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

A Varalli

S Rossi

C Panelli

G Goude

S W L Palstra

M Conventi

A Del Lucchese

D Arobba

A De Pascale

M Zavattaro

P Garibaldi

G Rossi

I Molinari

R Maggi

J Moggi-Cecchi

E Starnini

P Biagi

I Dori

Del Lucchese

Arene Candide

P E Perrando

Vii I Bb

Bernabò Brea

G Del Lucchese

Candide T1

Tinè Tiné

Dating the funerary use of caves in Liguria (northwestern Italy) from the Neolithic to historic times: Results from a large-scale AMS campaign on human skeletal series

Keywords:

The multidisciplinary research team of this new project aimed at the chronological, anthropological and funerary behavior characterization of the skeletal remains unearthed from various caves in western Liguria (northwestern Italy) between the mid-1800s and the 1990s. Most of the burials and scattered bone assemblages were excavated prior to the development of modern stratigraphic methods, or come from disturbed contexts, often resulting in a vague chrono-cultural attribution. We present here the results of a systematic dating project that produced 130 new AMS dates on human bone samples (documented burials or individuals from scattered remains) from sixteen Ligurian caves, including most of the skeletal series from renowned sites such as Arene Candide Cave and Grotta Pollera.

Results highlighted the funerary use of these caves from the last quarter of the sixth millennium BCE to the Common Era, with the majority of results clustering in the first half of the fifth millennium BCE. These dates allow for an initial assessment of patterns in Neolithic mortuary use of Ligurian caves, and aided in particular the characterization of funerary practices during the Square Mouthed Pottery culture.

Introduction

Few regions in the Mediterranean show a concentration of prehistoric funerary sites from the Upper Paleolithic, Neolithic, and Metal Ages like western Liguria ("Liguria di Ponente"), where hundreds of karstic caves open in a radius of a few kilometers around the Finale Ligure ("Finalese") and nearby municipalities [START_REF] Brandolini | Geomorphology and cultural heritage of the Ponci valley (Finalese karstic area, Ligurian Alps)[END_REF]. The history of archaeological and anthropological research in this area begun in the mid-1800s, at the dawn of the modern conceptualization of those disciplines. In the early decades, numerous naturalists, geologists, amateurs and looters explored the archaeological deposits of most caves, followed by more modern excavations beginning in the 1930s https://doi.org/10. 1016/j.quaint.2019.11 V.S. Sparacello, et al. and 1940s (De Pascale, 2007, 2008;2014[START_REF] De Vingo | Dopo la preistoria: uso e frequentazione del riparo dell'Arma dell'Aquila (Finale Ligure, Savona) tra età tardoromana e postmedioevo[END_REF][START_REF] Formicola | Tall guys and fat ladies: the Grimaldi's Upper Paleolithic burials and figurines in an historical perspective[END_REF]Maggi, 1997a;[START_REF] Rossi | Di una caverna ossifera di Finale": evidenze di archeologia ottocentesca nella Caverna delle Arene Candide[END_REF]. A survey of the literature and excavation reports indicates that, from the late 1800s to the 1990s, up to 200 burials and a myriad of scattered human remains possibly belonging to the Neolithic were excavated from several caves (e.g. Arene Candide, Pollera, Arma dell'Aquila, Bergeggi, and many others, see below), making the Ligurian skeletal series one of the most important for the understanding of the cultural and biological dynamics of the Neolithisation of the western Mediterranean (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] Bernabò Brea | Il Neolitico a Ceramica Impressa e la sua diffusione nel Mediterraneo[END_REF][START_REF] Biagi | La cultura della Ceramica Impressa nella Liguria di Ponente (Italia Settentrionale): distribuzione, cronologia e aspetti culturali[END_REF][START_REF] Binder | Mésolithique et Néolithique ancien en Méditerranée nord-occidentale entre 7000 et 5500 cal. BCE: questions ouvertes sur les dynamiques culturelles et les procès d'interaction[END_REF][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] Branch | The neolithisation of Liguria (NW Italy): an environmental archaeological and palaeoenvironmental perspective[END_REF].

Unfortunately, the methodology used to record earlier findings was far from the standards used today, and the resulting documentation, when present, is often lacking or fragmentary (e.g. De Pascale and [START_REF] De Pascale | Affinché non vada perduta la mia collezione. Fossili rinvenuti negli scavi eseguiti dal Sac. Morelli nella Caverna delle Arene Candide situata nel finalese[END_REF][START_REF] Issel | Note paletnologiche sulla collezione del sig. G. B. Rossi tavv. I-III[END_REF][START_REF] Issel | Liguria Preistorica XL Società Ligure di Storia Patria[END_REF][START_REF] Richard | Scavi nell'Arma dell'Aquila a Finale Ligure[END_REF]Sparacello et al., 2018aSparacello et al., , b, 2019)). The skeletal collections were often part of private collections, and are currently housed in various museums in Italy, but in part were lost or transferred abroad [START_REF] Almagro | Excavaciones de 1954 en la "Caverna dei Pipistrelli[END_REF][START_REF] Almagro | Gli scavi italo-spagnoli nella Caverna dei Pipistrelli (Finale Ligure)[END_REF][START_REF] Moggi Cecchi | Le collezioni antropologiche[END_REF]Panelli andRossi, 2015, 2017). As a result, the first significant attempt to create a complete catalogue of the Neolithic Ligurian anthropological material [START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF] already acknowledges the unknown fate or uncertain composition of some of the skeletal collections. In addition, following the bias of anthropologists of the time, the catalogue focused on complete burials of adults, only briefly and partially reporting on juveniles and on the composition of disarticulated remains. Subsequent attempts to provide a complete overview of prehistoric burials in Liguria could not overcome these limitations [START_REF] Delfino | Liguria Preistorica -sepolture dal Paleolitico Superiore all'Età del Ferro in Liguria e nell'area ligure[END_REF].

Furthermore, the chrono-cultural attribution to the Neolithic of most of the human remains was tentative and generic [START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF]. Human communities used Ligurian caves for various activities and funerary purposes throughout prehistory, from the Upper Paleolithic to the Roman Age, but especially during the Neolithic, as suggested by the stratigraphy and archaeological assemblages unearthed at Arene Candide Cave (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] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide. Parte Prima: gli Strati con Ceramiche[END_REF][START_REF] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide (Finale Ligure). Parte Prima: gli strati con ceramiche, Campagne di scavo 1948-50[END_REF]Maggi, 1997b;[START_REF] Tiné | Il Neolitico nella Caverna delle Arene Candide (scavi 1972-77)[END_REF]. In absence of more than vague information about the context, as is the case for several partial skeletons and scattered remains, human remains may in fact date back to any of these phases of occupation.

In addition, "Neolithic" comprises a succession of different cultural traditions. The western spread of the Neolithic from the Near East reached southern Italy c. 6000-5800 BCE, and by 5800-5600 BCE people belonging to the Impresso-Cardial cultural complex (ICC) were 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]. Beginning c. 5000 BCE, the Square Mouthed Pottery culture spread in Liguria and in northern Italy during the fifth millennium BCE (SMP; c. 5000-4300 BCE; [START_REF] Barfield | The first Neolithic Cultures of north eastern Italy[END_REF][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]Maggi, 1997a;[START_REF] Pearce | Radiocarbon chronology for the spread of the early Neolithic north through the Tyrrhenian and Ligurian Seas area[END_REF]. Later, Liguria was the gateway for the diffusion of the Chasséen in northern Italy from France (c. 4300-3700 BCE) [START_REF] Bagolini | The radiocarbon chronology of the Neolithic and Copper Age of northern Italy[END_REF][START_REF] Barfield | The first Neolithic Cultures of north eastern Italy[END_REF][START_REF] Binder | Épipaléolithique et Néolithique dans l'arc liguroprovençal: bilan et perspective de recherche[END_REF][START_REF] Crepaldi | Le Chasséen en Ligurie[END_REF]Maggi, 1997a). Most burials unearthed during early excavations were labelled as likely/probably/possibly "Neolithic", or as belonging to the SMP culture, depending on the information available about associated artefacts, and on stratigraphy when available (e.g. [START_REF] Canci | Resti scheletrici umani[END_REF][START_REF] Del Lucchese | Arene Candide: a functional and environmental assessment of the Holocene sequence (excavations Bernabò Brea-Cardini 1940-1950)[END_REF][START_REF] Formicola | Una sepoltura infantile del Neolitico Medio alle Arene Candide[END_REF]. In addition, several burials firmly associated to SMP layers present a funerary structure composed by a cist of stone slabs, with the individual laid down crouched on the left side [START_REF] Del Lucchese | Arene Candide: a functional and environmental assessment of the Holocene sequence (excavations Bernabò Brea-Cardini 1940-1950)[END_REF]. Therefore, information on body position, and on the presence/absence of the stone cist, allowed for a dubitative chronological attribution of some additional burials from early excavations.

