Salesse Kevin
email: kevin.salesse@u-bordeaux.fr

Élise Dufour

Matthieu Lebon

Chris M Wurster

Dominique Castex

Jaroslav Bruzek

Antoine Zazzo

K Salesse

Variability of bone preservation in a confined environment: the case of the catacomb of Sts Peter and Marcellinus (Rome, Italy)

Keywords: diagenesis, stable isotopes, collagen, apatite, radiocarbon, Fourier-transform infrared spectroscopy

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Introduction

Excavations conducted from 2004 to 2010 in the central area of the catacomb of Sts Peter and Marcellinus (hereafter the SSPM catacomb) located in the south-east of Rome revealed several mass graves dated to between the first and the third century AD based on radiocarbon and artefact (coins) dating [START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF]Castex et al., 2011). They contained tens to hundreds of articulated human skeletons laid together according to wellreasoned management [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF][START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF][START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]Castex et al., 2011). A significant number of these individuals received a specific funerary treatment characterized, in particular, by the use of gypsum to partially or entirely cover the corpses (Castex and Blanchard, 2011;[START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF][START_REF] Devièse | Détermination et fonction des substances organiques et des matières minérales exploitées dans les rites funéraires de la catacombe des Saints Pierre-et-Marcellin à Rome (Ier-IIIe siècle)[END_REF][START_REF] Vanhove | Pratiques et rites funéraires du monde romain méditerrannéen aux II ème et III ème siècles de notre ère. Caractérisation du matériel de la catacombe des Saints Pierre et Marcellin à Rome[END_REF]. A mortality crisis following an epidemic episode is thought to be at the origin of these mass graves suggesting that they have been used for a relatively limited period of time [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF]Castex and Blanchard, 2011;[START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]Castex et al., 2011;[START_REF] Kacki | Réévaluation des arguments de simultanéité des dépôts de cadavres: l'exemple des sépultures plurielles de la catacombe des Saints Pierre-et-Marcellin (Rome)[END_REF]. Unfortunately, the study of the biological identity (sex, age at death, stature, nonmetric traits, pathological conditions, etc.) of the individuals was restricted due to the state of skeletal preservation [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF]Castex and Blanchard, 2011;[START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF][START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]. Many bones and teeth have lost their anatomical integrity even though the preservation quality differs strongly among burial chambers, different deposit levels within a chamber, and different skeletons within the same level [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF]Castex and Blanchard, 2011;[START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF][START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]. Stable isotope approaches offer invaluable assistance when the macroscopic anthropological observations are limited by the poor quality of skeletal preservation (e.g. [START_REF] Katzenberg | Paleodiet reconstruction of Bronze age Siberians from the mortuary site of Khuzhir-Nuge XIV, Lake Baikal[END_REF][START_REF] Oelze | Multi-isotopic analysis reveals individual mobility and diet at the early Iron age monumental tumulus of Magdalenenberg, Germany[END_REF][START_REF] Wright | The children of Kaminaljuyu: isotopic insight into diet and long distance interaction in Mesoamerica[END_REF]. Measurements of the stable isotope composition of the SSPM population would allow us to better understand their life history, provided that in vivo isotopic compositions are preserved.

Checking whether the original isotope compositions of biomineral fractions are not altered by diagenesis is an essential prerequisite to interpret isotope analyses [START_REF] Bocherens | Une nouvelle approche pour évaluer l'état de conservation de l'os et du collagène pour les mesures isotopiques (datation au radiocarbone, isotopes stables du carbone et de l'azote)[END_REF][START_REF] Bocherens | Grotte Chauvet (Ardèche, France): a "natural experiment" for bone diagenesis in karstic context[END_REF][START_REF] Brady | Investigating intra-bone isotopic variations in bioapatite using IR-laser ablation and micromilling: implications for identifying diagenesis? Palaeogeography[END_REF][START_REF] Shin | Diagenesis in bone and enamel apatite carbonate; the potential of density separation to assess the original composition[END_REF]. Most of the studies investigating the diagenetic trajectory of fossil bones focus on open-air sites. To our knowledge, very little work has been published in confined environments. The advantage of such an approach was demonstrated by [START_REF] Bocherens | Grotte Chauvet (Ardèche, France): a "natural experiment" for bone diagenesis in karstic context[END_REF] in Chauvet cave. In this Palaeolithic site, the authors showed that the preservation of bear bones exposed on the floor of the cave varied widely probably due to spatial variation in local conditions. The Chauvet cave results cannot be directly translated to man-made cavities such as catacombs because the age and taphonomic contexts are quite different. Like in caves, the thermal history of catacombs is more stable than in open-air sites which should a priori favor their preservation.

On the other hand, the accumulation of corpses over a short period of time in catacombs could have an effect on the soil pH and therefore favor the destruction of human bones. The aim of this study is to describe the diagenetic trajectory of the human bone samples from the SSPM catacomb in order to assess their preservation and compare it with the preservation observed in contemporary open-air sites from the region of Rome. A multi-proxy approach (measurement of classical collagen preservation indicators; Fourier-transform infrared spectroscopy (FT-IR) based collagen preservation indicators, carbonate content and crystallinity, 14 C measurements, and stable isotope analysis of the collagen and carbonate fractions) was undertaken to provide a record of the preservation of the molecules. Our results show that both the mineral and the organic fractions are affected by diagenesis to various degrees, and that bones originating from the small burial chambers are the most diagenetically altered. While some of the bones were strongly recrystallized, the stable isotope values of bone carbonate remained reliable. Comparison with contemporary sites from the Latium showed that conditions prevailing in catacombs seem overall to favor, rather than disadvantage bone preservation

Study area and material

A total of 128 individuals were selected for this study. The material comes from six of the seven burial chambers discovered in 2003 in a previously unexplored area (region X) of the SSPM catacomb [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF][START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF] (Figure 1). These burial chambers are part of a vast funerary complex that covers approximately 3 ha with 4.5 km of underground galleries [START_REF] Guyon | Le cimetière aux deux lauriers. Recherches sur les catacombes romaines[END_REF]. Four of them (X78/T15, X80/T16, X81 and X82/T18) are small in size (< 7m 2 ), while the remaining two (X83 and X84) are larger in size (> 9m 2 ) [START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF][START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]. Different phases of multiple deposits separated by a layer of sediment were identified in the small chambers [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF]Castex et al., 2011;[START_REF] Kacki | Réévaluation des arguments de simultanéité des dépôts de cadavres: l'exemple des sépultures plurielles de la catacombe des Saints Pierre-et-Marcellin (Rome)[END_REF][START_REF] Sachau-Carcel | Sites à stratification complexe et modélisation tridimensionnelle : vers une nouvelle approche des sépultures multiples[END_REF]. At least three phases were recognized in X78/T15 [START_REF] Pagni | Gli inumati nella catacomba dei SS Pietro e Marcellino. Intervento di scavo antropologico[END_REF]) whereas 9 and 12 phases were distinguished in X80/T16 and X82/T18, respectively [START_REF] Blanchard | A mass grave from the catacomb of Saints Peter and Marcellinus in Rome, second-third century AD[END_REF][START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF]. In contrast, no stratum of soil was discovered between the skeletons in X81, X83 and X84 (Castex and Blanchard, 2011;[START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]Castex et al., 2011).

