Available online http://amq.aiqua.it ISSN (online): 2279-7335 Alpine and Mediterranean QuaternaryVol. 31 (Quaternary: Past, Present, Future - AIQUA Conference, Florence, 13-14/06/2018), 75 - 77 MIDDLE PLEISTOCENE HUMAN AND ANIMAL MOBILITY AT ISERNIA LA PINETA: A STRONTIUM AND OXYGEN ISOTOPE PERSPECTIVE Federico Lugli 1,2 , Anna Cipriani 2,3 , Julie Arnaud 4 , Marta Arzarello 4 , Giuseppe Lembo 4 , Renée Janssen 5 , Hubert Vonhof 5 , Carlo Peretto 4 , Stefano Benazzi 1,6 1 Department of Cultural Heritage, University of Bologna, Ravenna, Italy 2 Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Modena, Italy 3 Lamont-Doherty Earth Observatory, Columbia University, Palisades, New York, USA 4 Department of Humanities, Section of Prehistorical and Anthropological Sciences, University of Ferrara, Ferrara, Italy 5 Department of Climate Geochemistry, Max Planck Institute for Chemistry, Mainz, Germany 6 Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany Corresponding author: F. Lugli ABSTRACT: In this work, we explored the isotopic composition of faunal (rodents, rhinoceros and bison) and human skeletal remains from the Middle Pleistocene layers of Isernia la Pineta (Molise, Italy). We particularly focused on high spatial resolution isotope analyses of tooth enamel by laser ablation MC-ICP-MS for strontium isotopes and by micro-drilling sampling for oxygen isotopes. Results from bone specimens were compared with the isotope variability of modern plants collected in the area surrounding the site, in a radius of about 30 km. While the human group seems local, macro-mammals show a higher degree of mobility. KEYWORDS: Isotopes, middle Pleistocene, deciduous tooth, strontium isotopes, laser ablation, oxygen isotopes 1. INTRODUCTION In recent years, the use of isotope ratios (e.g. 87 Sr/ 86 Sr and δ 18 O) to decrypt past human and animal migratory patterns has been an important tool in ar- chaeological and paleontological research. In particular, the discovery and significance of natural variations in the isotopic composition of strontium in biological and inorganic materials, has made Sr isotopes one of the best geochemical indicators for provenance studies (Bentley, 2006). As an alkaline earth metal with chemi- cal-physical properties similar to those of calcium, stron- tium is commonly found in natural calcium carbonate (e.g. shells and speleothems) and phosphate (e.g. bones and teeth). The ratio between the isotopes 87 Sr and 86 Sr can be employed as a provenance marker typically deriving from a precise geological area depend- ing on the age and on petrochemical features of the local rocks. Thus, Sr enters the ecosystem reaching plants and animals, mainly through water, following erosion and weathering of the bedrock. Sr is mainly fixed in the hydroxyapatite of teeth and bone tissue sub- stituting calcium, with concentrations ranging between c.a. 50 to 1000 ppm. In this sense, tooth enamel be- comes the best target tissue to perform Sr isotope analysis. This is because of two different reasons. First, being a ~96% mineralized tissue (mainly hydroxyapatite Ca10(PO4)6(OH)2), enamel is quite resistant to diagenetic alterations, usually preserving the biogenic isotopic sig- nature of the individuals. Second, enamel forms during childhood and does not remodel during the life of the individual, therefore, its Sr isotopic composition should reflect the signature of the place where the individual has spent its youth. This makes teeth the perfect target to unravel human/animal migrations (Lugli et al., 2017a). Similarly, oxygen is fixed within carbonate and phos- phate groups of the hydroxyapatite. The δ 18 O composi- tion of tooth enamel reflects the isotope composition of the body water, which, in turn, varies linearly with the local meteoric water. Consequently, the δ 18 O value of skeletal remains is an important tool the decrypt the pa- laeoclimatic proxies, but also to track possible move- ments of an individual between places with different envi- ronmental conditions (Pellegrini et al., 2011). In this work, we used