Scattered human remains were generally attributed to collective burials ("grotticelle sepolcrali") that seem to characterize the mortuary behavior from the Copper (c. 3600-2200 BCE) and Early Bronze Age (2200-1600 BCE) (e.g. [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[END_REF][START_REF] De Marinis | L'età del Rame. La pianura padana e le Alpi al tempo di Ötzi[END_REF]Spadea, 2004, 2007;[START_REF] Del Lucchese | Dal diaspro al bronzo: l'Età del Rame e del Bronzo in Liguria: 26 secoli di storia fra 3600 e 1000 anni avanti Cristo[END_REF][START_REF] Delfino | Liguria Preistorica -sepolture dal Paleolitico Superiore all'Età del Ferro in Liguria e nell'area ligure[END_REF][START_REF] Maggi | Cronologia mineraria in Liguria[END_REF][START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF]. In some cases, this was confirmed by directly dated human remains found in association with material culture from the early Bronze Age (Del Lucchese and Maggi, 1998: 36). Still, a large number of skeletons and partial skeletons had no surviving information on funerary treatment, or were buried in a simple pit without grave goods, especially children (Bernabò Brea, 1946: 18-22;[START_REF] Del Lucchese | Arene Candide: a functional and environmental assessment of the Holocene sequence (excavations Bernabò Brea-Cardini 1940-1950)[END_REF]. It was often impossible to determine whether scattered remains resulted from disturbed burials, rather than belonging to collective inhumations from later periods.

Despite these uncertainties, and probably due to the poor quality of the historic documentation, until recently few human remains were directly dated, mostly coming from Arene Candide Cave (Table 1). Among these, three have an uncertainty too high to improve their generic attribution to the Neolithic (GX-16931-A, GX-16963-G and GX-16962-G), and are virtually unusable, while the dates obtained for other two individuals do not seem to fit with the stratigraphic relationships at the site. Two dates for Arene Candide V (KIA-28340; Le Bras- [START_REF] Bras-Goude | Stratégies de subsistance et analyse culturelle de populations Néolithiques de Ligurie : approche par l'étude isotopique (δ13C et δ15N) des restes osseux[END_REF]Beta-109797) from Bernabò Brea and Cardini excavations place this burial within the mid-sixth millennium BCE, but the stone cist in which the body is buried -thought to be typical of the SMP Neolithic -covers another cist belonging to individual VI, directly dated to 4360-3780 BCE (Table 1;Del Lucchese, 1997, p. 607;Maggi, 1997b, p. 36). The individual 2 from Tiné excavations, buried in a simple pit and covered with ochre, had been attributed to early Neolithic based on funerary behavior and stratigraphy [START_REF] Canci | Resti scheletrici umani[END_REF][START_REF] Tiné | Arene Candide[END_REF][START_REF] Tiné | Nuovi scavi nella caverna delle Arene Candide[END_REF], but the direct date placed it at the end of the fifth millennium BCE [START_REF] Biagi | La cultura della Ceramica Impressa nella Liguria di Ponente (Italia Settentrionale): distribuzione, cronologia e aspetti culturali[END_REF]. More recently, one child from Grotta Pollera was dated to the mid-fifth millennium BCE [START_REF] Sparacello | A 6,500-year-old Middle Neolithic child from Pollera Cave (Liguria, Italy) with probable multifocal osteoarticular tuberculosis[END_REF], and a number of AMS dates on human bone from Arma dell'Aquila highlighted the presence of burials and scattered human remains belonging to the sixth millennium BCE, contemporary with the ICC in Liguria [START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF]Sparacello et al., 2018b[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF], Table 1) (see Table 2).

Purpose of the study

The gap in knowledge regarding the effective number and biological profile of skeletal remains, their precise chronology, and funerary context, has constantly prevented anthropologists from providing more than general insights on "Neolithic" lifestyle (funerary practices, activity patterns, diet, dental and skeletal variation, pathology) sampling from a larger, probably heterogeneous, pool (e.g. [START_REF] Canci | I resti scheletrici umani del Neolitico ligure: una sintesi paleopatologica[END_REF][START_REF] Formicola | Neolithic transition and dental changes: the case of an Italian site[END_REF][START_REF] Formicola | Evidence of spinal tuberculosis at the beginning of the fourth millennium BC from Arene Candide Cave (Liguria, Italy)[END_REF][START_REF] Goude | Alimentation et modes de vie néolithiques en Ligurie[END_REF][START_REF] Bras-Goude | 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] Marchi | Biomechanical approach to the reconstruction of activity patterns in Neolithic Western Liguria, Italy[END_REF][START_REF] Marchi | Mobility and lower limb robusticity of a pastoralist Neolithic population from North-Western Italy[END_REF][START_REF] Sparacello | Mobility and subsistence economy: a diachronic comparison between two groups settled in the same geographical area (Liguria, Italy)[END_REF][START_REF] Sparacello | The importance of considering fibular robusticity when inferring the mobility patterns of past populations[END_REF]. In order to investigate questions on Neolithic biocultural adaptive strategies, in a diachronic framework linking material culture with funerary behaviors and biological traits, it was paramount to improve the chronological characterization of the Ligurian Neolithic skeletal series.

To begin overcoming these limitations, the authors of this study were involved in two projects: "Burial practices at the Pleistocene-Holocene transition: the changing role of pathology, violence, and exceptional events (BUR.P.P.H.; PI VSS)", and "Dental anthropology at the Pleistocene-Holocene transition -insights on lifestyle and funerary behavior from Neolithic Liguria (Italy) (DEN.P.H.; PI ID)". The joint project (BUR.DEN.), in collaboration with Museums and officers of the Italian Ministry of Cultural Heritage (see acknowledgements), has reexamined and catalogued the entire extant skeletal series, and collected comprehensive information on its biology (sex, age, pathology, osteometric and non-metric traits, dental anthropology), biochemical characterization (CNS stable isotopes), and information on the archaeological and funerary context when available.

In addition, the project undertook -to our knowledge for the first time in an Italian skeletal series of this size -a systematic, and as much as possible complete, campaign of direct radiocarbon dating of the human remains. We present here the results of 130 new AMS radiocarbon dates on human remain samples (from burials, individuals reconstructed from disturbed and/or undocumented contexts, and isolated scattered remains) aimed at an initial assessment of the chronology, possible temporal clustering, and chrono-cultural attribution of the skeletal series from western Liguria. The results will also set the bases for several anthropological studies on paleodemography and paleoepidemiology, diachronic changes in diet, habitual activities, life history, and funerary behaviors.

Materials and methods

The BUR.DEN. catalogued and studied skeletal remains from 16 western Ligurian caves/rock shelters in the Finalese area and from two caves in the nearby Val Pennavaira (Fig. 1), unearthed during excavations from the late 1800s to the 1990s (Table 2). These skeletal remains constitute the vast majority of the "historical" skeletal series unearthed in the area [START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF], and are curated in four Italian museums (Museo Archeologico del Finale, Finale Ligure; Museo di Archeologia Ligure, Genova Pegli; Museo di Storia Naturale dell'Università di Firenze -sezione di Antropologia ed Etnologia, Firenze; Museo Navale Romano, Albenga; Supplementary Information Table S1).

A detailed catalogue of the osteological composition of each skeletal assemblage goes beyond the scope of this paper, and will be included in further publications (cf. Sparacello et al., 2018aSparacello et al., , 2018b[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF]. In this study, we report whether the date was performed on bone belonging to three categories: "burial", "disarticulated/partial skeleton" and "scattered remain" (Table 2). The category "burial" comprised individuals for which information (of varying quality) on the depositional context is available and reasonably reliable. The category "disarticulated/partial skeleton" includes individuals -especially from earlier excavations -for which the attribution to a specific burial described in the excavation diaries is more tentative. The category "scattered remains" is composed by specimens from fragmentary and commingled contexts, for which we attempted to sample fragments from elements belonging to different individuals.

The 130 AMS measurement were performed on 120 individuals/ isolated remains, which were sampled for both AMS dating and isotopic analysis (Goude et al., in review, and in preparation); therefore, rib fragments, which are ideal for isotopic analysis, were preferred when available in burials and partial skeletons (details in Supplementary Information Table S1). The study of CN stable isotope ratios for each sample allows for the detection of individuals that consumed significant amount of marine resources, and, in case, correct the radiocarbon date accordingly [START_REF] Fontugne | De l'utilisation des isotopes stables du carbone dans la datation par la méthode du radiocarbone[END_REF]. However, none of the individuals in this study required such correction.

Several individuals in the skeletal series were restored and consolidated using glues and plaster, and often a coating of consolidant originating from organic matter was applied. This coating is water soluble, and easily removed by ultrasonic bath. Previous studies have demonstrated that, with a careful pre-treatment of the samples, the isotopic data is not affected by the presence of this modern collagen in the great majority of cases [START_REF] Goude | Détermination de l'état de conservation, du consolidant et dosages isotopiques (δ13C, δ15N) d'ossements d'anciennes collections anthropologiques Le cas de la grotte La Pollera (Ligurie, Italie)[END_REF]. However, the possible effect on radiocarbon dating is unknown. We tested the consistency of the results by dating, on the same individual (Pollera 1 excavation Issel-Morelli, 1892), a rib coated with consolidant and the dentine from an unworn molar, which was certainly not contaminated. The invasive nature of this procedure did not allow to perform this test on a larger sample.

The bone collagen used for both elemental composition analysis and radiocarbon dates was extracted at the LAMPEA (UMR 7269, Aix-en-Provence), according to the laboratory standard protocols. 14 C measurements on the collagen samples pretreated by LAMPEA were performed at the Centre for Isotope Research (CIO) in Groningen (the Netherlands; n = 114; Aerts-Bijma et al., in preparation), and the Artemis AMS facility in Saclay (France;n = 16;Moreau et al., 2013). Laboratory protocols and procedures are detailed in Supplementary Information 1.

