Environmental Archaeology The Journal of Human Palaeoecology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/yenv20 Dietary and Weaning Habits of the Roman Community of Quarto Cappello del Prete (Rome, rd 1st-3 Century CE) Flavio De Angelis , Virginia Veltre , Sara Varano , Marco Romboni , Sonia Renzi , Stefania Zingale , Paola Ricci , Carla Caldarini , Stefania Di Giannantonio , Carmine Lubritto , Paola Catalano , Olga Rickards & Cristina Martínez-Labarga To cite this article: Flavio De Angelis , Virginia Veltre , Sara Varano , Marco Romboni , Sonia Renzi , Stefania Zingale , Paola Ricci , Carla Caldarini , Stefania Di Giannantonio , Carmine Lubritto , Paola Catalano , Olga Rickards & Cristina Martínez-Labarga (2020): Dietary and Weaning rd Habits of the Roman Community of Quarto Cappello del Prete (Rome, 1st-3 Century CE), Environmental Archaeology, DOI: 10.1080/14614103.2020.1829297 To link to this article: https://doi.org/10.1080/14614103.2020.1829297 Published online: 13 Oct 2020. Submit your article to this journal Article views: 29 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=yenv20 ENVIRONMENTAL ARCHAEOLOGY https://doi.org/10.1080/14614103.2020.1829297 Dietary and Weaning Habits of the Roman Community of Quarto Cappello del Prete (Rome, 1st-3rd Century CE) Flavio De Angelisa, Virginia Veltrea, Sara Varanoa, Marco Rombonia, Sonia Renzia, Stefania Zingalea, Paola Riccib, Carla Caldarinic, Stefania Di Giannantonioc, Carmine Lubrittob, Paola Catalanod, Olga Rickardsa and Cristina Martínez-Labarga a a Centre of Molecular Anthropology for Ancient DNA Studies, University of Rome Tor Vergata. Via della Ricerca Scientifica 1, Rome, Italy; Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università degli Studi della Campania “Luigi Vanvitelli”, Caserta, Italy; cCollaborator of Servizio di Antropologia, Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, Rome, Italy; d Former Servizio di Antropologia, Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, Rome, Italy b ABSTRACT ARTICLE HISTORY This paper aims to provide the isotopic characterization of the diet consumed by people buried in a graveyard of the Imperial Rome Suburbium (1st–3rd centuries CE), where numerous children were buried. A sample of 50 human remains from Quarto Cappello del Prete was selected for carbon and nitrogen stable isotope analysis. Published data related to coeval faunal remains set the baseline of the diet. The results for humans were integrated with previously analyzed data from Quarto Cappello del Prete. The resulting sample of 71 people has been dissected for stratification according to demographics, focusing on the ability to ascertain the weaning process in children. Isotopic data are steady with an overall diet mainly based on terrestrial resources, where C3 plants played a pivotal role in the diet, though the δ13C range suggests that the foodstuff should have been heterogeneous. The remarkable amount of children allows us to evaluate the weaning process. Infants seem to be adequately weaned after 3 years, when they were considered as adults to what concerns the dietary habits. These data represent a valuable enhancement for understanding the weaning practices in ancient Rome, contributing to supporting the hypothesis about lifestyle and health in the Roman Imperial period. Received 30 January 2020 Revised 19 August 2020 Accepted 24 September 2020 Introduction The outstanding growth of the Biomolecular Archaeology (Brown and Brown 2013) makes this discipline currently able to address several archaeological questions. Indeed, the skeletal remains should be considered a plentiful archive of bio-cultural features related to human adaptations: bone tissue provides significant clues about several factors relating to the lifestyle of ancient populations. Isotopic evidence about organic and inorganic bone tissue components and archeogenomics are the leading biomolecular insights able to support interpretations about specific archaeological questions associated with dietary preferences, disease susceptibility, biodemography, mobility, and migration patterns. Recently, isotopic studies carried out on skeletal samples have provided an outstanding contribution to archaeology worldwide. Dietary habits reconstruction is one of the most focussed topics amid those that could be isotopically traced. Such a bioarcheological application relies on the analysis of bone collagen, the leading protein fraction of that tissue (Tzaphlidou 2008) or dentine (Sato et al. 2000). Dentine can trace CONTACT Flavio De Angelis flavio.de.angelis@uniroma2.it Vergata. Via della Ricerca Scientifica 1, 00133 Rome, Italy © Association for Environmental Archaeology 2020 KEYWORDS Diet; weaning practices; Romans; stable isotope analysis; Molecular Bioarcheology; Roman Suburbium the diet from a longitudinal perspective (Beaumont et al. 2013) through incremental sampling, though this strategy cannot always be accomplished for teeth preservation. Conversely, the carbon and nitrogen isotopic analysis of collagen recruited through a bulk sampling of the bones, reflects the latest years of life due to turnover rate (Fahy et al. 2017; Tsutaya and Yoneda 2013). The analysis of the ratios of carbon (13C/12C) and nitrogen (15N/14N) stable isotopes have been extensively used to identify dietary differences among groups (Ambrose 1986; Fernandes and Jaouen 2017; Katzenberg 1989; O’Brien 2015; Schoeninger 2014; Schwarcz 1991; Schwarcz and Schoeninger 2012; Tykot et al. 2009) in modern and archaeological communities. Carbon and nitrogen signatures derive primarily from consumed foodstuff and could, therefore, act as proxies for the identification of the diet regime. Specifically, carbon and nitrogen mostly derive from dietary proteins, even though carbon can also trace carbohydrates and lipids in low protein diets (Ambrose and Norr 1993; De Niro 1985). The carbon isotope ratio related to PDB (Pee Dee Belemnite) (δ13C) could be roughly used to Centre of Molecular Anthropology for Ancient DNA Studies, University of Rome Tor 2 F. D. ANGELIS ET AL. differentiate between the consumption of plants with different photosynthetic pathways (C3 vs. C4) (Tykot 2004). Furthermore, this marker enables the differentiation between terrestrial-based and marine-based resources in a C3 plant based-environment (Tykot 2004). Conversely, nitrogen isotope values related to AIR (δ15N) provide information about the trophic level of an individual