Late Upper Palaeolithic hunter-gatherers in the Central Mediterranean: New archaeological and genetic data from the Late Epigravettian burial Oriente C (Favignana, Sicily)

Journal Pre-proof Late Upper Palaeolithic hunter-gatherers in the Central Mediterranean: new archaeological and genetic data from the Late Epigravettian burial Oriente C (Favignana, Sicily) Giulio Catalano, Domenico Lo Vetro, Pier Francesco Fabbri, Swapan Mallick, David Reich, Nadin Rohland, Luca Sineo, Iain Mathieson, Fabio Martini PII:

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DOI:

https://doi.org/10.1016/j.quaint.2020.01.025

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JQI 8130

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Quaternary International

Received Date: 12 July 2019 Revised Date:

13 December 2019

Accepted Date: 28 January 2020

Please cite this article as: Catalano, G., Lo Vetro, D., Fabbri, P.F., Mallick, S., Reich, D., Rohland, N., Sineo, L., Mathieson, I., Martini, F., Late Upper Palaeolithic hunter-gatherers in the Central Mediterranean: new archaeological and genetic data from the Late Epigravettian burial Oriente C (Favignana, Sicily), Quaternary International, https://doi.org/10.1016/j.quaint.2020.01.025. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd and INQUA. All rights reserved.

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Late Upper Palaeolithic hunter-gatherers in the Central Mediterranean: new archaeological and genetic data from the Late Epigravettian burial Oriente C (Favignana, Sicily). Giulio Catalano1*, Domenico Lo Vetro2,3, Pier Francesco Fabbri4, Swapan Mallick5,6,7, David Reich5,6,7, Nadin Rohland5, Luca Sineo1, Iain Mathieson8, Fabio Martini2,3

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1) Dipartimento di Scienze e tecnologie biologiche, chimiche e farmaceutiche, Università degli studi di Palermo,

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Palermo 90128, Italy

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2) Dipartimento SAGAS – Unità di Archeologia Preistorica, Università di Firenze, Firenze 50121, Italy

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3) Museo e Istituto Fiorentino di Preistoria, Firenze 50122, Italy

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4) Dipartimento di Beni Culturali, Università del Salento, Lecce, Italy

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5) Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA

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6) Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA

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7) Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02442, USA

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8) Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia 19104, USA

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*Corresponding author. E-mail address: [email protected]

Abstract Grotta d’Oriente, a small coastal cave located on the island of Favignana (Sicily, Italy) is a key site

25

for the study of the early human colonization of Sicily. The individual known as Oriente C was

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found in the lower portion of an anthropogenic deposit containing typical local Late Upper

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Palaeolithic (Late Epigravettian) stone assemblages. Two radiocarbon dates on charcoal from the

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deposit containing the burial are consistent with the archaeological context and refer Oriente C to a

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period spanning about 14,200-13,800 cal. BP. Anatomical features are similar to those of Late

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Upper Palaeolithic populations of the Mediterranean and show some affinity with Palaeolithic

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individuals of San Teodoro (Messina, Sicily). Here we present new ancient DNA data from Oriente

32

C. Our results, confirming previous genetic analysis, suggest a substantial genetic homogeneity

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among Late Epigravettian hunter-gatherer populations of Central Mediterranean, presumably as a

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consequence of continuous gene flow among different groups, or a range expansion following the

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Last Glacial Maximum (LGM).

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Keywords: Late Glacial, Late Epigravettian, funerary practices, ancient DNA, Central-Western Mediterranean, Sicily.

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1.Introduction

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In the last few years, developments in sequencing techniques have enabled the generation of an

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unprecedented amount of genomic data from past populations. In particular, ancient genomes from

43

Upper Palaeolithic and Mesolithic periods have made possible to explore the early genetic makeup

44

of hunter-gatherers of the Mediterranean (Jones et al., 2015; Fu et al., 2016; Hofmanová et al.,

45

2016; Posth et al., 2016; Modi et al., 2017; Mathieson et al., 2018). Given its geographic location

46

and the presence of Upper Palaeolithic and Mesolithic human remains belonging to at least three

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individuals (Oriente A, B and C: Mannino et al., 1972; Lo Vetro and Martini, 2006; Di Salvo et al.,

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2012; Mannino et al., 2012; Martini et al., 2012a), Grotta d’Oriente, on the island of Favignana

49

(NW Sicily), is a key site for the study of the human colonization of Sicily during the Upper

50

Palaeolithic (Lo Vetro and Martini, 2012). Paleogenetic and morphological studies on the

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Mesolithic individual Oriente B indicated a close proximity with Late Epigravettians of the Italian

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peninsula (D’Amore et al., 2010; Mannino et al., 2012), while genome-wide single nucleotide

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polymorphism (SNP) data showed that the Upper Palaeolithic Oriente C clusters closely with other

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Western European Hunter-Gatherer (WHG) populations from Mesolithic and Late Palaeolithic

55

Western Europe (Mathieson et al., 2018), confirming previous morphological analysis (Henke,

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1989; Brewster et al., 2014).

57

Here we generated new genome-wide data in order to refine the genetic affinities of Oriente C to

58

other European hunter-gatherer populations. Our results and additional population genetic analyses

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provide insights into the origin and population structure of the hunter-gatherers that inhabited

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Europe during the Late Upper Palaeolithic and Mesolithic.