For each individual, a fragment of long (i.e. humerus, radius, ulna, metacarpus, femur and tibia), flat (i.e. cranial vault, rib and hip bone), irregular (i.e. mandible and maxilla) or short (calcaneus) bone was sampled. Due to the poor skeletal representation of bone remains of five individuals from the burial chamber X82/T18, several fragments of different skeletal parts were selected in order to obtain enough material to achieve the required analyses (hereafter named miscellaneous samples). Modern reference material required for FT-IR analysis was composed of bones of modern ox, calf, horse and sheep in order to take into account variation due to species and age at death.

Methods

Bone carbonate preparation and analysis

Between 30 and 36 mg of bone powder (grain size < 0.3 mm) was first soaked in 2 to 3% sodium hypochlorite at room temperature for 48 h to remove organic matter then rinsed five times with distilled water. The remaining fraction was treated with 1 M acetic acid for 1 h to remove exogenous carbonate then rinsed five times with distilled water. Sodium hypochlorite and acetic acid solutions were renewed half-way through each process. Samples were oven-dried for 18 h at 65°C following pretreatment. The purification process resulted in a weight loss comprising between 10 and 80%. Bone carbonate δ 13 C and δ 18 O values were measured at the Service de Spectrométrie de Masse Isotopique of the MNHN (SSMIM).

Samples weighing from 580 to 630 mg were reacted for 360 s with 100% phosphoric acid at 70°C in individual vessels in an automated cryogenic distillation system (Kiel IV device), interfaced with a ThermoScientific Delta V Advantage isotope ratio mass spectrometer. The stable isotope results are reported in per mil (‰) relative to VPDB. Over the period of analysis, the analytical precision calculated based on repeated measurements of our internal carbonate standard (Marble LM) was 0.02‰ for δ 13 Ccarb and 0.05‰ for δ 18 Ocarb values.

Bone collagen extraction and analysis

Bone collagen extraction followed [START_REF] Longin | New method of collagen extraction for radiocarbon dating[END_REF] protocol modified by [START_REF] Deniro | Influence of diet on the distribution of nitrogen isotopes in animals[END_REF] and [START_REF] Bocherens | Premières mesures d'abondances isotopiques naturelles en 13 C et en 15 N de la matière organique fossile de dinosaure. Application à l'étude du régime alimentaire du genre Anatosaurus (Ornithischia, Hadrosauridae)[END_REF][START_REF] Krueger | Exchange of carbon with biological apatite[END_REF]. Between 0.1 and 1.1 g of bone powder (grain size comprised between 0.3 and 0.7 mm) was soaked in 40 ml of 1 M hydrochloric acid for 20 min at room temperature then filtered for decalcification using 5 µm cellulose filters.

The residue was soaked into 0.125 M sodium hydroxide for 20 h at room temperature to oxidize fulvic and humic acids then filtered. The gelatin was then solubilized in 0.01 M hydrochloric acid at 100°C for 17 h, and filtered again to remove impurities. The solubilized collagen was then freeze-dried for 48 to 72 h and extraction yields calculated. Stable isotope composition was measured at the environmental isotope laboratory at James Cook University's Advanced Analytical Centre (Cairns, Australia) using samples weighing between 200 and 400 µg. Carbon and nitrogen abundance and isotope composition (δ 13 Ccol and δ 15 Ncol values) were determined using a Costech Elemental Analyzer 4010 fitted with a zero-blank auto-sampler coupled via a ConFlo IV to a Thermo Scientific Delta V PLUS using Continuous-Flow Isotope Ratio Mass Spectrometry. The δ 13 Ccol and δ 15 Ncol values are reported in per mil (‰) deviation relative to VPDB and AIR, respectively. Over the period of analysis, the carbon and nitrogen abundances were ±5% of the value for laboratory internal standards and the analytical precision for δ 13 C and δ 15 N was better than ±0.1‰ and ±0.2‰, respectively.

Weight percent carbon and nitrogen were used to calculate atomic C:N ratios.

FT-IR analysis

Attenuated total reflexion mode (ATR) FT-IR analysis was performed on bone powder (grain size < 0.3 mm) before and after pretreatment. Infrared spectra were collected by accumulation of 64 scans with a spectral resolution of 2 cm -1 on a Bruker Vector 22 FT-IR spectrometer equipped with a diamond-ATR accessory (Specac Golden Gate). Spectral data analyses were performed using OPUS software (Bruker). Crystallinity was evaluated using the Infrared Splitting Factor (IRSF) (Figure 2). The sum of the two ν4PO4 bands at 565 and 600 cm -1 were divided by the valley at 590 cm -1 following the protocol described by [START_REF] Weiner | States of preservation of bones from prehistoric sites in the Near East: a survey[END_REF]. Relative carbonate content was measured by the intensity ratio of the ν3CO3 band at 1415 cm -1 over the ν4PO4 band at 600 cm -1 (adapted from [START_REF] Wright | Infrared and isotopic evidence for diagenesis of bone apatite at Dos Pilas, Guatemala: palaeodietary implications[END_REF].

Relative collagen content was measured by the intensity ratio of the Amide I band at 1660 cm - 1 over the ν4PO4 band at 600 cm -1 (adapted from [START_REF] Trueman | Mineralogical and compositional changes in bones exposed on soil surfaces in Amboseli National Park, Kenya: diagenetic mechanisms and the role of sediment pore fluids[END_REF]. The precipitation of secondary calcite was monitored by the presence of its specific band at 712 cm -1 .

Radiocarbon dating

The collagen and apatite fraction of five individuals from burial chambers X80/T16, X82/T18 and X83 were radiocarbon dated. An aliquot of collagen (between 8 and 13 mg) was combusted for 15 to 60 min at 800°C. Carbonate was purified following a modified version of [START_REF] Krueger | Exchange of carbon with biological apatite[END_REF]. About 2 g of bone powder (grain size < 0.1 mm) was first soaked in 2 to 3% sodium hypochlorite at room temperature for six days to oxidize the organic matter.

Bone powder was then rinsed several times with distilled water and dried for 3 h at 100°C. It was then placed in 100 ml of 2 M acetic acid for 24 h at room temperature under weak vacuum. The evolution of fine gas bubbles usually slows down drastically after 1 h, and stops after 10+ h, suggesting that all secondary calcite as well as the smallest apatite crystals had been dissolved. The acetic acid treatment resulted in weight losses between 40 and 80%. An aliquot of purified bioapatite weighing between 220 and 280 mg was then reacted in orthophosphoric acid for 12 min at 70°C under vacuum. CO2 derived from the collagen and apatite samples was cryogenically trapped and about 1 mg C was sealed in a glass tube for radiocarbon dating. Graphitization and AMS measurement were conducted at Queen's University's 14 Chrono Centre (Belfast, Northern Ireland).

Bone diagenesis evaluation

Macroscopic preservation

Bone samples were classified into three categories according to their macroscopic characteristics as well as their strength and resistance when cutting: (1) "good preservation" characterizes a solid and heavy bone difficult to cut and crush; (2) "poor preservation" corresponds to a bone which is fragile, soft and easy to cut and crush; and finally (3) "medium preservation" refers to a bone with intermediate characteristics (Figure 3).