with an offset of 3‰-5‰ being detectable rising through the trophic levels (Katzenberg 2008). Aquatic resources (both marine and freshwater) typically produce high trophic levels and enriched nitrogen ratios compared with terrestrial ones (Katzenberg 2008). Although the isotope analysis provides reliable information about the foodstuff consumption, several confounding factors should be borne in mind. Metabolic and physiological processes could bias the straightforward relationship between stable isotopes and diet reconstruction (Bocherens, Fizet, and Mariotti 1994; Cherel et al. 2005; Pecquerie et al. 2010; Reitsema 2013). Even though both carbon and nitrogen signals could vary, the δ15N appears to fluctuate more with individual metabolism and physiological status. Specifically, recent evaluations point out that the nitrogen isotopic signature does not straightforwardly represent the diet. The nitrogen routing and the differential metabolic cycling for source and trophic amino acid nitrogen are starting to unveil a very complex scenario where the elemental sourcing should be appropriately accounted for (O’Connell 2017 and reference therein). Actually, the nitrogen signature is considered a complex proxy influenced by trophic position, metabolic offsets depending on the different amino acid carriers, and physiological and environmental factors. Several stressors and diseases could affect the isotope ratios, leading to a slight enrichment or depletion of a specific isotope. This bias is a well-known concern for pathology-influenced isotopic fractionation, primarily affecting the δ15N (Katzenberg and Lovell 1999; Reitsema 2013). A negative nitrogen balance during heavy nutritional stress also increases δ15N values because body proteins are enriched in 14N and are preferentially degraded (Hobson, Alisauskas, and Clark 1993; Mekota et al. 2006; Walter et al. 2020). Accordingly, tissue growth leads to a drop in δ15N as the human body prefers to store 14N as amino acids (Waters-Rist and Katzenberg 2010). Similarly, one of the leading physiological confounding factors could be the breastfeeding (Beaumont et al. 2015; Fogel, Tuross, and Owsley 1988; Fuller et al. 2006) which can boost nitrogen-enriched values (∼2–3‰ compared to the feeders, usually the mother). This enrichment tends to drop during the weaning process, when other supplementary and transitional food (Tsutaya and Yoneda 2013) progressively integrates and replaces the breast milk. Accordingly, it could be possible to estimate the breastfeeding and weaning practices in subadults by measuring the isotope ratios of their bone collagen. This approach relies on the notion that an individual isotope dataset obtained through bulk sampling represents only one stage of the individual’s lifespan, so the availability of a consistent subadult sample could allow for the reconstruction of these practices in a cross-sectional mode (Tsutaya and Yoneda 2015). This cross-sectional approach has been widely used for gain information on infant’s subsistence (reviewed in Tsutaya and Yoneda 2015), even though other tracers and sampling strategies on incremental tissues were also developed to address individual longitudinal approaches (Beaumont et al. 2013; Henderson, LeeThorp, and Loe 2014) Archeological Context Imperial Rome was by far one of the largest cities of the European Antiquity. Despite the plentiful archaeological, cultural, historical and iconographical records, the biological profiles of the Imperial Age Romans (1st-3rd centuries CE) is still a partially tackled topic (Catalano 2015; De Angelis, Pantano, and Battistini 2015), though recently its population genomics (Antonio et al. 2019) has been unveiled. The heterogeneous genetic makeup of the Rome population kept abreast of the complex social and cultural arrangements that people could establish in a vast territory such as the Rome area and it’s Suburbium. Several estates and productive facilities were scattered throughout this area. The necropolises were also located outside the city walls by law, and are often related to self-sufficient communities, mainly constituted by working-class people whose lifestyle and subsistence were founded upon different strategies. The way Romans tackle the food supply was and continues to represent a fruitful exploration field. Historical and iconographical records provide a great deal of evidence of the variety of foodstuffs at least some of the Roman populace could use for edible purposes. Many studies have provided isotopic data to reconstruct people’s diet in communities living at the western fringe of the Suburbium (O’Connell et al. 2019; Prowse et al. 2005; Prowse et al. 2007; Prowse et al. 2008) or in peri-urban Christian catacombs (Rutgers et al. 2009; Salesse 2015; Salesse et al. 2014). Similarly, mounting evidence about the diet of commoners living close to the Aurelian walls and in the western Suburbium has started to accumulate (De Angelis et al. 2020; Killgrove and Montgomery 2016; Killgrove and Tykot 2013; Killgrove and Tykot 2018), bridging the gap to the comprehensive analysis of the diet in ENVIRONMENTAL ARCHAEOLOGY Imperial Rome. The dietary landscape provided for the whole city area (De Angelis et al. 2020) is heterogeneous. This reflects the multifaceted reality of the capital of one of the most influential Empires in Antiquity. Even though the central supply could account for the fundamental dietary requirements such as grain, wine, and oil, the environment played a critical role in food exploitation, especially for lower social classes. Indeed, the topographical location of the settlements, and the necropolises where people were buried, forced the dietary preferences towards stuff that people could easily obtain from their neighbourhood (De Angelis et al. 2020). Both literary sources and isotopic evidence agree that grains were a source of staple foods for Romans as they were primarily used as bread or puls, a pottage that could also be mixed with vegetables, meat, and cheese (De Angelis et al. 2020; Garnsey 1999; Prowse et al. 2005). Historical records are plentiful providing information about a wide variety of vegetables, fruits, and legumes eaten by Romans (Garnsey 1999). C4 plants such as millet are mentioned, as they were basically considered as a relief for food shortages. Even though their consumption could be variously preferred throughout the Roman territory (Boivin, Fuller, and Crowther 2012), only a