone and incremental dentine collagen[END_REF]. We suggest that this individual experienced a different weaning process than the one apparent in SMP individuals, with an earlier cessation of EBF and TBF, perhaps using more vegetable-based weaning foods, possibly due to the unavailability of sufficient breast milk (eg. [START_REF] Moucheraud | Consequences of maternal mortality on infant and child survival: a 25-year longitudinal analysis in Butajira Ethiopia (1987-2011)[END_REF]. This may have decreased the chances of surviving during later childhood, when Ligurian Neolithic children tend to show growth faltering, as recognized by patterns of postcranial development [START_REF] Dori | Environmental correlates of growth patterns in Neolithic Liguria (northwestern Italy)[END_REF]. A relationship between weaning age and survivorship has been noted in modern groups (e.g. [START_REF] Caulfield | Interventions to improve intake of complementary foods by infants 6 to 12 months of age in developing countries: impact on growth and on the prevalence of malnutrition and potential contribution to child survival[END_REF][START_REF] Lindstrom | The effects of breastfeeding and birth spacing on infant and early childhood mortality in Ethiopia[END_REF][START_REF] Tenfelde | Risk of breastfeeding cessation among low-income women, infants, and children: a discrete time survival analysis[END_REF] and has been suggested in bioarchaeological studies (e.g. [START_REF] King | Linear enamel hypoplasia as indicator of systemic physiological stress: evidence from two known age-at-death and sex populations from postmedieval London[END_REF][START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF][START_REF] Pearson | Exploring the relationship between weaning and infant mortality: an isotope case study from Aşikli Höyük and Cayönü Tepesi[END_REF], with extremes of premature or extended BWPs being correlated with high mortality. However, our small sample and its composition does not allow the determination of optimal weaning strategies for survivorship in the local ecology of Neolithic Liguria. The individual with the smoothest dentine profile in our sample, Arene Candide V, died at the age of c. 15 years, potentially due to osteoarticular tuberculosis and displays evidence of growth faltering both in stature and long bone cortical development [START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF][START_REF] Dori | Environmental correlates of growth patterns in Neolithic Liguria (northwestern Italy)[END_REF].

Looking closer at individual Arene Candide V, the reconstruction of the dentine profile displayed a sharp incremental change in δ 15 N beginning around 11.5 years of age, and accelerating until the last microsection, around 14 years of age, i.e. close to the time of death. During the same period, the δ 13 C profile displayed a net decrease (although composed by an increase followed by a drop in the last section). As mentioned above, divergent patterns in δ 15 N and δ 13 C profiles are interpreted as indicators of malnutrition [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF][START_REF] Beaumont | The great Irish famine: identifying starvation in the tissues of victims using stable isotope analysis of bone and incremental dentine collagen[END_REF]). An isotopic signal of TB-induced 'consumption' was expected in this individual [START_REF] Macallan | Malnutrition in tuberculosis[END_REF][START_REF] Schwenk | Tuberculosis, malnutrition and wasting[END_REF][START_REF] Del Rey | Endocrine and cytokine responses in humans with pulmonary tuberculosis[END_REF], and its onset appears to be consistent with previous estimates of active TB duration, c. 2-3 years, based on skeletal gracilization and enamel hypoplasia [START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millenium BC from Arene candide cave (Liguria, Italy)[END_REF][START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF]. This finding appears to confirm that by the SMP Neolithic period, TB had become a slow, chronic condition, characteristic of diseases with a long history of hostpathogen coevolution [START_REF] Sparacello | Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)[END_REF].