isotope techniques to explore the mobility patterns of humans and animals from the Middle Pleistocene layers of Isernia la Pineta (Molise, Italy; Peretto et al., 2015). In particular, the most ancient human deciduous tooth ever found in Italy (~580 ka) years was studied employing the micro-destructivity of- fered by the laser ablation MC-ICP-MS. The mobility of the human was thus compared with macro-mammal (bison and rhinoceros) mobility patterns and with the local bioavailable Sr isotope composition obtained from the analysis of modern plants grown in areas away from Holocene contaminations and on and micro-mammals from the Middle Pleistocene layers of the site (i.e. ro- dents; Lugli et al., 2017b). Moreover, to get new insights https://doi.org/10.26382/AIQUA.2018.AIQUAconference into the Stephanorhinus hundsheimensis migratory pat- terns and to unravel possible routes taken by this ani- mal, we measured Sr and oxygen isotopes of the outer enamel surface in a high spatially resolved approach. 2. MATERIAL AND METHODS The 87 Sr/ 86 Sr ratios of the IS42 human and of a Stephanorhinus hundsheimensis tooth were determined by LA-MC-ICP-MS technique. We sampled several por- tions (linear transects) of the external enamel surface, following a cervical-incisal direction (human enamel n = 5; rhino enamel n = 25). Sr data were acquired with a double focusing MC-ICP-MS Neptune™ (Thermo Fisher Scientific), coupled to a 213 nm Nd:YAG laser ablation system (New Wave Research™), housed at the Centro Interdipartimentale Grandi Strumenti (CIGS) of the Uni- versity of Modena and Reggio Emilia. The detailed pro- tocol is described in Lugli et al. (2017a). In order to maximize the ion beam size stability, we chose to use dynamic linear ablation (500 x 100 µm), which provides more precise results compared to static spot ablation. We employed a laser frequency of 10 Hz and an energy of ~20 J/cm 2 . The following masses were collected us- ing nine Faraday detectors: 88 Sr, 87 Sr, 86.5, 86 Sr, 85 Rb, 85.5, 84 Sr, 83 Kr, 82 Kr. A 60 s background signal for each mass was collected before the analysis to correct for Kr interferences. The presence of doubly-charged Dy, Yb and Er was negligible, also suggesting a low content of rare earth elements within the enamel tissue. Data were acquired in single blocks of 200 cycles, with an integra- tion time of 0.5 s. To correct for the formation of 40 Ca 13 P 16 O and 40 Ar 13 P 16 O, we employed a set of in- house bioapatite reference materials (a swine tooth, a bovine tooth and a shark tooth) building a calibration curve. The analysis of our human tooth in-house refer- ence material (ROCht42) yielded an average (n = 10) LA 87 Sr/ 86 Sr ratio of 0.70865 ± 0.00040 (2σ) and an ac- curacy of 0.005% (16 ppm) compared with the true value obtained by dissolution MC-ICP-MS of 0.70864 ± 0.00001 (2se). Faunal enamel samples and modern plant speci- mens were analyzed after chemical dissolution and chromatographic separation of Sr. Samples preparation follows the protocol presented in Lugli et al. (2017a). 5- 10 mg of enamel and about 15 mg of plant ashes were digested in 1 ml of 14N HNO3 and re-dissolved in 3N HNO3 after evaporation. The Sr separation uses col- umns with a ~300µl volume filled with Eichrom Sr spec- resin. Sr isotope ratios were determined using the MC- ICP-MS Neptune at the CIGS - UNIMORE. A [blank/ standard/blank/sample/blank] bracketing sequence was adopted to monitor any instrumental drift. Data were normalized through exponential law to a 88 Sr/ 86 Sr ratio of 8.375209. During this analytical session, the standard yielded a 87 Sr/ 86 Sr ratio of 0.71025 ± 0.00002 (2σ; n = 33). The Sr ratios were corrected for instrumental bias to a NBS-987 value of 0.71026 ± 0.00002. High resolution (0.5 mm) sampling for stable iso- tope analysis was done on the outer enamel surface of a S. hundsheimensis tooth specimen (apex-cervix direc- tion), using a micro-drilling system. Fifty-eight samples were obtained this way. The δ 18 O isotope composition was thus determined at the Max Planck Institute for Chemistry (Mainz, Germany) on a Thermo V mass spectrometer, equipped with a GasBench II preparation device and a cold trap. In this configuration, tooth enamel samples of <100 microgram (equivalent of <5 microgram CaCO3) can be measured with <0.15‰ pre- cision (1SD). 