few isotopic glimpses for their exploitation have been found (Killgrove and Tykot 2013). Even though the role of pulses cannot be ruled out (Garnsey 1999), meat represented the bulk protein reservoir (Brothwell and Brothwell 1998; MacKinnon 2004). Domestic animals were the primary resources, although venison consumption was not widespread but locally consumed (O’Connell et al. 2019). The role of fish in ancient Rome mainly depends on the social and ecological contexts (Prowse et al. 2005; Purcell 2003), even though some specificities could be pointed out for an increased preference among early Christians (Rutgers et al. 2009). Despite these overall patterns, the extreme social heterogeneity of communities in the Roman suburbs can hardly be framed in fixed and configured categories. Nonetheless, they could provide useful knowledge about the relationship between humans and their cultural and natural environments. Accordingly, this paper aims to provide insights into the diet consumed by people now buried in an archaeologically-defined context in Rome Suburbium. Quarto Cappello del Prete archeological complex was established in the far eastern suburbs of Rome (Figure 1), near the ancient city of Gabii (Musco, Petrassi, and Pracchia 2001), a formerly independent town under Roman rule. Its population contracted and the town was eventually abandonment in the late Imperial Age and early Middle Age (Becker, Mogetta, and Terrenato 2009). The site is characterised by monumental structures related to a water management system dated over a 3 wide time range starting from the 2nd century BCE. Porches and fountains probably once surrounded a large cistern and a pool. A vast paved area putatively related to a production facility extended north to the tank, and the ruins of a forge and several semicircular rooms were recovered not far from the monuments (Musco et al. 2011; Caspio 2009). A burial ground was set close to the memorial and productive areas in the Imperial Age (1st-3rd centuries CE), spreading up to an artificial hypogeal poly-lobed cavity that was first used as a quarry. An explorative isotopic survey about the dietary habits of people buried in Quarto Cappello del Prete showed that the low trophic level suggested by nitrogen signature put Quarto Cappello del Prete at odds with respect to other suburban cemeteries in Rome (De Angelis et al. 2020). Indeed, the dietary habit was not the sole peculiarity for Quarto Cappello del Prete. Remarkably, more than 70% of the buried people were infants and juveniles, and 50% of them were between 0–6 years old (De Angelis, Pantano, and Battistini 2015). This demographic stratification seems to be at odds with the most applied demographic models (Coale, Demeny, and Vaughan 1983; Coale and Guo 1989), suggesting a unique status for this burial ground. This uniqueness could be due to social grounds that behold the selective burial for people with shared social or pathological characteristics (Catalano 2015; De Angelis, Pantano, and Battistini 2015; Musco et al. 2011). Subadult skeletons are often a challenging sample for bioarcheologists to analyse despite their relevance for ascertaining primary aspects of the lifestyle and health of ancient populations. They could often be buried in different places than adults or could be easily degraded because of their fragile nature and small size. The large-scale frequency of people who died in early childhood and are buried in Quarto Cappello del Prete could support the paucity of isotopic evidence of breastfeeding and weaning in Rome (Killgrove and Tykot 2013; Prowse et al. 2008; Rutgers et al. 2009). Even though several historical sources dealt with the use of wet nursing and addressed the breastfeeding in an allegorical or symbolic way rather than realistically (Challet 2017), the information of such practices in ancient Rome is quite rare. The isotopic data provided for Quarto Cappello del Prete could meaningfully improve our understanding of childhood lifestyle and health in the Roman Imperial period. Thus, the present paper aims to contribute to the reliable identification of dietary habits of people buried in Quarto Cappello del Prete by combining the data with previously collected ones (De Angelis et al. 2020), and focussing on the identification of the weaning period in the most extensive Roman children cohort so far analyzed. 4 F. D. ANGELIS ET AL. Figure 1. Topographic location of the necropolis of Quarto Cappello del Prete (QCP). Materials and methods Sample The present survey focuses on 50 skeletons from burials recovered at Quarto Cappello del Prete (QCP2) burial ground and dated back to the Imperial period (1st-3rd centuries CE). Despite the fact that socioeconomic composition of the burial community is unknown, most of the burials were made in the soil and just under half of them were buried with artifacts. A sample of 50 rib bones was selected for carbon and nitrogen stable isotope analysis. The visible preservation status of the ribs was the leading inclusion criterion for being selected. Information on individual sex and age at death were available from previous studies allowing us to select samples from 10 adults (5 males and 5 females) and 40 children (Catalano 2015). This sample will be integrated with a previously analyzed sample (QCP; De Angelis et al. 2020) to improve the representativeness of the diet reconstruction. The skeletally immature individuals were considered regardless of the sex as its estimation in children remains problematic, but a detailed age at death assessment has provided for each individual. The baseline for the terrestrial protein component of the diet was set using coeval faunal remains recovered from excavations at Rome and Portus for the same chronological frames. These published data were downloaded from the IsoArcH database in several queries performed on or before October 30th, 2019 (O’Connell et al. 2019; Prowse et al. 2005; Salesse et al. 2018). These data were supplemented by 17 coeval faunal remains recovered from recent excavations in Rome (De Angelis et al. 2020). Analytical Methods Carbon and nitrogen stable isotope analysis was performed on the organic fraction of human remains. The collagen extraction was individually performed following Longin’s protocol modified by Brown and colleagues (1988), 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 powder collected by drilling the bones. A concentration step was also carried out for