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2. Archaeological setting

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2.1. The site and its setting

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Grotta d’Oriente is a small coastal cave located on the island of Favignana, the largest (~20 km2) of

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a group of small islands forming the Egadi Archipelago, ~5 km from the NW coast of Sicily (Fig. 1

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A-B). The cave opens on the north-eastern slope of Montagna Grossa, at ~40 m. a.s.l. (Fig. 1 C-D)

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and is formed of two distinct areas: a small chamber at the left of the entrance and a large gallery on

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the right (Martini et al., 2012b) (Fig. 1 E). Early excavations were conducted, without a strict

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methodology, in the small chamber in 1972 (Mannino, 1972; 2002). New excavations were

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performed in 2005 as a part of an interdisciplinary project carried out by the University of Florence

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and Museo e Istituto Fiorentino di Preistoria (Colonese et al., 2011, 2014, 2018; Craig et al., 2010;

71

Lo Vetro and Martini, 2006; Martini et al., 2012a,b). A new trench was opened in 2005 next to the

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trench excavated in the 1970s and accurate recovery of materials and a microstratigraphic approach

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were followed. During the new excavations, a well-detailed archaeological sequence was

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documented (Fig. 1 F): the deposit, investigated up to a depth of about 2m, consists of 8 main

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sedimentological units (Layers); five of which contain evidence of human frequentation of the cave

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during prehistory: Late Upper Palaeolithic (Layer 7), Early Mesolithic (Layer 6), Late Mesolithic or

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Early Neolithic (Layer 5) and Bronze Age (Layers 4-3). These cultural deposits were further

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divided into sublayers each corresponding to different paleosurfaces which are often characterized

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by hearths and pits, and abundant artefacts and faunal remains (both terrestrial and marine) (Martini

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et al., 2012b; Colonese et al., 2018). These sublayers (AMS radiocarbon data are reported in Table

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1) are attributable to short-term episodes of human frequentation.

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At the top of the deposit there are two historical levels (Layers 1-2), with scarce pottery remains,

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which have been largely reworked. The lowermost level (Layer 8) contains Pleistocene fauna with

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no evidence of human activity.

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In addition to the Palaeolithic burial Oriente C, which is the object of this study and was brought to

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light during the excavations in 2005 (Lo Vetro and Martini, 2006; Martini et al., 2012a), two other

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burials have been unearthed during the 1972 excavation campaign: Oriente A and Oriente B

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(Mannino, 2002). Oriente A is a Mesolithic or, most likely, a Late Palaeolithic adult male (Lo Vetro

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and Martini 2006, for considerations about the chronological attribution of this burial); Oriente B is

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a Mesolithic adult female which has recently been directly dated (three AMS radiocarbon dates, all

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falling within the first half of the 10th millennium uncal. BP, Mannino et al., 2012). Other human

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bones recovered during 1972 excavations outside of the burial contexts (Mannino et al., 2012), have

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been recently identified at the Museo Archeologico Regionale “Antonio Salinas” (Palermo).

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2.2. The Oriente C burial

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The Oriente C funereal pit opens in the lower portion of Layer 7, specifically sublayer 7D. Two

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radiocarbon dates on charcoal from the sublayers 7D (12149±65 uncal. BP) and 7E, (12132±80

98

uncal. BP) are consistent with the associated Late Epigravettian lithic assemblages (see Table 1)

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(Lo Vetro and Martini, 2012; Martini et al., 2012b) and refer the burial to a period between about

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14,200-13,800 cal. BP, when Favignana was connected to the main island (Agnesi et al., 1993;

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Antonioli et al., 2002; Mannino et al., 2014). Several attempts to directly date Oriente C remains

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were performed by CEDAD laboratory (University of Salento) but all were unfortunately

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unsuccessful due to the lack of collagen preservation.

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The burial is located at the SW corner of the small chamber at the cave entrance, close to the rock

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wall. The skeleton is completely devoid of the lower limbs, large part of the pelvis and the hands.

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Based on the stratigraphic evidence, the lack of the lower portion of the skeleton could be possibly

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explained considering two events: 1) a small pit originated at the top of the Layer 7, during the

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Early Mesolithic, has partially affected the burial, perhaps removing the lower left skeletal

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elements; 2) the trench excavated by G. Mannino in 1972, intercepted the remaining part of

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skeleton at the level of the pelvis as is clearly visible in Fig. 2.

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A third event could have occurred without leaving clear traces in the stratigraphic succession: a

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very peculiar feature of this burial is, in fact, the presence of a femur (left) placed on the thorax

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between the shoulders. The taphonomic study did not detect any evident post-depositional

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disturbance of the skeleton that could have occurred in case of a reopening of the grave; the

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dislocations of some bones could therefore be attributed to post-burial movements inside the

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funerary pit. For this reason it is likely that femur could have been deposited during the interment

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and thus it could belonged to another individual (perhaps a relic?). However, since femur is

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compatible with the remaining part of the articulated skeleton (see below), it cannot be excluded the

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possibility that it belongs to Oriente C. In this case, we should assume a reopening of the grave (a

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violation?) during the Palaeolithic, after the decomposition of the body (no cut-marks or other

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traces linked to defleshing are evident on the femur), and the subsequent voluntary placement on the

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thorax of Oriente C of his own left femur. The reopening, which has no left clear traces on the rest

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of the skeleton, may have caused also a dislocation of the whole lower portion of the body. If this

124

event happened, it would have preceded the disturbance of the grave occurred during the Mesolithic

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(the small pit opened at the top of Layer 7A) as the grave was closed again after the deposition of

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the femur on the individual's chest and sealed by the Palaeolithic deposit (Layer 7C). This

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hypothesis could also explain why G. Mannino did not notice the presence of Oriente C burial,

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which he would have to intercept with his excavation trench, as the skeleton was already devoid of

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the lower limbs (Mannino, 2002; Lo Vetro and Martini, 2006).

