"Until death do us part". A multidisciplinary study on human- Animal co- burials from the Late Iron Age necropolis of Seminario Vescovile in Verona (Northern Italy, 3rd-1st c. BCE).

Animal remains are a common find in prehistoric and protohistoric funerary contexts. While taphonomic and osteological data provide insights about the proximate (depositional) factors responsible for these findings, the ultimate cultural causes leading to this observed mortuary behavior are obscured by the opacity of the archaeological record and the lack of written sources. Here, we apply an interdisciplinary suite of analytical approaches (zooarchaeological, anthropological, archaeological, paleogenetic, and isotopic) to explore the funerary deposition of animal remains and the nature of joint human-animal burials at Seminario Vescovile (Verona, Northern Italy 3rd-1st c. BCE). This context, culturally attributed to the Cenomane culture, features 161 inhumations, of which only 16 included animal remains in the form of full skeletons, isolated skeletal parts, or food offerings. Of these, four are of particular interest as they contain either horses (Equus caballus) or dogs (Canis lupus familiaris)-animals that did not play a dietary role. Analyses show no demographic, dietary, funerary similarities, or genetic relatedness between individuals buried with animals. Isotopic data from two analyzed dogs suggest differing management strategies for these animals, possibly linked to economic and/or ritual factors. Overall, our results point to the unsuitability of simple, straightforward explanations for the observed funerary variability. At the same time, they connect the evidence from Seminario Vescovile with documented Transalpine cultural traditions possibly influenced by local and Roman customs

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period

21openInternationalInternational coauthor/editorWe subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.openMaixner, Frank; Sarhan, Mohamed S; Huang, Kun D; Tett, Adrian; Schoenafinger, Alexander; Zingale, Stefania; Blanco-Míguez, Aitor; Manghi, Paolo; Cemper-Kiesslich, Jan; Rosendahl, Wilfried; Kusebauch, Ulrike; Morrone, Seamus R; Hoopmann, Michael R; Rota-Stabelli, Omar; Rattei, Thomas; Moritz, Robert L; Oeggl, Klaus; Segata, Nicola; Zink, Albert; Reschreiter, Hans; Kowarik, KerstinMaixner, F.; Sarhan, M.S.; Huang, K.D.; Tett, A.; Schoenafinger, A.; Zingale, S.; Blanco-Míguez, A.; Manghi, P.; Cemper-Kiesslich, J.; Rosendahl, W.; Kusebauch, U.; Morrone, S.R.; Hoopmann, M.R.; Rota-Stabelli, O.; Rattei, T.; Moritz, R.L.; Oeggl, K.; Segata, N.; Zink, A.; Reschreiter, H.; Kowarik, K

THE CELTUDALPS PROJECT: A MULTIFACETED PERSPECTIVE ON LATE IRON AGE TERRITORIAL MOBILITY AND GENETIC VARIATION IN NORTHERN ITALY AND SWITZERLAND

The steady increase of biogeochemical and biomolecular data is refining traditional anthropological and archaeological hypotheses about the genetic history and territorial mobility patterns of European human groups during the Late Iron Age. Compared with other geographic areas, however, only few data are yet available for those “Celtic” groups occupying the Northern Italian and Swiss territories during this period. Even fewer are collaborative research projects trying to compare these different types of information for these geographic areas. The resulting knowledge gap is especially relevant given the archaeologically and historically documented networks (cultural, commercial, and migratory) between the two sides of the Alps in this period. With CELTUDALPS we aim to address these issues by means of a multidisciplinary analysis of a large (ca. 300) individuals representing La Tène cultural groups inhabiting modern Switzerland and Northern Italy between the 4th -1st centuries BCE. By applying a range of methods including the analysis of ancient DNA and isotopic (δ15N, δ13C, δ34S, δ18O, 87Sr/86Sr) data, and the quantitative analysis of funerary patterns, CELTUDALPS aims to: (a) estimate the differences and similarities in territorial mobility between these groups; (b) reconstruct their genetic history and variation; (c) explore the possible links between mobility, kinship patterns, and social organization in each area. Here, we present the theoretical background and research design of the project, illustrate the state of the art of the project with examples selected from our most recent results, and discuss the planned next steps of our research

"Celts" down the Alps. New multi-isotopic data on territorial mobility in pre-Roman Verona (Seminario Vescovile, NE Italy, 3rd -1st c. BCE)