Collagen quality

Bone collagen preservation was evaluated based on three criteria: bone collagen extraction yield (wt%), carbon and nitrogen abundances in collagen (%), and atomic C:N ratio (Table 1). Collagen concentration in modern bone is around 20.4 wt% (SD ±3.9 wt%) [START_REF] Bocherens | Isotopic biogeochemistry ( 13 C, 15 N) of fossil vertebrate collagen: application to the study of a past food web including Neandertal man[END_REF], and values <1 wt% are usually considered as being unreliable [START_REF] Dobberstein | Archaeological collagen: why worry about collagen diagenesis?[END_REF][START_REF] Van Klinken | Bone collagen quality indicators for palaeodietary and radiocarbon measurements[END_REF]. In modern bones, carbon and nitrogen abundances range from 13 to 47% and from 4.8 to 17.3% respectively [START_REF] Ambrose | Preparation and characterization of bone and tooth collagen for isotopic analysis[END_REF], and values below or above these thresholds indicate alteration or contamination [START_REF] Garvie-Lok | Loaves and fishes: a stable isotope reconstruction of diet in medieval Greece[END_REF][START_REF] Iacumin | Stable carbon and nitrogen isotopes as dietary indicators of ancient Nubian populations (northern Sudan)[END_REF][START_REF] Reitsema | Stable carbon and nitrogen isotope analysis of human diet change in prehistoric and historic Poland[END_REF][START_REF] Van Klinken | Bone collagen quality indicators for palaeodietary and radiocarbon measurements[END_REF]. Average atomic C:N ratio in modern bones is 3.2 to 3.3 [START_REF] Ambrose | Preparation and characterization of bone and tooth collagen for isotopic analysis[END_REF][START_REF] Van Klinken | Bone collagen quality indicators for palaeodietary and radiocarbon measurements[END_REF], and vary between 2.9 and 3.6 [START_REF] Deniro | Postmortem preservation and alteration of in vivo bone collagen ratios in relation to palaeodietary reconstruction[END_REF]. Atomic ratios below or above these thresholds indicate alteration or contamination [START_REF] Ambrose | Preparation and characterization of bone and tooth collagen for isotopic analysis[END_REF][START_REF] Deniro | Chemical, enzymatic and spectroscopic characterization of ''collagen'' and other organic fractions from prehistoric bones[END_REF][START_REF] Grupe | Modeling protein diagenesis in ancient bone: towards a validation of stable isotope data[END_REF]. Samples that fail to pass any of these three criteria were considered to be poorly-preserved.

Infrared spectroscopy

Bone recrystallization promotes the formation of a more stable mineral phase characterized by a higher crystallinity (i.e. crystal size and lattice perfection) and lower carbonate content. These changes are related to both microbial activities (bioerosion) and interaction of bone mineral with the burial environment (i.e. pH) that control dissolutionrecrystallization processes [START_REF] Berna | Solubilities of bone mineral from archaeological sites: the recrystallization window[END_REF]Hedges and Millard, 1995;[START_REF] Smith | Bone diagenesis in the European Holocene : patterns and mechanisms[END_REF].

Crystallinity and carbonate content can be evaluated by FT-IR using the IRSF and CO3/PO4 ratio, respectively. IRSF values vary between 2.5 and 3.3 for modern bones [START_REF] Stiner | Differential burning, recrystallization, and fragmentation of archaeological bone[END_REF][START_REF] Wright | Infrared and isotopic evidence for diagenesis of bone apatite at Dos Pilas, Guatemala: palaeodietary implications[END_REF][START_REF] Nielsen-Marsh | Patterns of Diagenesis in Bone I: the Effects of Site Environments[END_REF][START_REF] Berna | Solubilities of bone mineral from archaeological sites: the recrystallization window[END_REF][START_REF] Munro | Burning and boiling of modern deer bone: effects on crystallinity and oxygen isotope composition of bioapatite phosphate[END_REF][START_REF] Asscher | Variations in atomic disorder in biogenic carbonate hydroxyapatite using the infrared spectrum grinding curve method[END_REF] and usually reaches values between 3.4 and 4.5 for archaeological samples [START_REF] Weiner | States of preservation of bones from prehistoric sites in the Near East: a survey[END_REF][START_REF] Stiner | Differential burning, recrystallization, and fragmentation of archaeological bone[END_REF][START_REF] Wright | Infrared and isotopic evidence for diagenesis of bone apatite at Dos Pilas, Guatemala: palaeodietary implications[END_REF][START_REF] Munro | Burning and boiling of modern deer bone: effects on crystallinity and oxygen isotope composition of bioapatite phosphate[END_REF]. Bones are usually considered altered for values above 3.8 to 4.0 [START_REF] Shemesh | Crystallinity and diagenesis of sedimentary apatites[END_REF][START_REF] Stiner | Differential burning, recrystallization, and fragmentation of archaeological bone[END_REF][START_REF] Berna | Solubilities of bone mineral from archaeological sites: the recrystallization window[END_REF]. In moderrn animal bones, CO3/PO4 ratio is comprised between 0.7 and 1.0. This ratios decreases to less than 0.2 for altered samples [START_REF] Lebon | Caractérisation par spectroscopie infrarouge à transformée de Fourier des ossements chauffés en contexte archéologique -Comparaison entre référentiel moderne et matériel fossile. Implications diagénétiques[END_REF]. The fate of the mineral phase is mainly dependent on the degradation of the organic phase [START_REF] Collins | The survival of organic matter in bone: a review[END_REF][START_REF] Lebon | Imaging fossil bone alterations at the microscale by SR-FTIR microspectroscopy[END_REF][START_REF] Trueman | Why do crystallinity values fail to predict the extent of diagenetic alteration of bone mineral? Palaeogeography[END_REF]. FT-IR analyses offer also the ability to measure the relative collagen content of a bone by measuring the AmideI/PO4 ratio. This ratio is comprised between 0.9 and 1.2 for modern bones and decreases as collagen decays. The AmideI/PO4 and CO3/PO4 ratios are usually correlated [START_REF] Lebon | Caractérisation par spectroscopie infrarouge à transformée de Fourier des ossements chauffés en contexte archéologique -Comparaison entre référentiel moderne et matériel fossile. Implications diagénétiques[END_REF][START_REF] Lebon | Imaging fossil bone alterations at the microscale by SR-FTIR microspectroscopy[END_REF].

Radiocarbon dating of bone collagen-carbonate pairs

The 14 C/ 12 C and 13 C/ 12 C ratios differ by several orders of magnitude. This results in a higher sensitivity of the 14 C/ 12 C ratio over the 13 C/ 12 C ratio to post-mortem isotope exchange.

