Antoine Zazzo

Rationale
Carbon and nitrogen stable isotope time series performed in continuously growing tissues (hair, tooth enamel) are commonly used to reconstruct the dietary history of modern and ancient animals. Predicting the effects of altitudinal mobility on animal δ13C and δ15N values remains difficult as several variables such as temperature, water availability or soil type can contribute to the isotope composition. Modern references adapted to the region of interest are therefore essential.

Methods
Between June 2015 and July 2018, six free‐ranging domestic horses living in the Mongolian Altaï were fitted with GPS collars. Tail hairs were sampled each year, prepared for sequential C and N isotope analysis using EA‐IRMS. Isotopic variations were compared with altitudinal mobility, and Generalized Additive Mixed (GAMMs) models were used to model the effect of geographic and environmental factors on δ13C and δ15N values.

Results
Less than half of the pasture changes were linked with a significant isotopic shift while numerous isotopic shifts did not correspond to any altitudinal mobility. Similar patterns of δ13C and δ15N variations were observed between the different horses, despite differences in mobility patterns. We propose that water availability as well as seasonal availability of N2 fixing type plants primarily controlled horse hair δ13C and δ15N values, overprinting the influence of altitude.

Conclusions
Our study shows that altitudinal mobility is not the main factor that drives the variations in horse tail hair δ13C and δ15N values and that seasonal change in the animal dietary preference also plays an important role. It is therefore risky to interpret variations in δ13C and δ15N values of animal tissues in terms of altitudinal mobility alone, at least in C3–dominated environments.

Because hard tissues can be radiocarbon dated, they are key to establishing the archaeological chronologies, palaeoenvironmental reconstructions and historical-biogeographical processes of the last 50,000 years. The advent of accelerator mass spectrometers (AMS) has revolutionized the field of archaeology but routine AMS dating still requires 60–200 mg of bone, which far exceeds that of small vertebrates or remains which hold a patrimonial value (e.g. hominid remains or worked bone artefacts). Here, we present the first radiocarbon dates obtained from minute amounts of bone (3–60 mg) using a MIni CArbon DAting System (MICADAS). An optimized protocol allowed us to extract enough material to produce between 0.2 and 1.0 mg of carbon for graphite targets. Our approach was tested on known-age samples dating back to 40,000 BP, and served as proof of concept. The method was then applied to two archaeological sites where reliable dates were obtained from the single bones of small mammals. These results open the way for the routine dating of small or key bone samples. Hard tissues (i.e. bones, teeth, antler and ivory) found in the fossil record have a tremendous informative potential relevant to the fields of archaeology, palaeoecology and the history of art and technology. Because they can be identified to the species level and radiocarbon dated, these fossil remains are key to establishing the archaeological chronologies, palaeoenvironmental reconstructions and historical-biogeographical processes (i.e. post-glacial recolonization events) of the last 50,000 years. In effect, they provide us with windows to past societies, and contribute to our knowledge of ancient human evolution and cultural development 1 , palaeoclimates 2 , paleoenvi-ronments 3 and past trade networks 4. Hard tissues contain an organic phase (mainly the protein collagen type I) embedded in a mineral phase (made of a non-stoichiometric biogenic apatite). While the exchange of inorganic carbon occurs much more readily 5, 6 , the relative chemical inertness of biopolymers makes them ideal for dating; therefore, the majority of bone radiocarbon dates are obtained from the collagen phase. The chemical integrity of this biomolecule can be assessed using simple biochemical criteria such as %C, %N and C/N ratio 7–9. The amount of collagen in fresh bone is approximately 20–25% 9, 10. As the diagenetic alteration proceeds, the quantity and quality of the collagen decreases; consequently, the sample size must increase in order to compensate for protein loss. Radiocarbon dating ancient bones can therefore prove challenging. The advent of accelerator mass spectrometers (AMS) in the eighties revolutionized the field of archaeology by allowing smaller samples to be measured. While it decreases the amount of carbon required for a radiocarbon measurement by several orders of magnitude, the AMS dating of bone collagen still requires at least 60–200 mg of bone 11–13 , depending on the protein preservation and the extraction protocol. However, this is still excessive for two classes of bone remains: (1) individual bones of small vertebrates which often weigh less than 60 mg; and (2) unique remains such as hominid bones or worked bone artefacts for which curators do not permit invasive sampling 14. The specification of sample weights used for dating is not considered necessary by the scientific community 15 and is seldom reported in publications, even when supplementary information is available (see for example refs 16–19). However, careful examination of the literature suggests that attempts at dating samples smaller than 60 mg are rare. Regarding small vertebrates, only two case studies were found: the Late Prehistoric dispersal of