During the Late Iron Age (4th - 1st c. BCE), continental Europe witnessed dense trade networks and significant population movements. The migration of transalpine human groups to the Italian peninsula is a testament to the dynamism of this era. This study, building on preliminary isotopic data (δ13C, δ15N, and δ18O) from a subset of individuals (n=49) in the Seminario Vescovile (SV-Verona, Italy, 3rd - 1st c. BCE), extends these previous analyses. It now includes a larger sample (n=127, encompassing approximately 70% of the buried individuals) and incorporates additional isotopic ratios. This expanded research aims to provide a more robust assessment of territorial mobility and investigate potential connections within this community between geographical origin, sex, and burial practices. To achieve these goals, we examined isotopic ratios of sulfur (δ34S; n=127 - 61 adults and 66 nonadults) and strontium (87Sr/86Sr; n=57 - 51 adults and 6 nonadults) from bone collagen and dental enamel, respectively. We utilized the standard deviation from the isotopic mean of archaeological faunal remains and modern plants as proxies for locally available δ34S and 87Sr/86Sr values, identifying individuals falling outside these ranges. Additionally, we conducted Mann-Whitney tests to explore variances in δ34S and 87Sr/86Sr concerning sex and burial features. Our analysis revealed that human δ34S and 87Sr/86Sr ratios averaged 5.7±1.7‰ VCDT and 0.709756±0.001430, respectively. Minimum estimates of non-local individuals range from 3 (2.5%) based on sulfur to 11 (19.3%) based on strontium. Notably, at least five cases displayed isotopic values suggestive of an alpine or transalpine origin. Interestingly, our isotopic data did not indicate any discernible associations with individual sex or funerary treatment. These findings prompt a reassessment of prior estimations of mobility in the Seminario Vescovile population and strongly suggest a connection between this human group and both alpine and transalpine regions

GENETIC HISTORY OF LATE IRON AGE (4TH – 1ST CENTURY BCE) HUMAN GROUPS FROM BOTH SIDES OF THE ALPS

During the Late Iron Age (La Tène, 4th-1st centuries BCE), various human groups sharing cultural elements, commonly referred to as “Celts”, were involved in migration processes and settled in the Italian Peninsula after crossing the Alps. The current knowledge of these people is largely drawn from osteological, isotopic, archaeological, and historical evidence. However, the genetic history of these groups from both north and south of the Alps remains unexplored. This paleogenomic study aims to analyze if the cultural similarity of these people is reflected in their genomic makeup. Moreover, it intends to study the genetic relationships between different La Tène groups from north and south of the Alps, and other ancient and present-day populations from Europe. We performed molecular analyses (shotgun and nuclear capture data – approx. 1.3 mio SNPs) on 194 individuals from 13 Swiss and North Italian Late Iron Age archaeological contexts. Ancient DNA data allows to determine the genetic sex in 119 individuals, comprising 76 XX and 43 XY, including 52 subadults. With the additional support of mitochondrial DNA data, we detected genetic relatedness among three individual pairs within two different Swiss archeological sites, and one case in the North-Italian site of Seminario Vescovile. Comparative analyses show a genetic similarity between the ancient Swiss and Italian individuals. Additionally, their genomic diversity overlaps with that of especially western and south-western European present-day populations. However, the results suggest a genetic discontinuity between Late Iron Age individuals from Northern Italy and present- day populations from the same geographic area. On the other hand, the comparison with published data infers a stronger genetic affinity among European Iron Age groups

„CELTS“ UP & DOWN THE ALPS. NEW ISOTOPIC INSIGHTS ON MOBILITY IN PRE-ROMAN VERONA (NE ITALY, 3RD -1ST C. BCE)

The European Late Iron Age (4th -1st centuries BCE) features intense trade networks and movements of people.The settling in the Italian peninsula of transalpine human groups exemplifies the dynamism of this period. Preliminary isotopic data (δ13C, δ15N and δ18O) from a subset (N=49) of individuals from Seminario Vescovile (SV-Verona,Italy 3rd -1st c. BCE) highlighted few nonlocals in this pre-Roman necropolis. Here, we expand our previous analysesof SV, by means of a larger sample (N=127, i.e., ca. 70% of buried individuals) and the inclusion of additional isotopicratios, in order to provide a more solid estimate of territorial mobility, and of the possible links in this community between provenance, sex, and funerary treatment. To this aim we analyzed the isotopic ratios of sulfur (δ34S; N=127 - 61 adults and 66 nonadults) and strontium (87Sr/86Sr; N=57: 51 adults and 6 nonadults) from respectively bone collagen and dental enamel. We used as proxy of locally bioavailable δ34S and 87Sr/86Sr ranges two standard deviations from the isotopic mean of archaeological faunal remains and modern plants, and checked for individuals falling outside this range. We then explored differences between sexes and funerary features in δ34S and 87Sr/86Sr by means of Mann-Whitney tests. Human δ34S and 87Sr/86Sr ratios average 5.7±1.7‰ V-CDT and 0.709756± 0.001430 respectively. Minimum estimates of nonlocals range from 3 (2.5%) for δ34S, to 8 (14%) based on 87Sr/86Sr. At least in two cases, these show isotopic values suggesting an alpine/transalpine origin. Isotopic data do not show an association with individual sex or funerary treatment. These new results call for a revision of previous estimates of mobility at SV, and supports a link between this human group and both alpine and transalpine contexts