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Beyond many disturbance episodes, the state of preservation of the human remains was generally

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very poor. For this reason, to allow the recovery of human remains avoiding irreparable damages, it

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was considered necessary to consolidate most of them several times with acrylic resin Paraloid B72

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(ethyl-methacrylate copolymer) mixed with acetone. Subsequently, the remains were removed and

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restored by gluing the parts after careful cleaning of the surfaces with acetone in order to remove

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the Paraloid film. Before any consolidation and restoration, during the burial excavation, numerous

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bone fragments were examined by morphological analysis in order to separate human bones from

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the animal ones which were accidentally in the pit filling, as usually happens in burials excavated in

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multi-layered deposits.

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Oriente C laid in dorsal decubitus oriented from South (the skull’s position) to North. The head

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rested on a large limestone chip with the face turned slightly to the left. The right upper limb was

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extended on the side of the trunk, while the left one was flexed (about 120°) with its lower end

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placed on the lower abdomen. All the bones, stored at the Laboratory of Prehistory-University of

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Florence (Table 2), show the same colour (red-brownish) and degree of fossilization except left ulna

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which is darker and left radius which is nearly black because of a small fire lit in the grave (see

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above). The skull, mandible and left elbow were slightly displaced, upper right limb long bones

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were articulated, and left iliac blade partially covered the flexed left forearm. These bones certainly

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belong to a single intentionally buried individual. A left femur was laid transversely above the

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upper part of the articulated skeleton, with the upper epiphysis on the left humerus (Fig. 2). No cut-

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marks or other traces linked to defleshing are evident on the femur. It is possible but not proven that

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the femur belongs to the same individual represented by the articulated bones (Lo Vetro and

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Martini, 2006).

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Many other human bones, often fragmentary, were also recovered during 1972 excavations.

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Approximately forty anatomical elements were recently identified among the material stored at the

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Museo Archeologico Regionale ‘‘Antonino Salinas’’ in Palermo (Mannino et al., 2012). Some of

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these, especially the hand bones, could belong to Oriente C but currently it is not possible to

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establish it. Moreover, a distal left humerus and radius, and a distal right ulna also occur. Mannino

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et al. (2012) excluded the attribution of these limb bones to both B and C individuals, but they

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could not ascertain the association with Oriente A or with a fourth individual. For this reason, they

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attributed these bones to Oriente X. In our opinion the distal left humerus does not belong to

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Oriente C, because a left humerus is present in the Florence collection. Conversely, the distal left

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radius and right ulna, absent in Florence, could belong to Oriente C but this is a hypothesis that

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should be confirmed after a direct observation of the remains and a comparative analysis.

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Age at death could not be accurately determined because of the lack of suitable anatomical parts.

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Nevertheless, we observe that exocranial sutures are not fused and there is a beginning of fusion on

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the endocranial aspect of the obelic suture; the lt. M3 and the fragment of upper molar (an M1 or

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M2) are unworn, the six preserved long bone epiphysis (inferior right and left humerus; upper right

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and left radius; upper and lower left ulna) are completely fused to the diaphysis and do not show

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traces of osteoarthritis. Hence, we conclude that the individual probably was a young adult, maybe

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25-30 years old. Oriente C lacks the diagnostic parts of the hip bones, but the long bone midshaft

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and epiphysis measurements – commonly used in sex determination of fragmentary human remains

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–indicated that the individual was most likely female (see Table S1 and Figure S1 in Supplementary

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material). This determination was later confirmed genetically (Mathieson et al., 2018). Stratigraphic

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and taphonomic features suggest that the funerary ritual of Oriente C consisted of a sequence of

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steps that can be summarized as follows:

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1- Excavation of the funerary pit. The pit originates in the lower part of the Layer 7 (sub-layer

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7D) and affects the base of the Layer 7 (sub-layer 7E) and the underlying Layer 8 (sterile

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yellowish sands); it is shallow (about 25 cm) and has a flat bottom. The original mouth of

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the pit may have been obliterated in the case of a subsequent reopening of the pit (see step

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5). The Northern portion of the pit was removed during 1972 excavations.

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2- Deposition of the body. The individual was placed into the grave, his skull rests almost on the western edge of the pit.

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3a- Burning action 1. After the deposition, when the soft tissues of the body were probably still

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present, a low-heat fire was lit at the bottom of the grave, in direct contact with the body, in

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the area of the lower left hemithorax. The short and weak combustion left traces on the left

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forearm and deposited charcoal, ashy soil, at the bottom of the pit. Following the hypothesis

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that femur was placed on the shoulders of the deceased during the interment, it must have

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occurred after the fire extinction, since femur lays on a thin layer of soil covering the

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charcoal and there are no traces of burning on it.

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3b- Burning action 2. A second low-heat fire was lit to the right of the skull.

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4- Closing of the grave. The individual was definitively interred.