Therefore, 14 C is a useful proxy to assess the influence of diagenesis on the carbon stable isotope composition of carbonate in apatite [START_REF] Shin | Diagenesis in bone and enamel apatite carbonate; the potential of density separation to assess the original composition[END_REF]Zazzo, in press). To quantify carbon isotope exchange between bone carbonate and dissolved inorganic carbon during diagenesis, we measured the radiocarbon age difference between the collagen and carbonate fraction ( 14 Ccol-carb) from the same bone. If bone collagen passes the three tests outlined above, any deviation should be the result of carbon isotope exchange in the carbonate fraction. This difference can then be transformed in percent of carbon isotope exchange assuming that exchange takes place with modern carbon (Hedges et al., 1995a). This is probably a reasonable approximation as an extensive review of the literature and a recent 14 C dating program of several archaeological sites worldwide demonstrated that diagenetically altered apatites are always younger than the reference age for the site (Zazzo, in press;[START_REF] Zazzo | Radiocarbon dating of biological apatites: a review[END_REF]. Radiocarbon ages are presented as BP and fraction modern (Fm).

Results

Collagen preservation

Collagen preservation varies greatly between different samples (Table 2). Collagen yields differ according to bone location within the catacomb, the anatomical part category and the macroscopic aspect (Figure 4). In total, 29 (23%) bone collagen samples do not pass at least one of the three tests defined (collagen yield, carbon and nitrogen abundances, and C:N ratio (Table 3). Within this group, all but two were classified as macroscopically poorlypreserved (Table 3). These samples all originate from three of the the four lowest levels of the chamber X80/T18 and from all the levels of the chamber X82/T18 (Table 3). They consist largely of long and flat bones (31 and 38%, respectively). Additionally, 46% of the flat bones and 100% of the miscellaneous samples have poorly-preserved collagen (Table 3).

Macroscopic preservation was variable among the 99 (77%) bone collagen samples with reliable collagen (e.g. see Figure 5 for the variations of atomic carbon and nitrogen abundances). Poor scores were obtained for macroscopically poorly-preserved specimens coming from two small chambers (X80/T16 and X82/T18).

FT-IR

The modern animal reference samples have relatively low IRSF values (Mean = 3.4; SD ±0.1) and high CO3/PO4 ratios (Mean = 0.85; SD ±0.10), consistent with values previously published [START_REF] Lebon | New parameters for the characterization of diagenetic alterations and heat-induced changes of fossil bone mineral using Fourier transform infrared spectrometry[END_REF]. With the exception of three samples, all the archaeological human bone samples present higher IRSF values and lower CO3/PO4 ratios than modern animal reference samples (Figure 6). IRSF values measured on the archaeological samples before pretreatment range from 3.6 to 5.1 (Mean = 4.3; SD ±0.4), while CO3/PO4 ratios vary between 0.15 and 1.01 (Mean = 0.49; SD ±0.20). IRSF values are negatively correlated with bone CO3/PO4 ratios (R² = 0.75) (Figure 6). No trace of calcite was detected (detection limit 1.5 %) in bone samples before pretreatment. Amide I/PO4 ratios vary from 0.07 to 0.86 (Mean = 0.35; SD ±0.25) and are lower for the archaeological bones than for the modern animal reference samples (Figure 7). Carbonate and collagen relative contents are positively correlated (R² = 0.76) (Figure 7). Small chamber samples are significantly different from large chambers samples with respect to IRSF values, CO3/PO4 and Amide I/PO4 ratios (Mann-Whitney U tests; p-values < 0.03). Samples from small chambers have lower relative carbonate content, lower relative collagen content, and higher IRSF values compared to samples from larger chambers. Following chemical pretreatment of bone carbonate, IRSF values increase (Mean = 4.6; SD ±0.3) and relative carbonate content decrease (Mean = 0.28; SD ±0.10). CO3/PO4 ratios remain negatively correlated to IRSF values (R² = 0.59) (Figure 8).

Stable isotope analysis

Bone collagen carbon and nitrogen isotope values were measured for all specimens that yielded a collagen extract (Table 2). There was no significant difference in δ 13 Ccol between the well-preserved and the poorly-preserved collagen samples (Mann-Whitney U test; p-value = 0.52). In contrast, the well-and poorly-preserved collagen samples have significantly different δ 15 Ncol values (Mann-Whitney U test; p-value = 0.00). There was no relationship between the δ 13 Ccol or δ 15 Ncol values and the collagen yield (R² < 0.05), the atomic carbon and nitrogen abundances (R² < 0.2) or the atomic C:N ratios (R² < 0.07). There was no significant difference between the isotope values and the burial chambers, the skeletal part categories, or the macroscopic appearances when either one of the two groups or the totality of the corpus is considered (Mann-Whitney U tests; p-values >> 0.05). We found one exception, however. When samples with well-preserved collagen were considered (n = 99), δ 15 Ncol values measured in macroscopically poorly-and well-preserved samples differed significantly (Mann-Whitney U test; p-value = 0.04).

Bone carbonate carbon and oxygen stable isotope values were measured for the 128 individuals (Table 2). Bone δ 18 Ocarb values in X82/T18 differed significantly from bone δ 18 Ocarb values in X80/T16 and X84 (Mann-Whitney U test; p-value = 0.01). When the totality of the corpus was considered, δ 13 Ccarb values of irregular and flat bones, irregular and long bone, and long and miscellaneous bones differed significantly (Mann-Whitney U tests; pvalues < 0.04). The same conclusion was reached (excepted for long vs. miscellaneous bones) when only samples with well-preserved collagen were considered (Mann-Whitney U tests; pvalues < 0.04). Moreover, when the totality of the corpus was considered bone macroscopically identified as well-preserved had δ 13 Ccarb and δ 18 Ocarb values that differed significantly from those measured in medium and poorly-preserved bones, respectively (Mann-Whitney U tests; p-values =< 0.015). The samples which yielded well-(n = 99) and poorly-preserved (n = 29) collagen samples also had significantly different δ 13 Ccarb and δ 18 Ocarb values (Mann-Whitney U test; p-value = 0.01 and 0.02, respectively). There was no relationship between the δ 13 Ccarb or δ 18 Ocarb values and the IRSF values (R² < 0.01), the CO3/PO4 ratio (R² < 0.01) or the AmideI/PO4 ratio (R² < 0.01) before or after bone carbonate pretreatment.

Radiocarbon dating

The 14 Ccol ages range from 1779 ± 27 BP to 1965 ± 31 BP whereas the 14 Ccarb ages range from 1690 ± 27 BP to 1848 ± 22 BP for the five individuals (Table 4). The 14 Ccol-carb age differences vary between -1 ± 40 and 153 ± 46 14 C yr (Table 4). Except for one individual in chamber X83 where the age measured in the two fractions are identical, the 14 Ccarb age is always younger than the 14 Ccol age. The 14 Ccol ages are positively correlated with the δ 13 Ccol values (R² = 0.95), negatively correlated with the δ 13 Ccarb values (R² = 0.68) but are not correlated with the δ 15 Ncol values (R² = 0.24). The 14 Ccarb ages are negatively correlated with the δ 13 Ccarb values (R² = 0.86) but are not correlated with the δ 18 Ocarb values (R² = 0.01). The 14 Ccol-carb age difference is not correlated with the δ 13 Ccol, δ 13 Ccarb, or δ 18 Ocarb values (R² < 0.42), but is negatively correlated with bone δ 15 Ncol values (R² = 0.88). In terms of 14 C activity, Fm values of bone collagen varies between 78.30 ± 0.31 and 80.13 ± 0.26 pMC while bone apatite is about 1% more active on average and varies between 79.45 ± 0.22 and 81.02 ± 0.27 pMC (Table 4).