all the samples to enhance the collagen yield through 30 kDa Amicon® Ultra-4 Centrifugal Filter Units with Ultracel® membranes. Each sample of collagen extract weighed 0.8-1.2 mg and was analyzed using an elemental analyzer isotope ratio mass spectrometer at the iCONa (isotope Carbon, Oxygen and Nitrogen analysis) Laboratory of the University of Campania. Carbon (δ13C) and nitrogen (δ15N) stable isotope ratios were measured in a single run on a Delta V Advantage isotope ratio mass spectrometer coupled to a Flash 1112 Elemental Analyser via a Conflow III interface (Thermo Scientific Milan, Italy). Results were expressed in δ notation (Coplen 1995) and reported in permille units. The measurements of δ13C were calibrated to the international standard VPDB with the standard reference materials IAEA-CH3, IAEA-CH6 and stable isotope ratio facility for environmental research at the University of Utah (SIRFER) yeast; δ15N measurements were calibrated to the international standard AIR 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 δ13C and 0.2‰ for δ15N. 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 (Ambrose 1990; DeNiro, 1985; Van Klinken 1999). ENVIRONMENTAL ARCHAEOLOGY Carbon content ranged from 15 to 45, nitrogen content between 5 and 15% (Ambrose 1990), and, finally, an atomic C/N ratio of between 2.9 and 3.6 (DeNiro 1985) represented the thresholds for considering collagen suitable for downstream evaluations. Descriptive statistics and comparison tests were performed by R v.3.6.1 (R Core Team 2017) The linear mixing model provided by Fraser et al. (2013) was used to detect the humans’ role compared to the available ecological resources. This model uses the midpoint and the offsets between consecutive trophic levels to identify the effect of putative prey on their predators. Thus, the information based on faunal remains is sorted according to typology (herbivores, omnivores, marine resources, freshwater organisms), and human data are plotted together in order to detect dietary habits. A Bayesian model for diet reconstruction (FRUITS v. 3.0; Fernandes et al. 2014) was also employed to compare the linear mixing model outcome. The model allows for dietary inputs estimation and incorporates food macronutrients, elemental composition, and isotopic composition. Average isotopic values represent the human (consumer) data. The total nitrogen in collagen was set up as deriving from dietary protein, and the carbon composition of bone protein was set to reflect an average of 3:1 ratio (74 ± 4% and 26%) of dietary protein to energy (Fernandes, Nadeau, and Grootes 2012). Three source groups were firstly hypothesised: meat, C3 plants, and freshwater resources. Meat isotope values were averaged for the faunal isotopic values considered in this study. There are not isotopic data for grains in the Rome area to the best of our knowledge, leading us to consider diachronic endmembers for diet reconstruction. Accordingly, we have considered the grain data from Gismondi et al. (2020) for an early medieval site close to Rome. Similarly, the freshwater fish remains in Rome are nearly totally absent, and their isotopic data are lacking. Data pertaining to this class could have been collected for two geographically far diachronic samples from preRoman Britain (Jay 2008) and the late-Roman Province of Pannonia (Hakenbeck et al. 2017), or for extant lacustrine species from the Alps (Dufour, Bocherens, and Mariotti 1999). However, the ecological differences among freshwater resources are known to drive their isotopic heterogeneity (Dufour, Bocherens, and Mariotti 1999). Accordingly, the unavailability of local endmembers and the lack of evidence for their consumption in QCP, lead us to exclude this class from the Bayesian mixing model analysis. Macronutrient composition for the stuff was estimated according to comparable food in the USDA National Nutrient Database for Standard Reference (Ahuja et al. 2017). Dry weights for each 100 g of reference food were estimated, and the protein and energy (lipids and carbohydrates) compositions of dried food 5 were calculated and converted to a percentage. The elemental composition of each macronutrient group was based on Morrison et al. (2000). Lastly, isotopic offsets between measured foodstuff isotope values and the isotopic values of specific macronutrients were established according to Tieszen (1991). Results The collagen extraction was performed for 50 samples, but the preservation status of the extracted collagen was unsuitable for 16 individuals (Table 1). The faunal δ13C values are related to a C3 ecosystem (De Angelis et al. 2020), and the δ15N signatures are consistent with those expected for the identified species (Figure 2). The descriptive statistics for δ13C and δ15N for humans were reported in Table 2. Discussion The present survey comprises the isotopic analysis of 50 individuals recovered in Quarto Cappello del Prete. The distinctive demographic feature of Table 1. Individual results for humans, with their demographic characteristics (De Angelis, Pantano, and Battistini 2015). F: female; M: male; ND: gender not available. Samples passing the quality control for collagen quality were listed. Tomb Sex Age class at death years QCP-4 QCP-5 QCP-10 QCP-11 QCP-19 QCP-30 QCP-32 QCP-33 QCP-42 QCP-46 QCP-47B QCP-51 QCP-54 QCP-55 QCP-56 QCP-59 QCP-63 QCP-64 QCP-65 QCP-67 QCP-72 QCP-73 QCP-82 QCP-4 QCP-5 QCP-6 QCP-7 QCP-11 QCP-13 QCP-US 118 QCP-8B QCP-14 QCP-15 QCP-17 ND ND ND ND ND M ND ND ND ND ND ND ND ND ND ND ND F ND ND ND ND ND F ND ND ND ND F M 0–6 0–6 0–6 0–6 0–6 20–29 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 13–19 20–29 0–6 0–6 0–6 0–6 07–12 30–49 07–12 0–6 07–12 0–6 30–49 30–49 38.3 33.3 31.7 46.2 41.8 41.3 41.8 40.8 32.4 39.0 57.0 36.9 46.7 44.2 31.3 33.0 33.3 40.9 39.9 36.3 43.6 32.1 46.7 35.2 33.8 31.4 33.9 45.4 42.9 43.2 13.8 12.2 10.2 15.9 14.7 14.8 15.2 14.7 11.2 14.0 20.3 12.9 16.6 15.9 11.3 11.4 12.0 13.4 14.5 13.0 14.9 11.5 16.0 12.7 12.2 11.2 11.7 16.3 15.2 15.0 3.2 3.2 3.6 3.4 3.3 3.3 3.2 3.3 3.4 3.2 3.3 3.3 3.3 3.2 3.2 3.4 3.2 3.6 3.2 3.3 3.4 3.3 3.4 3.2 3.2 3.3 3.4 3.3 3.3 3.4 −19.1 −18.4 −18.9 −18.4 −18.2 −19.3 −19.0 −18.8 −18.4 −18.4 −18.4 −18.9 −18.9 −19.4 −19.5 −18.7 −19.2 −19.6 −18.6 −18.7 −19.6 −19.4 −20.0 −19.5 −19.4 −18.9 −19.3 −19.3 −19.2 −19.1 11.0 12.6 13.1 11.4 12.9 8.4 10.8 12.7 12.5 13.5 13.5 12.2 10.5 10.5 8.4 13.2 10.0 11.2 10.5 11.7 9.9 10.1 8.6 7.4 8.4 13.0 9.3 6.4 10.5 10.0 ND ND