Neolithic vs Metal Ages subsistence patterns, adaptive strategies, and mobility

Despite the small sample size, this study highlighted a potential difference in weaning practices and diet during childhood between the SMP Neolithic and Metal Ages individuals, which may be important to understand evolving human adaptive strategies in later prehistory and protohistory. Obviously, grouping individuals under the label "Metal Ages" does not take into account the dramatic increase in socioeconomic complexity, urbanization, and demographic pressure that occurred from the Copper through the Iron Age (review in Bogucki and Crabtree, 2004;[START_REF] Cunliffe | The Oxford Illustrated Prehistory of Europe[END_REF][START_REF] Cunliffe | Europe between the oceans. 9000 BC -AD 1000[END_REF][START_REF] Guidi | Preistoria della complessità sociale[END_REF]. However, previous C and N isotopic analyses did not detect significant changes in diet until the beginning of the Iron Age in Liguria, when a significant consumption of C 4 plants were noted [START_REF] Varalli | Dal Neolitico all'Età del Ferro: indagine paleonutrizionale su alc:une popolazioni della Liguria attraverso un approccio isotopico[END_REF]. In the sample of adults used as a comparative baseline for child dentine profiles, the fact that all individuals were dated allowed for the detection of decreasing δ 15 N (r = 0.34; p < 0.05), but especially δ 13 C (r = 0.62; p < 0.0001 values, over time (up to the Iron Age, see below), which may be related to changing subsistence practices towards greater reliance on cultivated resources. In addition, the two Iron Age individuals display high δ 13 C values, which are compatible with C 4 plants being a staple in the diet. This change in crop consumption parallels the introduction of new agricultural practices, which in northeastern Italy appear to arrive earlier, with the transition between the Early and Middle Bronze Age [START_REF] Tafuri | Stable isotope evidence for the consumption of millet and other plants in Bronze Age Italy[END_REF]Varalli et al., 2016a). Dentine profiles and bone isotopic composition values confirm that the Iron Age child in our sample may have consumed a significant proportion of C 4 plants from the age of c. 8 years (enriched δ 13 C in later microsections and bone). However, they also suggest that significant dietary and life history changes had occurred already during the Copper Age, which shows a different pattern of weaning than SMP, especially at the end of the Copper Age, when the pattern of weaning appears similar to individuals from the Late Bronze and Iron Age. Intra-population variation in weaning strategies may be responsible for the differences observed (e.g. [START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF][START_REF] Howcroft | Infant feeding practices at the Pitted Ware Culture site of Ajvide, Gotland[END_REF], but the interpretation seems to be corroborated by the results obtained from the δ 34 S dentine profiles.

Although the data on δ 34 S dentine profiles are partial and could only be completed on a few individuals, three different temporal groups can be discerned. The ICC Neolithic and Copper Age individuals do not show substantial changes between c. 2.5-3.5 years, and show similar values that are consistent with data recorded in bone of individuals living close to the coast (Varalli et al., 2016b). The Late Copper Age individual displays values unique to the Mediterranean area, and more commonly found inland (Varalli et al., 2016b), sometimes in bone collagen of individuals consuming freshwater resources [START_REF] Drucker | Aquatic resources in human diet in the Late mesolithic in Northern and luxembourg: insights from carbon, nitrogen and sulphur isotope ratios[END_REF]. Finally, the Late Bronze Age and Iron Age children have a consistent δ 34 S dentine profile from ca. 2-4 years old, indicating that they were living more inland (e.g. [START_REF] Drucker | Aquatic resources in human diet in the Late mesolithic in Northern and luxembourg: insights from carbon, nitrogen and sulphur isotope ratios[END_REF]Rey et al., in press) than on the Ligurian coast. The Late Bronze Age individual shows an increase in δ 34 S between ca. 1 and 2 years old, which could reflect mobility between different inland locations and/or a modification of food protein intake due to BWPs.

Results, therefore, suggest that changes in BWPs, inferred by δ 15 N and δ 13 C profiles, might be explained, in part, by the migration to Liguria of people from different residences, different subsistence strategies and crops, and different technologies for land exploitation. Particularly interesting is the difference between 1) the Copper Age individual, who shows a different weaning experience than SMP, but shares isotopic profiles with Neolithic individuals in later childhood, and 2) the Late Copper Age individual, who suggests a completely different diet (δ 13 C values) and presumably provenience (δ 34 S values). These trends may be associated with technological changes and increased migration linked to the diffusion of the Bell Beaker culture throughout Europe, which in Liguria, occurred in the second half of the third millennium BCE [START_REF] Chiarenza | L'Eneolitico nell'Occidente Ligure[END_REF][START_REF] Chiarenza | Distribution et aspects du Campaniforme en Ligurie (Italie). Congrés Internacional Xarxes al Neolític -Neolithic Networks Rubricatum[END_REF]. A current project seeks to incorporate a larger sample size to untangle population mobility, diet and subsistence, and life history changes during the Metal Ages of Liguria (MetalLI project, JRF-Marie Curie COFUND, Durham University; PI AV).