“Celts” Up & Down the Alps: Origin and Mobility Patterns on Both Sides of the Alps During the Late Iron Age (4th – 1st century BCE)

The genetic history and the degree of mobility characterizing the “Celtic” population during the Late Iron Age (4th -1st century BCE) have become central topics in archaeology and anthropology. However, despite the steady increase of archaeological, biomolecular and biogeochemical studies on central European and British contexts, few data are available for the Northern Italian and Swiss territories. During the Late Iron Age the geographic areas corresponding to modern Switzerland and Northern Italy played a crucial role in connecting Central and Southern Europe. This connection involved not only forms of cultural transmission (e.g. diffusion of the La Tène material culture), but also the actual movement of people through the Alpine range. During the Late Iron Age the European area was affected by migrations of different human groups, with southward migratory processes documented historically and by archaeological traces in both Northern and Central Italy. Little is known however about the similarities and differences in genetic variability, mobility patterns, and social organization between the Late Iron Age populations distributed on the two sides of the Alpine range. The present project aims to address this issue, by means of a multidisciplinary (genetic, and isotopic) analysis of a large skeletal sample representing Late Iron Age populations of modern Switzerland and Northern Italy (Cenomani of Verona) between the 4th -1st centuries BCE. In particular, the present project aims to: 1) reconstruct the genetic history and variation of the “Celtic” groups inhabiting the Swiss and Northern Italian areas by analysing the genetic relationship among groups and the genetic structure of groups; 2) estimate the degree of mobility characterizing these populations, and the differences and similarities in mobility (e.g. frequencies of nonlocals, sex and age patterns, long-versus short distance mobility) between them; 3) test for the presence of relationships between mobility, kinship patterns, and social organization in each area, and the possible similarities and differences in social organization between the Swiss and Northern Italian communities. Analytical methods will include the study of ancient DNA (aDNA) and of stable isotopes (δ15N, δ13C, δ34S, δ18O, δ2H), in conjunction with a quantitative analysis of the archaeological evidence. This project will be the first to apply a multidisciplinary approach to the study of Late Iron Age populations from Continental Europe, and will generate new openly-accessible isotopic and genomic data for future studies on these populations from both sides of the Alps. The results will be disseminated across different target groups by means of: i) scientific papers, international conferences participation and open access data repository for scientists. ii) social networks and media platforms,didactic activities in schools, public conferences and interview/articles in local newspapers for the non-scientist audience

"Celts" down the Alps. New isotopic data on territorial mobility among the Late Iron Age Cenomani of Verona (NE Italy, 3rd -1st c. BCE)

The Late Iron Age (4th -1st centuries BCE) was characterized by frequent movements of people and goods across the Alpine range resulting in the settlement of "Celtic" groups in the Italian peninsula. Explorative stable isotope data of oxygen and carbon of the Cenomani of Seminario Vescovile (SV-Verona, Italy 3rd -1st c. BCE) suggested the presence of few non-local individuals in this community. Here, we build on these preliminary results and expand them by analyzing a larger sample from SV and additional isotopic ratios. Our aim is to clarify the degree of mobility in this group, the potential origin of non-locals, and the correlation between mobility, sex, and funerary treatment. We analyze the ratios of sulfur (δ34S; N=127) and strontium (87Sr/86Sr; N=57) from bone collagen and dental enamel respectively. We establish the local isotopic range based on published soil, plants and water values, as well as on the human and animal variability by using as criterion the median ± 3MAD (three times the median absolute deviation). We check for differences between sexes and funerary features in δ34S and 87Sr/86Sr by means of Mann-Whitney tests. The highest frequency of non-locals (10/57: 17.5%) is found when considering 87Sr/86Sr, with at least two individuals showing values consistent with an alpine/transalpine origin. No trend characterizes isotopic values based on sex or funerary treatment. Our data allow revising previous estimates about a reduced mobility at SV, while supporting at the same time a link between this population and both alpine and transalpine contexts