A range of archaeological and palaeoclimatic studies use isotopic analyses of ungulate hypsodont tooth enamel. Such studies commonly assume a constant growth rate, though this has not been fully tested. Here, we use stable isotope analyses of sequential enamel samples to study horse tooth growth. We fit the data using models corresponding to constant and exponentially decreasing rates of growth, and compare the results to metrical data showing the geometry and timing of apposition. The results indicate enamel apposition and maturation advances at an exponentially decreasing rate. An understanding of this variable growth rate is crucial for interpreting isotopic data from equid teeth.

Only the inorganic fraction of bone survives the high temperatures reached during cremation, so that it remains the sole material available for isotopic analyses. In order to assess the amount of information that can be extracted from such material, we measured carbon and oxygen isotope ratios in the remaining carbonate fraction of experimentally heated modern bone, and cremated bone from several archaeological sites. The results show that the isotope composition of cremated bone is strongly altered, but some information can nevertheless be extracted. First, we find very little evidence of post-burial alterations on the isotope composition of calcined bone. More importantly, it appears possible to obtain information about the way bodies were burned (with or without fuel, oxygen availability) giving the opportunity to improve our knowledge regarding funerary practices in places and times where cremation was practiced.

Ra's al-Hamra 6 (RH-6) is one of the earliest stratified archaeological sites along the eastern littoral of the Arabian Peninsula. This shell midden was radiocarbon dated to the 6th–5th millennium cal BC, but the majority of the dates were obtained before the advent of accelerator mass spectrometry (AMS) 14 C dating and suffer from large uncertainties. In addition, most of these dates were obtained on marine and mangrove shells and required correction for local variations from the global average marine 14 C reservoir age (MRA). This proved difficult because no consensus value exists for this period in the area. Recent excavations at RH-6 offered the opportunity to redate this important site in order to precisely determine its occupation history and later use as a graveyard, and establish the marine reservoir effect for this time period. Thirty-eight samples of charcoal, shells, and human bone apatite were selected for 14 C dating. Bayesian modeling of the 14 C dates suggests that the formation of the shell midden spanned ~1 millennium, between the mid-6th and the mid-5th millennium cal BC. Positive and consistent ΔR values were calculated throughout the entire sequence, ranging from 99 ± 27 to 207 ± 43 14 C yr. At the beginning of the 4th millennium cal BC, RH-6 was used as a graveyard, as suggested by the 14 C dating of a shell in strict association with an individual buried at the surface of the site. 14 C dating of human bone apatite allowed us to calculate that 89% of this individual's diet derived from marine resources. This finding confirms previous observations showing the overwhelming presence of marine and mangrove-dwelling species in the faunal and charcoal assemblage, and implies a low mobility, or mobility restricted to the coast for this population during the 4th millennium cal BC.

Bone remains of small vertebrate fossils provide valuable information for paleoenvironmental and paleoclimatic reconstructions. However, direct radiocarbon dating of small vertebrates remains challenging as the extraction of sufficient good quality collagen is required. The efficiency of eight collagen extraction protocols was tested on seven samples, representative of different ages and burial environments, including both macro and small vertebrate taxa. First, the samples were prescreened using attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) to quantify collagen content in archaeological bones, revealing that one should be discarded for 14 C dating. Then, the quantity of protein extracted (yield) and collagen integrity were checked using conventional elemental analysis. The results show that one protocol was not able to accurately extract collagen from the samples. A soft HCl-based protocol seems more appropriate for the pretreatment of archaeological small mammal bones, whereas a harsher protocol might be more efficient to extract a higher amount of collagen from large mammals as well as amphibian bones. The influence of the tested protocols on carbon and nitrogen isotope values was also investigated. The results showed that isotopic variability, when existing, is related to the interindividual differences rather than the different protocols.