Discussion

Diagenetic trajectory

The high IRSF values displayed by bone apatite from the SSPM catacomb, ranging between 3.6 and 5.1, are indicative of a moderate to high degree of recrystallization for these samples (Table 2, Figure 6). The presence of a PO4 peak at 1093 cm -1 , and of shoulders at 3570 cm -1 and 630 cm -1 in the most severely altered samples argue for a partial recrystallization in hydroxylapatite. The inverse relationship between mineral crystallinity and carbonate content is typical of dissolution/recrystallization processes that take place following burial (Nielsen-Marsh and [START_REF] Nielsen-Marsh | Patterns of Diagenesis in Bone I: the Effects of Site Environments[END_REF]. Such an alteration of the mineral phase is frequently linked to the loss of the organic matter. This is confirmed by the correlation observed between collagen and carbonate relative contents (Figure 7). This relationship can be explained by the intimate association of the apatite crystals and collagen fibrils at the nanoscale in the bone structure. Mineral dissolution exposes the collagen to microbial or physico-chemical alterations while, in turn, the loss of the organic matter increases microporosities and interactions of bone minerals with surrounding water [START_REF] Collins | The survival of organic matter in bone: a review[END_REF]. The combination of collagen loss, decrease in carbonate content and increase in crystallinity could have been driven by collagen hydrolysis and/or microbial activity [START_REF] Smith | Bone diagenesis in the European Holocene : patterns and mechanisms[END_REF].

Bacterial attacks generally result in a loss of the chemical integrity of collagen and a loss of collagen in bone [START_REF] Balzer | In vitro decomposition of bone collagen by soil bacteria: the implications for stable isotope analysis in archaeometry[END_REF][START_REF] Child | Towards an understanding of the microbial decomposition of archaeological bone in the burial environment[END_REF][START_REF] Grupe | Archaeological microbiology[END_REF][START_REF] Harbeck | Experimental chemical degradation compared to natural diagenetic alteration of collagen: implications for collagen quality indicators for stable isotope analysis[END_REF] and begin quickly following burial [START_REF] Hedges | Bone diagenesis: an overview of processes[END_REF]. Chemical hydrolysis of collagen is another, slower mechanism [START_REF] Child | Towards an understanding of the microbial decomposition of archaeological bone in the burial environment[END_REF][START_REF] Smith | The strange case of Apigliano: early 'fossilization' of medieval bone in southern Italy[END_REF]. The lack of data regarding the histological preservation and the porosity of samples does not allow us to distinguish between the two processes. However, archaeological evidence suggests that both could have occurred.

The simultaneous or quasi-simultaneous decomposition of the high number of bodies in the mass graves from the SSPM catacomb could have been conducive to microorganism development. Moreover, the burial chambers from the SSPM catacomb were carved in a pozzolan [START_REF] Funicielllo | La geologia di Roma dal centro storico alla periferia[END_REF], which presents an alkaline pH ranging from 7.5 to 7.9 (measurements carried out by [START_REF] Zitelli | Étude de la cinétique de dépôt de corps par analyses physico-chimiques des parois de tombes[END_REF] on samples from ceiling of X82/T18 and X83).

These results are consistent with the pH of environmental water recorded in other catacombs in Rome varying between 7.5 and 7.8 (Sánchez-Moral et al., 2005a). However, a much lower pH ranging from 5.9 to 6.4 has been found at the floor level of the X82/T18 and X83 chambers [START_REF] Zitelli | Étude de la cinétique de dépôt de corps par analyses physico-chimiques des parois de tombes[END_REF]. This decrease of the rock pH at the bottom of the stratigraphic sequences could be caused by the decomposition fluids (principally organic acids; [START_REF] Vass | Decomposition chemistry of human remains: a new methodology for determining the postmortem interval[END_REF]) released from the corpses in putrefaction [START_REF] Carter | Cadaver decomposition and soil: processes[END_REF][START_REF] Gill-King | Chemical, ultrastructural aspects of decomposition[END_REF][START_REF] Schotsmans | Long-term effects of hydrated lime and quicklime on the decay of human remains using pig cadavers as human body analogues: field experiments[END_REF]. In addition, the gypsum used as part of the funerary practices inside these graves could have also contributed to the acidification of the burial environment. In environmental management, gypsum is frequently incorporated alone or with other amendments in alkaline soils in order to reduce soil pH [START_REF] Carter | Microbial biomass and mineralizable nitrogen in solonetzic soils: influence of gypsum and lime amendments[END_REF][START_REF] Courtney | Gypsum amendment of alkaline bauxite residue -Plant available aluminium and implications for grassland restoration[END_REF][START_REF] Kordlaghari | The role of gypsum in the reactions of phosphate with soils[END_REF][START_REF] Wong | Carbon dynamics of sodic and saline soils following gypsum and organic material additions: a laboratory incubation[END_REF]. Bone apatite is known to have a low solubility in alkaline environments (pH > 7.5) while its solubility becomes relatively high in acidic environments (pH < 6.5) [START_REF] Berna | Solubilities of bone mineral from archaeological sites: the recrystallization window[END_REF][START_REF] Nielsen-Marsh | Patterns of Diagenesis in Bone I: the Effects of Site Environments[END_REF][START_REF] Pate | Ionic exchange between soil solution and bone: toward a predictive model[END_REF]. The drop in pH of the burial environment in the chambers could have favored dissolution/recrystallization processes leading to the high IRSF values that we have measured.

Finally, the high porosity of the pozzolan (Sánchez-Moral et al., 2005a;Sánchez-Moral et al., 2005b) could have facilitated water drainage. These particular conditions, associated with the absence of sediments between the different strata of corpses in certain burial chambers may have limited the capacity of the burial environment to buffer water and increase interaction of bone with groundwater [START_REF] Nielsen-Marsh | Bone diagenesis in the European Holocene II: taphonomic and environmental considerations[END_REF][START_REF] Smith | The strange case of Apigliano: early 'fossilization' of medieval bone in southern Italy[END_REF]. These conditions could then have accelerated the recrystallization of bone apatite and the subsequent alteration of the collagen matrix.

Spatial heterogeneity of bone preservation

Bone preservation in the SSPM catacomb is highly variable. While some of the archaeological samples present IRSF values and CO3/PO4 ratios very close to those observed for the modern samples, most of them display higher IRSF values and lower CO3/PO4 ratios.