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5-Possible reopening of the grave and deposition of a femur (if the femur belonged to Oriente

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C individual). A femur was placed between the shoulders of the body after the reopening of

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the grave. In this case the original mouth of the grave may have been partially obliterated

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and pit limits detected during the excavation may refer to the reopening of the burial. The

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reopening, if there was any, took place during the Late Upper Palaeolithic (top of the Layer

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7D), since Layer 7C, which covers the mouth of the pit, still refers to the Late Epigravettian.

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6-Deposition of stones. Along the eastern edge of the grave, and also inside it, limestone blocks

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were deposited. Some of these blocks protruding from the pit were probably placed as a

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marker for the identification of the location of the burial.

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The available anatomical features of Oriente C are similar to those of Late Upper Palaeolithic

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populations of the Mediterranean and show affinity with other Palaeolithic individuals of Sicily, but

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the fragmentary cranial remains of Oriente C do not permit a deeper morphological analysis. As

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suggested by Henke (1989) and Fabbri (1995) the hunter-gatherer populations were

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morphologically rather uniform. This interpretation is further supported by the low or negligible D2

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distance demonstrated by D’Amore et al. (2009) in the comparison between San Teodoro

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(individuals 1-2-3-5-7) craniofacial morphometrics and other Upper Palaeolithic individuals.

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Like other Late Epigravettian burials in Sicily and Italy (Palma di Cesnola, 2003; Giacobini, 2007),

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Oriente C is a simple burial with little or no grave goods and personal ornaments. The only items in

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the pit were a pierced shell of Cerithium sp. (perhaps a clothing ornament) and a few small lumps of

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red ochre, next to the skull and the femoral head. Stable isotope analysis suggested a largely

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terrestrial diet with low-level consumption of marine foods which is comparable to other Late

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Upper Palaeolithic individuals from Sicily and Italy (Craig et al., 2010; Mannino et al., 2012).

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3. Genetic analysis

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A first attempt to analyse mitochondrial DNA from a rib fragment of Oriente C, performed in 2006

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by University of Rome ‘‘Tor Vergata” (Lo Vetro and Martini, 2006), was unsuccessful likely

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because made on a fragment too compromised by restoration procedures. The current ancient DNA

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analysis was done on a single long bone fragment that was not exposed to the substances used for

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consolidation and restoration (see above section 2.2). Sample preparation, DNA extraction and

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library construction were carried out in dedicated ancient DNA facilities in Boston as described in

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(Mathieson et al., 2018). To increase coverage compared to the previously reported data, we

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generated two additional libraries from the same extract, performed in-solution enrichment

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(“1240k”) and sequenced the product on an Illumina NextSeq500 using v.2 150 cycle kits for 2 ×

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76 cycles and 2 × 7 cycles. We merged these data with the data from the original library and made

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pseudo-haploid calls by selecting a single sequence at each single nucleotide polymorphism (SNP).

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The resulting dataset contained information on 288,223 SNPs covered at least once, compared to

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61,547 in a previous publication (Mathieson et al., 2018), allowing for higher resolution analysis.

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To investigate the genetic affinities of Oriente C, we used the qp3Pop program from

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ADMIXTOOLS (Patterson et al., 2012) to compute f3-statistics and to estimate the amount of

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shared genetic drift between Oriente C and 98 published Mesolithic and Late Palaeolithic hunter-

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gatherers (with coverage at a minimum of 20,000 of 1240k positions) from 30 sites across Europe

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(Gamba et al., 2014; Gonzales-Fortes et al., 2017; Gunther et al., 2018; Haak et al., 2015; Jones et

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al., 2015; Lazaridis et al., 2014; Lipson et al., 2017; Mathieson et al., 2015, 2018; Olalde et al.,

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2014). We used the qpDstat program to estimates D-statistics to test whether pairs of populations

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form a clade. The statistic D (outgroup, A, B, C) is zero if A is an outgroup to the clade (B, C),

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positive if A is closer to C, and negative if it is closer to B. For D- and f3-statistics, we estimated

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standard errors using the default block jackknife procedure implemented in ADMIXTOOLS

240

(Patterson et al., 2012).

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We confirmed the originally reported mitochondrial haplogroup assignment of U2'3'4'7'8'9. This

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haplogroup is present in both pre- and post-LGM populations, but is rare by the Mesolithic, when

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U5 dominates (Posth et al., 2016). We further confirmed that the new genome-wide data was

244

consistent with the original data by computing D-statistics (Patterson et al., 2012) of the form D

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(Mbuti, X, Original Oriente data, Merged Oriente data). None of these statistics were significantly

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non-zero when X ranged over other European Mesolithic hunter-gatherers (maximum |Z| = 1.8 in 34

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tests), and present-day French (Z = -0.35) and Sardinian (Z = -0.13) populations.

248

Lipson et al. (2018) (their supplementary Figure S5.1) and Villalba-Mouco et al. (2019) (their

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Figure 2A) showed that European Late Palaeolithic and Mesolithic hunter-gatherers fall along two

250

main axes of genetic variation. Multidimensional scaling (MDS) of f3-statistics shows that these

251

axes form a “V” shape (Fig. 3). At the root of the “V” lie the individuals that have been described as

252

belonging to the “Western hunter-gatherer” (WHG) population, clustering closely with the 8,000

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BP Loschbour individual (Lazaridis et al., 2014). One arm represents a cline of ancestry that links

254

WHG with “Eastern hunter-gatherer” (EHG) populations who carry ancestry related to the “Ancient

255

North Eurasian” (ANE) population represented by the 24,000 BP Mal’ta individual (Raghavan et

256

al., 2014). Along this cline lie Eastern European hunter-gatherer populations such as those from the

257

Balkan Peninsula, present-day Ukraine (Mathieson et al., 2018), the Baltic (Jones et al., 2017;

258

Mathieson et al., 2018) and Scandinavia (Haak et al., 2015, Gunther et al., 2018). The other arm of

259

the “V” is a cline containing Late Upper Palaeolithic and Mesolithic individuals from Iberia (for

260

example the individuals from El Mirón and La Braña), and Late Upper Palaeolithic individuals

261

from Central Europe (Goyet and Hohle Fels). As shown by Fu et al. (2016), this cline reflects an

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ancestry contribution from a population related to the 35,000 BP Aurignacian Goyet Q116-1

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individual. In this analysis, Oriente C falls at the tip of the “V”, at the extreme end of the WHG

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grouping.