Neolithic life history patterns, environmental correlates, limitations of the study, and conclusions

Although the precise timing of EBF and TBF is difficult to assess, this study has highlighted changes in BWPs accompanying subsistence changes between SMP Neolithic and the Metal Ages in Liguria. As expected, all the individuals analyzed here appear to have had shortened TBF when compared to the 3-5 years reported for hunter-gatherers, but SMP individuals show more gradual changes in isotopic ratios during growth. Overall, SMP BWPs appear to be at the later end of the variation displayed by early prehistoric agriculturalists [START_REF] Cienkosz-Stepanczak | Nitrogen and strontium isotopes as tools for reconstruction of breastfeeding practices and human behavior -a Neolithic collective grave in Bronocice (Poland)[END_REF][START_REF] Fernández-Crespo | Infant and childhood diet at the passage tomb of Alto de la Huesera (north-central Iberia) from bone collagen and sequential dentine isotope composition[END_REF][START_REF] Howcroft | Studies of the Infant Diet in Prehistory[END_REF][START_REF] Howcroft | Infant feeding practices at the Pitted Ware Culture site of Ajvide, Gotland[END_REF][START_REF] Pearson | Exploring the relationship between weaning and infant mortality: an isotope case study from Aşikli Höyük and Cayönü Tepesi[END_REF][START_REF] Pearson | Stable carbon and nitrogen isotope analysis and dietary reconstruction through the life course at Neolithic Çatalhöyük, Turkey[END_REF]Scharlotta et al., 2018b) and modern non-industrial agriculturalists [START_REF] Dettwyler | When to wean: biological versus cultural perspectives[END_REF][START_REF] Kennedy | From the ape's dilemma to the weanling's dilemma: early weaning and its evolutionary context[END_REF][START_REF] Marlowe | Hunter-gatherers and human evolution[END_REF]Sellen, 2001aSellen, , 2001b[START_REF] Sellen | Lactation, complementary feeding, and human life history[END_REF][START_REF] Sellen | Evolution of infant and young child feeding: implications for contemporary public health[END_REF][START_REF] Sellen | Evolution of human lactation and complementary feeding: implications for understanding contemporary cross-cultural variation[END_REF][START_REF] Sellen | Relationship between subsistence and age at weaning in "preindustrial'' societies[END_REF]. This may offer further evidence to inform the current debate on factors affecting female fertility among early agriculturalists [START_REF] Bentley | Fertility changes with the prehistoric transition to agriculture. Perspectives for reproductive ecology and paleodemography[END_REF][START_REF] Ellison | Introduction. Ecology, reproduction, and human evolution[END_REF][START_REF] Page | Reproductive trade-offs in extant huntergatherers suggest adaptive mechanism for the Neolithic expansion[END_REF].