Collagen extraction yields are also variable (from 0 to 100%). Time, burial environment (temperature, hydrogeology, soil pH, etc.) and taphonomic processes (inhumation type, filling of the grave, anthropogenic or animal disturbance, etc.) are the three main parameters controlling the state of preservation of bones in archaeological contexts [START_REF] Collins | The survival of organic matter in bone: a review[END_REF][START_REF] Hedges | Bone diagenesis: an overview of processes[END_REF]. These different parameters interplay in a complex manner between and within archaeological sites (Hedges et al., 1995b;[START_REF] Nielsen-Marsh | Patterns of Diagenesis in Bone I: the Effects of Site Environments[END_REF][START_REF] Pestle | Bone collagen preservation in the tropics: a case study from ancient Puerto Rico[END_REF]. In the SSPM catacomb the organization and taphonomic evolution of the deposition levels suggests massive accumulation of corpses [START_REF] Castex | Les ensembles funéraires du secteur central de la catacombe des Saints Pierre et Marcellin (Rome, I er -III e siècle) : caractérisation, hypothèses d'interprétations et perspectives de recherches[END_REF][START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]Castex et al., 2011). Despite the fact that contemporary inhumation of 2000-3000 corpses stricto senso is unlikely given the dimensions of the burial chambers [START_REF] Sachau-Carcel | Apport de la modélisation tridimensionnelle à la compréhension du fonctionnement des sépultures multiples[END_REF][START_REF] Sachau-Carcel | Sites à stratification complexe et modélisation tridimensionnelle : vers une nouvelle approche des sépultures multiples[END_REF], sepulchral areas appear to have been used during a relatively short period of time (Castex and Blanchard, 2011;[START_REF] Kacki | Réévaluation des arguments de simultanéité des dépôts de cadavres: l'exemple des sépultures plurielles de la catacombe des Saints Pierre-et-Marcellin (Rome)[END_REF]. Therefore, time cannot be invoked to account for the strong heterogeneity of the conservation of the remains and other parameters need to be discussed.

The heterogeneity of bone preservation is not randomly distributed. With the exception of the small chamber X81 in which bone samples were well-preserved, most of the well-preserved samples come from the large chambers (X83 and X84) while the poorlypreserved samples are found mostly in the small chambers (X80/T16 and X82/T18). In chamber X78/T15, the limited sampling (n= 2) did not allow us to conclude on the quality of collagen preservation in this room. This result is in keeping with anthropological observations performed during the excavation although local variations can occur depending on the disposition of the bones within a chamber (see below). Like in other great Roman catacombs, the SSPM catacomb has a relatively stable climate throughout the year. Sánchez-Moral et al.

(2005a) reported a number of micro-environmental parameters in the catacombs of Domitilla and St. Callistus that can easily be extrapolated to the SSPM catacomb given the similar characteristics and geographical proximity (a few kilometers away) between these sepulchral complexes. A high humidity close to saturation (≥ 97%) and a constant temperature of 15-17°C were recorded in the two catacombs. Given the small size of the central sector of the SSPM catacomb and the existence of large openings between chambers, differences in microclimate between rooms are highly unlikely. During the excavation, visual inspection of bones in the large chambers indicated that bones close to the chamber walls were more altered than in the center [START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]. We propose that runoff against the walls and higher humidity locally could explain this difference in preservation between the center and the periphery of the large chambers. In the smaller chambers, all the bones remain relatively close to the walls and would be equally affected by the water runoff. In the large chambers, buffering conditions (constant humidity and temperature) would have favored exceptional bone collagen preservation for bones situated further away from the walls. Due to its central position, the small chamber X81 has several openings (Figure 1). This has probably restricted runoff against the walls, which could explain the better preservation of the bones in this chamber in general. Finally, trampling of the human remains by the grave diggers, families of the deceased and/or pilgrims along the walls of the burial chambers, especially in the large ones, could be another explanation. A circulation area along the west wall of the chamber X83 has been identified causing the loss of the anatomical integrity of the bone in this sector of the chamber [START_REF] Castex | Les sépultures du secteur central de la catacombe des Saints Pierre-et-Marcellin (Rome). État des analyses bio-archéologiques et perspectives[END_REF]. 6.3. Testing for the preservation of the isotopic composition of bone apatite using radiocarbon dating A crucial point for the use of bone apatite isotope values to infer diet and mobility patterns of past populations is to establish whether bone recrystallization was associated with carbon and oxygen isotope exchange. We found apatite to be slightly more 14 C active (between 0 and 1.5 pMC) than collagen from the same individual. This is in keeping with differences calculated for bones in archaeological sites ranging in age between 0 and 9.5 kyr BP (Zazzo, in press). The positive 14 Ccol-carb age difference together with the correlation between 14 Ccarb and δ 13 Ccarb values, indicate that carbon isotope exchange between bone apatite and dissolved inorganic carbon took place with a younger source of carbon with higher δ 13 C values. The 14 Ccol age offset can be used to calculate a percent contamination of the carbonate fraction with modern carbon if this value represents the true age of an individual.

However, the positive correlation found between δ 13 Ccol and 14 Ccol values suggests that the individuals with the highest δ 13 C values could have consumed marine foods as these usually have more positive δ 13 C values than terrestrial foods [START_REF] Craig | Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of Vesuvius[END_REF][START_REF] Richards | Isotope evidence for the intensive use of marine foods by late Upper Palaeolithic humans[END_REF][START_REF] Schoeninger | Stable nitrogen isotope ratios of bone collagen reflect marine and terrestrial components of Prehistoric human diet[END_REF]. The lack of correlation between the δ 15 Ncol values and the 14 Ccol ages could be explained by the consumption of low trophic level fish (e.g., anchovies, sardines, mackerel, etc.) or bivalves (e.g., oysters, cockles, clams, etc.) [START_REF] Carlier | Benthic trophic network in the Bay of Banyuls-sur-Mer (northwest Mediterranean, France): an assessment based on stable carbon and nitrogen isotopes analysis[END_REF][START_REF] Garvie-Lok | Loaves and fishes: a stable isotope reconstruction of diet in medieval Greece[END_REF]. This result is coherent with the significant (ca. 15-20%) proportion of marine food found in the diet of the victims of the 79 AD Vesuvius eruption at Herculaneum [START_REF] Craig | Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of Vesuvius[END_REF]. According to the equation in Figure 3A from [START_REF] Craig | Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of Vesuvius[END_REF], δ 13 Ccol values measured for the five individuals could correspond to 14 C age excesses between 60 (for Individual US216/Mand1) and 133 (for Individual 64) 14 C yr. This would affect the collagen age more than the apatite age because collagen records the protein fraction of the diet, while apatite carbon records the whole diet, leading to an overestimation of the age differences between the two fractions. A large dependence on marine food in individual 64 could explain the large age difference (180 ± 40 14 C yr) measured between the two individuals dated in the burial chamber X82/T18 (Table 4). This hypothesis is also supported by the fact that in chamber X80/T16, the lack of 14 Ccol age difference between individuals 24 and 79 corresponds to identical δ 13 Ccol values for these specimens, suggesting a similar amount of marine food in their diet. For these reasons, the value of 1.5 pMC contamination must be considered as a maximum estimate.