For all dates, calendar ages were determined using OxCal v 4.3 (Bronk Ramsey, 2009) and calibrated using IntCal13 curve [START_REF] Reimer | IntCal13 and Marine 13 radiocarbon age calibration curves 0-50,000 Years cal BP[END_REF]. Reported calibrated age ranges correspond to 95.4% probability.

Results

Results of the direct dating are reported in Appendix 1 at the end of this paper. In order to identify the specimens, we used an individual identifier based on the museum catalogues and on notes accompanying the material, but also an individual code created by the BUR.DEN. project (Appendix 1). A more detailed version of Appendix 1 is available as Supplementary Information (Table S1), which reports the sample number, the type of skeletal element analyzed, and information on collagen preservation. The table also reports the six samples, in addition to the 130 reported here, for which collagen extraction failed.

The two dates performed on the same individual (Pollera 1 excavation Issel-Morelli, 1892) to check for the possible influence of consolidant on radiocarbon determination are GrM-13669 (dentine from an unworn molar, most likely not contaminated) and GrM-14517 (rib coated with consolidant). Results are identical (5839 ± 25 and 5840 ± 25 uncal BP; Appendix 1), suggesting that pre-treatment was effective in removing the potential effect of consolidant.

However, a careful pre-treatment and sample selection has revealed to be fundamental, especially when measurements involve thin cranial bones covered in plaster for reconstruction purposes. Results obtained for the cranium labelled as AC 6625.1 (GrM-13676, 5647 ± 25; remeasuring the same collagen give similar results: GrM-18889, 5671 ± 28) did not overlap with what we determined to be its postcranium, labelled as AC 6626.2 (GrM-13686, 5840 ± 25). However, the selection of a different cranial fragment -distant from the plastered area and displaying thicker cortical bone -gave results compatible with the postcranium (GrM-18885, 5829 ± 28).

Remains attributed to the same individual, PIPI 001 (Grotta dei Pipistrelli, burial n°3 from Almagro, 1954-55 excavations; Appendix 1), were divided in three different museum boxes, and fragments from each box were dated before realizing the common origin of the materials. Two dates (GrM-13618: 5772 ± 25, and GrM-13620: 5799 ± 25) are compatible using the function R_Combine in OxCal 4.3, while the third (GrM-13621, 5882 ± 25) gave a significant Χ 2 Test (p < 0.05). We re-measured the remaining collagen of GrM-13620-21, and obtained virtually identical results, but did not give a significant Χ 2 Test when combined (GrM-18891, 5799 ± 28;GrM-18940, 5860 ± 28). The remains from Grotta dei Pipistrelli do not appear consolidated, most are still covered in dirt, and are very fragmented. Further research will determine the possible reasons for this small discrepancy, which may also include the third bone fragment not actually belonging to the same individual.

In another instance, two cranial fragments of same individual (PO 034) were dated twice (Pollera 6677a, and a mislabeled fragment from Pollera 6677b, from Issel-Morelli excavations 1892) giving nearly identical results: GrM-13438, 5897 ± 25; GrM-13493, 5901 ± 25 (Appendix 1, Supplementary Information S1).

Finally, in another case the osteological analysis suggested that two dated catalogue numbers (PO 6690bis.1 and PO 6687.1) belonged to the same individual (now coded as PO 032). They gave very similar results (GrM-13435, 5811 ± 25; GrM-13501, 5851 ± 25; Appendix 1, Supplementary Information S1), which did not give a significant Χ 2 Test when combined.

Discussion

Over the decades, scholars have highlighted the importance of systematic and complete campaigns of radiocarbon determinations aimed at a better understanding of the cultural and population dynamics in the Italian Neolithic [START_REF] Pearce | Radiocarbon chronology for the spread of the early Neolithic north through the Tyrrhenian and Ligurian Seas area[END_REF][START_REF] Skeates | Towards a radiocarbon chronology for the Neolithic in Central Italy[END_REF][START_REF] Skeates | Radiocarbon dating and Interpretations of the Mesolithic-Neolithic transition in Italy[END_REF][START_REF] Skeates | Neolithic Italy at 4004 BC: people and places[END_REF]. This study had undertaken a large-scale direct dating of the skeletal series from Liguria excavated since the late 1800s, and has allowed for an initial assessment of temporal patterns in the mortuary use of caves and variability of funerary behaviors. In addition, the direct dating has restituted importance to a large portion of the skeletal series with little or no contextual information beyond the site of provenience, which is now available for taphonomic re-evaluations through the cross-referencing excavation reports with osteological data (e.g. Sparacello et al., 2018a;b, 2019).

Results indicate that most Neolithic remains, i.e. chronologically overlapping with the successions of Neolithic cultural traditions of Liguria, belong to the period c. 5000-4300 BCE, when the Square Mouthed Pottery Culture (SMP) was attested in this area [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]Maggi, 1997a). A few burials whose 95.4% calibrated 14 C range spans across the ending of the sixth and beginning of the fifth millennium BCE were tentatively attributed to the early phases of this culture, as suggested in Del Lucchese and Starnini (2015). In the two largest assemblages from Arene Candide Cave (n = 38, including the two dates for Tiné's burials, Table 2) and Grotta Pollera (n = 43, including the date for Pollera 21; Sparacello et al., 2017, Table 2), the vast majority of burials and partial skeletons belong to this phase (84% and 95%, respectively). In other sites such as Bergeggi, all dates refer to this period (n = 7). This datum alone would aid the chrono-cultural attribution of other remains from the same caves and excavation campaigns which could not be dated, or for which the collagen extraction failed (see Supplementary Information Table S1).

In addition, there is now further and definitive evidence of the introduction of a specific kind of burial, the rectangular stone cist formed by flat slab of stones, with the SMP culture [START_REF] Del Lucchese | Arene Candide: a functional and environmental assessment of the Holocene sequence (excavations Bernabò Brea-Cardini 1940-1950)[END_REF]. Based on the dated individuals for which we have information about the depositional context (Table 4, details in Supplementary Information Table S2; see also Sparacello et al., 2018b[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF], it is clear that this funerary structure is absent in earlier and later periods, and is almost exclusively present in SMP adults (both sexes) and in adolescents. The same chrono-cultural attribution can be therefore extended to the other individuals buried in a stone cist that could not be dated due to lack of collagen (Arene Candide 6 PE, excavations Perrando, 1874; Arene Candide II, excavations Bernabò Brea, 1940-50; Supplementary Information Table S2), or that are reported in the literature but could not be found (e.g. Bernabò Brea, 1946: 220), or accessed (e.g. Issel, 1908: 383-384;Parenti and Messeri, 1962: Tav. 19/IX-21/XI). Future research will take advantage of the new chronological framework to further contextualize the material culture often associated with funerary behavior during the SMP of Liguria, such as eclogite stone axes [START_REF] Pedrotti | La pietra levigata nei corredi delle sepolture neolitiche dell'Italia settentrionale[END_REF][START_REF] Garibaldi | Industria in pietra verde dalla grotta Pollera (SV)[END_REF], and bone and shellfish artefacts [START_REF] Mazzieri | Tradizioni funerarie e ornamenti personali. Alcune osservazioni dalla sfera VBQ emiliana alla luce delle ultime scoperte[END_REF]. In addition, comparisons with the numerous burials documented in the Po plain (northern Italy), spanning the different phases of the SMP [START_REF] Bernabò Brea | Testimonianze funerarie della gente dei Vasi a Bocca Quadrata in Emilia occidentale[END_REF], 2014), will inform on the regional and temporal variation of funerary behaviors within the same cultural tradition.