Population and individual variation in the timing of BWPs are considered to be the result of the cultural mediation of parental strategies to maximize the outcome of the "weanling dilemma", i.e. the balancing between immunological protection and adequate nutrition [START_REF] Mcdade | The weanling's dilemma reconsidered: a biocultural analysis of breast-feeding ecology[END_REF][START_REF] Rowland | The weanling's dilemma[END_REF][START_REF] Waterlow | Observations on the suckling's dilemma: a personal view[END_REF], without affecting reproductive fitness within a specific local ecology. Delayed BWPs are more common in marginal environments, where appropriate weaning food resources are scarce, when growth is perceived as unsatisfactory, and when mothers maximize immunological protection in the presence of high pathogen loads [START_REF] Mcdade | The weanling's dilemma reconsidered: a biocultural analysis of breast-feeding ecology[END_REF]Sellen, 2001b[START_REF] Sellen | Evolution of infant and young child feeding: implications for contemporary public health[END_REF][START_REF] Cantrelle | Breast feeding, mortality in childhood and fertility in a rural zone of Senegal[END_REF][START_REF] Lindstrom | The effects of breastfeeding and birth spacing on infant and early childhood mortality in Ethiopia[END_REF]. All motivations, especially the latter, may be appropriate for the SMP Neolithic of Liguria, which appears to have had a high frequency of tuberculosis. However, it should be noted that no individual studied here reached adulthood, and one SMP individual actually died with active osteoarticular TB, which affected growth and metabolism for at least two years, as reconstructed from δ 15 N profiles. It is therefore possible that, instead of reconstructing living population life-history parameters and optimal BWPs in the Ligurian Neolithic local ecology, our sample depicts the pattern of stress observed when unsuccessful, suboptimal strategies were applied, leading to later susceptibility to environmental hardships and low survivorship. The negative relationship between longer weaning and EBF timing and later survivorship has been explored in medieval Nubia, where survivors were weaned earlier than non-survivors [START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF]. In the Neolithic of Turkey (9th-8th millennium cal BC), mortality rates after 24 months are significantly higher at Çayönü Tepesi, where isotopic evidence suggests that EBF was discontinued at about 2 years, compared to Aşikli Höyük where food supplementation commenced at 1 year of age [START_REF] Pearson | Exploring the relationship between weaning and infant mortality: an isotope case study from Aşikli Höyük and Cayönü Tepesi[END_REF].

The problem with inferring life history parameters and health of the living population from skeletal assemblages of non-survivors is a wellknown issue in bioarchaeology (i.e. the "osteological paradox"; DeWitte and [START_REF] Dewitte | The osteological paradox 20 years later: past perspectives, Future Directions[END_REF][START_REF] Wood | The osteological paradox: problems of inferring prehistoric health from skeletal samples[END_REF][START_REF] Wright | Recent progress in bioarchaeology: approaches to the osteological paradox[END_REF]. Unfortunately, this problem is unavoidable when using the incremental dentine methodology on teeth coming from a context with intense dental wear, such as the Neolithic. Another problem with the methods used here is the necessity to compare bone and dentine isotopic ratios to make inferences about trophic shifts. Bone growth is less canalized than dental growth and possibly fails to record isotopic levels during periods of stress [START_REF] Beaumont | Comparing apples and oranges: why infant bone collagen may not reflect dietary intake in the same way as dentine collagen[END_REF]. In addition, isotopic fractionation and collagen turnover in bone are complex processes that have not been completely understood yet (e.g. [START_REF] Szulc | Biochemical measurements of bone turnover in children and adolescents[END_REF][START_REF] Sandberg | Intra-tooth stable isotope analysis of dentine: a step toward addressing selective mortality in the reconstruction of life history in the archaeological record[END_REF][START_REF] Herrscher | Longitudinal study of stable isotope compositions of maternal milk and implications for the palaeo-diet of infants[END_REF][START_REF] Matsubayashi | Collagen turnover and isotopic records in cortical bone[END_REF]. More research is necessary to understand whether these limitations may have influenced the interpretations made here. In addition, parental strategies and BWPs are heavily influenced by cultural factors (e.g. Sellen, 2001a, b), which are difficult to untangle in a bioarchaeological context. Thanks to recent development in the amelogenin test from enamel [START_REF] Stewart | Sex determination of human remains from peptides in tooth enamel[END_REF], future research will explore nutritional differences and BWPs of prehistoric children based on sex [START_REF] Fernández-Crespo | Infant and childhood diet at the passage tomb of Alto de la Huesera (north-central Iberia) from bone collagen and sequential dentine isotope composition[END_REF].

