Isotopic evidence for population dynamics in the Central Italian Copper Age and Bronze Age

The mobility patterns in the Italian peninsula during prehistory are still relatively unknown. The excavation of the Copper Age and Bronze Age deposits in La Sassa cave (Sonnino, Italy) allowed to broaden the knowledge about some local and regional dynamics. We employed a multi-disciplinary approach, including stable (carbon and nitrogen – C and N, respectively) and radiogenic (strontium-Sr) isotopes analyses and the identification of the cultural traits in the material culture to identify mobility patterns that took place in the region. The Sr isotopic analyses on the human bones show that in the Copper Age and at the beginning of the Bronze Age, the cave was used as a burial place by different villages, perhaps spread in a radius of no more than 5 km. Stable isotopes analyses suggest the introduction of C4 plants in the diet of the Middle Bronze Age (MBA) communities in the area. Remarkably, in the same period, the material culture shows increasing influxes coming from the North. This evidence is consistent with the recent genomic findings tracing the arrival of people carrying a Steppe-related ancestry in Central Italy in MBA.


Introduction
La Sassa cave (Sonnino, Latina province in Southern Latium) was explored since 2015 in multiple archaeological campaigns 1,2 . The cave holds a multi-stratified archaeological archive from the Late Pleistocene to present days (Fig. 1). The cave area, subdivided in progressively numbered rooms, is articulated in at least 8 Rooms. The osteological material in La Sassa constitutes multiple bone assemblages reflecting naturally occurred arrangements of the pristine primary depositions, as several tiny bones originally tied in weak diarthroses, such as metacarpals and phalanges, were recovered. Human remains were found in three soundings (L, WD, and N) in Room 1 (Fig. 1, b). No diagnostic potsherds associated with these remains have been found, but the stratigraphy and the radiocarbon dates from human bones constraint these layers to the Copper Age (CA) and Early Bronze Age (EBA) 3 . Some human remains have also been collected in Room 2. Again, no associated diagnostic potsherds were found, but radiocarbon dates fall in the CA 3,4 . Few human bones in Room RA belong to an infant whose femur was radiocarbon dated to the Middle Bronze Age (MBA), sub-phase 2 3 .
An extensive set of isotopic and preservation analyses has been performed on bones and teeth enamel to characterize individual mobility and dietary patterns of people buried in La Sassa. Soil and water samples, as well as faunal remains, were analyzed to obtain the local isotopic baselines. The results were put in the context of recent genomic and isotopic findings, suggesting the area surrounding the Pontine plain (also known as Agro Pontino) as a complex stage for the population dynamics in prehistoric Central Italy. Most of archaeological items testifying for settlements of the CA around La Sassa were found by chance ( Fig. 2). Some isolated items come also from the Lepini and Ausoni Mounts 6 . Burial places have been recognized in two nearby caves: the Scalelle and Vittorio Vecchi caves. The burial goods collected at the Scalelle cave 7 parallelize with the southern Italian CA culture named Gaudo 8 . The oldest radiocarbon dates for this southern culture in Latium ranges from 3330 to 3210 BCE 9 .
In Vittorio Vecchi cave, hundreds of disarticulated human bones have been found together with abundant EBA and MBA potsherds, and a few CA potsherds 10 . Despite the lack of radiocarbon dates, it is possible the narrowing of the funerary use of the cave to the BA, which starts between 2100 and 2000 BCE in the central Italian chronological framework 11 . No EBA evidence has been found so far in the Lepini and Ausoni Mounts.
In the nearby Pontine plain, only a few finds have been collected 12 : an isolated bronze axe in the Sabaudia lagoon 13 , and some potsherds below the tephra of the Avellino eruption 14 , recently dated around 1900 BCE 11,15,16 . The lack of CA and EBA occurrence in the mountains might be due to the sparse unsystematic research in the area. In contrast, the paucity of evidence in the nearby Pontine plain seems to reflect a low population density in the CA. However, multiple sites arose along the coastline and at the foot of the Ausoni Mounts in the next MBA sub-phases 1 and 2 13 .