Table 3

Chrono-cultural attribution of the human remains included in this study, based on the results of the radiocarbon age determination. Number of samples outside of parentheses, number of individuals in parentheses. For most of the remains, a direct association with artefacts belonging to the different cultural traditions is absent, therefore the attribution should be considered a hypothesis. ICC: Impresso-Cardial Complex [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] Arobba | Cereal remains, plant impressions and 14C direct dating from the Neolithic pottery of Arene Candide cave (Finale ligure, NW Italy)[END_REF]; SMP: Square Mouthed Pottery (Maggi, 1997a;[START_REF] Pearce | Radiocarbon chronology for the spread of the early Neolithic north through the Tyrrhenian and Ligurian Seas area[END_REF]; Chasséen (Maggi, 1997a;[START_REF] Crepaldi | Le Chasséen en Ligurie[END_REF][START_REF] Binder | Épipaléolithique et Néolithique dans l'arc liguroprovençal: bilan et perspective de recherche[END_REF]. 1 All remains pertain to the last quarter of the sixth millennium BCE; based on current archaeological evidence, it is not possible an attribution to a specific cultural tradition within the Impresso-Cardial cultural complex. 2 One individual from Arene Candide has 4 dates; 3 Includes individuals having part of the 95.4% range falling into the fifth millennium BCE (Appendix 1). 4 One individual from Pipistrelli has 5 dates. 5 While the skeletal material contemporaneous to the SMP culture is abundant, few individuals date to the sixth millennium BCE, confirming the substantial rarity of human remains from the earlier phases of the Neolithic of northern Italy. An exception to this pattern is the site of Arma dell'Aquila, in the Finalese, where recent research has shown that most burials and skeletal remains belong to the sixth millennium BCE, and several to its first half [START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF][START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF]. In the other Ligurian sites reported here, only nine individuals have a calibrated 95.4% range spanning into the last quarter of the sixth millennium BCE. These do not include Arene Candide V, an adolescent buried in a lithic cist, previously directly dated to the mid-sixth millennium BCE (see Introduction), for which we obtained a more coherent date placing it in the fifth millennium BCE (Appendix 1). Among the nine individuals chronologically spanning into the sixth millennium, only four have information about their depositional context. The best documented is Arene Candide 2 (Tiné excavations 1973-77), an adult male flexed on his left side, covered with abundant ochre, and deposited in a simple pit opened in ICC layers (Layer 14; [START_REF] Tiné | Arene Candide[END_REF][START_REF] Tiné | Nuovi scavi nella caverna delle Arene Candide[END_REF]. Previous direct AMS dating on this individual suggested a later chrono-cultural attribution, possibly Chasséen [START_REF] Biagi | La cultura della Ceramica Impressa nella Liguria di Ponente (Italia Settentrionale): distribuzione, cronologia e aspetti culturali[END_REF]. The date obtained here is more compatible with the stratigraphic position of the burial reported by the excavators [START_REF] Tiné | Arene Candide[END_REF][START_REF] Tiné | Nuovi scavi nella caverna delle Arene Candide[END_REF]. Unfortunately, Arene Candide 2 Tiné and the contemporaneous depositions from Arma dell'Aquila (Sparacello et al., 2018[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF] do not have material culture directly associated with the skeleton: further archaeological research beyond direct dating is necessary to attribute more precisely these burials to a specific cultural tradition within the Impresso-Cardial cultural complex [START_REF] Binder | La séquence de l'Impresso-Cardial de l'abri Pendimoun et l'évolution des assemblages céramiques en Provence[END_REF].

Even rarer are the human remains chronologically overlapping with the diffusion in Liguria of the Chasséen tradition (c. 4300-3700 BCE; [START_REF] Crepaldi | Le Chasséen en Ligurie[END_REF]Maggi, 1997a), consisting of a perinatal from Grotta Pollera (excavations Rossi, 1885Rossi, -1892;;Issel, 1908: 338), and a partially disturbed deposition of a late adolescent or young adult from Arma di Nasino (excavations Anfossi, 1961-74; Appendix 1; Supplementary Information Table S1). Also in this case, a direct connection of the skeleton with material culture present at Arma di Nasino -spanning from the Upper Paleolithic to the Bronze Age -is not present [START_REF] Scotti | Il riparo dell'Arma di Nasino (Albenga, SV) -scavo Anfossi 1961-1974: revisione della stratigrafia sulla base dello studio delle industrie (livelli con ceramiche)[END_REF]. Interestingly, Nasino 1 is the only burial in the skeletal series that is surrounded by stones (Scotti, 1999: 155), rather than being in a simple pit or enclosed in a stone cist. More research is necessary to determine whether this detail adds to the characterization of diachronic patterns in Neolithic funerary behaviors in Liguria. In general, the presence of a gap between the latest SMP burials and the two burials pertaining to the end of the fifth/beginning of the fourth millennium BCE highlights the need for further research in order to clarify the funerary and population dynamics with the spread of the Chasséen tradition in Liguria.

After this quasi-hiatus of over five hundred years in the evidence of funerary use of caves between c. 4300-3700, human remains become more common, with decreasing frequency from the Copper Age to the Iron Age (Table 3 andAppendix 1). The findings confirm the long-term use of certain caves for funerary activity (Arene Candide, Camere, Pollera, and Pipistrelli), and are compatible with the numerous reports of commingled assemblages directly dated to the Copper and Bronze Age in Finalese caves (e.g. [START_REF] Arobba | Le Età dei Metalli[END_REF][START_REF] Del Lucchese | Datazioni radiometriche su resti scheletrici umani dal Buco del Diavolo (Triora) e da altre grotticelle sepolcrali[END_REF][START_REF] Del Lucchese | Dal diaspro al bronzo: l'Età del Rame e del Bronzo in Liguria: 26 secoli di storia fra 3600 e 1000 anni avanti Cristo[END_REF]. However, our results add new information to the characterization of funerary variability in the Copper Age of Liguria: in addition to commingled assemblages, remains dated here comprise a secondary burial "a pozzetto" ("well burial"; Grotta dell'Acqua; Cornaggia Castiglioni, 1961), and four partial skeletons, including an almost complete perinatal from Arene Candide (AC 6635.1; Appendix 1, and Supplementary Information Table S1). Similarly, finding non-combusted human remains from the Iron Age was not expected, and deserves further investigation given the prevalence in Liguria of the practice of cremation at the end of the Bronze Age (e.g. [START_REF] Paltineri | La necropoli di Chiavari[END_REF].

The double burial from Boragni, "surrounded by large stones", and including two skeletons without grave goods (Bernabò Brea, 1947: 85), had originally a Neolithic attribution [START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF], but now it dates to historic times, when the Byzantine Empire dominated over Liguria. The use of Finalese caves and rock shelters at the time is well-known, but never attested for funerary purposes [START_REF] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide (Finale Ligure). Parte Prima: gli strati con ceramiche, Campagne di scavo 1948-50[END_REF][START_REF] De Vingo | Dopo la preistoria: uso e frequentazione del riparo dell'Arma dell'Aquila (Finale Ligure, Savona) tra età tardoromana e postmedioevo[END_REF]Murialdo, 1989). Further research will investigate how this double burial relates to the chrono-cultural context of Late Antiquity in Liguria.

Overall, this dating campaign has significantly improved our knowledge of the chronology and composition of the skeletal series from Liguria. Previously, only a dozen burials from a few excavation campaigns, virtually all from Arene Candide, could be reasonably attributed to the SMP Neolithic based on stratigraphic evidence and associated finds [START_REF] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide. Parte Prima: gli Strati con Ceramiche[END_REF][START_REF] Bernabò Brea | Gli scavi nella Caverna delle Arene Candide (Finale Ligure). Parte Prima: gli strati con ceramiche, Campagne di scavo 1948-50[END_REF][START_REF] Canci | Resti scheletrici umani[END_REF][START_REF] Canci | Una sepoltura infantile neolitica delle Arene Candide (SV) recuperata nel 1986[END_REF][START_REF] Formicola | Una sepoltura infantile del Neolitico Medio alle Arene Candide[END_REF][START_REF] Odetti | Saggio nei livelli neolitici della Grotta Pollera[END_REF][START_REF] Tiné | Arene Candide[END_REF][START_REF] Tiné | Nuovi scavi nella caverna delle Arene Candide[END_REF][START_REF] Tiné | Il Neolitico nella Caverna delle Arene Candide (scavi 1972-77)[END_REF]. As explained above, for two of them the radiocarbon dating gave inconsistent results (Le Bras- [START_REF] Bras-Goude | 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] Biagi | La cultura della Ceramica Impressa nella Liguria di Ponente (Italia Settentrionale): distribuzione, cronologia e aspetti culturali[END_REF], which have been corrected here. In general, the attribution to the SMP, when proposed in those studies, was correct. However, in contrast with that dozen, more than a hundred individuals were labelled as "uncertain" (see [START_REF] Parenti | I resti scheletrici umani del Neolitico Ligure[END_REF], including virtually all individuals from large skeletal series such as Pollera and most of the children. The work done here, and in recent re-assessments [START_REF] Mannino | Analisi isotopiche e datazioni sul collagene osseo degli inumati dell'Arma dell'Aquila[END_REF]Sparacello et al., 2018b[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF], has returned to the scientific community one of the largest Neolithic skeletal collections in Europe, composed by more than a hundred individuals from all classes of age, which is now being better characterized in terms of demography, activity, diet, life history, and paleopathology (e.g. Dori et al., in review.; Goude et al., in review; Orellana-Gonzales et al., in review; Varalli et al., in review). In addition, this study has clarified the chronological status of several commingled assemblages and isolated findings.

Conclusions

The history of anthropological research in Liguria is almost as old as the discipline itself, and the skeletal remains from this region always had an important place in the debate on the Neolithic peopling of the western Mediterranean. However, the often poor quality of the documentation associated with these remains significantly limited the potential of this skeletal series for modern anthropological studies. This study is part of a renewed collaborative attempt towards obtaining higher-resolution information through the comprehensive survey of all the available funerary and osteological data from the extant skeletal series from Liguria, re-analyzing the available documentation from past excavations, and cross-referencing the resulting information with a refined absolute chronology. The 130 new dates on human bone presented here significantly improve the chronological characterization of the skeletal series unearthed between the late 1800s and the 1990s in the Finalese area, allow for an initial assessment of patterns in Neolithic funerary use of Ligurian caves, and set the basis for upcoming multidisciplinary studies.

Results indicate that few remains can be attributed to the last quarter of the sixth millennium BCE, with uncertain attribution within the Impresso-Cardial cultural complex, and that the great majority of burials and partial skeletons (n = 88) chronologically overlap with the Square Mouthed Pottery culture, which characterizes the fifth millennium BCE in Liguria. The cultural or demographic implications of this emphasis in the funerary use of caves in this period will be explored in future research, but the present paper aided the characterization of its funerary practices.