ND ND 0–6 0–6 0–6 07–12 48.8 35.6 41.2 30.1 16.9 12.9 14.7 10.2 3.4 3.2 3.3 3.6 −19.2 −18.6 −18.9 −19.4 9.4 10.2 12.2 8.6 %C %N C/ N δ13C ‰ δ15N ‰ 6 F. D. ANGELIS ET AL. Figure 2. Plot for δ13C than δ15N values for humans from QCP2 and faunal remains collected in the Roman area. the necropolis results in a skewed ratio between 10 adults and 40 children to resemble the general buried population. Collagen preservation precludes 16 samples from further analysis. Because the overall demographic distribution of people buried in the necropolis is reflected by the high proportion of immature people, it is difficult to determine if the Table 2. Descriptive statistics for the 34 individuals analyzed in this survey showing suitable collagen quality. These statistics are the minimal value (min), the maximal value (max), the range (range, that is, max-min), the median (median), the mean (mean), the standard error on the mean (SE.mean), the confidence interval of the mean (CI.mean) at the p=0.95 level, the variance (var), the standard deviation (Std.dev) and the variation coefficient (Coef.var) defined as the standard deviation divided by the mean. δ13C ‰ size min max range median mean SE.mean CI.mean var Std.dev Coef.var δ15N ‰ 34 −20.0 −18.2 1.8 −19.0 −19.0 0.1 0.2 0.2 0.4 0.0 6.4 13.5 7.1 10.5 10.8 0.3 0.7 3.5 1.9 0.2 diet of the whole population is represented therein. Accordingly, a previously analyzed sample from Quarto Cappello del Prete (hereafter named as QCP; De Angelis et al. 2020) comprising 20 skeletally immature individuals and 17 adults was appended to the present dataset to depict a more robust diet reconstruction for the people pertaining to that burial ground (Figure 3). This explorative survey (QCP) identified the dietary habits of people buried in Quarto Cappello del Prete and suggested that their diet was mainly based on C3 plant foodstuff or pulses (De Angelis et al. 2020). A statistical evaluation was performed in order to establish the differences between the two samples. The δ15N distributions are significantly similar (t= −1.67, p=0.05), while a different distribution could be highlighted for δ13C datasets (t=−2.12, p=0.02). Since the samples pertain to the same archaeological context and the sampling strategy was driven only by the preservation status of the ribs, these differences seem to be apparently unusual to address. Nevertheless, the different diet between adults and children could be one of the confounding factors responsible for these differences. Thus, the observed diversity was analyzed with respect to the demographic composition of the two samples in adult/children ratios. ENVIRONMENTAL ARCHAEOLOGY 7 Figure 3. Values distribution, where the shape of the indicator is dependent on the age class of each individual: the squares are referred to skeletally immature individuals while the triangles related to adults. QCP: data from De Angelis et al. 2020; QCP2: data for current sample. Only 5 adults are analyzed in QCP2 but they highlight a narrow dispersion of δ13C values (δ13C range=0.49) that is not consistent with a broad δ15N distribution (δ15N range=3.81). The skeletally immature fractions seem to be roughly overlapping for both the dietary proxies, even though the bimodal distribution of QCP2 δ15N values (N=29) seems to be shift towards more positive values. A proper evaluation of these differences was performed to compare the variances in δ13C and δ15N between QCP and QCP2 through the Levene’s test. This test suggests that the two variances are comparable, and consequently, we could move to test the influence of a single factor (site or dichotomic age class -adults vs. children) through ANOVA. This analysis shows that age classes (adults vs. children) do not determine the difference on their own (F=2.80; p=0.10), while the sample (QCP vs. QCP2) represents a critical factor (F=4.63; p=0.03) for δ13C differences. Otherwise, the age class underpins the differences in δ15N comparison between the samples (F=5.12; p=0.03). Accordingly, we have compared the data related to both the adults and the skeletally immature people of each sample. The distributions of the two adult fractions have been reliably compared due to the homogeneity of their variances (Levene’s F: 3.05; p=0.1 for δ13C; Levene’s F: 0.09; p=0.76 for δ15N) and the pairwise test comparisons definitively support that the adults in QCP and QCP2 pertain to a unique sample without stratification (F=0.00, p=0.98 for δ13C; F=0.01, p=0.92 for δ15N). The adult sample’s gender dissection due to osteological data does not support a different diet between males and females in the whole adult sample (F=0.89; p=0.36 for δ13C; F=0.74; p=0.40 for δ15N), even though the restricted samples size (12 males and 10 females) could bias this suggestion. Similarly, the age at death estimation for each adult individual leads us to cluster people according to age classes (20-29 years, 30–49 years and more than 40 years), but this clustering does not return significant 8 F. D. ANGELIS ET AL. differences both in δ13C and δ15N (F=0.10; p=0.99 for δ13C; F=1.20; p=0.36 for δ15N). Thus, the adult people from the two Quarto Cappello del Prete sub-samples should be considered as a whole sample, and no differential clustering according to sex or age at death could be established. The same approach was carried on for the skeletally immature people of the two sub-samples (20 children in QCP vs. 29 children in QCP2). Both δ13C and δ15N distributions show similar variances between QCP and QCP2 (Levene’s F = 0.10, p=0.76 for δ13C; Levene’s F = 0.18, p=0.67 for δ15N) and appear to be similar (Ttest p value=0.1 for δ13C and T-test p value=0.34 for δ15N). Therefore, since the adults and children are homogeneous in the sub-samples respectively, we can proceed to merge QCP and QCP2. Accordingly, a whole sample of 71 individuals from Quarto Cappello del Prete (37 from QCP and 34 from QCP2) has been achieved, with basic descriptive statistics listed in Table 3. The adult and skeletally immature people show different values distribution both in δ13C and δ15N (Figure 5), and the higher values for children could represent the legacy of the breastfeeding. The skeletally immature sample was split into two classes according to the possibility of being related to breastfed infants rather than already weaned children. According to historical and bioarcheological data for ancient Rome, the threshold to consider the children dependent on