Evidence of regional mobility in the Italian context
Multiple pottery items were found in the local contexts. From a ceramic typological perspective, during the CA and the EBA, the area around La Sassa cave seems to be strongly influenced by (or even part of) the southern cultures of Gaudo (CA) and Palma Campania (EBA). At the beginning of MBA 1-2, the potsherds so far collected in the Pontine plain show influences both from the northern Grotta Nuova 22 and the southern Protoappeninico cultures 23 . Indeed, the boundary between the latter had already been hypothesized in the Pontine plain 23,24  These stylistic differences suggest that the local boundary between the two cultural contexts might be placed around the Amaseno valley (Fig. 2), and their contacts increased starting from the MBA1B. Recent genomic findings 4 confirm the area surrounding the Pontine plain as a liminal zone not only for cultural circulation but rather for mobility of people with different genomic characteristics that could have moved throughout that area in the BA. Indeed, even considering a few CA individuals from La Sassa cave, the signature of BA movement from genomically Steppe-related people has been demonstrated through Italy, emerging in the EBA in Northern Italy and moving southward through time 4 .
So far, the in-depth analysis of the individuals buried in La Sassa appears pivotal in broadening the knowledge about the population dynamics occurring in that area at the CA/BA boundary.
To date, the mobility in Italian CA and BA communities has only been partially explored through Sr and O isotopic analysis. Recently, De Angelis and colleagues 25 found evidence of a sedentary lifestyle rather than extensive mobility in Central Italian CA burials, like pointed out in some Southern Italian sites 26 .
Conversely, the mobility practices of the northern Italian communities across a timeframe ranging from the EBA to the Late Bronze Age (LBA) suggested that people spurted their mobility 27 .

Carbon and nitrogen stable isotopes background in the Copper Age and Bronze Age
The analysis of carbon and nitrogen stable isotopes of bone proteins was widely advocated to investigate further ancient bio-cultural characteristics such as diet 28 . Often, a change in diet might be suggestive for people moving from elsewhere carrying novel cultural and biological features. This theoretical framework was already applied to explain the dietary differences in multiple Italian BA funerary contexts 29,30 . 29,30 Indeed, recent surveys related to CA and BA communities in Central Italy underlined that this chronological and cultural transition represented a critical step for the Italian populations, characterized by economic changes and technological improvements 29,31