After this widespread use of caves for funerary purposes, the dates suggest a dramatic decrease until the Copper Age, when caves were used for collective inhumations. Few remains from the Bronze Age, Iron Age, and Byzantine period, confirm the long-term tradition of use of Ligurian caves for funerary purposes from the Upper Paleolithic to historic times.

Future research will build upon the framework created here to further characterize funerary practices, bio-cultural adaptations, and social reproduction in the Neolithic of Liguria. The cultural attributions are necessarily broad in this work, and will be refined through further archaeological studies of the material evidence associated to burials. Future improvements will include Bayesian modelling on the dates presented here. This can only be achieved by incorporating information from ongoing systematic reviews of old excavation contexts (similarly to the one recently conducted at Arma dell'Aquila, Sparacello et al., 2018b[START_REF] Sparacello | The re-discovery of Arma dell'Aquila (Finale Ligure, Italy): new insights on Neolithic funerary behavior from the sixth millennium BCE in the north-western Mediterranean[END_REF] and the completion of the study of the stratigraphic series recently investigated on the key-site of Arene Candide Cave (excavations Maggi, Panelli andRossi, 1997-2012). Finally, we will attempt to extend this work of recovery and valorization of old collections and documentation to the material that is still considered lost, or was not accessible for the present study.

Declarations of competing interest

None. Vitale Sparacello, on behalf of the Authors of the manuscript. 1 ICC: Impresso-Cardial Complex [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] Arobba | Cereal remains, plant impressions and 14C direct dating from the Neolithic pottery of Arene Candide cave (Finale ligure, NW Italy)[END_REF]; SMP: Square Mouthed Pottery (Maggi, 1997a;[START_REF] Pearce | Radiocarbon chronology for the spread of the early Neolithic north through the Tyrrhenian and Ligurian Seas area[END_REF]; Chasséen (Maggi, 1997a;[START_REF] Crepaldi | Le Chasséen en Ligurie[END_REF][START_REF] Binder | Épipaléolithique et Néolithique dans l'arc liguroprovençal: bilan et perspective de recherche[END_REF].

2 Remains from the same individual, PIPI 001, were divided in three boxes with different museum codes. 3 Same individual.

Fig. 1 .

 1 Fig. 1. Top: geographical position of Liguria and the Finalese area in northern Italy and the northwestern Mediterranean. Bottom: The location of the sites included in this study: 1) Arene Candide; 2) Battorezza, Mandurea, Parmorari; 3) Arma delle Anime; 4) Grotta dell'Acqua, Arma del Morto, Caverna della Matta o del Sanguineto; 5) Pollera; 6) Arma dell'Aquila; 7) Strapatente; 8) Boragni; 9) Pipistrelli; 10) Pian del Ciliegio; 11) Bergeggi; 12) Grotta delle Camere; 13) Arma di Nasino.

Fig. 2

 2 Fig. 2 shows the stack plot, and Fig. 3 the curve plot, of the 120 individuals 95.4% probability calibrated BCE ranges. When multiple dates were available for the same individual, they were combined using the function R_combine (OxCal 4.3). Confidence intervals for dates span from c. 5300 BCE to c. 650 CE, with an apparent concentration between 5000 and 4300 BCE. We divided the results based on the traditional chrono-cultural subdivisions in Liguria (Table 3): most individuals [n = 88 (98 dates)] fall in the period during which the Neolithic SMP culture was attested in Liguria. Only 6 individuals have ranges falling completely in the sixth millennium BCE, with uncertain cultural attribution (see Discussion), and 2 fall during the period in which the Chasséen cultural tradition was present in Liguria. Fifteen individuals have been dated to Copper Age, 6 to different moments of the Bronze Age, and 3 to after 900 BCE.

Fig. 2 .

 2 Fig. 2. Stack plot of the AMS dates for the 120 individuals included in this study. The ranges are 95.4% probability calibrated.

  .034 Received 2 August 2019; Received in revised form 19 November 2019; Accepted 19 November 2019

	4782-4502	4779-4587	4724-4552		5361-5220		5208-5003		5202-4962				5475-5374	5208-4956		5658-5536		5657-5533	5646-5527	5720-5631	4686-4501		5644-5528		5207-4940	5764-5640	5777-5642	5484-5372	5206-4911
	SMP	SMP	SMP		ICC		ICC		ICC				ICC	ICC		ICC		ICC	ICC	ICC	SMP		ICC		ICC	ICC	ICC	ICC	ICC
		5820 ± 30	5804 ± 33	5800 ± 90	6318 ± 33		6155 ± 34	6125 ± 35	6029 ± 33	6114 ± 32	5985 ± 35	6315 ± 35	6447 ± 18	6118 ± 33		6678 ± 33		6675 ± 35	6670 ± 35	6770 ± 30	5738 ± 33		6669 ± 34		6105 ± 30	6825 ± 35	6830 ± 35	6470 ± 30	6095 ± 30
		MAL	MAF		MAF		MAF		MAF				MAF	MAF		MAF		MAF	MAF	MAF	MAF		MAF		MAF	MAF	MAF	MAF	MAF
		Rossi 1885-1892	Zambelli 1934		Richard 1938, 1942		Richard 1938, 1942		Richard 1938, 1942				Richard 1938, 1942	Richard 1938, 1942		Richard 1938, 1942		Richard 1938, 1942	Richard 1938, 1942	Richard 1938, 1942	Richard 1938, 1942		Richard 1938, 1942		Richard 1938, 1942	Richard 1938, 1942	Richard 1938, 1942	Richard 1938, 1942	Richard 1938, 1942
		Pollera 21	Arma dell'Aquila Z1		Arma dell'Aquila R1		Arma dell'Aquila R2		Arma dell'Aquila R3				Arma dell'Aquila R4	Arma dell'Aquila R5		Arma dell'Aquila R6		Arma dell'Aquila R7	Arma dell'Aquila R8	Arma dell'Aquila RS1	Arma dell'Aquila RS2		Arma dell'Aquila RS3		Arma dell'Aquila RS4	Arma dell'Aquila RS5	Arma dell'Aquila RS6	Arma dell'Aquila RS7	Arma dell'Aquila RS9
		Sparacello et al. (2017)	Mannino et al. (2018); Sparacello et al.	(2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Mannino et al. (2018); Sparacello et al.	(2019)			Sparacello et al. (2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Sparacello et al. (2019)	Sparacello et al. (2019)	Sparacello et al. (2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Mannino et al. (2018); Sparacello et al.	(2019)	Sparacello et al. (2019)	Sparacello et al. (2019)	Sparacello et al. (2019)	Sparacello et al. (2019)	Sparacello et al. (2019)
		Beta-409341	Combined (OxA-V-2365-37; GrN-17730)		OxA-V-2365-36		Combined (OxA-V-2365-35; GrA-38258)		Combined (OxA-V-2365-34; OxA-V-2365-33; GrA-38328; GrA-	38257)			GrM-14531	OxA-V-2365-32		OxA-V-2365-31		Lyon-14594	Lyon-14595	Lyon-14590	OxA-2365-51		OxA-2365-50		Lyon-14591	Lyon-14592	Lyon-14593	GrM-15910	Lyon-14596

Table 2

 2 Number of burials/partial skeletons/scattered remains analyzed (number of samples in parentheses), by site and excavation campaign.

	Site	Excavation	Burials or disturbed burials	Disarticulated or partial skeletons	Scattered remains
	Arene Candide	Amerano 1887-1897		1
		Morelli and Morelli-Issel 1884-87	2	16 (19)
		Rossi (uncertain)		1
		Bernabò Brea 1940-50	8	3	3
		Tiné 1973-76	1
		Del Lucchese 1986	1
	Grotta dell'Acqua	Cornaggia Castiglioni 1958	1
	Arma delle Anime	Giuggiola 1963-64			2
	Battorezza	Dentella 1950-60			5
	Bergeggi	Modigliani 1880	5	1
		Giuggiola 1968			1
	Boragni	Crowfoot 1907-1909	1
	Camere	Anfossi 1961-74			3
	Mandurea	Silla 1932			3
	Arma del Morto	Amerano 1890s		2
	Caverna della Matta o del Sanguineto	Rossi 1890?		2
	Arma di Nasino	Anfossi 1961-74	1
	Arma dei Parmorari	Richard 1931-33			3
	Pian del Ciliegio	Del Lucchese 1995	2
	Grotta dei Pipistrelli	Almagro 1954-55	6 (10)		2
	Grotta Pollera	Morelli 1885-1886	2	2
		Rossi 1885-1892		26
		Issel-Morelli 1892	1 (2)	6 (8)	1
		Amerano 1890s	1
		Tiné 1972	1
		Gruppo Speleologico Alassio 1981		1
		Uncertain		1
	Arma Strapatente	Franconi 1960s	2

Table 4

 4 Three individuals from Pollera have 2 dates. The directly dated individuals divided by class of age and type of burial (details in Supplementary Information TableS2).