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Focusing further on Oriente C, we find that it shares most drift with individuals from Northern

266

Italy, Switzerland and Luxembourg, and less with individuals from Iberia, Scandinavia, and East

267

and Southeast Europe (Fig. 4A-B). Shared drift decreases significantly with distance (Fig. 4C) and

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with time (Fig. 4D) although in a linear model of drift with distance and time as a covariate, only

269

distance (p=1.3×10-6) and not time (p=0.11) is significant. Consistent with the overall E-W cline in

270

hunter-gatherer ancestry, genetic distance to Oriente C increases more rapidly with longitude than

271

latitude, although this may also be affected by geographic features. For example, Oriente C shares

272

significantly more drift with the 8,000 year-old 1,400 km distant individual from Loschbour in

273

Luxembourg (Lazaridis et al., 2014), than with the 9,000 year old individual from Vela Spila in

274

Croatia (Mathieson et al., 2018) only 700 km away as shown by the D-statistic (Patterson et al.,

275

2012) D (Mbuti, Oriente C, Vela Spila, Villabruna); Z=3.42. Oriente C’s heterozygosity was

276

slightly lower than Villabruna (14% lower at 1240k transversion sites), but this difference is not

277

significant (bootstrap P=0.12). The low coverage of the sample meant that its genotype at any

278

particular site–for example those directly related to phenotypic traits–could not be reliably

279

ascertained. However, at sites with coverage, Oriente C appears consistent with other Western

280

Hunter Gatherers. For example, it carries the ancestral allele (2 reads) at the SLC45A2

281

pigmentation-associated SNP rs16891982 and (3 reads) at the lipid metabolism-associated SNP

282

rs174546 in the FADS gene cluster. Oriente C’s genotype at other phenotypically associated sites

283

including the LCT lactase persistence SNP and the immune-associated TLR cluster could not be

284

determined, however.

285 286

4. Discussion

287

Sicily falls within the area of expansion of the Epigravettian model widespread after the LGM in

288

Mediterranean Europe, from Provence to the eastern Balkan border up to the Black Sea and the SW

289

Anatolia. This “cultural province” is characterized by peculiar features, rooted in the local

290

Gravettian traditions, which concern not only lithotechnics but also artistic production and burial

291

customs (Kozlowski, 2005 and reference therein; Fontana et al., in press). The Epigravettian (about

292

21.0-11.5 cal. ka BP) is a quite homogeneous cultural phenomenon despite the paleo-environmental

293

differences occurring in a wide territory and some differentiations in resources exploitation

294

strategies and human-environment interactions which were, perhaps, responsible for the formation

295

of regional variants (Palma di Cesnola, 1993; Kozlowski, 2005). In Italy a regionalization of

296

Epigravettian stone assemblages is more clearly recognizable at the end of the Late Glacial (on this

297

topic see Palma di Cesnola, 1993 and the several contributions in Martini, 2007). Recently the

298

regionalist hypothesis has been criticized by Tomasso (2017) in a paper where the evidence from

299

the southernmost part of the Italian peninsula and Sicily was not considered.

300

In Sicily, where the only reliable evidence belongs to the Late Epigravettian (about 16-11.500 cal.

301

BP), this culture presents very specific local features, especially in lithic assemblages (Martini et al.,

302

2007; Lo Vetro and Martini, 2012). The figurative production is the element that more than others

303

seems to connect Sicily to the peninsular Epigravettian sphere; the iconographic languages of

304

Sicilian Late Epigravettian fit very well in the so-called “Mediterranean style” sensu Graziosi

305

(1956). Although this definition is no longer appropriate to the current archaeological context

306

(Vigliardi, 1996, 2005; Bicho et al., 2007; Martini, 2016), it highlights the existence of stylistic

307

affinities in the Upper Palaeolithic art of some areas of Central-Western Mediterranean Europe. A

308

further element that connects Sicily to the Italian peninsula and more generally to the Western

309

Europe consists in the so-called “Azilian” artistic productions (i.e. painted pebbles from Grotta di

310

Cala del Genovese in Levanzo island), a phenomenon that seems to have crossed the Tyrrhenian

311

side of the Italian peninsula from Liguria to the southern regions (Martini, 2016 and reference

312

therein). Even the funeral practices (i.e. Grotta di San Teodoro) show strong affinity with the

313

peninsular evidence (Palma di Cesnola, 2003; Giacobini, 2007), from Calabria (i.e. Grotta del

314

Romito) (Martini and Lo Vetro, 2018 and reference therein) up to Veneto (i.e. Riparo Tagliente and

315

Riparo Villabruna) (Broglio, 1995; Bartolomei et al., 1974). Industrial and symbolic evidence,

316

therefore, suggest that during the Late Glacial period Sicily was fully included in the “Epigravettian

317

cultural province” and for this reason we can consider Sicily as the most continental of the

318

Mediterranean islands.