Strontium isotopic results and FT-IR analyses
We determined the Sr isotopic compositions of spring water and soil samples in a radius of c.a. 5km around the cave to characterize the "local baseline" (Table S3) Table S4). The highest value belongs to the LS419 Equus sp. The Sr isotope ratios for human teeth enamel and bones vary from 0.70822 to 0.70955, and from 0.70810 to 0.70894, respectively (Table S4). It is evident the difference between the isotope compositions of water samples and those of the biological material. In order to determine the bone preservation status and to verify the occurrence of post-depositional alteration FTIR analyses have been performed on 14 bones and, as an exploratory sample, one tooth (more details in Supplementary, chapter 3). LS151, LS418, and LS2176 have 87 Sr/ 86 Sr similar to the cave soil, while LS2177, LS2203, LS865, LS120, LS2208 have higher Sr isotope ratios (Tab S2 and Tab S3).
Moreover, some of the analysed individuals have enamel teeth and bones with similar 87 Sr/ 86S r (LS896, LS2203, LS2208, LS2176, and LS2177), while others -LS150, LS1956 and LS882 -have teeth and bone with different Sr isotope compositions (Fig. S4). Interestingly, all of them return similar FT-IR spectra (Fig.   S5), showing an enlarged hump in the OH-stretching region overlapped by signals at ca. 2960 and 2880 cm −1 from ν(CH-)Lipids, a strong peak at ca. 1990-2000 cm −1 from cyanamide, and several absorption bands between ca. 1794 cm −1 and 400 cm −1 . These characteristics can be assigned to the most important functional groups in bone, e.g., HPO 4 2-, PO 4 3-, CO 3 2and amide [39][40][41][42][43][44][45] . Spectra are also characterized for an evident ν1(PO 4 3-) absorption peak at ca. 1045 cm -1 and the occurrence of fluorapatite (ca. 1090 cm -1 ), suggesting an expected (but not complete) deproteinization with reference to fresh (modern) bones 43 . Since the young age 46 , the LS 2176 infant lacks of a clear peak at 1415 cm -1 coupled to the 1450 cm -1 one (although a value can be assigned; see section 3 of Supplement). The C/P index, suitable both for pellets and DRIFT FT-IR techniques 43 is above 0.34 and significantly higher than 0.15, which is the considered threshold for altered bones. In addition, the order degree in phosphate minerals (CPI) varies between 2.7 and 3.4. Although these values could be slightly overestimated by DRIFT, they are generally lower than 3.4, which usually sets the limit between modern and archaeological bones. Therefore, the bones could not be considered diagenetically altered. Even though there are de-proteinized and de-phosphatized samples such as LS2208, LS151, LS120, LS2161, (Supplementary, Fig. S6), any contribution of type-A carbonate can be hypothesized except for the LS2176 bone 39 . Overall, the FT-IR indexes are not correlated with stable isotope compositions, whereas correlations between the C/P index and 87 Sr/ 86 Sr ratio (Supplementary, Fig S6d) are pointed out. These results, suggest that the 87 Sr/ 86 Sr teeth-bone pairs as reliable markers for hypothesizing the mobility pattern of people from La Sassa.

Carbon and Nitrogen isotopic results
Forty human samples (87%) fitted the quality criteria related to collagen preservation (Table 1).       IQR EBA bones : 0.0003; Mann-Whitney U=49.5; p=0.24 for teeth enamel and U=25 p=0.042 for bones), suggesting a slightly dissimilar mobility pattern between the differential periods.

Samples
Overall, EBA data are more scattered than CA values, supporting the few isotopic studies about mobility for Italian prehistoric communities, suggestive for the development of a mobile behaviour starting from the BA [25][26][27] .
Remarkably, human data seem to be unrelated to the hydrographic net identified by the sampled springs, suggesting that people exploited water sources other than those nowadays flowing around La Sassa, or that unsampled springs were exploited. These springs could be possibly fed by water isotopically different from the exploitation area. Indeed, thermal waters in the aquifers of Mt. Massico ridge, at the south of Mt.
Aurunci, are featured by variable Sr ratio, ranging from 0.7080 to 0.7096) 56 , because the isotopic signatures of the water are impacted also by the rocks of the catchment area, which could be isotopically different from those in the exploitation area 57 . Accordingly, the signature for humans was compared to the isotopic fingerprint of multiple soils. The isotope variability of soils prevents for identifying some specific areas where the geographical origin of both CA and EBA communities could be hypothesized. The overall drop in 87 Sr/ 86 Sr between teeth and bones suggests a certain degree of mobility for people between childhood and adulthood (Fig. 4). Therefore, people buried in La Sassa spent their childhood elsewhere from the area surrounding the cave. Whatever being the difference between teeth and bone values, it should be noted that the samples do not return isotopic fingerprint matching that obtained for the cave soil ( Supplementary Fig.   S4). This evidence suggests that the dwellings for both CA and EBA people were established in areas other than La Sassa geological setting. Accordingly, we can assume that the sampled individuals lived away from La Sassa cave, and they did not depend upon the sampled hydrographic net. However, some individuals return similar signatures in teeth and bones, suggesting a sedentary lifestyle rather than extensive mobility behaviours, scattering their values around 0.7085 and 0.7089. We cannot determine the exact geographic origin for CA and BA individuals analysed, as their isotopic values match those of wide areas around the cave, as suggested by the map of the distribution of the soils (Fig. 4). Overall, it seems reasonable that people moved around La Sassa in adulthood from sites characterized by soil, food, water etc. with slightly different isotopic ratios. This speculation can be corroborated considering the variability in the Sr isotope composition of a ca. 78 km 2 area around the cave (corresponding to ca. 5 km-radius area from the cave, selected to characterize the local baseline), where the significant soils were geochemically characterized ( Fig. 5 and Supplementary Table S3).