	Date cal BCE		Neolithic			Metal Ages			Historic Times
			5300-5000	5000-4300	4300-3700	3700-2200	2200-900	900-180	180 BCE-643 CE
	Chrono-cultural attribution	ICC 1	SMP	Chasséen	Copper Age	Bronze Age	Iron Age	Roman and Byzantine
	Arene Candide		2	33 (30) 2		3		1
	Grotta dell'Acqua					1
	Arma delle Anime					1		1
	Battorezza					1	4
	Bergeggi			7
	Boragni								1
	Camere		1			1	1
	Mandurea					3
	Arma del Morto		1	1
	Matta-Sanguineto		2
	Arma di Nasino				1
	Arma dei Parmorari					2	1
	Pian del Ciliegio			2
	Grotta dei Pipistrelli			10 (6) 3,4		2
	Grotta Pollera			43 (40) 5	1	1
	Arma Strapatente			2 3
	Total		6	98 (88)	2	15	6	2	1
		Burial Type
	ICC-POSTC
		Stone cist	Simple pit	Stone circle
	Adult		6
	Adolescent
	Infant		5
	SMP	Stone cist	Simple pit	Stone circle
	Adult	17	3
	Adolescent	3	1
	Infant	1	15
	Chasséen	Stone cist	Simple pit	Stone circle
	Adult				1
	Adolescent
	Infant		1

Acknowledgements

Access to the skeletal collections was granted by the Soprintendenza Archeologia, Belle Arti e Paesaggio per la città metropolitana di Genova e le provincie di Imperia, La Spezia e Savona, the Museo di Archeologia Ligure, Genova, the Museo Archeologico del Finale, Finale Ligure, and the Museo di Storia Naturale -Sezione di Antropologia e Etnologia, Università degli Studi di Firenze. Thanks to the Superintendent Vincenzo Tiné, and to the Officers of the Soprintendenza Nico Radi and Stefano Costa. Thanks to Camillo Costa and the staff of the Museo Navale Romano di Albenga. We are grateful to Vincenzo Formicola, Giuseppe "Cisque" Vicino, Maria Tagliafico, Elisa Bianchi, Simona Mordeglia, Walter Siciliano, Giovanna Stefania, Luca Bachechi, Chiara Bullo, and Brunetto Chiarelli for assistance during data collection. Thanks to Giovanni Murialdo for his scientific input.

For continuous assistance during the analysis, we are grateful to all the staff of the Centre for Isotope Research (CIO), University of Groningen, to Christine Oberlin, Centre de Datation par le RadioCarbone, Université Claude Bernard Lyon 1, and to Lucile Beck, Responsable de la Plateforme Nationale LMC14 Laboratoire de Mesure du Carbone 14 (CEA/CNRS/IRD/IRSN/MCC)-LSCE CEA Saclay. The AMS dates performed at "Centre de Datation par le RadioCarbone benefitted from the support of the program "ARTEMIS". Thanks to Sara Bernardini (PhD student, UMR 7269 LAMPEA) for laboratory work.

The project BUR.P.P.H: Burial practices at the Pleistocene -Holocene transition: the changing role of pathology, violence, and "exceptional events" (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 behavior from Neolithic Liguria (Italy) (ID) is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 752626. ES was co-funded by PRA 2018-Università di Pisa, "Paesaggi funerari tra rito e società. Nuovi approcci allo studio delle necropoli nel mondo antico".

Appendix B. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.quaint.2019.11.034.

Appendix 1

Lab Code BUR.DEN. Indiv code


Luminita Fritea

Andrew J Gross

Bertrand Reuillard

Karine Gorgy

Serge Cosnier

Alan Le Goff

A diethyleneglycol-pyrene-modified Ru(II) catalyst for the design of buckypaper bioelectrodes and the wiring of glucose dehydrogenases

A novel water-soluble Ru complex bearing phenanthrolinequinone ligands, diethyleneglycol linkers and pyrene anchoring groups was synthesized. Electrical wiring of NAD-and FAD-dehydrogenases is demonstrated via NADH oxidation and mediated electron transfer respectively. Bioelectrocatalysis is facilitated by the flexible and hydrophilic PEG groups, and strong binding of pyrene groups with the carbon nanotubes (CNTs) of the electrodes. Furthermore, owing to the surfactant properties of the PEG-modified complex, high quality CNT dispersions were obtained for homogeneous buckypaper preparation.

Introduction

Flavin adenine dinucleotide-dependent glucose dehydrogenase dehydrogenase (FAD-GDH) has been recently envisioned as a promising alternative for catalytic oxidation of glucose at the anode of enzymatic fuel cells (EFCs). [1][2][3][4][5][6] FAD-GDH offers important advantages as compared to the more extensivelystudied NAD-dependent GDH or glucose oxidases (GOx). [7][8][9][10][11][12] On the contrary to GOx, FAD-GDH does not use O2 as a co-substrate and therefore does not generate H2O2 which can inhibit cathodic enzymes and degrades biofuel cell performance. [8] Unlike NAD-GDH, NADH cofactor is not required for the oxidation of glucose with FAD-GDH because the FAD cofactor is tightly bound to the enzyme. However, the commercially-available fungal FAD-GDH is not easily wired at an electrode surface and therefore requires the use of an artificial redox mediator. Different types of redox partners have been investigated for the wiring of fungal FAD-GDH such as quinones, [11,13,14] polyoxometalates [15] , osmium, [7,9,12,16] ruthenium [17] or iron complexes [8] . These redox partners have mainly been directly adsorbed on electrodes or covalentlyattached to a polymer backbone. Carbon nanotubes (CNTs) have been intensively studied in the design of EFC electrodes. [2,3,[18][19][20] In particular, the emerging use of buckypaper (BP) electrodes has recently improved the processability and flexibility of CNT-based electrodes in the design of flexible and wearable EFCs. [21][22][23][24][25][26] BPs are commonly prepared from the filtration of a concentrated dispersion of surfactant-wrapped CNTs using a nonionic surfactant bearing PEG groups such as Triton X-100. [27] However, the presence of residual surfactant within the BP is highly undesirable for electrochemical applications as it will result in hindered conductivity and catalysis, as well as raising serious biocompatibility concerns. [26] Thus, surfactant-free methods have been developed, although dispersion quality can strongly vary depending on the type and concentration of CNTs used in the process. [13,28,29] Taking advantage of the large specific surface area and high conductivity of CNT films, BPs also provide easily-scalable and flexible film-like electrodes with quasi-ideal properties for EFC applications. [26] Another great advantage of using CNTs is the large library of chemical modification strategies that have been made available for the functionalization of CNT sidewalls. [30,31] In particular, pi-pi stacking of pyrene molecules represents a reliable, soft and stable method for the non-covalent attachment of redox mediators and enzymes. A broad range of redox mediators have recently been modified with pyrene groups for stable attachment to CNTs while acting as a redox partner for enzyme wiring. [32][33][34][35][36] Here we report the synthesis of an original Ru(II) complex bearing two phenanthrolinequinone (phendione) ligands and a bipyridine ligand substituted with a diethyleneglycol-type linker and a pyrene anchoring group (RuPEG). Multi-walled CNT (MWCNT) electrodes were first modified with this complex and then by either a FAD or NAD-dependent oxidoreductase enzyme. NADdependent dehydrogenase was used in a combined electroenzymatic process leading to NADH and glucose oxidation. The enzymes were chemically immobilized on MWCNTs through the formation of an amide linkage with 1-pyrenebutyric acid Nhydroxysuccinimide ester (pyreneNHS) (Figure 3A and5A). Furthermore, a functional free-standing BP bioelectrode was designed by taking advantage of the surfactant properties of the RuPEG complex stemming from the hydrophilic diethyleneglycol chains and hydrophobic pyrene moieties present on the phendione ligand. The electrical wiring of FAD-GDH via RuPEG was studied and compared with a parent Ru complex lacking the PEG chains (Ru2).

Results and Discussion

The ligand and the complex RuPEG were synthesized following a multi-step procedure as described in Figure 1. All of the intermediates and the final complex were fully characterized by 1 H NMR and mass spectroscopy. The complex RuPEG was then immobilized on MWCNT electrodes via pi-pi stacking on CNT sidewalls by soaking the electrode in a solution of 2.5 mM RuPEG in DMF.