A remarkable new site consisting of a concentration of as many as forty or more camel skeletons has been discovered in Abu Dhabi’s Western Region in the United Arab Emirates. Eight camel bone samples (from six individuals) from the site have been AMS radiocarbon dated by the Leibniz Laboratory for Radiometric Dating and Stable Isotope Research, Kiel University, Germany, revealing that they date from the second half of the fifth millennium BC. The site is located in an interdunal area to the south-east of the Baynunah Plantation, not far from the Ruwais–Habshan pipeline. The spread of camel bones extends over an area of about 10,000 m2. Preliminary analysis of the bones suggests that they are from wild dromedaries. Other archaeological finds associated with the camel bones include a finely made flint arrowhead. This important newly discovered site will provide a valuable opportunity to examine a large sample of wild camel bones during the later prehistory of south-eastern Arabia. Future detailed investigations at the site will throw fresh light on the early interactions between the communities inhabiting late prehistoric Arabia and the camel.

The site of Wakrita is a small Neolithic establishment located on a wadi in the tectonic depression of Gobaad in Djibouti in the Horn of Africa. The 2005 excavations yielded abundant ceramics that enabled us to define one Neolithic cultural facies of this region, which was also identified at the nearby site of Asa Koma. The faunal remains confirm the importance of fishing in Neolithic settlements close to Lake Abbé , but also the importance of bovine husbandry and, for the first time in this area, evidence for caprine herding practices. Radiocarbon dating places this occupation at the beginning of the 2nd millennium B.C., similar in range to Asa Koma. These two sites represent the oldest evidence of herding in the region, and they provide a better understanding of the development of Neolithic societies in this region.

Les microvertébrés sont d'excellents marqueurs environnementaux et climatiques. Par ailleurs, leur histoire phylogéographique récente n'est pas toujours bien cernée. La datation directe de leurs restes retrouvés en contexte archéologique permettrait d'inscrire les reconstructions associées à leur découverte dans une chronologie absolue. La datation 14 C de microéchantillons osseux représente aujourd'hui un défi car cela nécessite de pouvoir extraire suffisamment de collagène purifié de toute contamination. Les os de ces taxons pèsent typiquement 20 mg alors qu'un échantillon osseux datable aujourd'hui pèse au moins 150 mg. Ils ne contiennent donc, au mieux, que quelques milligrammes de collagène pour un os frais. En conséquence, la quantité de carbone que l'on peut extraire de tels os pour la datation rend la mesure délicate sur un AMS conventionnel. Le cas des os archéologiques, pour lesquels le collagène est souvent dégradé est encore plus problématique. Pour cette raison, il était nécessaire d'une part d'optimiser la quantité de collagène extrait et d'autre part d'optimiser la mesure du 14 C restant dans les échantillons. Dans un premier temps, huit protocoles d'extractions différents, décrits dans la littérature ont été testés sur onze échantillons représentatifs. Les résultats de l'optimisation des protocoles d'extraction en termes de rendement et de qualité du collagène extrait seront rapidement évoqués. Nous nous attarderons ici sur la deuxième étape du projet c'est-à-dire l'optimisation de la dernière étape de préparation et de la datation sur le nouveau spectromètre de masse par accélérateur ECHoMICADAS. Les difficultés liées à la manipulation de microquantités de collagène, les risques de contamination ainsi que la graphitisation pour la mesure en mode « solide » seront exposés. Les résultats des tests effectués sur des os de macromammifères d'âges consensus (VIRI) seront ensuite présentés. Enfin, l'application au cas d'échantillons réels de musaraignes