The central area of the entire SSPM catacomb and numerous others roman catacombs (e.g. the Domitilla and St Callixtus catacombs) is carved through pozzolan, a tuff layer of the Villa Senni formation resulting from an eruptive event of the Colli Albani volcano [START_REF] Funicielllo | La geologia di Roma dal centro storico alla periferia[END_REF][START_REF] Giordano | Stratigraphy, volcano tectonics and evolution of the Colli Albani volcanic field[END_REF]. Bulk measurements on samples coming from the Domitilla and St Callixtus catacombs indicate that pozzolan can contain 1-5% calcite (Sánchez-Moral et al., 2005a;Sánchez-Moral et al., 2005b). To our knowledge, there is no published stable isotope data for this ignimbrite. However, δ 13 C of carbonate in lavas and ejecta from the same volcanic complex have been measured and range from -19.8 to 3.5‰ [START_REF] Fornaseri | Carbon and oxygen isotopic composition of carbonates in lavas and ejectites from the Alban Hills, Italy[END_REF]. If we use the highest δ 13 C value as an estimate for the diagenetic endmember, a simple mass balance calculation taking into account the intra-individual differences in 14 C activity allows us to estimate a δ 13 C values shift between 0.00‰ (SD ±0.07‰) and 0.27‰ (SD ±0.08‰) as a result of diagenesis. Therefore, the intake of exogenous carbon in bone carbonate is limited and does not modify its carbon isotope composition to any great extent. This is in keeping with the slightly acidic conditions in the SSPM catacomb. As previously mentioned, the correction corresponds to a maximum estimate. Thus, the isotopic composition of the carbonate fraction of bone can be considered as reliable as that of the collagen fraction and can be used to reconstruct the life histories of the individuals buried in the central area of the catacomb. This is beyond the scope of this study and will be the focus of another article.

Finally, the significant carbon and oxygen isotope difference of apatite found between samples whose collagen was well-and poorly-preserved is intriguing and deserves attention.

Samples which did not pass the collagen preservation tests had δ 13 Ccarb values 0.3‰ lower and δ 18 Ocarb 0.3‰ higher on average than those with well-preserved collagen (Table 2). The difference between the two groups, although small, was significant for δ 13 Ccarb and δ 18 Ocarb (Mann-Whitney U tests; p-values << 0.05). Because of the negative correlation found between 14 Ccarb ages and δ 13 Ccarb values, lower δ 13 Ccarb are an unlikely result of a contaminant with a more negative δ 13 C value. We propose a different interpretation for the difference found between the two groups. It is striking that the direction and magnitude of the isotopic shifts that we observed are similar to those measured by [START_REF] Koch | The effects of sample treatment and diagenesis on the isotopic integrity of carbonate in biogenic hydroxylapatite[END_REF] between untreated and acetic-acid treated modern enamel and bone samples. The two sub-groups of bones from the SSPM mimic the untreated and treated samples from the [START_REF] Koch | The effects of sample treatment and diagenesis on the isotopic integrity of carbonate in biogenic hydroxylapatite[END_REF] experiment.

Moreover, bones with low collagen yields are also highly recrystallized and have lost part of their structural carbonate. Carbonates in apatite are present in two different positions, A and B, A-bonds being the weakest. We found OH-ions in some of the spectra, possibly substituting for A-carbonates. As postulated by [START_REF] Koch | The effects of sample treatment and diagenesis on the isotopic integrity of carbonate in biogenic hydroxylapatite[END_REF] it is plausible to assume that these two sites with different chemical bonds discriminate stable isotopes differently.

Since A/B type carbonate ratios are likely different in recrystallized and preserved apatites, the slight isotopic difference measured at the population level could well reflect this difference in isotope fractionation. This difference would then result from recrystallization, not isotope exchange. 6.4. Regional variability in bone preservation: catacombs vs. open-air sites [START_REF] Rutgers | Stable isotope data from the early Christian catacombs of ancient Rome: new insights into the dietary habits of Rome's early Christians[END_REF] hypothesized that the microclimate of catacombs affects negatively bone collage preservation. To further investigate this issue, we compared bone collagen preservation in the catacombs and in contemporary open air sites from Italy (Figure 9). The choice of sites included in this comparison is dependent on the collagen extraction protocol used on each site. Some methods, such as ultra-filtration, are known to drastically reduce collagen extraction yields. Because of this possible methodological bias, our comparison only includes sites from central Italy -all located in Rome's region, also known as Latium - [START_REF] Killgrove | Food for Rome: a stable isotope investigation of diet in the Imperial period (1 st -3 rd centuries AD)[END_REF][START_REF] Prowse | Isotopic paleodiet studies of skeletons from the imperial Roman-age cemetery of Isola Sacra, Rome, Italy[END_REF][START_REF] Prowse | Isotopic and dental evidence for diet from the necropolis of Isola Sacra (1 st -3 rd centuries AD), Italy, Department of Anthropology[END_REF][START_REF] Prowse | Isotopic evidence for age-related immigration to imperial Rome[END_REF][START_REF] Prowse | Isotopic evidence for age-related variation in diet from Isola Sacra, Italy[END_REF][START_REF] Rutgers | Stable isotope data from the early Christian catacombs of ancient Rome: new insights into the dietary habits of Rome's early Christians[END_REF], but excludes sites from southern Italy [START_REF] Craig | Stable isotopic evidence for diet at the imperial Roman coastal site of Velia (1 st and 2 nd Centuries AD) in southern Italy[END_REF][START_REF] Craig | Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of Vesuvius[END_REF]. With an average collagen extraction yield close to 50%, bones from the SSPM appear to be better preserved than most of the contemporary sites of the Latium. Comparison indicates that the quantity of extracted collagen is similar (50% or below) in the small chambers (with the exception of X81) but much higher (between 60-90% on average) in the large chambers. It is also interesting to note that the two sites presenting the highest (although highly variable) average extraction yields in the Latium are catacombs.

Despite the limitations described above, the relatively stable climatic conditions prevailing in catacombs seem overall to have favored, rather than disadvantaged bone collagen preservation and therefore contradicts [START_REF] Rutgers | Stable isotope data from the early Christian catacombs of ancient Rome: new insights into the dietary habits of Rome's early Christians[END_REF]'s assumption. However, an opposite pattern seems to emerge if one looks at the mineral fraction of bone. The comparison of the crystallinity values calculated for the bone samples from the SSPM catacomb with those published for open air sites from different Roman Empire provinces clearly shows that the mineral phase of bones is much more altered in the SSPM catacomb than in the other sites (Figure 10). Although we are aware that the comparison of IRSF values reported in different studies can be problematic due to variances in sample preparation protocols and measurement methods (KBr vs. ATR mode), the IRSF values calculated for the SSPM catacomb are similar to those observed for highly degraded bones [START_REF] Berna | Solubilities of bone mineral from archaeological sites: the recrystallization window[END_REF][START_REF] Lebon | New parameters for the characterization of diagenetic alterations and heat-induced changes of fossil bone mineral using Fourier transform infrared spectrometry[END_REF]. Overall, bones found in catacomb contexts seem to present a Janus face1 , showing both a wellpreserved organic and a poorly-preserved mineral phase. But as previously shown, this high degree of recrystallization is not correlated with isotope exchange of the mineral phase with the surrounding environment, possibly due to the relatively low pH reigning in the catacomb.

Based on this extensive study, we can conclude that it will be possible to examine traits of the life history of the individuals buried in the catacomb based on the combined analysis of bone collagen (when preserved) and apatite isotope values.