[a] Dr L. Fritea, Dr A. Supporting information for this article is given via a link at the end of the document. The immobilization of the RuPEG complex on MWCNT-coated glassy carbon (GC) electrodes was initially compared with the immobilization of the previously-described Ru(II) complex without the diethyleneglycol linker (complex Ru2, Figure 2). [37] The redox behavior of the modified electrode was characterized using cyclic voltammetry in 0.2 M potassium phosphate buffer, pH 7.0 (Figure 2). A reversible bielectronic peak system could be observed at +0.040 and +0.015 V vs. Ag/AgCl for RuPEG-and Ru2functionalized MWCNT electrodes, respectively, corresponding to the two protons/two electrons (Q/QH2) phendione ligands electroactivity in aqueous solution. [37][38][39] Oxidation and reduction peak currents remained highly stable upon multiple cyclic voltammetry (CV) scans (95% of the initial response retained after 100 cycles). The linear dependence of the peak currents towards the scan rate further confirmed the stable immobilization of both complexes on the MWCNT electrodes. Surface concentrations of 1.0 nmol cm -2 and 0.84 nmol cm -2 , for RuPEG and Ru2 complexes, respectively, were estimated by integration of the charge under the anodic or the cathodic peak current. The similar surface concentration for the two types of functionalized electrode likely signifies a similar association constant for both complexes via pyrene-CNT interactions. [37] Owing to the simple modification of MWCNTs using different types of pyrene derivatives, [37] a mixture of RuPEG and pyreneNHS (1:1 ratio) was explored to perform the double functionalization of CNTs and allow the covalent attachment of the enzyme via amide coupling. In the first approach, NADdependent GDH was combined with the Ru(II) catalyst and compared with the previously-designed bioelectrodes based on Ru2 (Figure 3). [37] The properties of RuPEG as a MWCNT surfactant were also investigated to facilitate the preparation of functionalized CNT dispersions and the fabrication of BPs. This configuration was also studied in order to address the ability of RuPEG to act as a redox mediator for FAD-GDH, co-immobilized via the pyreneNHS linker. As shown in Figure 4A, the addition of RuPEG to a suspension of MWCNTs improved the dispersion of MWCNTs after sonication for 10 min and centrifugation, underlining the surfactant properties of the Ru complex towards MWCNTs. Improved solubility of modified CNTs has already been demonstrated using polypyridyl-ruthenium complexes bearing piextended ligands [40] and pyrene anchors [41] but never with a diethyleneglycol chain which can improve the hydrophilicity and biocompatibility of the formed CNT composite. Thus, highly stable and flexible BPs could be obtained following vacuum filtration of a 6 mg mL -1 RuPEG-modified MWCNTs dispersion onto a PTFE membrane. Laser scanning microscopy (LSM) and scanning electron microscopy (SEM) images, respectively, demonstrated homogeneity of the BP surface at the macroscale and a typical dense, intimately-intricate network of MWCNTs at the nanoscale (Figure 4B). The homemade BPs had thicknesses of 180 (±10) µm and were accompanied with reasonable conductivities of 6 (±1) S cm -1 . These characterization experiments indicate the formation of a well-defined free-standing MWCNT film due to the presence of RuPEG. Following the immobilization of the FAD-GDH on the RuPEGfunctionalized homemade BP, CV experiments performed in the presence of glucose revealed a clear electrocatalytic wave starting at -0.1 V vs. Ag/AgCl (Figure 5B). The latter was ascribed to a mediated electron transfer (MET)-based bioelectrocatalytic oxidation of glucose by the FAD-GDH that is able to directly transfer electrons to the quinone moieties of RuPEG. A maximum current density of 0.06 mA cm -2 could be reached at 0.2 V vs. Ag/AgCl. These results demonstrate that diethyleneglycol -based Ru complexes can not only be used as redox mediators for FAD-GDH, but also as CNT surfactants for the fabrication of robust BP electrodes.

In order to benchmark this homemade BP electrode, we also investigated the modification of a commercially available MWCNT BP (from NanoTechLabs Inc) with the RuPEG catalyst and the FAD-GDH enzyme. The non-covalent modification was performed by first drop coating 40 µL of a 1:1 mixture of RuPEG and pyreneNHS at 2.5 mM in DMF onto a 6 mm diameter BP disk followed by drop coating of a 2.5 mg mL -1 of FAD-GDH in phosphate buffer at pH7. LSM and SEM images presented in Figure 6A show homogeneity of the commercial BP surface at the macroscale and a significantly more porous MWCNTs network with pores of larger diameters than the RuPEG modified homemade BP (Figure 4). Figure 6B shows CV scans for the commercial BP electrodes modified with Ru2 and RuPEG, after the immobilization of FAD-GDH, in the absence and presence of glucose. In the presence of the enzyme's substrate, a bioelectrocatalytic wave is observed for both BP bioelectrodes with an onset potential of about -0.15 V vs. Ag/AgCl. Maximum current densities of 0.095 and 1.36 mA cm -2 were obtained at 0.2 V with 150 mmol L -1 glucose for Ru2 and RuPEG-modified BP, respectively. These results confirm that RuPEG can act as an efficient redox mediator for glucose oxidation by FAD-GDH. Furthermore, the large difference of catalytic current density observed between RuPEG and Ru2 modified BP highlights the crucial finding that the PEG chain readily improves the electrical wiring of the FAD-GDH enzyme to the electrode surface. This enhanced wiring effect observed with RuPEG can be attributed in part to the improved flexibility of the PEG chain that helps to promote electronic communication with the buried FAD site. The strong influence of the chain length over the MET efficiency, and the importance of having a flexible linker, have already been demonstrated by different groups in the engineering of osmium hydrogels for the wiring of glucose oxidases and multicopper enzymes [42] or ferrocene-PEG derivatives for the mediated oxidation of NADH, [43,44] but not for the emerging FAD-GDH enzyme. The higher performances observed at the RuPEGfunctionalized commercial BP (1.36 mA cm -2 ) compared to the RuPEG-modified homemade BP (0.06 mA cm -2 ) may be attributed to the limited interaction between the Ru complex and the enzyme in the more compact homemade BP structure. The difference in the type of CNTs used, a mixture of ca 5 nm and ca 80 nm diameter MWCNTs, may also contribute to the improved catalytic performance observed with the commercial BP electrodes, for example, via hierarchical porosity effects. Moreover, the diethyleneglycol groups provide regions of hydrophilicity at the electrode which, when combined with the "open" porous structure of commercial BP, facilitate enzyme immobilization. Overall, these experiments demonstrate that the RuPEG-modified commercial BP electrode combines the advantages of an efficient redox mediator within a porous environment thus optimizing the electrical wiring of the FAD-GDH and the diffusion of glucose.

Conclusions

This work underlines the benefit of molecularly engineering redox centers for the wiring of redox enzymes confined at porous electrode surfaces. The multifunctional ruthenium complex bearing pyrene-terminated diethyleneglycol chains, RuPEG, allowed for the simple functionalization of CNT-coated and BPbased electrodes while generating an efficient redox wiring of NAD-and FAD-dependent dehydrogenases for glucose oxidation.

In the general context of the development of enzyme based bioelectrodes, this strategy of rationally designing single-molecule redox centers stands as a viable alternative to the use of redox hydrogels for efficient redox enzyme wiring. This type of versatile transition-metal complex bearing pyrene-terminated polyethyleneglycol-type chains could be extended to other types of enzymes and redox partners for the building of robust and catalytically powerful bioelectrodes. Future work will focus on the optimization of the loading of the enzyme and the redox mediators in buckypapers to further increase current densities.

Experimental Section

Materials and methods

All chemical products were purchased from Sigma Aldrich. Fungal flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, 1150 U mg -1 solid) was purchased from Sekisui Diagnostics (UK). NAD-dependent GDH from Pseudomonas sp. (235 U mg -1 ) was purchased from Aldrich. Commercial grade thin multi-walled carbon nanotubes (MWCNTs, 9.5 nm diameter, purity > 95%, 1.5 µm length) were obtained from Nanocyl. Commercial MWCNT proprietary blend buckypaper with reference (NTL-12218, 60 gsm) was obtained from NanoTechLabs, Inc (Buckeye Composites). Carbon nanomaterials were used as received without any purification. When not used, the enzyme was stored at -20°C. All the reagents were used without further purification. All solvents were of analytical grade. Distilled water was passed through a Milli-Q water purification system to obtain ultrapure water at 18.2 MΩ cm - 1 . Glucose solutions were left to mutarotate overnight to β-Dglucose prior to use. 3D and profile images were taken using a Keyence VK-X200 laser microscope. 0.1 M, pH 7 phosphate buffer solution was prepared from Milli-Q water. NMR spectra were recorded on a Bruker AVANCE 400 operating at 400.0 MHz for 1H. ESI mass spectra were recorded with a Bruker APEX-Qe ESI FT-ICR mass spectrometer. Conductivity measurements were performed using a Keithley 2450 sourcemeter with an S-302-4 mounting stand and SP4 fourpoint probe head, taking into account the thickness of each sample. 6 measurements were recorded per sample from a minimum of 2 independent samples.

Electrochemical measurements

The electrochemical experiments were carried out in a three electrode electrochemical cell using an Autolab PGSTAT100 potentiostat and a Biologic VMP3 Multi Potentiostat. A Pt wire was used as the counter electrode and the Ag/AgCl (KCl sat.) served as the reference electrode. All experiments were conducted at room temperature in air. All current densities are normalized towards the geometrical surface of the electrode. All simulated curves were obtained via Origin Pro 9.0. Synthesis [Ru(Phendion)2Cl2] and (1,10-phenathroline-5,6-dione)2((4,4'bis(4-pyrenyl-1-ylbutyloxy)-2,2'-bipyridine) Ru(II)] (PF6)2) Ru2 were synthesized according to previously described procedures [37,38,45,46] . BpyPEG-NHBOC: solution of 100 mg of 1, Next, RuPEG/pyrene-NHS (1:1, 250 µmol L -1 ) were added into the dispersion and the resulting mixture was agitated by hand and vortex for 10 min. The dispersion was subsequently passed through a Millipore PTFE filter (JHWP, 0.45 μM pore size) under high vacuum using a MZ 2C NT Vacuubrand GMBH membrane pump, rinsed with ultrapure water, then left under vacuum for a further 30-60 min. The BP coated filter papers were left overnight, peeled off the filter to obtain the free-standing BP, then cut into 6 mm diameter circular disk electrodes. For enzyme immobilization, casted onto the electrode then dried overnight in the fridge. The electrodes were rinsed several times with deionized water before use.

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Figure 1 .