the mothers or the nurses was set to 3 years (Dupras, Schwarcz, and Fairgrieve 2001; Fulminante 2015). This clustering suggests two groups comprising 18 individuals who putatively share weaning (3+ years at death) and 31 children that could be breastfed yet at the time of their decease (Table 4) and results in Table 3. Descriptive statistics for the whole sample for Quarto Cappello del Prete (34 individuals from this survey and 37 from De Angelis et al. 2020). These statistics are the minimal value (min), the maximal value (max), the range (range, that is, maxmin), the median (median), the mean (mean), the standard error on the mean (SE.mean), the confidence interval of the mean (CI.mean) at the p=0.95 level, the variance (var), the standard deviation (Std.dev) and the variation coefficient (Coef.var) defined as the standard deviation divided by the mean. δ13C ‰ Size min max range median mean SE.mean CI.mean var Std.dev Coef.var δ15N ‰ 71 −20.5 −18.2 2.4 −19.2 −19.1 0.1 0.1 0.2 0.5 −0.03 6.4 14.3 7.9 10.2 10.4 0.2 0.4 3.1 1.7 0.2 significant differences both in δ13C and δ15N (Ttest=2.78, p=0.01 for δ13C; T-test=5.40, p=0.01 for δ15N). Thus, the skewed isotopic data in the Quarto Cappello del Prete sample seem to be due to the breastfeeding effect (Fuller et al. 2006) and influencing a large fraction of the analyzed individuals (31 over 71 samples) (Figure 4). These results agree with the previous studies, which suggests the weaning rarely occurred later than 4 years of age in pre-industrialized societies (Beaumont et al. 2013; Britton et al. 2015 and reference therein). The small amount of isotopic data pertaining the nursing in ancient Rome and the neighbouring eastern suburbs are consistent with this age threshold (Killgrove and Tykot 2013; Killgrove and Tykot 2018; Rutgers et al. 2009) while the western Table 4. Age of death for the skeletally immature samples. + indicates people more than 3 years old, - stands for people less than 3 years. The midpoint of the Age range was set as detailed estimation. Site QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 QCP2 Sample 3+ years at death QCP3 QCP5 QCP7 QCP10 QCP13 QCP15 QCP16 QCP18 QCP19 QCP20 QCP23 QCP24 QCP25 QCP27 QCP30 QCP31 QCP34 QCP36 QCP37 QCP39 4 5 10 11 19 US898 32 33 42 46 47B 51 54 55 56 59 63 65 67 72 73 82 5 6 7 11 8B 14 15 17 + + + + + + + + + + + + + + + + + + Age in years, if not specified 0–6 7–12 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 0–6 13–19 0–6 0–6 7–12 13–19 0–6 months 0–1 0–6 months 3–9 months 12–16 months 1–2 6–9 months 1–2 8–9 months 0–3 months 3–9 months 0–6 months 1–2 2–6 1,5-2,5 13–19 9–12 months 0–6 months 3–5 3–4 10–15 7–12 1,5-2,5 10–12 4–6 0–6 months 0–5 months 3–4 6–8 ENVIRONMENTAL ARCHAEOLOGY 9 Figure 4. a) Violin plots for adult and children δ13C distributions in each sample. QCP: De Angelis et al. 2020, QCP2: current sample. b) Violin plots for adult and children δ15N distributions in each sample. QCP: De Angelis et al. 2020, QCP2: current sample. Figure 5. a) Violin plots for adult and children isotopic δ13C values distributions for the whole sample from Quarto Cappello del Prete, with the box plot embedded in the violin. b) Violin plots for adult and children isotopic δ15N values distributions for the whole sample from Quarto Cappello del Prete, with the box plot embedded in the violin. The line inside the boxes represents the median value. communities seem to wean the children earlier (Prowse et al. 2008; Prowse et al. 2005). Even though the nitrogen fractionation relies on the above mentioned extremely complex assumptions (see the introduction section and O’Connell 2017), in this survey, we could not leverage the single amino acid nitrogen signature to dissect their metabolic implications, so we were forced to consider the bulk nitrogen data. The comparison of the infant δ15N values and those from the female adult individuals could be meaningful for the weening practices reconstruction. The median for 0–3 years infants is higher than that of adult females (11.7 vs. 9.1; interquartile range 2.3 vs. 1.7: Wilcoxon rank-sum test W: 270; p<0.01) supporting the above interpretation. Infants of 0–3 years are also different than children of 3+ for the δ15N distributions (median 11.7 vs. 8.8; interquartile range 2.3 vs. 1.5: Wilcoxon rank-sum test W: 492; p<0.01). Remarkably, the 3+ children values are aligned to those related to adult females (8.8 vs. 9.1; interquartile range 1.5 vs. 1.7: Wilcoxon rank-sum test W: 91; p=0.9). This evidence supports that breastfeeding was performed for up to 3 years. Then the children were soon aligned to the adult dietary habits, where supplemental and transitional food progressively replaced the breastmilk. The mother’s breastmilk is known to represent a critical aid for both the mother and kids, both in the short term and the longer term, as it represents a critical factor for the nursing infant’s endurance and wellbeing during its early life. Indeed, the breastmilk 10 F. D. ANGELIS ET AL. Figure 6. Linear model for identify prey-predator relationship. th: terrestrial herbivore; thc: terrestrial herbivore consumer; to: terrestrial omnivore; toc: terrestrial omnivore consumer; fwE: freshwater fish form England; fwP: freshwater fish form Pannonia; fish: marine fish. The dashed lines define consumers’ boxes. QCP: whole sample from Quarto Cappello del Prete. should not be considered only a food resource, but rather a complex fundamental nutritional compound providing antibodies, enzymes, hormones, and vitamins, all of which have health benefits for the infants (Binns, Lee, and Low 2016). At the same time, the children feeding practice could represent one of the most important health indicators in ancient human populations (Lewis 2007). In this perspective, the broadening of the knowledge about the timing of breastfeeding and weaning practices in ancient Rome could underpin the discussion about the lifestyle and health of the Roman population. However, several confounding factors should be born in mind when dealing with this topic in an archeological frame. Firstly, the ‘osteological paradox’ (Wood et al. 1992) advises against the analysis from archaeological skeletal series that are not integrally comparable to the health status and life history of past populations. The inter-individual heterogeneity in the risk of death and disease susceptibility could represent a misleading factor, as the analyzed samples represent individuals who