Conclusion

This study -the first focusing on diagenesis in a catacomb context -illustrates the effect of post-depositional processes on bone preservation. Our multi-proxy approach showed that extreme differences in preservation can be found between contemporary individuals within a small area, highlighting the influence of localized variations on taphonomy. We found that the heterogeneity of bone preservation was not randomly distributed in the SSPM catacomb and that bone was better preserved in the large chambers than in the small ones. This is in keeping with anthropological observations performed during the excavation and we propose that the combination of acidic conditions, together with condensation of air humidity and runoff against the walls could explain this differential preservation. Comparison with contemporary sites from the Latium indicates the two sites presenting the highest (although Portus Romae and dated from the 1 st to 3 rd c. AD [START_REF] Prowse | Isotopic paleodiet studies of skeletons from the imperial Roman-age cemetery of Isola Sacra, Rome, Italy[END_REF][START_REF] Prowse | Isotopic and dental evidence for diet from the necropolis of Isola Sacra (1 st -3 rd centuries AD), Italy, Department of Anthropology[END_REF][START_REF] Prowse | Isotopic evidence for age-related immigration to imperial Rome[END_REF][START_REF] Prowse | Isotopic evidence for age-related variation in diet from Isola Sacra, Italy[END_REF]. The ANAS cemetery is located between Ostia and Rome and dated from the 1 st to 3 rd c. AD [START_REF] Prowse | Isotopic paleodiet studies of skeletons from the imperial Roman-age cemetery of Isola Sacra, Rome, Italy[END_REF][START_REF] Prowse | Isotopic and dental evidence for diet from the necropolis of Isola Sacra (1 st -3 rd centuries AD), Italy, Department of Anthropology[END_REF]. The necropolises of Casal Bertone and Castellaccio Europarco are located in Rome and dated from the 2nd to 3rd and from the 1 st to 2 nd c. AD, respectively [START_REF] Killgrove | Migration and mobility in imperial Rome[END_REF][START_REF] Killgrove | Food for Rome: a stable isotope investigation of diet in the Imperial period (1 st -3 rd centuries AD)[END_REF]. The St Callixtus catacomb is located in Rome and dated from the 3 rd to 5 th c. AD [START_REF] Rutgers | Stable isotope data from the early Christian catacombs of ancient Rome: new insights into the dietary habits of Rome's early Christians[END_REF]. Boxplots represent the 25 th , 50 th (median) and 75 th percentiles in the distribution of Oasis in Egypt and dated from the 1 st to 5 th c. AD [START_REF] Dupras | Dining in the Dakhleh Oasis, Egypt: determination of diet using documents and stable isotope analysis[END_REF][START_REF] Dupras | Strangers in a strange land: stable isotope evidence for human migration in the Dakhleh Oasis, Egypt[END_REF]. The Leptiminus cemeteries are located in the modern day town of Lamta in Tunisia and dated from the 2 nd to 6 th century AD [START_REF] Keenleyside | Stable isotopic evidence for diet in a Roman and late Roman population from Leptiminus, Tunisia[END_REF]). The Poseidonia

Figure Captions Figure 1 :

 Captions1 Figure Captions

Figure 2 :

 2 Figure 2: FT-IR spectrum of a fresh bone showing the regions of interest as well as the IRSF

Figure 3 :Figure 4 :

 34 Figure 3: Photographs showing examples of good (A), medium (B) and poor (C) preservation

Figure 5 :

 5 Figure 5: Carbon and nitrogen abundances of the SSPM bone samples. Samples which fail

Figure 6 :

 6 Figure 6: IRSF plotted against CO3/PO4 ratio of the SSPM bone samples. Modern bone

Figure 7 :

 7 Figure 7: CO3/PO4 ratio plotted against Amide1/ PO4 ratio of the SSPM bone samples.

Figure 8 :

 8 Figure 8: IRSF plotted against Amide1/ PO4 ratio of the SSPM bone samples. Modern bone

Figure 9 :

 9 Figure 9: Inter-site comparison of the collagen extraction yields measured on contemporary

  scores. Whiskers indicate the variability outside the upper and lower quartiles. Outliers are identified by individual points. The shaded area corresponds to the collagen yield measured in modern bones (after Bocherens et al. 1991). OAS stands for open air site and SS stands for subterranean site.

Figure 10 :

 10 Figure 10: Inter-site comparison of the IRSF values measured on human bone apatite

named after the Roman deity Janus who had two faces, one looking forward, the other looking backward. By extension, this term refers to a deceitful person or an object having or containing contrasting characteristics.

highly variable) average extraction yields are catacombs, and that bone collagen in large chambers is much better preserved than in other open air contemporary sites. Therefore, the relatively stable climatic conditions prevailing in catacombs seem overall to have favored, rather than disadvantaged bone preservation.

Despite high rates of recrystallization observed for some of the bone samples, coherent radiocarbon ages measured in the carbonate and collagen fractions of several individuals demonstrates a lack of significant (>1%) isotopic exchange. We noticed a slight (0.3‰) difference in both δ 13 C and δ 18 O values between the well-preserved and the most severely recrystallized samples. This difference is similar in magnitude and direction with differences observed on modern samples pretreated with acetic acid and could be due to selective leaching of A vs. B-type carbonate in apatite during recrystallization. This shift is close to analytical precision and does not preclude reliable reconstructions of the individual history of these specimens based on stable isotope analysis of the carbonate fraction. Therefore, further dietary reconstructions can include all the individuals of the population, even those for which bone collagen is no longer preserved.

Acknowledgements

The authors wish to express their gratitude to R. Giuliani, chief inspector of the Vatican's Pontifical Commission for Sacred Archaeology. The study has profited largely from extensive discussions with the members of the scientific team of the SSPM catacomb project, P. Blanchard, G. Sachau-Carcel, S. Kacki and E. Schotsmans. We thank O. Tombret (Labex BCDiv) and J. Ughetto-Monfrin (SSMIM) for their help with the FT-IR and stable isotope analysis. Analyses benefited from the financial help of the Action Thématique du Muséum (ATM) "Biomineralization" and from Archeosphère Inc. (Bordeaux, France) thanks to F. Lacrampe-Cuyaubère and C. Beauval. We are also grateful to the Maison des Sciences de necropolises are located near Capaccio-Paestum in Italy and dated from the 7 th c. BC to the 2 nd c. AD [START_REF] Alfano | Detection of diagenetic alterations by spectroscopic analysis on archaeological bones from the necropolis of Poseidonia (Paestum): a case study[END_REF]). The Isola Sacra necropolis is located near Ostia and Portus Romae and dated from the 1 st to 3 rd c. AD [START_REF] Prowse | Isotopic paleodiet studies of skeletons from the imperial Roman-age cemetery of Isola Sacra, Rome, Italy[END_REF][START_REF] Prowse | Isotopic and dental evidence for diet from the necropolis of Isola Sacra (1 st -3 rd centuries AD), Italy, Department of Anthropology[END_REF][START_REF] Prowse | Isotopic evidence for age-related immigration to imperial Rome[END_REF][START_REF] Prowse | Isotopic evidence for age-related variation in diet from Isola Sacra, Italy[END_REF]. Boxplots represent the 25 th , 50 th (median) and 75 th