3. RESULTS The human tooth yielded an average 87 Sr/ 86 Sr ratio of 0.70914 ±0.00038 (2σ; 5 ablation lines). Rodent teeth, analyzed by dissolution MC-ICP-MS, showed an 80 Lugli F. et al. Fig. 1 - (a) Strontium isotope data from human, fauna and modern plant specimens of the Isernia La Pineta site and surroundings. (b) High spatial resolution analyses of strontium isotopes and oxygen isotopes of a single S. hundsheimensis tooth. 81 average Sr isotope composition of 0.70924 ±0.00013 (2σ; n =16). Seven bison teeth yielded 87 Sr/ 86 Sr ratios ranging between 0.70927 and 0.70954 (average 0.70938 ±0.00018; 2σ; n =7). Rhinoceros teeth, meas- ured by dissolution MC-ICPMS, showed an 87 Sr/ 86 Sr ratio ranging from 0.70941 and 0.70979 (average 0.70958 ±0.00030; 2σ; n =9). One rhinoceros tooth was also analyzed by LA-MC-ICP-MS to explore its intra- tooth variability, yielding 87 Sr/ 86 Sr ratios ranging be- tween 0.71019 to 0.70956 (0.70982 ±0.00038; 2σ; n = 25). On the same tooth, high resolution oxygen isotope analyses yielded δ 18 OC (SMOV) values between 24.6 and 26.6‰. These values were then converted in δ 18 O phosphate values and in δ 18 O water values using regres- sion from Pellegrini et al (2011) and D’Angela & Longinelli (1990). 4. DISCUSSION AND CONCLUSION When compared with micro-mammals and local plants, the human Sr isotope composition falls within the supposed local isotopic variability (Fig. 1). This suggests that the human group from Isernia likely exploited local resources, within a limited supply area (c.a. 10-15 km), similarly to modern hunter-gatherer collectors (Lugli et al., 2017b). On the contrary, given that the Sr isotope composition of macro-mammals seems affected by a higher radiogenic Sr signature, we infer that both rhinos and bisons grazed on a different supply area. We sug- gest that the higher 87 Sr/ 86 Sr ratio derives from the con- tamination with volcanic soils and are likely related to seasonal migrations in the south-west direction, toward the fertile grounds of the Roccamonfina volcano area (Conticelli et al., 2009). The high-resolution isotope analysis of a S. hundsheimensis tooth agrees with this interpretation. In fact, both Sr and O isotope ratios show clear fluctuations correlated with the growth of the tooth enamel within a period of ca. 400 days (Fig. 1). These variations can be related to seasonal movements be- tween places with different oxygen and Sr isotope com- position (e.g. from Isernia to Roccamonfina/Albani Hills and vice-versa). ACKNOWLEDGEMENTS This project was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement No 724046 - SUCCESS awarded to Prof. Stefano Benazzi - erc-success.eu). Additional funding was provided through the Programma Giovani Ricerca- tori Rita Levi Montalcini (to AC). REFERENCES Bentley R.A. (2006) - Strontium isotopes from the earth to the archaeological skeleton: a review. Journal of Archaeological Method and Theory, 13, 135- 187. Conticelli S., Marchionni S., Rosa D., Giordano G., Boari E., Avanzinelli R. (2009) - Shoshonite and sub-alkaline magmas from an ultrapotassic vol- cano: Sr-Nd-Pb isotope data on the Roccamonfina volcanic rocks, Roman Magmatic Province, South- ern Italy. Contribution to Mineralogy and Petrol- ogy, 157, 41-63. D'Angela D., Longinelli A. (1990) - Oxygen isotopes in living mammal's bone phosphate: further results. Chemical Geology, Isotope Geoscience Section, 86, 75-82. Lugli F., Cipriani A., Peretto C., Mazzucchelli M., Brunelli D. (2017a) - In situ high spatial resolution 87 Sr/ 86 Sr ratio determination of two Middle Pleisto- cene (ca. 580 ka) Stephanorhinus hundsheimen- sis teeth by LA-MC-ICP-MS. 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Human and animal mobility at Isernia la Pineta Ms. received: May 7, 2018 Final text received: May 19, 2018 82