319

Numerous archaeological sites related to the human frequentation of Sicily in the Late Upper

320

Palaeolithic reveal that Late Epigravettian hunter-gatherer groups inhabited intensively the island

321

during the Late Glacial period (Lo Vetro and Martini, 2012). The considerable record of

322

radiocarbon dates proves that they reached Sicily not before 16-14 ka cal. BP, several millennia

323

after the LGM peak, during a period of sudden sea level rise which caused a dramatic

324

transformation of the coastal morphology of the island (Lambeck et al., 2011; Lo Presti et al.,

325

2019). In our opinion, in fact, the hypothesis about an early colonization of Sicily by Aurignacians

326

(Laplace, 1964; Chilardi et al., 1996) must be rejected on the basis of a recent re-interpretation of

327

the techno-typological features of the lithic assemblage from Riparo di Fontana Nuova which has

328

been re-assigned to the Late Epigravettian (Martini et al., 2007; Lo Vetro and Martini, 2012; on this

329

topic see also Di Maida et al., 2019).

330

The Late Upper Palaeolithic Oriente C is a simple burial, and its sober ritual and the modality of

331

deposition fit very well in the context of the Late Epigravettian burials of Sicily and Central-

332

Southern Italy (Palma di Cesnola, 2003; Giacobini, 2007). Regarding the funerary ritual, an

333

interesting issue, unique in the panorama of the Palaeolithic burials in Europe, concerns the peculiar

334

occurrence of a femur placed on the shoulders of the individual and its possible belonging to the

335

skeleton found in place.

336 337

Many sources of evidence indicate that the LGM may have had a major role in shaping the genetic

338

and phenotypic variation of Upper Palaeolithic populations. A recent study based on complete

339

mitochondrial genomes has revealed a genetic homogeneity between European hunter-gatherers. A

340

significative predominance of the U lineage was detected with most of the sequences belonging to

341

U5 haplotypes (Posth et al., 2016). The finding of the haplogroup U2'3'4'7'8'9 in the Oriente C

342

individual, previously recovered in the Upper Palaeolithic humans from Grotta Paglicci (Posth et

343

al., 2016) provides additional evidence for the hypothesis that Epigravettian culture might have

344

reached Sicily during the migration of Upper Palaeolithic groups from Southern Italy after the LGM

345

(Palma di Cesnola, 2006; Lo Vetro and Martini, 2012; Mannino et al., 2012), which accords with

346

the morphological similarity of Late Upper Palaeolithic and Early Mesolithic populations in the

347

region (Henke, 1989; Brewster et al., 2014). The find of genetic similarity of Oriente C with Late

348

Upper Palaeolithic and Mesolithic individuals from Northern Italy (i.e. Villabruna) and Central

349

Europe (i.e. Bichon, Loschbour) (Fig. 3) is also in line with previous studies according to which

350

Sicilian hunter-gatherers were found to be morphologically closely related to Late Epigravettian

351

individuals of the Italian peninsula and continental Europe (Fabbri, 1995; D’Amore et al., 2010).

352 353 354

5.Conclusions

355

These analyses have implications for the understanding of the origin and diffusion of the hunter-

356

gatherers that inhabited Europe during the Late Upper Palaeolithic and Mesolithic. Our findings

357

indicate that Oriente C shows a strong genetic relationship with Central and Western European Late

358

Upper Palaeolithic and Mesolithic hunter-gatherers, suggesting that the “Western hunter-gatherers”

359

was a genetically homogeneous population widely distributed in the Central Mediterranean. In our

360

opinion this geographic structure may reflect a continuous gene flow among different groups or at

361

least partly may represent the legacy of South to North migration events after the Late Glacial

362

period which replaced almost all of the pre-LGM ancestry in Central and Western Europe. At the

363

same time, it is risky to force a correlation between evidences that work on different resolution

364

scales. The archaeological record available for the final Epigravettian of Southern Italy is

365

considerable in terms of absolute chronology. On the other hand, it is not possible to elaborate

366

reliable hypotheses based on genetic data only, given the paucity of Epigravettian genomes

367

analyzed so far. Hence, we believe that more paleogenomic data for the chrono-cultural segment

368

spanning between 20 to 16 ka BP (ancient and evolved Epigravettian) will allow a more

369

comprehensive understanding of the dynamics of European hunter-gatherer populations in the

370

period immediately after the LGM.

371 372

In conclusion, the DNA study of Oriente C is particularly relevant to improve the knowledge about

373

the peopling of the Central Mediterranean by Anatomically Modern Humans after the LGM. The

374

data support the hypothesis that hunter-gatherer groups arrived in Sicily from the Italian peninsula,

375

confirming results derived from anatomical studies on human fossil remains of Grotta di San

376

Teodoro and from material productions (lithic and figurative) whose characteristics fall within the

377

Late Epigravettian physiognomy of Southern Italy, albeit with some peculiar features, especially in

378

lithic productions, which reveal a regional identity.

379 380 381 382

Data Availability:The 1240k capture sequencing data for Oriente C (merged new and existing data) has been deposited in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under accession number PRJEB33231.

