Exploring prehistoric plant use by molecular analyses of Neolithic grave goods

Vegetation History and Archaeobotany https://doi.org/10.1007/s00334-023-00910-8 ORIGINAL ARTICLE Exploring prehistoric plant use by molecular analyses of Neolithic grave goods Alessia D’Agostino1,3 · Gabriele Di Marco1 · Mario Federico Rolfo2 · Antonella Canini1 · Angelo Gismondi1 Received: 25 July 2022 / Accepted: 19 December 2022 © The Author(s) 2023 Abstract At the site of Grotta Mora Cavorso (Lazio, Italy), an unusual archaeological find, made of two coarse pottery vessels, was recovered from burial levels radiocarbon dated to 6,405–6,275 bp. These artefacts were analysed using several methods, for interpretation of the cultural practices of the earliest inhabitants in central Italy. This first molecular evidence about the potential processing and storage of poppy-based products in Neolithic pottery was obtained by detecting ancient DNA (aDNA) and chemical compounds. This study represents the second evidence from the Mediterranean area of the use of Papaver L. (poppies), although the actual use(s) of these plants then, for example as sedatives, drugs, or food, remains uncertain. Also, the employment of Olea europaea L. (olive) derivatives in foods or for other purposes was suggested, in agreement with the recovery of fruit stones at the site. The results of the present archaeobotanical investigation show the environmental knowledge of the first prehistoric communities living in central Italy, who might have shared their ethnobotanical practices. Keywords Pottery · Ancient DNA · DNA barcoding · Olea europaea · Papaver sp. · Prehistory · Secondary metabolites Introduction The arrival of farming in the central and western Mediterranean area and the possible medicinal or ritual uses of plants are key issues of European prehistory; however, these topics are still relatively poorly understood, especially the level of interaction between different groups of people and diversity in the subsistence economy. In this context, ancient pottery surely represents an interesting source of data, and finding a way to successfully analyse this archaeological record allows scientists to provide important insights about past cultural traditions. Communicated by F. Antolín. * Angelo Gismondi 1 Laboratory of Botany, Department of Biology, University of Rome “Tor Vergata”, Via Cracovia 50, 00133 Rome, Lazio, Italy 2 Department of History, Culture and Society, University of Rome "Tor Vergata", Facoltà di Lettere e Filosofia Building B—4th Floor, Via Columbia 1, 00133 Rome, Italy 3 PhD Program in Evolutionary Biology and Ecology, Department of Biology, University of Rome "Tor Vergata", Rome, Italy Shape, archaeological context and association with other items are the main elements to suggest the uses of pottery (Evershed 1993; Eerkens 2007; Radford 2019). However, in the last few decades, new analytical approaches such as the use of chromatography for studying the organic residues preserved in archaeological artefacts have provided data on human activities as quantitative and qualitative results (Evershed 1993; Eerkens 2007; McGovern and Hall 2016; Dunne et al. 2017; Luong et al. 2017, 2018; Roffet-Salque et al. 2017; Smith et al. 2018; Radford 2019; Demirci et al. 2020; Pecci et al. 2020). One of the most critical issues in such types of investigation is the degradation process which affects biomolecules (Whelton et al. 2021). Chemical and physical factors of the depositional environment may affect the organic residues in different ways. Among all organic compounds, lipids are the most common to be found, due to their great resistance to deterioration. However, the study of fatty acids provides a limited amount of information and their level of identification is usually restricted (Evershed 1993; Eerkens 2007; Radford 2019). The potential of genetic analyses for solving long-standing questions in archaeology is increasingly being used. Ancient DNA (henceforth aDNA) has been found to be well preserved in ancient remains and on artefacts, including stone tools and surfaces of unglazed pottery (Pääbo 1989; 13 Vol.:(0123456789) Vegetation History and Archaeobotany Hofreiter et al. 2001; Shanks et al. 2005; McGovern and Hall 2016). Pottery, in particular, can absorb compounds and then protect them from outside contamination (Evershed 1993; Eerkens 2007; Hansson and Foley 2008; Foley et al. 2012; Robinson et al. 2017; Radford 2019). To date, no research has been done on plant aDNA extracted from early Neolithic pottery assemblages from the Mediterranean area, in contrast to more recent periods (Hansson and Foley 2008; Foley et al. 2012). On the other hand, several studies have been carried out on other ancient biomolecules, mainly lipids and proteins, isolated from similar materials (Dunne et al. 2017; Chowdhury et al. 2021; Drieu et al. 2021; Tanasi et al. 2021). The present contribution attempts to address this gap by investigating the prehistoric pottery recovered from Grotta Mora Cavorso (Jenne, central Italy). This archaeological site, at UTM coordinates (ED50) 33 T UG (03)48570(46)38010 and 715 m a.s.l., is a multi-tunnel karst cave system, located above the upper valley of the river Aniene in south-eastern Lazio. The complex stratigraphy of the site spans from the Late Pleistocene up to the present (Rolfo et al. 2009, 2016; Achino et al. 2016). The multidisciplinary data obtained from Grotta Mora Cavorso have been published in Silvestri et al. (2020), for overall information about this fascinating cave. It was on an important route between the Adriatic and Tyrrhenian coasts of Italy and has one of the most important early Neolithic burial deposits in Mediterranean Europe (Rolfo et al. 2016). The cave was considered as a sacred environment connected with the underworld; indeed, in the inner chambers, human skeletons have been found together with animal bones and pottery, which have been interpreted as grave goods (Fig. 1; Silvestri et al. 2020). Among the latter, fragments of an ovoid vessel and a hemispherical bowl were selected and analysed to obtain any clues about the plants used by the people living around the cave (Fig. 1c). This research succeeded in isolating plant aDNA from the powdered pottery, while the chromatography results support this genetic evidence. The data obtained from these potsherds provides some information about the cultural practices and living environments of the Neolithic people in central Italy, knowledge not previously obtained from archaeobotanical or anthropological analyses (Gismondi et al. 2012; Scorrano et al. 2019; D’Agostino et al. 2022). Materials and methods Archaeological artefacts and sampling procedures The archaeological pottery investigated in this study was dated to the early Neolithic (Table 1). It was found in a layer below an upper flowstone (a sheet of carbonaceous mineral 13 deposited by flowing water), on the surface of the burial together with bones, charcoal and ash, above a lower flowstone. One of the ancient pots (...truncated)