Finally, this paper presents, to our knowledge, the first δ 34 S dentine profiles. Although results are currently incomplete, the data obtained integrated dietary/weaning reconstructions and highlighted possible differences in migration in the Neolithic and the Metal ages, which may be a consequence of changing landscape exploitation during prehistoric times. Future technical improvements, such as the new generation of EA-IRMS (EA IsoLink™ IRMS System ThermoFischer Scientific), will further lead to promising discoveries due to the decreased amount of collagen necessary for analysis.

Fig. 1 .

 1 Fig. 1. Geographical location of the sites included in this study. Top: the Finalese area within the Liguria region in northwestern Italy. Bottom: the sites in the municipality of Finale Ligure: 1) Arene Candide Cave; 2) Arma dei Parmorari; 3) Arma delle Anime; 4) Arma dell'Aquila.

Fig. 2 .

 2 Fig. 2. Nitrogen stable isotope ratio profile of dentine microsections for all individuals. Bone values for each individual are indicated when present. Adult variability is provided by period.

Fig. 3 .

 3 Fig. 3. Carbon stable isotope ratio profile of dentine microsections for all individuals. Bone values for each individual are indicated when present. Adult variability is provided by period.

Fig. 4 .

 4 Fig. 4. Sulfur stable isotope ratio profile of dentine microsections for all individuals. Bone values for each individual are indicated when present. Adult variability is provided by period.

Table 1

 1 Individuals

	C date, and chrono-cultural
	Individual ID, name of the specimen, 14
	AlQahatani et al., 2010; 3

analyzed in this study. Teeth legend: I: incisor; P: premolar; M: molar; U: upper; L: lower; R: right; L: left; 2

Acknowledgements

The authors would like to thank the Soprintendenza Archeologia, Belle Arti e Paesaggio per la città metropolitana di Genova e le provincie di Imperia, La Spezia e Savona, for granting access to skeletal collections, especially the Superintendent Vincenzo Tiné and the Officers Marta Conventi, Stefano Costa, and Nico Radi. We are grateful to the directors, curators, and staff of the museums where the skeletal collections are preserved, for continuous assistance during the data collection: Daniele Arobba, and Andrea De Pascale (Museo Archeologico del Finale, Finale Ligure), Patrizia Garibaldi, Guido Rossi, Irene Molinari (Museo di Archeologia Ligure, Genova), and Monica Zavattaro (Museo di Storia Naturale -Sezione di Antropologia e Etnologia, Università degli Studi di Firenze). Thanks to Chiara Panelli, Stefano Rossi, Roberto Maggi, Vincenzo Formicola, Paolo Biagi, Giovanni Murialdo, Elisa Bianchi, Simona Mordeglia, Giuseppe "Cisque" Vicino, Maria Tagliafico, Elisa Bianchi, Simona Mordeglia, Walter Siciliano, Jacopo Moggi, Giovanna Stefania, Luca Bachechi, Chiara Bullo, and Brunetto Chiarelli for their invaluable help during the data collection, and for their scientific input. Thanks to the Editor of this journal and to the reviewers for the comments that improved the earlier version of this paper.

The AMS dates performed at "Centre de Datation par le RadioCarbone, UMR 5138 "Archéométrie et Archéologie", Lyon" benefitted from the support of the program "ARTEMIS". We are grateful to Sanne Palstra and all the staff of the Centre for Isotope Research (CIO), University of Groningen, for continuous assistance during the analysis.

The project BUR.P.P.H: Burial practices at the Pleistocene -Holocene transition: the changing role of pathology, violence, and "exceptional events" (PI: VSS) has received financial support from the French State in the framework of the "Investments for the future" Program, IdEx Bordeaux, reference ANR-10-IDEX-03-02. The project DEN.P.H.: Dental anthropology at the Pleistocene-Holocene transitioninsights on lifestyle and funerary behaviour from Neolithic Liguria (Italy) (PI: ID) is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 752626. ES is co-funded by PRA 2018-Università di

Pisa, "Paesaggi funerari tra rito e società. Nuovi approcci allo studio delle necropoli nel mondo antico".

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ijpp.2019.12.007.