died at an early age. Accordingly, the isotopic signals from infant skeletons could determine the dietary patterns in dead individuals rather than those in people who survived into adulthood. Thus, since breastfeeding and weaning practices should be considered closely connected with health and survival, they could be biased by putative atypical diet and weaning strategies. Secondly, some of the weaned people could retain some prior Figure 7. Bayesian diet reconstruction for adults and putatively weaned children by FRUITS. Boxes represent a 16th to 84th percentiles credible interval (corresponding to 68%) while the whiskers represent a 95% credible interval (corresponding to the 2.5th and 97.5th percentiles). The horizontal solid line represents the estimated mean while the dashed line represents the estimated median (50th percentile). ENVIRONMENTAL ARCHAEOLOGY isotopic signature due to the lag times originating from collagen turnover in bones. To tackle this issue, we sampled rib bones that show high turnover rate being bones with a greater surface to volume ratios (Tsutaya and Yoneda 2013). These bones show an extremely high turnover rate for both collagen and bone minerals until the late-teens ages. It has been suggested that a children’s rib bone collagen can be used to proficiently address the nitrogen signatures with the shortest lag time respect to the adults (Tsutaya and Yoneda 2013, and reference therein). Thus, it appears reasonable considering the difference between infants 0–3 and 3+ in Quarto Cappello del Prete to be stem from the dietary shift people experienced with the weaning. The isotopic data of Quarto Cappello del Prete is consistent with the suggestions provided by the explorative sample previously analyzed (De Angelis et al. 2020) and it indicates that people consumed a roughly varied diet based on C3 plant backbone resources. In contrast, exclusive C4 plant exploitation seems to be excluded. This outcome confirms the overall disregard of the C4 plants in the human diet in the ancient Rome area, where only a few data suggest people could have used the millet as a dietary source (Killgrove and Tykot 2013). Indeed, no C4 plants remains have been ever found in archeological surveys at Rome, even in massive trading place such as Portus, the maritime harbour of Rome (O’Connell et al. 2019). No direct evidence of exclusive marine or freshwater resource intake could be determined due to the δ13C distribution values. The model proposed by Fraser et al. (2013) and subsequently used by Fontanals-Coll et al. (2017) and De Angelis et al. (2019) was used to gain information about the diet reconstruction based on the bulk elemental signatures. It can define the average and variance values in the dietary proxies for herbivores and omnivores from available faunal data. The dispersion measures enable us to draw boxes where all the faunal values fall. These boxes are then moved in order to account for the predator-prey offsets, which were estimated as +1‰ for δ13C and +4‰ for δ15N, the latter deriving from the rough median value between two trophic levels in the bulk elemental analysis (Fraser et al. 2013). Data on freshwater resources have been recovered in the literature (De Angelis et al. 2020, and references therein) and are only tentatively used due to the known isotopic variation related to different ecological parameters which could lead to misinterpretation (Dufour, Bocherens, and Mariotti 1999). The determination of the consumers’ boxes indicates that the majority of individuals fall inside the boxes built for herbivore consumers and omnivore consumers (dashed lines in Figure 6). 11 The values from Quarto Cappello del Prete suggest a diet that was based on C3 plants and their consumers, even though the prey’s signatures are not provided by local faunal remains, that were not recovered at the site. To the best of our knowledge, the coeval faunal data pertaining to foodstuff consumed in western Rome and Ostia territory could proficiently represent the ecological background of eastern Suburbium, where no faunal data have been obtained to date. Indeed, no evidence for ecological or culture-derived faunal dispersal could be claimed among the different sites in the Rome area. Despite the presence of a large amount of freshwater and suitable structures for managing it (Musco, Petrassi, and Pracchia 2001), the clues for the exploitation of lacustrine or riverine species seem to be totally lacking. Furthermore, fish remains are missing in the necropolis as well as any fishing equipment to support the exploitation of this putative prey. The diet reconstruction based on Bayesian modelling agrees with the linear mixing model. In order to account for different resources, we have collected isotopic data to enrich the endmembers to be used in the diet reconstruction. Specifically, we used the grain data collected by Gismondi et al. (2020) for an early medieval site close to Rome. The diet reconstruction via FRUITS software was performed for the adults and 3+ children, which could be considered as dietary-consistent with them. The Bayesian evaluation enhances the pivotal role of a C3 plant-based diet complemented by faunal-derived protein (Figure 7). Nevertheless, the results presented for Quarto Cappello del Prete, along with its skewed demographic, and the presence of valuable grave goods and equipment scattered throughout the graveyard (Caspio 2009; Musco et al. 2011), pave the way to consider the burial ground not related to a community strictly deployed in local farming activity. The archaeological records suggest the necropolis was established in a cultic site (Catalano 2015; Musco, Petrassi, and Pracchia 2001). People from different communities could have been come therein from different locations and assimilated into Quarto Cappello del Prete necropolis after their death. The recovery of specific pottery items in the area as well as some specific miniaturised grave goods, and the identification of funeral structures such as the cupae burials support the proposed interpretation (Caspio 2009; Musco et al. 2011; Musco and Catalano 2010; Tomei et al., 2006). Mounting genomic pieces of evidence in an ancient DNA analysis for some individuals are consistent for proposing that people buried in Quarto Cappello del Prete could also share some biological characteristics that could be related to genetic-related osteo-dysmorphias (De Angelis, Pantano, and Battistini 2015). 