Material and methods
The archaeological excavation The relative chronology is based on typo-chronological parallels and stratigraphic considerations, the absolute chronology is based on radiocarbon dates 3,4 .

Biological remains selection
The analysed skeletal series consists of over 800 human finds mainly recovered in Room 1 and Room 2 in a chaotic bone assemblage, probably due to taphonomic constraints, preventing the identification of individual burials. However, a comprehensive classification of each informative remain was performed according to bone or tooth, side, and, whenever possible, osteological markers suggestive for sex and age at death estimations.
The age of death was addressed by the wear of the teeth occlusal surface, the level of formation of the dental roots, and the synostosis degree of the tubular bones 58,59 .
Sex identification was performed on dimorphic skeletal districts, mainly focusing on hipbones and skull traits 60,61 .
The mandible was the most recovered anatomical district that allows for identifying at least 17 individuals (Minimum number of individuals; MNI). However, the joint information about sex, age at death, osteological characterization and osteometric analysis of the whole osteological material pushed us to consider a reliable sample consisting of at least 27 individuals.
Fifteen mandibles, a maxillary bone, six femur and 25 teeth were selected for tracing the 87 Sr/ 86 Sr ratio across the individuals. The selection was driven by macroscopic preservation (Supplementary Table S4).
Due to the impossibility in identifying whether some anatomically unrelated bones pertain to a single individual, we selected 46 bones representative for the at least the whole sample identified to explore the carbon and nitrogen stable isotopes ratios, even though multiple districts could be redundant. To account for the ecological background, 7 faunal remains were also characterized isotopically ( Table 2).
To set the isotopic baseline within and around La Sassa cave, 6 groundwater and 7 soils samples were analysed for their Sr ratio (Supplementary Table S3).

Carbon and nitrogen isotope analyses
The collagen extraction from bones was individually performed following Longin's protocol modified by Brown and colleagues 65 , which was also simultaneously applied to a modern bovine sample as a reference.
In order to obtain an acceptable yield of collagen, the extraction was performed on about 500 mg of bone with the standard reference materials USGS-34, IAEA-N-2 and SIRFER yeast. Typical analytical precision, evaluated from a repeated measurement of an internal standard, was 0.1‰ for δ 13 C and 0.2‰ for δ 15 N. The reliability of the procedure and the exclusion of exogenous contamination were accounted for through a comparison against established criteria to ascertain the percentages of carbon and nitrogen, atomic C/N ratios, and collagen yields [67][68][69] .
The carbon and nitrogen contents and C/N ratio are listed in Table 1. To assess the preservation state of the extracted collagen, we considered carbon and nitrogen contents between 15-51% and 5-18%, respectively 67 , and C/N ratios within the range 2.9 to 3.6 68 . The extraction yield was not used as a criterion 67

Sample preparation and analytical conditions for FT-IR analyses
FT-IR spectroscopy applies in investigation of human remains in archaeological and palaeontological contexts [39][40][41][42][43][44][45]73 . Phosphate and carbonate minerals, as well as the collagen, that constitute bones and teeth can differ for structure and abundance based on freshness, aging maturation and post-mortem processes  Table 3 were calculated by using the line between 1900-900 and 800-500 (750-400) cm - 1 39,41,43 . The relative amount of carbonate vs.
phosphate (CC/PP) was also calculated from the ratio between the sum of absorbances at 1460 and 1425 cm -1 and the sum of absorbances at 605 and 568 cm -1 by using the baseline defined between 500 and 2000 cm -