 1 Figure 1. Synthesis of the complex RuPEG: (i) O-(2-Aminoethyl)-O′-[2-(Bocamino)ethyl]diethylene glycol, Et3N, DMF, 80°C, overnight, 69% (ii) trifluoroacetic acid (TFA), H2O, triisopropylsilane (TIPS), RT, 30min, 100% (iii) Pyrene-NHS, Et3N, DMF, 80°C, overnight, 88% (iv) [Ru(Phendion)2Cl2], ethylene glycol, reflux, 1h, 54%

Figure 2 .

 2 Figure 2. Cyclic voltammetry of (a) RuPEG-and (b) Ru2-functionalized MWCNT electrodes performed in 0.2 M PB (pH 7, 25°C, v = 10 mV s -1 ) vs. Ag/AgCl. Experiments were performed on at least 3 three electrodes.

Figure 3 .

 3 Figure 3. (A) Schematic representation of the RuPEG-functionalized MWCNT electrode with immobilized NAD-GDH. (B) Cyclic voltammograms of (a) RuPEG-and (b) Ru2-functionalized MWCNT electrodes with immobilized NAD-GDH in the absence (dashed line) and presence (solid line) of glucose (160 mmol L -1 ) in phosphate buffer (0.2 M, pH 7) containing 10 mM NAD + at 37°C (v = 10 mV s -1 ). (C) Calibration curve for glucose between 0 and 160 mmol L -1 and Michaelis-Menten-type fitting model (dashed line) for (a) RuPEG and (b) Ru2 MWCNT electrodes. Applied potential 0.15 V vs. Ag/AgCl. Experiments were performed on at least 3 three electrodes. The error bars correspond to the standard deviation.

Figure 4 .

 4 Figure 4. (A) Photographs of supernatant aqueous solutions obtained from dispersions after centrifugation of (a) RuPEG, (b) MWCNT+RuPEG and (c) MWCNT; (B) LSM and SEM images of the RuPEG-functionalized BP

Figure 5 .

 5 Figure 5. (A) RuPEG-functionalized MWCNT electrode with immobilized FAD-GDH. (B) CVs of RuPEG-functionalized BP in the absence (black line) and presence (blue line) of glucose (150 mmol L -1 ) in PB (0.2 M, pH 7, 25°C, v = 1 mV s -1 ) vs. Ag/AgCl; inset: photograph of the RuPEG-functionalized BP. Experiments were performed on three electrodes.

Figure 6 .

 6 Figure 6. (A) LSM and SEM images of the RuPEG-functionalized commercial BP (B) Cyclic voltammograms of commercial BP bioelectrodes functionalized with pyreneNHS/FAD-GDH and Ru2 (gray lines) or RuPEG (black lines) complexes in the absence (dashed line) and presence (solid line) of 150 mmol L -1 glucose in PB (0.2 M, pH 7, 25°C, v = 1 mV s -1 ) vs. Ag/AgCl. Experiments were performed on at least 3 three electrodes.

  1'-[2,2'-Bipyridine-4,4'-diylbis(carbonyloxy)]di(2,5-pyrrolidinedione) in 4 mL DMF was slowly added to a solution of 0.11 mL of O-(2-Aminoethyl)-O′-[2-(Boc-amino)ethyl]diethylene glycol (2 eq) and 0.08 mL Et3N (3 eq) in 8 mL DMF . The mixture was stirred overnight at 80°C. The solvent was evaporated and the crude product was washed twice with H2O and Et2O, affording a white powder (111 mg, 69 % yield). 1 H NMR: δH/ppm (400 MHz, DMSO): 1.35 (s, 18H, 6 x CH3), 3.06 (m, 4H, 2 CH2), 3.40 (m, 4H, 2 CH2), 3.46-3.58 (m, 16 H, 8 x CH2), 6.71 (m, 2H, NH), 7.85 (m, 2H, NH), 8.82 (m, 2H, bpy), 8.87 (m, 2H, bpy), 9.00 (m, 2H, bpy) BpyPGE-NH2: A solution of 50 mg of BpyPEG-NHBOC and 9.5 mL of TFA (95 %) was prepared in 0.25 mL of H2O and 0.25 mL of TIPS. The mixture is stirred at room temperature for 30 min. The solvent is evaporated and the crude oil is washed twice with Et2O, affording 35 mg of BpyPGE-NH2 (100 % yield). 1 H NMR: δH/ppm (400 MHz, DMSO): 2.97 (m, 4H, 2 CH2), 3.49 (m, 4H, 2 CH2), 3.60 (m, 16 H, 8 x CH2), 7.73 (m, 4H, NH2), 7.86 (m, 2H, NH), 8.79 (m, 2H, bpy), 8.87 (m, 2H, bpy), 9.00 (m, 2H, bpy) ; MS (ESI + ): 253.2 (BpyPEG-(NH3 + )2) BpyPEG-pyrene: A solution of 55 mg of pyrene-NHS in 3 ml DMF was slowly added to a mixture of 36 mg of BpyPGE-NH2 and 0.03 mL Et3N (3 eq). The mixture is stirred overnight at 80°C. The DMF is evaporated and the crude product is washed with H2O and Et2O, yielding 66 mg of a light-brown powder (88% yield). 1 H NMR: δH/ppm (400 MHz, DMSO): 1.97 (m, 4H, 2x CH2), 2.22 (m, 4H, 2 x CH2), 3.24 (m, 8H, 4 x CH2), 3.43 (m, 8 H, 4 x CH2), 3.53 (m, 12 H, 6 x CH2), 7.82 (m, 4H), 7.90 (m, 2H), 8.01 (m, 2H), 8.05 (m, 4H), 8.18 (m, 4H), 8.34 (m, 2H), 8.76 (m, 2H), 8.82 (m, 2H), 8.95 (m, 2H) ; MS (ESI+): 1045.7 Complex RuPEG: A solution of BpyPEG-pyrene (53mg) and [Ru(Phendion)2Cl2] (30 mg) in ethylene glycol (3 mL) was refluxed for 1 h under argon. After cooling down to room temperature, a 10mL aqueous solution of saturated NH4PF6 was added, allowing the as-formed product to precipitate. The orange brown precipitate was filtrated, then washed with water and Et2O, yielding 53 mg of product (54% yield). 1 H NMR: δH/ppm (400 MHz, DMSO): 1.88 (m, 8H, 4 x CH2), 2.22 (m, 4H, 2 x CH2), 3.20 (m, 8H, 4 x CH2), 3.40 (m, 20 H, 10 x CH2), 7.50 (m, 4H), 7.70 (m, 6H), 8.01 (m, 14H), 8.36 (m, 6H), 8.37 (m, 2H), 8.47 (m, 2H), 8.97 (m, 2H); MS (ESI + ): 783.1 (M-2PF6 2+ ) Fabrication of the MWCNT-coated GC electrodes The MWCNT-coated electrodes were obtained by drop casting 20 µL of a MWCNT dispersion in 1-methyl-2-pyrrolidinone (NMP) (5 mg mL -1 ) onto polished glassy carbon (GC) rod electrodes (3 mm diameter), affording a 5-µm-thick homogeneous MWCNT film on the GC electrode. The RuPEG/GDH and Ru2/GDH MWCNT enzyme-modified electrodes were prepared by a first incubation step in a 1:1 ratio of 2.5 mM RuPEG/Pyrene-NHS in DMF. After solvent removal under vacuum, the bi-functionalized electrodes were then incubated for at least 6 h in 20 µL of a 2.5 mg mL -1 solution of GDH in phosphate buffer pH 7 at 4°C. After each incubation, the electrodes were rinsed several times with deionized water. Preparation of commercial BP modified with pyreneNHS, RuPEG and FAD-GDH (drop-casting only) For preparation of the RuPEG-based commercial BP, high conductivity MWCNT buckypaper from NanoTechLabs Inc was first cut into 6 mm diameter circular disk electrodes. Next, 40 µL of 2.5 mmol L -1 RuPEG/pyrene-NHS (1:1) in DMF was dropcoated onto the electrode and left to dry overnight at room temperature. For enzyme immobilization, 40 µL of 2.5 mg mL -1 FAD-GDH in 0.1 mol L -1 PB pH 7 was drop-casted onto the electrode then dried overnight in the fridge. Electrical contact to the BP was obtained via a metal wire with carbon paste. The back and sides of the electrode were sealed with silicone paste. The electrodes were rinsed several times with deionized water before use. Preparation of homemade BP from a MWCNT dispersion modified with pyreneNHS, RuPEG and FAD-GDH (bulk functionalization and drop-casting) For preparation of the RuPEG-based homemade BP, 6 mg of MWCNTs was added into 3 mL of DMF and dispersed by 60 min sonication using a Bandelin sonorex RK100 ultrasonication bath.

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Acknowledgements

This work was supported by the Labex ARCANE ((ANR-11-LABX-0003-01) and the Graduate School on Chemistry, Biology and Health of Univ Grenoble Alpes CBH-EUR-GS (ANR-17-EURE-0003). The authors wish to acknowledge the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and Yannig Nedellec for BP conductivity measurements. SEM was performed at the CMTC characterization platform of Grenoble INP supported by the Centre of Excellence of Multifunctional Architectured Materials "CEMAM" n°AN-10-LABX-44-01 funded by the "Investments for the Future" Program.

Keywords: glucose • biofuel cell • ruthenium complex • carbon nanotubes • pyrene

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