12 F. D. ANGELIS ET AL. The biomolecular evidence related to both isotopic results and ongoing genomic hints could be consistent with the interpretation of Quarto Cappello del Prete archeological complex. Accordingly, it should be definitively considered a necropolis that could have served as a burial site for selected people. Their diets would have been varied, as suggested by the δ13C range. The premature death of several children buried in this cemetery seems to be related to endogenous factors rather than poor lifestyles. Indeed, they were characterised by a high trophic level that could be ascribed to breastfeeding. It is well known that breastmilk is, and it was in the past too, a nearly perfect mix of nutrients (protein, vitamins, and fatty acids) that babies need to grow (Nolan, Parks, and Good 2020), as well as immunological factors that help them for fighting off viruses and bacteria (Abrams and Chan 2019). The large number of the skeletally immature people buried in Quarto Cappello del Prete lacked specific osteological markers related to infectious diseases but several hypovitaminosis markers could be detected as bone thickness (De Angelis, Pantano, and Battistini 2015; Pasqualini et al. 2016). A farming-related diet should have quickly fulfilled the nutritional requirements, so the etiological factors related to those deficiencies I could relate to other characteristics, and among them, the genetic alterations cannot be ruled out. Conclusions The paper outlines the dietary habits of people buried in a monumental area in the eastern fringe of the Roman Suburbium, dating back to the 1st-3rd century CE. Despite the necropolis located outside the city walls, it seems to be devoted to hosting people that seem unfamiliar with intensive farming activities such as those characterising the ancient villae rusticae. The demographic profile and the presence of peculiar grave goods, along with the distinctive location, make Quarto Cappello del Prete a worthwhile skeletal sample to be well studied. The biomolecular evaluation provides a unique glimpse into their lives and supplies critical information about their relationships with food, weaning practices, and the neighbouring environment. The dietary landscape we provide is heterogeneous but almost entirely founded on C3 plants and their primary consumers. Despite the presence of a large amount of freshwater and suitable structures for managing it, any clues for the exploitation of lacustrine or riverine species are totally lacking. A significant bulk of data related to breastfeeding and weaning practices has been provided. Infants underwent to breastfeeding, and then they were suddenly shifted to an adult diet, with short lag time for the introduction of specific transitional food. The pivotal role of breastfeeding seems to be noticeable in skeletally immature people under 3 years. This data supports the small amount of isotopic information about the Roman area in the Imperial Age, providing the largest children cohort at Rome up to date. The adults shared the same diet, whatever being their gender or age. Indeed, heterogenous C3-based foods were eaten due to the range in δ13C, paving the way to consider the burial ground as a cemetery for people characterised by multiple diets and perhaps multiple origins. The information provided herein represents a step toward the catching on the organisation of this ancient cemetery. This information will be complemented by strontium and oxygen isotopic data that could bring to light a real collective nature of this burial ground, where people suffering from osteological alterations seem to be hosted. Acknowledgements Authors would acknowledge the Superintendent Daniela Porro on behalf of the Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, and Cristina D’Agostini as current Director of the Anthropology Section of this Superintendence for granting the access to skeletal material. Authors are grateful to the reviewers for the suggestions aimed to improve the quality of the manuscript. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This work was supported by the Italian Ministry of Education, Universities and Research (MIUR) through PRIN 2015 (Diseases, health and lifestyles in Rome: from the Empire to the Early Middle Age, Grant ID: 2015PJ7H3K) allotted to CML and Project N.: 85201715143. ‘Genomica e dieta: evoluzione e obiettivi della valutazione nutrizionale (GEDEON)’ funded by Regione Lazio – Bando regione Lazio progetti di gruppi di ricerca allotted to OR. Notes on contributors Flavio De Angelis is a bioarchaeologist and molecular anthropologist at the University of Rome Tor Vergata, Rome, Italy. Virginia Veltre is a Ph.D. candidate in Evolutionary Biology and Ecology at the University of Rome Tor Vergata, Rome, Italy. Sara Varano is a molecular anthropologist at the University of Rome Tor Vergata, Rome, Italy. Marco Romboni is a bioarchaeologist at the University of Rome Tor Vergata, Rome, Italy. Sonia Renzi is an MSc candidate at the University of Rome Tor Vergata, Rome, Italy. ENVIRONMENTAL ARCHAEOLOGY Stefania Zingale is an MSc candidate at the University of Rome Tor Vergata, Rome, Italy. Paola Ricci is an archaologist at Università degli Studi della Campania “Luigi Vanvitelli”, Caserta, Italy. Carla Caldarini is an anthropologist collaborator of Servizio di Antropologia, Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, Rome, Italy. Stefania Di Giannantonio is an anthropologist collaborator of Servizio di Antropologia, Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, Rome, Italy. Carmine Lubritto is Professor of Applied Physics at Università degli Studi della Campania “Luigi Vanvitelli”, Caserta, Italy. Paola Catalano is former Director of Servizio di Antropologia, Soprintendenza Speciale Archeologia, Belle Arti e Paesaggio di Roma, Rome, Italy. Olga Rickards is Full Professor of Molecular Anthropology at the University of Rome Tor Vergata, Rome, Italy. Cristina Martinez–Labarga is bioarchaeologist and Professor of Forensic Anthropology at the Department of Biology of the University of Rome Tor Vergata. ORCID Cristina Martínez-Labarga 0439-0379 http://orcid.org/0000-0003- References Abrams, E. M., and E. S. Chan. 2019. “It’s Not Mom’s Fault: Prenatal and Early Life Exposures That Do and Do Not Contribute to Food Allergy Development.” Immunology and Allergy Clinics of North America 39: 447–457. 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