383 384 385 386 387 388

Acknowledgments: The authors thank and remember with affection their friend and colleague Sebastiano Tusa, prematurely died in the plane crash of the Ethiopian Airlines in 2019. Dr. Tusa did so much for the understanding of the prehistoric heritage of Sicily.

389 390 391 392 393 394

Authors' Contributions: GC and DLV designed the paper, DLV and FM conducted the excavation at Grotta d’Oriente in 2005, PFF conducted the anthropological study of the human remains, DLV, FM, PFF provide information about the taphonomy of the burial, LS collected the sample, IM, SM, NR and DR conducted the DNA analysis, GC, DLV, PFF, IM wrote the manuscript with input from all co-authors.

395 396 397 398 399 400 401 402 403

Funding: The excavation of Grotta d’Oriente in 2005 was funded by the Regional Operational Programme for Sicily 2000/2006, II, 2.0.1. (European Commission), and performed with the permission of the Soprintendenza ai Beni Culturali e Ambientali (Regione Siciliana, Assessorato ai Beni Culturali e Ambientali). The study of Oriente C individual was part of the MIUR-PRIN 2010– 2011 action (EPIC Project: Biological and cultural heritage of the central-southern Italian population through 30 thousand years. Grant ID: 2010EL8TXP). DR is an Investigator of the Howard Hughes Medical Institute.

404 405

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592

593 594 595 596 597 598

Table 1. Grotta d’Oriente, radiocarbon dates on charcoals from the stratigraphic sequence (2005 excavations). 14C ages are reported as conventional and calibrated years BP, the dates were calibrated with OxCal v4.3 using the IntCal13 calibration curve (Reimer et al., 2013). The dates were performed at the CEDAD (Centre for Dating and Diagnostics), University of Salento, Italy. 14C yr cal. BP 14C yr cal. BP (68%) (95%)

Lab. Code

Layer

14C yr BP

Cultural period

Reference

LTL877A

5A

7040 ± 55

7940 – 7829

7969 – 7741

Late Meso-Early Neolithic

Martini et al., 2012b

LTL876A

6B

8619 ± 65

9660 – 9530

9762 – 9485

Early Mesolithic

Martini et al., 2012b

LTL874A

6C

8608 ± 65

9658 – 9526

9737 – 9480

Early Mesolithic

Martini et al., 2012b

LTL875A

6D

8699 ± 60

9732 – 9551

9888 – 9542

Early Mesolithic

Martini et al., 2012b

LTL14260A

7D

12149 ± 65

14136 – 13932

14195 – 13791

Upper Palaeolithic

Unpublished

LTL873A

7E

12132 ± 80

14107 – 13853

14198 – 13765

Upper Palaeolithic

Lo Vetro and Martini, 2006; Martini et al., 2012b

599 600 601 602 603 604

Table 2. Oriente C skeletal remains housed at the Museo e Istituto Fiorentino di Preistoria in Florence. Anatomical region

Bone cap mandibula

Skull teeth right humerus left humerus right radius Upper limbs

left radius right ulna left ulna

Lower limbs

left iliac bone left femur

Preservation with fragmentary base fragments of mandibular rami -left M3 -fragment of an upper molar larger than M3 -dental fragments -fragmentary lower third diaphysis -fragments of lower epiphysis lower epiphysis head -upper epiphysis -fragments of diaphysis upper 2/3 of diaphysis -upper and lower epiphysis missing styloid process -fragmentary diaphysis fragment comprising the anterior superior Iliac spine missing lower epiphysis

left 3rd metatarsal

-

605 606

607 608 609 610 611 612 613 614 615

Figure 1. A-B) Geographic location of Grotta d’Oriente; C-D) The cave entrance on the slope of Montagna Grossa; E) Excavation areas; F) Stratigraphic sequence (2005 excavations) showing the layers and sublayers.

616 617 618

619 620 621 622 623

Figure 2. The burial Oriente C during excavations: a wide view of the burial on the left, a close-up of the skeleton on the right (Photo D. Lo Vetro).

624 625 626 627 628 629 630 631 632 633 634 635 636 637

Figure 3. Multidimensional scaling of outgroup f3-statistics for Late Upper Palaeolithic and Mesolithic hunter-gatherers.

638

639 640 641 642 643

Figure 4.A) Shared drift, estimated using f3-statistics between Oriente C and 98 Mesolithic and Late Palaeolithic hunter-gatherers from 30 sites; B) The same statistic as in A plotted with geographic position; C) Decay of shared drift with distance from Oriente C; D) Decay of shared drift with absolute difference in date from Oriente C.

644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677

678

Supplementary material Skeletal Sex identification of Oriente C Age at death cannot be quantified lacking suitable anatomical parts, nevertheless we observe that: exocranial sutures are not fused and there is a beginning of fusion on the endocranial aspect of the obelic suture; lt. M3 and a fragment of upper molar (an M1 or M2) are unworn, six preserved long bone epiphysis (inferior right and left humerus; upper right and left radius; upper and lower left ulna) are completely fused to the diaphysis and they don’t show traces of osteoarthritis. The individual probably was a young adult, maybe 25-30 year old. Oriente C lacks hip bones, hence we tried to have an indication of sex using preserved long bone midshaft and epiphysis measurements which are commonly used in sex determination in fragmentary human remains (Tab. S1). Measurements of articulated bones, humerus and ulna, are lower than male means (z-scores -1.62 and -0.85) and close to female ones (z-scores -0.28 and 0.11); the unarticulated femur is very small, its z-score is -2.70 compared to males and -1.18 compared to females. In order to establish if such a difference between upper and lower limb bones could be found in a single individual, we computed the ratio of humeral lower epiphysis breadth (articulated bone) to femoral AP head breadth (unarticulated bone) found in the Oriente C burial and in a sample of European Upper Palaeolithic articulated individuals (Fig.S1). In Oriente C the ratio (1.393) is high, but still within the UP range (1.116-1.429) and it is intermediate between two female individuals, G.d.Enfants 3 (1.375) and Paglicci 25 (1.402). We can conclude that size proportions observed between articulated (humerus) and unarticulated (femur) bones found in Oriente C burial can be found in fully articulated UP female individuals. Hence we cannot exclude that articulated and unarticulated bones found in the Oriente C burial might belong to the same individual. In this case Oriente C would be a female as the femoral measurement is lower than the male minimum and close to the female minimum observed in our UP sample. In case the unarticulated female femur represents a different individual from articulated bones found in Oriente C burial, the sex of the latter would be uncertain, but more probably female, as far as upper limb bone measurements are concerned.

Table S1. Selected skeletal measurements of Oriente C compared to European Upper Palaeolithic male and female samples. Sex-bone

Measure

Martin N°

n

mean

sd

min

max

Oriente C

z-score

Male humerus

Low. Epiph. Br.

M4

27

61.4

2.2

57.0

66.0

57.8

-1.62

Male ulna

Min. perimeter

M3

25

34.7

4.4

27.0

45.0

31.0

-0.85

Male femur

A-P head br.

M19

27

48.5

2.6

43.0

53.0

41.5

-2.70

Female humerus

Low. Epiph. Br.

M4

10

58.7

3.0

52.0

62.0

57.8

-0.28

Female ulna

Min. perimeter

M3

11

30.6

4.2

27.0

40.0

31.0

0.11

Female femur

S-I head height

M19

10

44.5

2.5

40.0

48.0

41.5

-1.18

D. D. V G G. G. A. A Pr Pr Pr S Pre es .d.E Ves O C . C d ed h ed ha ed .Te dm to nf to Rom Ro A.C d.E ed Pa .En an Can R a R n m an nf m glic fa did di Bic m o nce om mo om lo I m o od os ice ant nic I H s t o r t 1 1 s e 1 i t o i t o d i d an t o s ci n t e d e ho s t it o s t l i a t 3 4 o 7 t9 2 s3 5 e s5 de 2 5 n 4 4 3 4 4 4 4 3 o (f; 5 (f (f; (m; (m (m (m ; (? (f; (m; 4 (f (m (m (f; (m (m (m; (m (m; (m 3 (f (? rt ; lt rt rt ; lt ; lt rt ; lt rt rt ; lt ; lt ; lt rt ; lt ; lt rt ; lt rt ; lt ; lt ; lt .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .) .)

Hum M4/Fem M19

1,150

1,200

1,250

1,300

1,350

1,400

1,450

Pr

1,100

679 680 681 682 683 684 685 686 687 688 689 690 691

Figure S1. Humeral lower epiphysis breadth (M4) / femoral head supero-inferior height (M18) ratio in Upper Palaeolithic individuals (sex; side). Comparison sample. A.Candide: Paoli et al. (1980). Bichon:Chauvière (2008). Chancelade: Billy (1969). D.Vestonice:Trinkaus and Svoboda (2006). Ohalo:Hershkovitz et al. (1995). Ostuni (S.M. Agnano): Vacca et al. (2013). Predmost:Matiegka (1938).Sunghir: Alexeeva and Bader (2000). G. d. Enfants, Paglicci, Romito, San Teodoro and Vado all’Arancio measured by PFF.

692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718

References Alexeeva, T.I., Bader, N.O., 2000. Homo Sungirensis: Upper Palaeolithic man: ecological and evolutionary aspects of investigation. Moscow: Scientific World. Billy, G., 1969. Le squelette post-crânien de l'homme de Chancelade. L'Anthropologie 73, 207–246. Chauvière, F.-X., 2008. La grotte du Bichon : un site préhistorique des montagnes neuchâteloises. Archéologie neuchâteloise 42, pp. 164. Hershkovitz, I., Speirs, M.S., Frayer, D., Nadel, D., Wish-Baratz, S., Arensburg, B., 1995. Ohalo II H2: a 19000-year-old skeleton from a water-logged site at the Sea of Galilee, Israel. Amer. Jour. Physical Anthropology 96, 215–234. Matiegka, J., 1938. L'homme fossile de Predmosti en Moravie (Tchécoslovaquie). II. Autres parties du squelette. Prague, Nàkladem Ceské Akademieveda Umeni. Paoli, G., Parenti, R., Sergi, S., 1980. Gli scheletri mesolitici della caverna delle Arene Candide. Memorie dell'Istituto di Paleontologia Umana, N.S. 3, 33–154. Trinkaus, E., Svoboda, J., 2006. Early modern human evolution in central Europe: the people of Dolni Vestonice and Pavlov. New York: Oxford University Press. pp. 489. Vacca, E., Formicola, V., Pesce Delfino, V., Coppola, D., 2013. I resti scheletrici umani delle sepolture paleolitiche di Grotta Santa Maria d'Agnano - Ostuni (BR). In D. Coppola (a cura) "Il riparo di Agnano nel Paleolitico Superiore, la sepoltura di Ostuni 1 ed i suoi simboli" Ed. Un. di Roma Tor Vergata.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: