Megalithic Tombs in Western and Northern Neolithic Europe Were Linked to a Kindred Society Supplement

Home , Carrowmore, Court cairn, Dolmen (miniseries), Haplogroup I (mtDNA), Holm of Papa, Lengyel culture

Megalithic tombs in western and northern Neolithic Europe were linked to a kindred society Supplement

Federico Sánchez-Quinto1*, Helena Malmström1,2*, Magdalena Fraser1,3*, Linus Girdland- Flink4, Emma M. Svensson1, Luciana G. Simões1, Robert George5,10, Nina Hollfelder1, Göran Burenhult3, Gordon Noble6, Kate Britton6,7, Sahra Talamo7, Neil Curtis6, Hana Brzobohata8, Radka Sumberova8, Anders Götherström9, Jan Storå10#, Mattias Jakobsson1,2#

* Equally contributed to this work

#corresponding authors

Affiliations

1 Human Evolution, Department of Organismal Biology, Uppsala University, SE-752 36 Uppsala, Sweden 2Centre for Anthropological Research and Department of Anthropology and Development Studies, University of Johannesburg, P.O. Box 524, Auckland Park, 2006, South Africa 3Department of Archaeology and Ancient History, Uppsala University-Campus Gotland, SE-621 67 Visby, Sweden 4Research Centre in Evolutionary Anthropology and Paleoecology, School of Natural Sciences and Psychology, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK 5Royal Prince Alfred Hospital, Sydney Australia 6Museums and Special, Collections, University of Aberdeen, Sir Duncan Rice Library, Bedford Road, Aberdeen, AB24 3AA, Scotland 7Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103, Leipzig, Germany 8Institute of Archaeology of Czech Academy of Sciences, Letenská 4, CZ-11801 Prague, Czech Republic 9Archaeological Research Laboratory, Department of Archaeology and Classical Studies, Stockholm University, SE-106 91 Stockholm, Sweden 10Osteoarchaeological Research Laboratory, Department of Archaeology and Classical Studies, Stockholm University, SE-106 91 Stockholm, Sweden

Data Availability

Raw sequencing reads produced for this study have been deposited in the European Nucleotide Archive (ENA) under accession number ENA: PRJEB31045.

www.pnas.org/cgi/doi/10.1073/pnas.1818037116 Supplement: Megalithic tombs in western and northern Neolithic Europe

Contents S1 Archaeological Background ...... 3

S2 The ancient individuals, provenance and site description ...... 5

Carrowmore, County Sligo, Ireland ...... 6

Primrose Grange, County Sligo, Ireland ...... 9

Midhowe, Orkney, Scotland...... 13

Lairo, Orkney, Scotland ...... 14

Balintore, Ross and Cromarty, Scotland ...... 14

Ansarve, Gotland, Sweden ...... 15

Kolin, Bohemia, Czech Republic ...... 18

S3 Sample preparation, DNA extraction and Library preparation ...... 20

S4 Processing of NGS data ...... 22

S5 Contamination estimation ...... 24

S6 Calling mtDNA haplogroups ...... 26

S7 Calling Y chromosome haplogroup ...... 28

S8 Molecular sexing of individuals ...... 36

S9 Reference panels and group labels ...... 38

S10 Estimating kinship relationships among Megalith individuals ...... 40

S11 Population genetic analyses ...... 55

S12. Primrose Grange, Tomb. Original field documentation...... 70

References supplement ...... 75

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S1 Archaeological Background On farmer migrations routes along the Atlantic facade

Recent archaeogenetic research has shown the importance of human migrations and mobility in the dispersal of Neolithic lifeways over the European continent. Apart from the Early Neolithic inland and the Mediterranean coastal expansion routes, archaeological research suggests a western European Atlantic maritime route connecting Neolithic cultures via the Atlantic, the North Sea, and the Baltic littoral (1–4), of which some routes may have been important already during the Mesolithic period as well as the Early Neolithic (5, 6). Genetic patterns are generally consistent with archaeological data (7–14), although the processes of population expansion and migration resulted in some degree of genetic differentiation between different Neolithic groups (15). While available genetic data from Neolithic farmers that were buried on the northwestern fringes of Europe (i.e. the British Isles and Scandinavia) show strong affinities to Central European farmers (16–21), individuals buried in Megalithic chambered tombs in Ireland (Ballynahatty), and Sweden (Gökhem) had strong affinity to Neolithic and Chalcolithic Iberian farming groups (19, 22). However, the extent to which coastal and maritime migration routes existed along the islands and the coastal regions of western Europe remains unknown.

Megalithic burial tradition

Megalithic tombs (from the Greek megas, great and lithos, stone) are large burial constructions usually built from rock, soil and wood (23). Based on both materials and design, different types of megalithic tombs can be identified, e.g. dolmens, passage graves, and court cairns to name a few. All types may collectively be categorized as chambered tombs and/or megalithic tombs. These types of burials are often found along the former coastlines and on islands, suggesting maritime connections (24–26); however, inland locations also occur. The geographic and chronological distributions of the different types of chambered tombs vary. While dolmens and passage graves are commonly found across the whole Atlantic façade and some Mediterranean islands (24–30), chambered cairns are mainly found in the British Isles (31, 32). However, the explanation behind the compelling similarities in the construction and design of dolmens and passage graves from Iberia to southern Scandinavia, including the British Isles, remains elusive. The inter-regional interaction linking the west European megalithic sites, consistent also with the dispersal of domesticated resources, raw materials and artefacts, has often been attributed to shared social and cultural systems, and also cosmology (23, 31, 33).

The European monumental burials, which accompanied funerary rites, have been studied at least since early medieval times (23). The stone structures are problematic to date, and many burials do not contain organic remains that can be directly radiocarbon dated. However, the earliest dates seem to fall in the middle of the 5th millennium BCE in Brittany and the Channel Islands, in Catalonia, in 3

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west and central southern France, on Sardinia, in northern Italy, and possibly in Apulia and also on Corsica (33). An important insight to the complexity regarding the dating of various phases in megalithic tombs in Ireland is a recent study and re-examination of the find materials from a multi- phase passage tomb on Baltinglass Hill, Co. Wicklow, excavated in the 1930s (34). The study is important and presents evidence of unexpectedly early megalithic construction, possibly as early as 3900 Cal BCE, challenging conventional views of the emergence of the Irish Neolithic. At least three phases between c. 3900-2900 Cal BCE can be determined at Baltinglass Hill (34). Also, a comprehensive study of all available dates from the Mesolithic-Neolithic interface, the Neolithic and the Bronze Age in Ireland was recently published, showing that traces of Neolithic activities are scarce prior to 3750 Cal BC (35).

Megalithic tombs are, as a rule, multiperiod sites. They have been built, used, rebuilt and reused again. These burials were often collective graves, and their usage also continued for centuries and/ or millennia, including secondary burials in later time periods (21, 24, 26, 36–41). During additional deposits, earlier material and human remains have often been pushed aside or probably even thrown out from the chambers. Consequently, the dating of the primary construction is most often difficult but very important when it comes to understanding the underlying settlement-subsistence system and economy of the tomb-building society in question. There are today several archaeogenetic studies on human remains recovered in megalithic burial structures in Europe, such as the Gökhem passage grave (16–18, 42) and the Ansarve dolmen (20, 21) in Scandinavia, the La Mina Passage grave in Iberia (43), the Ballynahatty dolmen on Ireland (19), as well as several recently published megalithic burials from Scotland, Britain, and Wales (44). However, the megalithic tradition and the social dynamics of the groups that use them in the European Middle Neolithic have not yet been investigated as a topic of its own based on the genetic patterns of the human burials. The focus of previous studies has mainly been on general demographic characteristics and large-scale migrations during the Neolithic. Here we investigate internal genetic structure, kinship, and origins of people buried in megaliths. Below we present new data from megalithic burials in Ireland (Carrowmore and Primrose Grange) and Scotland (Midhowe and Lairo), as well as previously published data from the Ansarve dolmen in Sweden (20, 21). We also present reference data from a newly-identified Neolithic cist from Balintore in Scotland, and also the previously published Kolin rondel in the Czech Republic (21, 45, 46).

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S2 The ancient individuals, provenance and site description All individuals presented here have been directly AMS radiocarbon dated, and also isotope ratio mass spectrometer (IRMS) analysed for stable isotopes (13C and 15N) (except for the Balintore and Kolin samples). New radiocarbon dates, Carbon and Nitrogen values were obtained for the Irish samples (Primrose Grange and Carrowmore) at BETA analytic Inc. (Miami, Florida) using standard methods of the lab, while the dating and isotope analyses for the Midhowe, Lairo, Ansarve, and the Kolin samples have been published previously (see Table S1). All dating results have been calibrated using Oxcal online software version 4.2.4 (47) based on the IntCal13 atmospheric curve (48). Some samples were dated twice (prs009, ans003, and ans005) these dates were combined and recalibrated using the R_combine function in Oxcal (Table S1). All dates are presented in cal BCE, 95% CI and have been rounded to the nearest tenth value. The stable isotope analyses were made to infer dietary habits in the individuals as well as to investigate if the radiocarbon dating results needed to be adjusted for marine reservoir effect (see individual sample presentations below). All individuals presented here revealed either a strict-, or a slightly varied, terrestrial diet consistent with a farmer life style, except for Ansarve16 who displayed a mixed diet that also included marine protein. Thus, the corresponding radiocarbon dating was corrected for marine reservoir age (see sections below and Table S1).

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Table S1. The ancient individuals analyzed in this study including radiocarbon dates and stable isotope data.

AMS radiocarbon dating & IRMS Radio- R. Age Calibrated stable isotope carbon Age R_Comb. dates Sample name Lab ID Site Location Type Material analysis Lab No. (BP) (BP) (BCE,95.4%)1 δ13C ‰ δ15N‰ Ref Carrowmore4 car004 Carrowmore Ireland Dolmen Tooth Beta-468275 4770 ± 30 3640-3380 -22.8 10.9 PS Primrose2 prs002 Primrose Grange Ireland Court tomb Tooth Beta-446171 4950 ± 30 3790-3660 -20.8 8.9 PS Primrose17 prs017 Primrose Grange Ireland Court tomb Tooth Beta-446181 4940 ± 30 3780-3650 -20.9 9.7 PS Primrose18 prs018 Primrose Grange Ireland Court tomb Tooth Beta-446182 4920 ± 30 3770-3650 -20.5 10.0 PS Primrose12 prs012 Primrose Grange Ireland Court tomb Tooth Beta-446178 4930 ± 30 3770-3650 -21.2 10.9 PS Primrose3 prs003/015 Primrose Grange Ireland Court tomb Tooth Beta-468277 4920 ± 30 3770-3650 -20.4 9.4 PS Primrose16 prs016 Primrose Grange Ireland Court tomb Tooth Beta-446180 4830 ± 30 3690-3530 -21.0 11.0 PS Primrose10 prs010 Primrose Grange Ireland Court tomb Tooth Beta-446176 4780 ± 30 3640-3520 -21.3 10.3 PS Primrose6 prs006/011 Primrose Grange Ireland Court tomb Tooth Beta-446177 4740 ± 30 3640-3380 -21.5 11.7 PS Primrose13 prs013/014 Primrose Grange Ireland Court tomb Tooth Beta-446179 4690 ± 30 3630-3370 -21.5 12.6 PS Primrose7 prs007 Primrose Grange Ireland Court tomb Tooth Beta-448276 4640 ± 30 3510-3360 -20.6 10.9 PS prs008/ 4670±30/ -21.4/ 9.8/ Primrose9 Primrose Grange Ireland Court tomb Teeth Beta-446174/5 4630±22 3500-3360 PS 009 4590±30 -21.4 10.9 Midhowe1 mid002 Midhowe Scotland Chamber cairn Skull frag. SUERC-46400 4700 ± 30 3630-3370 -20.5 10.3 71 Midhowe2 mid001 Midhowe Scotland Chamber cairn Skull frag. SUERC-46401 4531 ± 28 3360-3100 -20.6 10.3 71 Lairo1 lai001 Knowe of Lairo Scotland Chamber cairn Skull frag. SUERC-45833 4537 ± 34 3360-3100 -20.8 11.2 71 Balintore4 bal004 Balintore Scotland Short cist Skull frag. MAMS-21254 4543 ± 24 3370-3110 -19.2 Nd PS Ua-3785/ 4640±70/ 20.4 Ansarve5 ans005 Ansarve Sweden Dolmen Tooth 4589±28 3500-3130 -19.1 14.0 Beta-432257 4580±30 9 Ua-3783/ 4595±65/ 20.4 Ansarve3 ans003 Ansarve Sweden Dolmen Teeth 4566±28 3490-3110 -18.6 11.1 Beta-432256 4560±30 9 Ansarve8 ans008 Ansarve Sweden Dolmen Tooth Ua-45398 4480 ± 31 3340-3030 -21.1 8.6 20 Ansarve14 ans014 Ansarve Sweden Dolmen Mandible Beta-402962 4450 ± 30 3330-2950 -18.8 10.8 20 Ansarve17 ans017 Ansarve Sweden Dolmen Mandible Beta-402965 4430 ± 30 3330-2930 -18.8 11.1 20 Ansarve6 ans006 Ansarve Sweden Dolmen Tooth Ua-45394 4388 ± 31 3090-2920 -21.7 9.8 20 Ansarve7 ans007 Ansarve Sweden Dolmen Tooth Ua-45395 4310 ± 31 3010-2890 -21.7 9.6 20 Ansarve9 ans009 Ansarve Sweden Dolmen Tooth Ua-45400 4164 ± 32 2880-2630 -19.5 11.5 20 Ansarve16 ans016 Ansarve Sweden Dolmen Mandible Beta-402963 4160 ± 30 2810-25802 -17.5 13.0 20 Kolin6 kol006 Kolin Czech Rondel Radius UGAMS-9615 5950 ± 25 4910-4740 -19.9 Nd 45 Kolin2 kol002 Kolin Czech Rondel Radius UGAMS-9614 5710 ± 25 4650-4460 -20.4 Nd 45

Nd=not done. 1Calibration using OxCal 4.2.4 and IntCal13 (47-48). 2Corrected for radiocarbon age. PS = present study.

Carrowmore, County Sligo, Ireland The Carrowmore megalithic cemetery is situated on the Knocknarea Peninsula in Co. Sligo, Ireland (Fig. S1), and covers an area of about one square kilometre. It consists of approximately 30 tombs which were placed in an oval shape around an area where only one central monument was placed, Tomb No. 51 (also named Listoghil). The entrances to all preserved tombs at Carrowmore are facing this monument showing the importance of the location. The tombs at this site, with the exception of Tomb No. 51, have been grouped with the Irish Passage Tomb tradition, with simple dolmens containing passages, surrounded by stone circles. Unburnt human bones are occasionally found in Irish passage tombs, although cremation is more common, as is also the case at Carrowmore, except for Tomb No. 51. Thus, the cemetery has traditionally been classified as a passage tomb cemetery,

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although almost no features in tomb morphology correspond to other passage tomb cemeteries in Ireland. The low altitude of the cemetery (about 50 meters above sea level) and its position near the sea are also unusual features in the Irish passage tomb tradition. Since none of the tombs at Carrowmore display the kind of passage normally featured in Irish passage tombs, and only Tomb No. 51 was originally covered with a cairn, the only connection between Carrowmore and the Irish passage tomb tradition, apart from the concentration of monuments and cremated human remains, lies in the finds of passage tomb artefacts; such as the mushroom headed antler pins, stone and antler beads, and stone balls that were found in several of the tombs during the nineteenth-century excavations. Apart from the primary usage of the monuments, many of the tombs at Carrowmore also contain secondary deposits from the Late Neolithic, Bronze-, and Iron Ages (50). Various other stone monuments occur outside the central part of the cemetery, mainly to the north, and altogether 45 sites still exist in the area.

There may originally have been more tombs present at Carrowmore that have since been destroyed due to quarrying and field clearance during the last 300 years. Many of the tombs had previously also been dug into and robbed in the early 1830s by R.G. Walker, and several were also excavated in the early 1870s by W.G. Wood-Martin (51). Parts of the cemetery has also been excavated during the 1880s, as well as in 1977-1982, and in 1994-1998, by the Swedish Archaeological Excavations team lead by Göran Burenhult (52–61).

Tomb No. 51 (Listoghil)

The positioning in the centre of the cemetery and the original features of Tomb No. 51 (SRM No. SL14-20922) gives the monument a significant focal role in the Carrowmore megalithic cemetery context. The size of the burial site, with a diameter of c. 40 metres, as well as the existence of the remains of a cairn, marks a sharp difference between Tomb No. 51 and all other preserved monuments in the cemetery. Also, the chamber itself, constructed as a rectangular cist, or chamber, covered with a flat limestone roof-slab makes the monument unique among the Carrowmore tombs (Fig. S1). Megalithic art, typical passage-tomb circular carvings, were found on the front of the roof-slab.

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Figure S1. Tomb No. 51, Carrowmore, Co. Sligo, Ireland. Photo Göran Burenhult.

Tomb No. 51 was partially excavated by the Swedish team in 1996-1998. The complete central chamber area as well as three test trenches through the cairn, were investigated. Also, the well- preserved boulder circle, built of more than a hundred stones, was exposed. A series of radiocarbon dates (9 charcoal samples) indicate that the construction took place sometime between 3650 and 3450 cal BCE (61–66). A number of pits had also been dug into the layer during the erection of the monument, and one pit contained a large concentration of charcoal. Radiocarbon dates from these pits confirm that all these activities were more or less contemporary, centring around 3550 cal BCE (61, 65, 66).

Scattered finds and concentrations of burnt and unburnt human remains were found both inside and outside the central chamber. However, some of these remains were possibly dislocated during the treasure hunts and early excavations in the 19th century. Bones of at least five adult individuals and two children, approximately three and seven years old, were identified. Three unburnt bone pieces (frontal, temporal and malar bone), likely belonging to the same individual, showed clear cut-marks resulting from de-fleshing or possibly scalping (64). The frontal bone was radiocarbon dated to 3630- 3110 cal BCE, 95% CI (Ua-11581: 4625 ± 60 BP) (64). An additional tooth (car004 analysed here) 8

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revealed similar dating (see below). This shows that inhumations took place within the determined initial building period. These dates are slightly younger than the samples underneath the cairn and fit well within the proposed timespan for the construction of the limestone chamber and the cairn (see above). Although no cremated remains have been dated at Tomb No. 51 there are radiocarbon dated cremated remains from other tombs (No. 1, 19 and 55) at this site which show similar dating results (61, 64–66). This indicates that inhumation and cremation burials occurred at the same time-period at the Carrowmore megalithic burial site.

The sampling for DNA analyses were conducted under the Export Licence September 22, 1997, Licence to Alter 1996, October 1, 1997 and August 6, 1998 and Excavation Licence 96E0020, The Office of Public Works and The National Museum of Ireland.

Carrowmore4 (car004) – This individual was represented by an incisive tooth, ID60183) found inside the central chamber (X-0.83 Y-1.55 Z+58.20). This individual was dated to 3640-3380 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-468275: 4770 ± 30 BP, IRMS: δ13C -22.8 ‰, δ15N 10.9 ‰). The tooth was biologically inferred to belong to a male (Tables 1, and section S8). The crown exhibits slight wear, indicative of a young adult individual.

Primrose Grange, County Sligo, Ireland Primrose Grange Tomb 1 (SMR No. SL014-166) is a court tomb located about 2 kilometers southwest of Carrowmore, on the Knocknarea Peninsula in Co. Sligo, Ireland (Fig. 1). This tomb was excavated in 1996-1998 by the Swedish Archaeological Excavations team, led by Göran Burenhult and site director Maria Davidsson (61, 64–67). The excavations revealed that this monument was in use during the same period as the Carrowmore tombs (see below), although fundamental differences in tomb morphology, burial practices grave-goods could be documented. The Primrose Grange court tomb is a twelve meters long rectangular stone-cist, divided two separate sections, Cists A and B (Fig. S2, see also appendix S12). While inhumation is dominating the burials at Primrose Grange, cremation is the rule in the passage tombs at Carrowmore. Also, the grave-goods in the tomb depositions differ markedly between the sites. While mushroom-headed antler pins and stone/ antler beads dominate in the passage-tombs, extraordinary pieces of chert, mainly leaf-shaped or pointed arrow-heads, were found in the Primrose court cairn burial. The human remains found in the Primrose Grange tomb are almost all inhumation; very few cremated bones were located. Several concentrations of unburnt human bones were found, often mixed with animal bones. Most of these remains were disarticulated although a few had a higher degree of anatomical relationship. The small amounts of burnt human bones found were generally scattered over the whole area. The mixed state of the remains may indicate regular clearance of the bones to accommodate new burials. Osteological analyses indicate that Cist A contained at least 21 individuals; two males, three possibly males, two females and one possibly female, and two adults of undetermined sex. Eleven subadult individuals were also determined; one infant, two Infans I, five Infans II and three juveniles (68). Although Cist B has not 9

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undergone osteological analysis, the amount of bones found there indicates a similar number of individuals, meaning that possibly >40 individuals were buried in the tomb.

Figure S2. Primrose Grange, Tomb 1 before, during, and after excavation. Photos: Göran Burenhult. 10

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Three charcoal samples from the bottom layer (Layer III) in Cists A and B of the chamber have been dated c. 4300 and 4000 Cal BCE which is early for these types of monuments in Ireland and could indicate earlier Mesolithic activity at the site. While the majority of the so far dated bone and charcoal samples from depositions in the various layers, most of them from Layer II, show a continuous use between c. 3800 – 3000 Cal BCE (60, 61).

The fact that the Carrowmore tombs and Primrose Grange were used contemporaneously, in spite of such great differences when it comes to tomb morphology, burial traditions and grave goods, raises questions if this can be interpreted as symbols of status, group affiliation or hereditary rank in the local society, rather than successive stages of usage. This poses questions about social structure and even elitism. There is reason to believe that the builders of court tombs and portal tombs in Ireland had a close and formally organized relation with those building the passage tombs, maybe as hereditary-, or lineage-linked, chieftains or village chiefs. As in any chiefdom, possibly powerful leaders forming sub-groups in the megalithic society. The excavation of Primrose Grange Tomb 1 within the Carrowmore project revealed important data to suggest such an organization. The results of the genetic studies now presented are of fundamental importance in understanding this social complexity and the origin and economy of the tomb builders in the Mesolithic-Neolithic interface.

In total, 15 loose teeth from Primrose Grange were analysed in this study. Because of the mixed state of the bones in the tomb, each tooth was initially treated as a separate individual. Later, inferences of genetic relatedness (READ, (69)) between the samples, revealed that some of the teeth belonged to the same individual, thus the final consensus was a total of 11 individuals (see section S10, Tables 1, and S1-10). The 11 individuals analysed in this study are described below and were named according to the tooth ID that generated the largest amount of genetic data. All individuals were dated for this study and revealed dates between 3790 and 3360 cal BCE, 95% CI (see below, Tables 1, and TableS1).

Primrose2 (prs002) – This individual was represented by a canine tooth found in Cist A, layer II, in the bone concentration in the northeast corner. The tooth was radiocarbon dated to 3790-3660 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta-468275: 4950 ± 30 BP, IRMS: δ13C -20.8 ‰, δ15N 8.9 ‰). The tooth was biologically inferred to belong to a female (section S8). The tooth crown exhibited slight wear only, indicative of a (young) adult individual.

Primrose3 (prs003) – This individual was represented by a right maxillary 1st molar (prs003) found in Cist A, layer II, in the bone concentration in the southeast corner, as well as a left maxillary 2nd molar (prs015) from the same cist, in layer I, X+4 Y+6. Both teeth were biologically inferred to belong to a male and displayed the same mitochondrial haplotype (section S4, S6 and S8). They were assessed by READ (69) to belong to the same individual, now named prs003. The latter tooth was dated to 3770-3650 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals

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(Beta-468277: 4920 ± 30 BP, IRMS: δ13C -20.4 ‰, δ15N 9.4 ‰). The first molar exhibits moderate wear, the second exhibits slight wear, indicative of an adult individual.

Primrose6 (prs006) – This individual was represented by a right maxillary 1st incisive (prs006), as well as a left maxillary 1st incisive (prs011), both from Cist A, layer II, in the bone concentration in the northeast corner. Both teeth were biologically inferred to belong to a female, and they shared the same mitochondrial haplotype (section S4, S6 and S8). They were assessed by READ to belong to the same individual, now named prs006. The latter tooth was dated to 3640-3380 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-446177: 4740 ± 30 BP, IRMS: δ13C - 21.5 ‰, δ15N 11.7 ‰). Both crowns exhibit slight wear, adult individual.

Primrose7 (prs007) – This individual was represented by a left mandibular 2nd molar that was found in Cist A, layer II, in the bone concentration in the northeast corner. The tooth was dated to 3510-3360 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-448276: 4640 ± 30 BP, IRMS: δ13C -20.6 ‰, δ15N 10.9 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits slight wear, probably (young) adult individual.

Primrose9 (prs009) – This individual was represented by a (left?) mandibular premolar (prs008), as well as a (right?) mandibular 1st molar (prs009) which were both found in Cist B, layer II, square 13. Both teeth were biologically inferred to belong to a male and displayed the same mitochondrial haplotype (section S4, S6 and S8). They were assessed by READ to belong to the same individual, now named prs009. Both samples were radiocarbon dated displaying a combined calibrated date of 3500-3360 cal BCE, 95% CI (R-Combine: 4630 ± 22 BP), and stable isotopes revealed strict terrestrial dietary signals although slightly different (prs008 Beta-446174: 4670 ± 30 BP, δ13C -21.4 ‰, δ15N 9.8 ‰, and prs009 Beta-446175: 4590 ± 30 BP, IRMS: δ13C -21.4 ‰, δ15N 10.9 ‰). Both teeth exhibit slight wear, (probably young) adult individual.

Primrose10 (prs010) – This individual was represented by a (left?) maxillary premolar found in Cist A, layer I, X+3 Y+6. The tooth was dated to 3640-3520 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta-446176: 4780 ± 30 BP, IRMS: δ13C -21.3 ‰, δ15N 10.3 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits slight wear, probably (young) adult individual.

Primrose12 (prs012) – This individual was represented by a left maxillary 3rd molar that was found in Cist A, layer II, X+7 Y+7, in the bone concentration in the northeast corner. The tooth was dated to 3770-3650 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta- 446178: 4930 ± 30 BP, IRMS: δ13C -21.2 ‰, δ15N 10.9 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits slight wear only, adult individual.

Primrose13 (prs013) – This individual was represented by a right maxillary deciduous 2nd molar (Pd2) (prs013), as well as a left maxillary 1st incisive (prs014), both found in Cist A, layer II, in the 12

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bone concentration in the northeast corner, X+7 Y+7. Both teeth were inferred to belong to a male and displayed the same mitochondrial haplotype (section S4, S6 and S8). They were assessed by READ to belong to the same individual, now named prs013. The former tooth was dated to 3630-3370 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-446179: 4690 ± 30 BP, IRMS: δ13C -21.5 ‰, δ15N 12.6 ‰. Both teeth exhibit slight wear, the deciduous molar more advanced, sub-adult individual.

Primrose16 (prs016) – This individual was represented by a maxillary (1st?) molar (found in Cist A, layer II, in the bone concentration in the northeast corner. The tooth was dated to 3690-3530 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta-446180: 4830 ± 30 BP, IRMS: δ13C -21.0 ‰, δ15N 11.0 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits slight wear, probably (young) adult individual.

Primrose17 (prs017) – This individual was represented by a mandibular 3rd molar that was found in Cist A, layer II, in the bone concentration in the northeast corner. The tooth was dated to 3780-3650 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta-446181: 4940 ± 30 BP, IRMS: δ13C -20.9 ‰, δ15N 9.7 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits slight wear only, adult individual.

Primrose18 (prs018) – This individual was represented by a left maxillary 2nd molar found in Cist A, layer II, X+7 Y+7, in the bone concentration in the northeast corner. The tooth was dated to 3770- 3650 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Beta-446182: 4920 ± 30 BP, IRMS: δ13C -20.5 ‰, δ15N 10.0 ‰). The tooth was biologically inferred to belong to a male (section S8). The crown exhibits moderate wear, adult individual.

Midhowe, Orkney, Scotland Midhowe is a long horned stalled chambered cairn located on the southern coast of the island of Rousay, Orkney (Fig. 1). Prior to excavation in the early 1930s the cairn (covering mound) measured around 33m in length and 13m in breadth. Midhowe represents a so-called Orkney-Cromarty chambered cairn, which is a type of chambered cairn defined partly by their organization into stalled compartments placed on each side of a linear, central passageway (70, 71). The Midhowe burial chamber was organized into 12 cells, or compartments, on each side of the gallery, separated from each other by narrow slabs of stone (72). The remains of 25 individuals were originally excavated from Midhowe, although only two skulls remain in the museum collections at the University of Aberdeen. The excavation revealed that several individuals had been deposited onto shelves in some of the stalls or compartments; some individuals were anatomically intact (although sometimes headless) and placed in a crouching or sitting positions, while others had been disturbed and re- deposited either into commingled heaps of bone (disarticulated bone) or as disarticulated, individual

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skulls, suggesting a continued use (or reuse) of the chambered cairn over some period of time (71). Both individuals are included in this study (see below, Tables 1, and S1).

Midhowe1 (mid002) – This individual was an adult male and was represented by a skull (ID25898) in this study (museum number ABDUA:25898). The skull was dated to 3630-3370 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (GU-30636S/SUERC-46400: 4700 ± 30 BP, IRMS: δ13C -20.5, δ15N 10.3). The individual was biologically inferred to be male (section S8).

Midhowe2 (mid001) – This individual was a juvenile (in the late teens) and was represented by a skull (ID25899) in this study (museum number ABDUA:25899). The skull was dated to 3360-3100 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (GU-30637/SUERC- 46401: 4531 ± 28 BP, IRMS: δ13C -20.6, δ15N 10.3). The individual was biologically inferred to be male (section S8).

Lairo, Orkney, Scotland Lairo is a long horned chambered cairn of the Orkney-Cromarty style located on the island of Rousay, Orkney (Fig. 1). The cairn was partly excavated in the mid-1930s and revealed a tripartite chamber that was divided into three parts/cells by two pairs of narrow slabs of stone (in similarity to the chambered cairn of Midhowe, Orkney). The slabs were placed in pairs on each side of a narrow passage that leads through the chamber. Grant and Wilson (73) report that two skulls alongside ‘bones’ were found in two different parts of the cairn; one sealed off by a 14-inch secondary wall, and one placed in a recess of one of the compartments. One skull is currently held at the Marischal Museum at the University of Aberdeen and is included in this study (see below, Tables 1, and S1).

Lairo1 (lai001) – The sample for DNA analysis was taken from the skull (ABDUA:14761) of this individual. The skull was dated to 3360-3100 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals, (SUERC-45833: 4537 ± 34 BP, IRMS: δ13C -20.8 ‰, δ15N 10.2 ‰). The individual was biologically inferred to be male (section S8).

Balintore, Ross and Cromarty, Scotland This burial is situated in the village of Balintore in northern Scotland (Fig. 1). Development in the village in 1976 led to the discovery of a number of short cists, including one containing two partially preserved skeletons: one originally identified as an adult male based on morphology (INVMG 1985.022/bal004) and the other a small child aged approximately 2 years ± 8 months (INVMG 1985.021/bal005). The remains of the child were found overlying the adult skeleton. Although short cists generally date to the Early Bronze Age in Scotland (as with other cists found at Balintore), the individuals in this tomb has been dated to 3370-3030 cal BCE, 95% CI, thus belonging to the

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Neolithic period. The human remains are held at Inverness Museum. Both individuals yielded DNA, but only the adult individual was sequenced further (Table 1, and S1).

Balintore4 (bal004) – This adult individual (INVMG 1985.022) had previously been determined to be a male on the basis of morphological assessment but has been biologically inferred to be female based on genetic analysis of the right mandibular second molar root (see section S8). Bone from the skull of bal004 was dated to 3370-3110 cal BCE, 95% CI. The AMS dating result revealed a δ13C- value of -19.2 ‰ thus suggesting mainly a terrestrial diet (MAMS-21254: 4543 ± 24 BP).

Ansarve, Gotland, Sweden The Ansarve dolmen is located in Tofta Parish on the central west coast of the Swedish Island of Gotland in the Baltic Sea (Fig. 1). It is the north-eastern most confirmed megalithic burial belonging to the Funnel beaker culture (North group complex), where the dating of 17 individuals revealed continuous use between 3500 and 2580 cal BCE, 95% CI (20, 49). Secondary burials were also performed inside the dolmen during the Scandinavian Late Neolithic period (20). The Ansarve dolmen (Fig. S3) is the only confirmed megalithic burial from this time-period on the island, and individuals in the burial have been shown to be descendants of the European Early Neolithic expansion and show genetic affinity with other Middle Neolithic farmer groups in Scandinavia and continental Europe (20, 21). A test pit was excavated in the centre of the chamber by School Principal Hans Hansson and Army Doctor Karl Bolin in 1912 where the partial remains of at least eight individuals were recovered; three adult males, two adult females, plus three children; 5-14 years of age (49). Three (adult) mandibles were dated between 3630 and 3030 cal BCE, 95% CI (49). A second excavation was performed in 1984 in association with the “Archaeological Exploration Methods” framework Göran Burenhult, and the “Stone Age Gotland” project Inger Österholm (Stockholm University), in which the grave was analysed in detail. At least 31 individuals were identified in the osteological assemblage: sixteen adults (18-60 years, male and female), plus four juveniles (13-17 years), eight children (5-12 years), and three infants (0-4 years) (74), but the individuals from both excavations have not been osteologically analysed together. The dolmen had been partially destroyed and disturbed prior excavation and the human remains were very fragmented and comingled, partly also because of the excavation in 1912. Apart from the human remains, four pieces of amber, several hundred flint flakes (three of south Scandinavian flint), four round butted pecked axes, and a carved sandstone with a zig-zag pattern was located within and surrounding the tomb, but no pottery of any type was found.

The human remains from the 1912 excavation had been stored at the Swedish History Museum, and the remains from the 1984 excavation had been stored at Gotland University (now Uppsala University - Campus Gotland), as well as at the Gotland Museum. Two partial mandibles from the 1912 excavation, and three additional partial mandibles, as well as several loose maxillary, right 1st molars were included in the analyses of which nine individuals gave results for mitochondrial DNA (20) and 15

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six of those also gave enough results for nuclear DNA analyses (21). The nine individuals are included in this study (see below, Tables 1, and S1).

Figure S3. Left: Photo of the Ansarve dolmen with stone packing facing southeast (Photo by M. Fraser). Right: Plan drawing of the rectangular dolmen with entrance stones and limestone edging (from (75)). The angle corresponds to the photograph to the right.

Ansarve3 (ans003) – This individual was represented by a partial mandible with teeth from the 1912 excavation osteologically determined to be a female (49). Two 1st molars were radiocarbon dated which gave a combined calibrated date of 3490-3110 BCE, 95% CI (R_combine: 4566 ± 28 BP) and stable isotopes revealed mainly terrestrial dietary signals (Ua-3783: 4595 ± 65 BP, AMS δ13C-value: -19.3 ‰, and Beta-432258: 4560 ± 30 BP, IRMS: δ13C -18.6 ‰, δ15N 11.1 ‰) (20). The osteological sex determination was also confirmed biologically (21).

Ansarve5 (ans005) – This individual was represented by a partial mandible with teeth from the 1912 excavation, osteologically determined to be 17-25 years old (49). One 1st molar was radiocarbon dated twice which gave a combined calibrated date of 3500-3140 BCE, 95% CI (R_combine: 4594 ± 28 BP) and stable isotopes revealed mainly terrestrial dietary signals with possible some input of freshwater fish (Ua-3785: 4640 ± 70 BP, AMS δ13C-value: -18.6 ‰, and Beta-432259: 4580 ± 30 BP, IRMS: δ13C -19,1 ‰, δ15N 14.0 ‰) (20). The individual was biologically inferred to be female (21).

Ansarve8 (ans008) – This individual was represented by a right maxillary 1st molar from an approximately individual retrieved from the 1984 excavation (74).. The molar was radiocarbon dated to 3340-3030 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Ua- 16

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45398: 4480 ± 31 BP, IRMS: δ13C -19.2 ‰, δ15N 12.3 ‰) (20). The individual was biologically determined to be male (21).

Ansarve14 (ans014) – This individual was represented by a partial mandible with teeth from an approximately individual retrieved from the 1984 excavation (20). The mandible was radiocarbon dated to 3330-2940 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-402962: 4450 ± 30 BP, IRMS: δ13C -18.8 ‰, δ15N 10.8 ‰) (20). The individual was biologically determined to be male (21).

Ansarve17 (ans017) – This individual was represented by a partial mandible with teeth from an approximately individual retrieved from the 1984 excavation (20). The mandible was radiocarbon dated to 3330-2930 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Beta-402965: 4430 ± 30 BP, IRMS: δ13C -18.8 ‰, δ15N 11.1 ‰) (20). The individual was biologically determined to be male (21).

Ansarve6 (ans006) – This individual was represented by a right maxillary 1st molar from an approximately individual retrieved from the 1984 excavation (74). The molar was radiocarbon dated to 3090-2920 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Ua- 45394: 4388 ± 31 BP, IRMS: δ13C -21.7 ‰, δ15N 9.8 ‰) (20). The tooth was biologically determined to be male (This study, section S8).

Ansarve7 (ans007) – This individual was also represented by a right maxillary 1st molar from an approximately individual retrieved from the 1984 excavation (74). The molar was radiocarbon dated to 3010-2890 cal BCE, 95% CI, and stable isotopes revealed strict terrestrial dietary signals (Ua- 45395: 4310 ± 31BP, IRMS: δ13C -21.7 ‰, δ15N 9.6 ‰) (20). The tooth was biologically determined to be male (This study, section S8).

Ansarve9 (ans009) – This individual was represented by a right maxillary 1st molar from an approximately individual retrieved from the 1984 excavation (74).. The molar was radiocarbon dated to 2880-2630 cal BCE, 95% CI, and stable isotopes revealed mainly terrestrial dietary signals (Ua- 45400: 4164 ± 32 BP, IRMS: δ13C -19.1 ‰, δ15N 11.5 ‰) (20). The tooth was biologically determined to be female (This study, section S8).

Ansarve16 (ans016) – This individual was represented by a partial mandible with teeth from an approximately individual retrieved from the 1984 excavation (20). The mandible was radiocarbon dated to 2810-2580 cal BCE, 95% CI, and as stable isotopes revealed slightly elevated marine dietary signals and the dating result was corrected for marine reservoir effect (Beta-402963: 4160 ± 30 BP, (Reservoir correction 70 ± 40), IRMS: δ13C -17.5 ‰, δ15N 13.0 ‰) (20). The individual was biologically determined to be male (21).

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Kolin, Bohemia, Czech Republic Significant changes took place in central Europe between the culturally homogenous Linear Band Keramik (LBK) and subsequent archaeological cultures. The cultures that followed LBK were more distinct and dispersed on a smaller regional scale. In the west the Rössen culture was formed, in the southeast the Lengyel culture, and the Stichbandkeramik (STK) or Stroked Pottery culture replaced LBK in the east (46). The two STK individuals analysed here come from the site of Kolín in Bohemia, Czech Republic (These two individuals are also referred as “Czech_MN” for demographic analysis i.e. sections 11.1-4; see Extended Dataset 1.3 for details). The site is located on the Elbe river terrace, where four Neolithic rondels have been discovered, two of them together with human remains (45, 46). Two individuals from this site previously presented in Fraser et al. (21) are included in this study (see below, Tables 1, and S1).

Kolin2 (kol002) – This individual (skeleton 265) was morphologically determined to be a 20-25-year- old female with a stature of 148 cm (46) and is represented here by a right mandibular Incisor (I2). She was buried in a flexed position on her right side, in a ditch of the STK rondel accompanied by ceramics (see Fig. S4). She has previously been dated to 4650-4460 cal BCE, 95 but was not specifically IRMS analyzed for stable isotopes, the AMS dating result revealed a δ13C-value of -20.4 ‰ thus mainly a terrestrial diet (UGAMS-9614: 5710 ± 25 BP) (45). The osteological sex determination was also confirmed biologically (21).

Kolin6 (kol006) – This individual (skeleton 5160) was morphologically determined to be a female with a gracile body construction and moderately-developed muscular insertions (46) and is here represented by a lower right molar (M2). She was buried in a shallow construction pit along the wall of a long Neolithic house (see Fig. S5). She has previously been dated to 4910-4740 cal BCE, 95 but was not specifically IRMS analyzed for stable isotopes, the AMS dating result revealed a δ13C-value of -19.9 ‰ thus mainly a terrestrial diet (UGAMS-9615: 5950 ± 25 BP) (45). The osteological sex determination was also confirmed biologically (21).

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Figure S4. Excavation photograph of the kol002 female (skeleton 265). Photo by Radka Sumberová.

Figure S5. Excavation photograph of the kol006 female (skeleton 5160). Photo by Radka Sumberová.

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S3 Sample preparation, DNA extraction and Library preparation

The samples were prepared in three facilities all dedicated to the analyses of ancient DNA (aDNA): the DNA Laboratory DBW on Campus Gotland, the Human Evolution aDNA lab at the Evolutionary Biology Centre (EBC) both at Uppsala University, and at the Archaeological Research Laboratory (AFL) at Stockholm University. These laboratories are equipped with air-lock separating the lab from the outside corridor, positive air pressure, UV lamps in the ceiling (254nm) and HEPA-filtered laminar flow hoods. They are frequently cleaned with bleach (NaOH) and UV-irradiation and all equipment and non-biological reagents are regularly decontaminated with bleach and/or DNA-away (ThermoScientific) and/ or UV irradiation. The work followed strict standards for working with degraded samples as outlined in (76–80). Prior to sampling the samples were wiped with sterile cotton swabs soaked in 0.5% NaClO solution, then with distilled water, air dried, and irradiated with UV- light at 6 Joule/cm2. The outer surface of the tooth roots or bone was carefully removed and discarded.

Between one and seven DNA extractions were carried out for each individual (Table S2) and between 40 -120 mg of bone powder was used for each DNA extraction. The powder was obtained either through drilling using a round Meisinger ISO 021 dental burr or a Dremel drill with either cutting discs or dental burs, or through cutting off tooth roots and grinding them in the a Starbeater (VWR). DNA was extracted using a silica spin column method (81) modified as in Malmström et al. (82), Günther et al. (79) and Schlebusch et al. (78), or using the method from Dabney et al. (83) (Table S2). A negative extraction control accompanied every 6-10 samples. Some of the samples were extracted using the previously mentioned methods but using the QIAcube from Qiagen.

Different library building strategies were used to generate sequencing data for different samples in this study, for details on which samples were processed with a given strategy see Table S2. In brief, Illumina multiplex sequencing libraries were created with a blunt end (BE) ligation method following Meyer and Kircher (84) as described in Günther et al. (79). Further for some samples damage-repair (DR) libraries were generated following Briggs and Heyn (85) and Günter et al. (79). For some of the Irish samples single strand libraries (SS) were also produced following the method according to Gansuage and Meyer (86). Some of the BE libraries from the Irish samples were also enriched for human genomic DNA using Caucasian baits in the MYbait Human Whole Genome Capture (WGC) Kit (MYcroarray, Ann Arbor) following the manufacturer’s instructions (MYbait manual version 2.3.1) with hybridization for 36-44 hours at 55ºC (see Table S2).

Four to six parallel PCRs were set up for each library at 9 to 15 cycles dependent on the sample extract and library. Quantification and library pooling were performed at the Evolutionary Biology

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Centre, Uppsala University, and at AFL, Stockholm University. Libraries were quantified on the Bioanalyzer 2100 using the High Sensitivity DNA chip (Agilent) or the 2200 TapeStation using High sensitivity DNA screentape (Aligent) following manufacturer’s protocols. Libraries were pooled in equimolar concentrations and sequenced on the Illumina Hiseq 2500 or the HiseqX at the SNP & SEQ technology platform at SciLife Sequencing Centre at Uppsala University, or at Sci Life NGI in Stockholm.

Table S2. Extraction and library information for the ancient samples.

Library Library Yang Dabney Library Damage- single- MYbaits Individual Bone element Lab Extraction Extraction Blunt-end repair stranded capture prs002 Tooth EBC 4 1 1 6 1 1 prs017 Tooth EBC 1 1 2 prs018 Tooth EBC 1 1 1 1 2 prs012 Tooth EBC 3 1 1 2 1 2 prs003 Teeth (prs003+prs015) EBC 3 2 1 1 2 prs016 Tooth EBC 2 1 1 prs010 Tooth EBC 1 1 1 1 2 prs006 Teeth (prs006+prs011) EBC 2 2 2 2 4 prs013 Teeth (prs013+prs014) EBC 4 2 2 7 2 3 prs009 Teeth (prs008+prs009) EBC 5 2 2 8 2 3 prs007 Tooth EBC 1 1 0 1 2 car004 Tooth EBC 3 1 0 1 2 mid002 Skull fragment AFL 1 1 mid001 Skull fragment AFL 1 1 lai001 Skull fragment AFL 1 1 bal004 Tooth AFL 1 1 ans003 Tooth DBW 7 11 ans005 Tooth DBW 4 5 ans006 Tooth DBW 1 1 ans007 Tooth DBW 4 4 ans008 Tooth DBW 3 12 ans009 Tooth DBW 3 3 ans014 Teeth DBW 5 14 ans016 Tooth DBW 1 4 ans017 Teeth DBW 13 13 kol006 Tooth EBC 3 4 6 kol002 Tooth EBC 1 2

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S4 Processing of NGS data Each sequencing experiment was processed as in (79). Paired-end reads were merged using MergeReadsFastQ_cc.py (87), when an overlap of at least 11 base pairs was found the base qualities were summed up together, remaining adapter sequences were trimmed. Merged reads were then mapped single ended to the human reference genome hg19 (build 37) with bwa aln 0.7.17 (88) using the non-default parameters: seeds disabled -l 16500 -n 0.01 -o 2 (17, 89). In order to remove PCR duplicates, reads with identical start and end positions were collapsed using a modified version of FilterUniqSAMCons_cc.py (87). Finally, only reads with less than 10% mismatches to the human reference genome, and reads longer than 35 base pairs, were kept for downstream analyses and for estimating contamination (79).

In general, genetic data from the Ansarve, Scottish individuals, and Kolin2 was produced using only the BE strategy. In order to maximize the yield and improve the quality of the aDNA extracted for some of the individuals from Primrose as well as Carrowmore4 and kolin6, additional data was produced using the DR library-building strategy, SS retrieval and/ or WGC, as described above (see Table S2). Given that for most of our analyses only transversions were used (demographic analyses) or data is analysed independently (characterization of biological sex and uniparental markers) we decided to combine all types of data available for the all Primrose and Kolin6 individuals for which data was generated using different types library-building (see Table S2, section S6-8,11).

Given the commingled nature of the Primrose Grange archaeological material used in this study (see section S2 above), and the fact that some of these remains harboured the same mitochondrial haplotype (see Extended Dataset 1.1), we hypothesized that some of these remains might belong to the same individual. We ran READ (69), a tool to estimate kin-relationship from ancient DNA, using the genetic data from all fifteen Primrose Grange bone elements to identify the total number of different individuals.

We found that genetic data from remains from the following pairs: Primrose 3 and 15, Primrose 6 and 11, Primrose 8 and 9, and Primrose 13 and 14 were predicted to be from the same individuals (or identical twins), and thus, these samples actually belonged to only four different individuals. In all cases the mitochondrial lineages from each pair of bone elements matched to each other (see section S6 and Extended Dataset 1.1). Data was merged using Samtools v1.3 merge command, first per each type of library-building data, and was then named after the bone element that had generated most data (i.e. Primrose 3, 6, 9, 13). Bone element data belonging to the same individual was merged accordingly for each type of library-building data (i.e. BE, WGC, DR) independently. In the end genetic data from the Primrose Grange archaeological site was composed of 11 unique individuals

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(section S2 and Table S2). For the Y-chromosome lineage characterization, as well as the demographic analyses (see section S7 and S11, respectively), all available data per individual was merged at the library-building individual level. For all samples analysed, except the Primrose individuals, kinship analyses were undertaken for the library-building strategy type of data available. For the Primrose individuals, familiar relationships were inferred using both BE and WGC data independently (see section S10).

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S5 Contamination estimation Contamination was estimated using three different sources of data: (I) the mitochondrial genome, (II) the X chromosome if the individual was male, and (III) the autosomes as described in Günther et al. (79). Low contamination estimations over the three different approximations were interpreted as data mapping to the human genome being largely endogenous.

Mitochondrial contamination

We used ContaMix (90) to estimate contamination in the samples from the present-study using mtDNA data. In brief, ContaMix models sequencing reads per each sample as coming from a mixture of a set of full-length mitochondrial genomes (311 mitochondrial genomes as reported in Green et al. (91)) and the 'endogenous' consensus with unknown proportions. Such proportions are in turn inferred using a MCMC method. Thus, the contamination fraction is the sum of the proportions corresponding to 'non- endogenous' mitochondrial sequences. ContaMix assumes that contamination is present in less than 50% when doing the estimations per sample, so accurate estimations are obtained. ContaMix contamination estimates for each of the samples of the present study are reported in the Extended Dataset 1.2. Extended Dataset 1.4 contains mitochondrial contamination estimates for each type of libraries build for the Primrose individuals (i.e. BE, WGC and DR; see section S4).

X chromosome contamination

Given the high number of male individuals from the present study, we estimated contamination in these individuals using a method that examines heterozygous sites within the X chromosome (92, 93). The ANGSD (v.0.902) X chromosome contamination module was run with two steps as described in the software manual. In the first step, a binary count file was built using the command “angsd -r X:5000000-154900000 -doCounts 1 -setMinDepth 3 -setMaxDepth 100 -iCounts 1 -minMapQ 30 - minQ 30”. In the second step the contamination estimate was obtained with the command “contamination -d 3 -e 100”. In this latter step, only transversion polymorphisms were screened to avoid bias due to post-mortem DNA damage. We report contamination estimates and the confidence interval from method 1, which samples all reads from each site, thereby producing a more precise and sensitive estimate compared to method 2, which randomly samples one read per site. Given the low- coverage of the Midhowe2, Lairo1, Primrose4, Primrose3, Primrose12, Primrose10, Primrose17, and Primrose18 individuals, X contamination estimations were assessed at filtered sites with at least one overlapping read per screened position. For the rest of the male individuals only filtered sites with more than three overlapping reads were included (Extended Dataset 1.2).

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Contamination in the nuclear genome

The level of autosomal contamination in the ancient samples was estimated using verifyBamID (94, 95). We assume that if contamination is present, it would most likely come from a European genetic background. Therefore, we used allele frequencies from a population of Central European descent, namely CEU 1000 genomes project data to model contamination. To estimate contamination at the sample level, we ran VerifyBamID (v.1.1.2) as described in Günther et al. (79) and restricted to estimate contamination on transversion sites only, to avoid overestimation of point estimates due to post-mortem damage. The program reports the contamination estimates in the FREEMIX column. As stated in Günther et al. (79), we note that while this is a powerful approximation to estimate contamination directly to nuclear data in main analyses, its accuracy to estimate contamination in ancient low coverage samples has not been formally tested. Nonetheless, the consistency and low contamination estimates obtained using the three approximations above (when possible) advocated for the authenticity of our data (Extended Dataset 1.2).

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S6 Calling mtDNA haplogroups In order to assess the mitochondrial haplogroup to which each bone element/ individual belonged to, we generated a mtDNA consensus sequence, in fasta format, for each type of data bamfile generated for each of the bone elements/individuals. Ancient DNA deaminations could introduce artificial variation that might (slightly) modify the mtDNA lineage assignation however, and complicate the inference of mtDNA private mutations, particularly of samples with a low mtDNA average coverage. In order to account for this potential source of error, MapDamage (V2.0.2-15) (96, 97) was used with default parameters to rescale the base qualities of potentially deaminated sites to non-significant base quality value (i.e. “#”). ANGSD (v.0.902) (93) was used to generate a majority-called consensus mitochondrial sequence, employing the following command: ”angsd -i infile.bam -minQ 30 - minMapQ 30 -doFasta 2 -doCounts 1 -setMinDepth 3 -basesPerLine 60 -out outfile.MT”. Such commands filter for sites that have a minimum coverage of 3, and which have a base and mapping Phread quality score of at least 30. Haplogroups were assigned to each mitochondrial consensus sequence using Haplogrep2 (98, 99). The mitochondrial coverage, haplogroups, mutations supporting the called haplogroup, and private mutations are reported in Extended Dataset 1.1.

We found that two mitochondrial lineages were overrepresented among the Primrose Grange individuals: K1a (Primrose6, Primrose10, Primrose17 and Primrose18), and K1a4a1 (Primrose7 and Primrose16). A similar pattern was observed among the Ansarve individuals, where Ansarve8 and Ansarve14 shared lineage J1c5, and Ansarve7 and Ansarve9 shared haplotype K2b1a. Interestingly, the Primrose17-Primrose18 pair shared the same private mutations (see Extended Dataset 1.1). To assess the diversity of mitochondrial lineages per site we classified the mtDNA lineages into 20 major mitochondrial groups, as described in (20, 100). All megalithic burial groups investigated to date (16, 17, 19, 21, 44), as well as non-megalithic burial groups from the British Isles, either from inhumations or cave burials, were used as controls.

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Figure S6. MtDNA haplogroup frequencies (defined from) megalithic burials across Europe, plus non-megalithic burials from the British Isles (BI); either from cave burials (NM_Caves_BI), single inhumations (NM_SI_BI), or the latter two groups together (NM_BI).

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S7 Calling Y chromosome haplogroup All single base substitutions from the International Society of Genetic Genealogy (ISOGG; http://isogg.org (version 11.110, April 21, 2016)) were called using BAM files mapped to hg19 and SAMTools mpileup (version 1.3). Sites with a mapping quality and a base quality of at least 30 were extracted and insertions/ deletions, and sites that displayed multiple alleles, were excluded. We investigated both transitions, transversions, plus A>T and G>C sites and report derived alleles within the called haplogroup. For low coverage individuals, we further report key ancestral alleles within the haplogroup to display the certainty of the call. The marker name, nucleotide position, type of substitution (ancestral>derived state), and coverage, are shown in brackets. When more than 20 haplogroup defining markers display derived allele states, we report three of them in the text. We note that there were some derived alleles scattered all over the phylogeny, these were however, contradicted by upstream alleles.

Because of this, we only call sub-haplotypes if > two derived alleles support that particular lineage. We used the updated ISOGG haplotype nomenclature (from version 11.329, 22 Dec 2017). We note that several of the individuals (Primrose9, Primrose13, Primrose16, Primrose17, Ansarve8, Ansarve14 and Ansarve17) displayed derived alleles for the I2a2a2a marker L1228. This observation was, however, refuted by several ancestral allele states at upstream markers, as well as by the wide support for the haplotypes actually called. The possible explanations we can find for this is that this mutation is either a recurrent mutation or that the allele reported as ancestral (C) and derived (G) at ISOGG is erroneous. We rule out contamination as the cause as the samples are from different countries and have been handled and processed by different people in different laboratories. We report presence of derived or ancestral alleles per informative lineages in the form of: (Name_markers:pos, mutation, coverage) as displayed below.

Carrowmore4

Car004 had low amounts of data (the genome coverage was 0.04X) and he likely belongs to haplogroup I. He displayed derived alleles for five markers defining I (PF3758:17245841, T>C, 1; PF3771:17940414, G>A, 1; PF3795: 21077471, C>T, 1; PF3815:21841289, G>T, 1 and FGC7049:22459264, G>A, 1) but also the ancestral state for one I marker (P38:14484379, A>C, 1). He could possibly belong to I2a2a1 (CTS616:6906332, G>C, 1) but as no data was available for other markers leading to this lineage, we adopt a stringent strategy and only assign him to haplogroup I. It may be possible to exclude a lineage calling within I2a1b (S2632:7317227, G>A, 1), but again, although he is ancestral for this marker there is not enough data to be certain.

Primrose3

Prs003 was assigned to haplogroup I based on 11 mutations (PF3878:23401471, C>T, 1; PF3722:15089989, T>C, 1; PF3734:15793946, G>A, 1; PF3738:15960476, C>G, 1; 28

Supplement: Megalithic tombs in western and northern Neolithic Europe

PF3739:16039881, C>T, 1; PF3653:8267857, G>A, 1; PF3689:13642029, A>C, 1; L503:21359407, C>G, 1 and PF3800:21402723, A>G, 1). A downstream transversion mutation may indicate a belonging in I2a2a (S119:24475669, G>T, 1) although more data would be needed to confirm that call. Ancestral allele states downstream of I2a2a was further explored, and due to lack of data, we cannot exclude that this individual belong to the I2a2 subclades that other individuals from Primrose Grange belong to. We could only exclude some other sub-lineages (I2a2a1a1a1-S165:14901633, C>T, 1; I2a2a1a1a2a-CTS4922:15903939, T>C, 2; I2a2a1a2a1a1a-Y9443:6371561, A>G, 1; I2a2a1a2a1a1c1-Y4760:15025945, A>T, 1; I2a2a1a2a2-S18331:16449714, C>T, 1; I2a2a1a2b- Y7240:2688254, T>G, 1 and I2a2a1b1-L701:6753316, C>T, 1). Prs003 displayed ancestral alleles (and could thus be excluded) for markers defining other I-lineages, such as I1 (S64:17766762, T>C, 1; S109:7548915, G>A, 1; P30:14496753, G>A, 1; L118:15500110, A>C, 1; S108:6681479, T>G, 1 and L80:14640715, A>G, 1), some I2a1 subclades (I2a1a1a1a-S5312:8681127, C>A, 1; I2a1b1b- PF4135:18981938, G>A, 1 and I2a1b2a1 -L147.2:6753258, T>C, 1) and I2c (S12355:9403861, G>A, 1; S6728:16526276, T>C, 1; SK1266:8634040, C>A, 1).

Primrose7

The haplogroup could not be called for prs007 due to lack of data and we could only conclude that belongs within macro haplogroup BT (as he had derived alleles for M9076: 14018605, G>A, 1; M9130:15147332, T>C, 1; M9131:15147334, A>G, 1; M9195:17063750, T>A, 1; M9249:18133150, G>C, 1; M9367:22879049, T>C, 1 and M8949:2750722, G>C, 1).

Primrose9

Prs009 was assigned to I2a2a1a1a (L1195:18865320, G>A, 5), and displayed derived alleles also for I2a2a1a (Y3721: 22547472, C>T, 3), I2a2a1 (CTS616:6906332, G>C, 3), I2a2a (PF3857:7716262, A>C, 4; S152:17570599, C>T, 3; M223:21717307, G>A, 3; L59:7113556, C>T, 2; S24:15517851, T>G, 3; S119: 24475669, G>T, 2; PF3858:8353707, C>A, 2; U250:18888200, C>G, 4 and S117:16699334, C>G, 2), I2a2 (S150:22725379, C>A, 4; S153:17516123, T>C, 4; S33:18747493, G>C, 5; L181:19077754, G>T, ; L368:6931594, C>T, 4; S30:13992338, C>G, 2; S23:7628484, C>T, 1 and S32:17493630, T>G, 4), I2a (PF3647: 7879415, A>C, 5), I2 (PF3781:18700150, C>T, 2; S31:16638804, A>G, 3 and Z2638:8567995, G>A, 3) as well as for 166 markers defining I (e.g. PF3715:14847792, A>C, 5; PF3721:15023364, T>C, 11 and PF3742:16354708, G>A, 4). As he was ancestral for I2a2a1a1a1 (S165:14901633, C>T, 4; S166:9791250, G>A, 1 and L369:14850314, T>C, 4), I2a2a1a1a2 (L1193:9448484, C>A, 3) and I2a2a1a1a2a (CTS4922:15903939, T>C, 9; Y3713:19106058, G>A, 6 and Y3684:6760498, C>T, 6) he belongs to I2a2a1a1a-L1195 (xI2a2a1a1a1, I2a2a1a1a2). Like several of our samples, prs009 carried the derived allele for an I2a2a2a marker (L1228:15446045, C>G, 5). It is, however, unlikely that he belongs to this lineage as he displayed ancestral alleles for the upstream I2a2a2-lineage (Y6098:9647453, C>T, 5 and SK1254:6742730, T>C, 3).

Supplement: Megalithic tombs in western and northern Neolithic Europe

Primrose10

Prs010 displayed derived alleles for 17 markers defining haplogroup I (PF3878:23401471, C>T, 2; PF3706:14337364, T>C, 1; PF3707:14352669, G>A, 1; PF3712:14646409, C>T, 1; PF3716:14884646, C>T, 1; PF3717:14884659, A>C, 1; PF3747:16548548, G>A, 1; PF3757:17090238, C>G, 1; PF3776:18172947, A>G, 1; Y1880:13835003, T>C, 1; YSC0000284:8485677, C>A, 1; Z16984:13442439, C>T, 1; PF3694:13900590, T>C, 1; CTS5263:16171560, G>A, 1; CTS8963:18582617, C>T, 1; PF3794:21067903, C>T, 1 and PF3649:8046731, A>C, 1). This individual was also derived for I2a2a1a (Y3721: 22547472, C>T, 1).

As this is a C to T transition, that could potentially be caused by ancient DNA damage, we denote him conservatively to haplogroup I although he could potentially belong to a similar I2a2a-lineage as many of the other individuals from Primrose Grange. We also note that he is less likely to belong to I2a1b, the group which several of the Scottish and Ansarve males belongs to, as he displays an ancestral read for such a marker (S2702: 17359886, A>C, 1). We could further exclude downstream haplogroups I2a2a1a2, I2a2a1a2a and I2a2a1a2b (Z2083:15379927, T>C, 2; L1229:14937828, C>A, 1 and Y7240:2688254, T>G, 1 respectively) as well as other I-lineages such as I1 (L187:18077297, A>T, 1 and M253:15022707, C>T, 1), I2a2a1b1 (L702: 7629205, C>T, 1), I2a2b2b1 (Y13074: 14094675, A>C, ), I2a2b (S156: 16202267, T>C, 2) and I2c (PF3895:8638358, T>G, 2 and S6659:13891261, G>A, 1).

Primrose12

Prs012 could be assigned to I2a2a1a1a2 (L1193: 9448484, C>A, 1) and further displayed upstream mutations for I2a2a (U250:18888200, C>G, 1), I2a2 (S32:17493630, T>G, 1) as well as 12 mutations for I (PF3712:14646409, C>T, 1; PF3752:16826642, G>A, 1; PF3758:17245841, T>C, 3; PF3773:18018313, C>A, 1; PF3786:18992894, T>C, 1; PF3788:19097563, T>C, 1; Y1903:21155653, C>T, 2; YSC0000298:23154034, C>T, 3; PF3811:21627180, C>T, 1; PF3642:7712917, A>T, 1; PF3666:8728974, T>G, 1 and YSC0000256.1:9827411, G>A, 1). He was ancestral for the downstream marker defining I2a2a1a1a2a (Y3684:6760498, C>T, 1) as well as for other I-lineages such as I1 (S66:22914378, T>C, 1; S65:18759669, T>C, 1 and L187:18077297, A>T, 1), I2c (PF3889:8001008, T>C, 1), I2a1a1 (S169.1: 15810964, T>G, 1), I2a1b (S2715: 17893806, A>G, 2), I2a2b (L272.3:3436239, A>T, 1) and I2a2a1a2 (Z2083:15379927, T>C, 2).

Primrose13

Prs013 belonged to I2a2a1a1a (L1195: 18865320, G>A, 1) and further displayed derived allele states for I2a2a1a (Y3721:22547472, C>T, 1), I2a2a1 (CTS9183:18732197, A>G, 6 and CTS616:6906332, G>C, 2), I2a2a (PF3857:7716262, A>C, 2; S152:17570599, C>T, 2; M223:21717307, G>A, 1; L59:7113556, C>T, 6; S24:15517851, T>G, 1; S119:24475669, G>T, 2; PF3858:8353707, C>A, 2 and U250:18888200, C>G, 2), I2a2 (S150:22725379, C>A, 8; S153:17516123, T>C, 4; S33:18747493, 30

Supplement: Megalithic tombs in western and northern Neolithic Europe

G>C, 2; L181:19077754, G>T, 4; L368:6931594, C>T, 3; S23:7628484, C>T , 2and S32:17493630, T>G, 6), I2a (PF3647: 7879415, A>C, 2), I2 (PF3781:18700150, C>T, 5; S31:16638804, A>G, 3 and Z2638:8567995, G>A, 1) and 139 mutations for I (e.g. PF3715:14847792, A>C, 1; PF3721:15023364, T>C, 1 and PF3742:16354708, G>A, 1). As prs013 displayed ancestral allele states downstream of I2a2a1a1a, for I2a2a1a1a1 (S165:14901633, C>T, 1; S166:9791250, G>A 6 and L369:14850314, T>C, 2) and I2a2a1a1a2a (CTS4922:15903939, T>C, 3; Y3713:19106058, G>A, 1 and Y3684:6760498, C>T, 4), we could exclude that sublineage. Like several of the other individuals, prs013 further displayed the derived allele for I2a2a2a (L1228:15446045, C>G, 3) although being ancestral for two upstream markers defining I2a2a2 (Y6098:9647453, C>T, 1 and SK1254:6742730, T>C, 2). He was also derived for a marker defining I1b (Z131:5845252, G>A, 1), most likely caused by ancient DNA damage, as ancestral allele states were displayed for 21 upstream markers defining haplogroup I1 (e.g. M253:15022707, C>T, 2; L80:14640715, A>G, 5 and P203.2:22750951, G>A, 3).

Primrose16

Prs016 could be assigned to haplogroup I2a2a1a1a (L1195:18865320, G>A, 4) and to upstream I2a2a1 (CTS616:6906332, G>C, 3), I2a2a (PF3857:7716262, A>C, 1; S152:17570599, C>T, 3; M223:21717307, G>A, 1; L59:7113556, C>T, 1; S24:15517851, T>G, 4; S119:24475669, G>T, 3; PF3858:8353707, C>A, 4; U250:18888200, C>G, 2 and S117:16699334, C>G, 4), I2a2 (S150:22725379, C>A, 4; S33:18747493, G>C, 4; L181:19077754, G>T, 8; L368:6931594, C>T, 2; S30:13992338, C>G, 1; S23:7628484, C>T, 3 and S32:17493630, T>G, 1), I2a (PF3647:7879415; A>C, 2), I2 (PF3781:18700150, C>T, 3 and S31:16638804, A>G, 2) and was also derived for 171 markers defining I (e.g. PF3715:14847792, A>C, 4; PF3721:15023364, T>C, 4 and PF3742:16354708, G>A, 3). Like some of the other males, prs016 displayed the derived allele for a marker defining I2a2a2a (L1228:15446045, C>G, ) but was ancestral for two upstream I2a2a2 markers (Y6098:9647453, C>T and SK1254:6742730, T>C). Similar to prs013, prs016 displayed ancestral allele states downstream of the called I2a2a1a1a, for I2a2a1a1a1 (S165:14901633, C>T, 7; S166:9791250, G>A, 2 and L369:14850314, T>C, 1) and I2a2a1a1a2a (CTS4922:15903939, T>C, 4; Y3713:19106058, G>A, 3 and Y3684:6760498, C>T, 4), indicating that such sublineages could be excluded.

Primrose17

Prs017 belongs to haplogroup I and displayed derived allele states for 15 markers that defines the haplogroup (PF3703:14214481, G>T, 1; PF3712:14646409, C>T, 1; PF3725:15377802, G>A, 1; PF3735:15799074, C>T, 1; PF3739:16039881, C>T, 1; PF3753:16836079, C>A, 1; PF3761:17525137, A>G, 1; PF3768:17818847, G>A, 1; PF3773:18018313, C>A, 1; Y1841:4974832, A>G, 1; YSC0000275.1:2884029, T>C, 1; L503:21359407, C>G, 1; PF3800:21402723, A>G, 1; PF3815:21841289, G>T, 1 and PF3649:8046731, A>C, 2). Prs017 could possibly belong to I2a2 (L368:6931594, C>T, 1), but as this transition could be caused by ancient DNA damage, we denote

Supplement: Megalithic tombs in western and northern Neolithic Europe

him conservatively to haplogroup I. Similar to several other males from Primrose Grange, this individual was derived for I2a2a2a (L1228:15446045, C>G, 1), although ancestral for upstream I2a2a2 (Y6098:9647453, C>T,1). As the coverage was low (0.18X genome coverage) we further report the ancestral allele states for I-lineages and could thus exclude I1 (S63:16478520, A>G, 1; P203.2:22750951, G>A, 1 and L118:15500110, A>C, 1), some I2c sublineages (I2c1a1- S21750:19076690, C>T, 2, I2c1a2a-CTS4092:15391894, G>A, 1 and I2c2-Z26388:16756192, G>A, 1; PH3539:17949064, G>A, 1; Z26404:21594210, C>T, 1; Y16672:22155834, C>T, 1; Y16659:23998554, T>C, 1 and Y16680:9796337, C>G, 1), some I2a1 sublineages (I2a1a1a1a- S5312:8681127, C>A, 1 and I2a1b2a1a3-Z16983:17558968, T>C, 1), and some I2a2 sublineages (e.g. I2a2a1a1a2a-Y3684:6760498, C>T, 1 and I2a2a1a2-Z2083:15379927, T>C, 1).

Primrose18

Prs018 belongs to haplogroup I and has ten haplogroup defining mutations (PF3778:18257568, G>A, 1; PF3786:18992894, T>C, 1; Y1865:7642823, G>T, 1; YSC0000285:8536868, C>G, 1; YSC0000275.1:2884029, T>C, 1; YSC0000280:7856500, C>T, 1; CTS7329:17424807, C>T, 1; PF3828:22458430, C>T, 1; PF3596:4403308, T>G, 1 and PF3605:5217196, A>G, 1). He could possibly belong to a different sublineage compared to the other Primrose Grange males as he displayed a transversion mutation for I2a1b (S2702:17359886, A>C, 1). As there were no more data supporting this lineage, we adopt a conservative approach and denote him to haplogroup I. As the coverage was quite low for this individual (0.09X genome coverage), we further investigated which I- lineages we could exclude (i.e. which markers he displayed an ancestral allele state for), and conclude that he does likely not belong to either I1 (S65:18759669, T>C, 1), I2c1a1 (S16199:14601211, T>C, 1), I2c1a2a (CTS4092:15391894, G>A, 1), I2a2 (S150:22725379, C>A, 1) and I2a1a1 (PF4056:21865821, G>A, 1).

Ansarve8

Ans008 belongs to I2a1b1a1a. The haplotype call was supported by derived alleles for I2a1b1a1a (S2703:17361387, C>A, 2) and for upstream I2a1b1a1 (L1498:18668472, C>T, 1), I2a1b (S2768:22905944, G>C, 5; S2687:16594452, A>G, 1; S2715:17893806, A>G, 5; S2722:18049134, C>T, 2 and M423:19096091, G>A, 1), I2a (PF3647:7879415, A>C, 2) as well as for 63 markers defining haplogroup I (e.g. PF3721:15023364, T>C, 1; PF3742:16354708, G>A, 1 and PF3730:15595624, G>A, 5). Although Ans008 also displayed a derived allele state for a marker defining another sub-clade within I, I2a2a2a1 (L1228:15446045, C>G, 1), this was refuted by ancestral allele states for 13 upstream mutations (I2a2a2-Y6098:9647453, C>T, 1 and SK1254:6742730, T>C, 1; I2a2a-PF3857:7716262, A>C, 1; S152:17570599, C>T, 1; S24:15517851, T>G, 3; PF3858:8353707, C>A, 1; U250:18888200, C>G, 1 and S117:16699334, C>G, 2 and I2a2- S150:22725379, C>A, 1; S153:17516123, T>C, 1; S33:18747493, G>C, 3; L368:6931594, C>T, 1 and S23:7628484, C>T, 1).

Supplement: Megalithic tombs in western and northern Neolithic Europe

Ansarve14

Ans014 belongs to I2a1b1a1a. This individual was derived for I2a1b1a1a (S2703:17361387, C>A, 1) and for upstream I2a1b1a1 (L1498:18668472, C>T, 2), I2a1b (S2768:22905944, G>C, 2; S2638:14074218, A>T, 2; S2679:16233135, A>G, 2; S2687:16594452, A>G, 3; S2702:17359886, A>C, 1; S2715:17893806, A>G, 1; S328:15574052, G>A, 2 and M423:19096091, G>A, 3), I2a (PF3647:7879415, A>C, 2), I2 (PF3781:18700150, C>T, 1; S31:16638804, A>G, 3 and Z2638:8567995, G>A, 2) and for 117 markers defining haplogroup I (e.g. PF3721:15023364, T>C, 1; PF3742:16354708, G>A, 2 and PF3753:16836079, C>A, 6). Like Ans008 and Ans017, Ans014 had a derived allele state for a marker defining another sub-clade within I, namely I2a2a2a1 (L1228:15446045, C>G, 2). There was no further support for this sub-clade as this individual was ancestral for I2a2a2 (SK1254:6742730, T>C, 2), I2a2a (M223:21717307, G>A, 4; L59:7113556, C>T, 4; S24:15517851, T>G, 2; S119:24475669, G>T, 3 and S117:16699334, C>G, 2) and I2a2 (S150:22725379, C>A, 2; S33:18747493, G>C, 2; L181:19077754, G>T, 1; L368:6931594, C>T, 1 and S32:17493630, T>G, 3).

Ansarve16

Ans016 likely belongs to I2a1b as he was derived for 28 markers defining haplogroup I (e.g. PF3742:16354708, G>A 1; PF3569:2688442; T>A, 1 and PF3574:2974782, A>C, 2) and further for an A to G mutation defining I2a1b (S2715:17893806, A>G, 2). He could possibly belong deeper into the I2a1b sub-clade, but the lack of data only made it possible to exclude I2a1b2 (S392:18760081; G>A, 1), I2a1b2a (CTS10936:22844090, T>C, 2) and I2a1b2a1a1c (Z17855:18102497, G>A, 1). Based on ancestral allele states for markers defining other sub-clades within I, we could exclude assignment within I1 (S66:22914378, T>C, 1; P203.2:22750951, G>A, 1; P40:14484394, C>T, 1 and L840:20834703, C>G, 1), I2b (S332:8426321, T>C, 2), I2c (S11759:8638918, C>T, 1; PF3896:9419125, A>G, 1 and S6694:22861120, A>G, 2) and I2a2 (S153:17516123, T>C, 1; L368:6931594, C>T, 1 and S30:13992338, C>G, 1).

Ansarve17

Ans017 belongs to I2a1b1a1a and displayed derived allele states for the following; I2a1b1a1a (S2703:17361387; C>A, 6), I2a1b1a1 (L1498:18668472, C>T, 3), I2a1b1 (S185:22513718, C>T, 5), I2a1b (S2768:22905944, G>C, 4; S2632:7317227, G>A, 4; S2621:2785672, A>G, 1; S2638:14074218, A>T, 1; S2679:16233135, A>G, 10; S2687:16594452, A>G, 8; S2702:17359886, A>C, 7; S2715:17893806, A>G, 6; S2722:18049134, C>T, 3; S328:15574052, G>A, 3 and M423:19096091, G>A, 1), I2a (PF3647:7879415, A>C, 4), I2 (PF3781:18700150, C>T, 6; S31:16638804, A>G, 4 and Z2638:8567995, G>A, 4), and for 166 markers defining haplogroup I (e.g. PF3715:14847792, A>C, 2; PF3721:15023364, T>C, 3 and PF3742:16354708, G>A, 3). Similar to Ans008 and Ans014, Ans017 was derived for one marker defining another sub-clade within I, I2a2a2a1 (L1228:15446045, C>G, 8) although this was not further supported as upstream markers 33

Supplement: Megalithic tombs in western and northern Neolithic Europe

were ancestral for I2a2a2 (Y6098:9647453, C>T, 3 and SK1254:6742730, T>C, 5), for I2a2a (PF3857:7716262, A>C, 3; S152:17570599, C>T, 6; L622:13718315, C>A, 7; M223:21717307, G>A, 6; L59:7113556, C>T, 7; S24:15517851, T>G, 1; S119:24475669, G>T, 1; PF3858:8353707, C>A, 4; U250:18888200, C>G, 2 and S117:16699334, C>G, 4), I2a2 (S33:18747493, G>C, 6; L181:19077754, G>T, 3; L368:6931594, C>T, 4; S30:13992338, C>G, 3; S23:7628484, C>T, 4 and S32:17493630, T>G, 2).

Lairo1

Lai001 had 18 mutations placing him first within haplogroup I (PF3719:14986989, T>G, 2; PF3726:15479899, T>A, 1; PF3752:16826642, G>A, 1; PF3754:16836548, G>A, 1; PF3757:17090238, C>G, 1; PF3571:2723755, G>A, 1; Y1903:21155653, C>T, 1; YSC0000281:8382265, C>G, 1; YSC0000291:18394743, A>G, 1; YSC0000263:15615533, C>A, 1; Z16985:13804066, G>C, 1; PF3780:18404486, C>T, 1; PF3806:21525069, G>A, 1; PF3811:21627180, C>T, 1; PF3822:22200336, G>A, 1; Z16987:22243817, A>G, 1; PF3642:7712917, A>T, 1 and YSC0000300:23479970, A>C, 1) and further into I2a1b (S2722:18049134, C>T, 2) and I2a1b1 (S185:22513718, C>T, 1).

Due to displaying ancestral alleles, we could exclude that he belonged in sublineage I2a1b2a1 (CTS5966:16579499, T>A, 1) or in the lineages I1 (S66:22914378, T>C, 1; L187:18077297, A>T, 1; L118:15500110, A>C, 1 and L81:22513726, A>C, 1), I2c (PF3931:23413444, G>T, 1; S6659:13891261, G>A, 1 and SK1266:8634040, C>A, 1) and I2a2 (S150:22725379, C>A, 1).

Midhowe1

Midhowe1 (mid002) was derived for 18 markers defining haplogoroup I (PF3838:22845794, A>G, 1; PF3844:23267211, G>A, 1; PF3878:23401471, C>T, 1; PF3737:15937959, C>T, 2; PF3747:16548548, G>A, 1; PF3772:17949402, C>G, 1; YSC0000283:8465165, C>T, 1; YSC0000263:15615533, C>A, 1 ; YSC0000275.1:2884029, T>C, 1; YSC0000298:23154034, C>T, 1; PF3796:21119888, G>T, 1; L503:21359407, C>G, 1; CTS1006:7137088, C>T, 1; PF3641:7688470, T>C, 2; PF3645:7853028, C>A, 1; PF3864:7898045, A>G, 1; PF3672:9376351, T>C, 1 and YSC0000300:23479970, A>C, 2) as well as for two markers defining I2a1b (S2679:16233135, A>G, 1 and S2702:17359886, A>C, 1). The only markers downstream of I2a1b that we have data for are ancestral, so we can exclude I2a1b2a1a3 (Z16983:17558968, T>C, 1) and I2a1b2a1b1 (Y3118- 23548545, T>C, 1). We could further exclude the following I-lineages; I1 (L187:18077297, A>T, 1), I2c (Y5332:7956096, G>T, 1; PF3893:8571993, T>C, 1 and S6659:13891261, G>A, 1), some I2a1a sublineages (I2a1a1-PF4073:23496560, G>A, 1; PF4056:21865821, G>A, 1 and I2a1a2a1- L1287:21970862, G>T, 1 and I2a1a2a2-L880:3436270, C>T, 1) and I2a2 (L368:6931594, C>T, 1 and S30:13992338, C>G, 1).

Midhowe2 34

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Midhowe2 (mid001) belongs to haplogroup I as he was derived for 13 markers defining the haplogroup (PF3712:14646409, C>T, 1; PF3868:15389836, T>C, 1; PF3871:16780748, C>G, 1; PF3629:6926038, T>A, 1; PF3766:17692855, T>A, 1; Y1869:8262092, C>T, 1; YSC0000260:14974451, C>T, 1; YSC0000285:8536868, C>G, 1; YSC0000280:7856500, C>T, 1; CTS10058:19233673, A>G, 1; PF3800:21402723, A>G, 1; PF3804:21465033, C>A, 1 and PF3574:2974782, A>C, 1). There is not enough data to further subtype Midhowe2 (mid001). We can, however, likely exclude the following lineages due to ancestral allele states: I1 (S64:17766762, T>C, 1; S65:18759669, T>C, 1; L840:20834703, C>G, 1; L118:15500110, A>C, 1 and S108:6681479, T>G, 1), I2c (PF6907:14197631, G>A, 1 and PF3889:8001008, T>C, 1), I2a2a (S152:17570599, C>T, 1), I2a2b (S154:15668070; A>G, 1) and for some I2a1 sublineages (I2a1a1a1a1-PF4194:23075481, C>T, 1; I2a1a1a1b-F1295:8545494, C>T, 1 and I2a1b1b-PF4135:18981938, G>A, 1).

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S8 Molecular sexing of individuals As first analysis towards characterizing the genetic structure of people buried in Megaliths, we investigated whether there was a significant ratio of genetically sexed males to females buried at these burial monuments. We used data from the present study, as well as previous publications (see Extended Dataset 1.3). We use Ry to sex individuals from genetic data (101), since sexing individuals morphologically can be complicated in young individuals or from using fragmentary bone elements. Ry’s biological sex prediction for Midhowe1, Midhowe2, Ansarve6, Ansarve7 and Ansarve9 individuals was inconclusive due to low coverage (Extended Dataset 1.3). Therefore, we decided to use Rx (102), which is more robust in prediction of biological sex for low coverage samples.

Assuming equal probabilities for a male or a female to be buried in these tombs, we estimated the binomial probability of finding exactly (x) or more males per megalithic tomb (n>=6) or in broad geographical regions (Table S3). We included non-megalithic burial data (single inhumations and cave burials) from the British Isles (44) to assess the social structure of individuals with a similar demographic history as the British megalith builders but buried in different funerary traditions, following the same sampling scheme (Table S4). Assignation to a megalithic funerary tradition vs. the British Isles non-megalithic burial traditions (44) are displayed in Extended Dataset 1.3.

In Britain we find a significant excess of males buried at megalithic funerary monuments. This is exemplified by the ratio observed within the Primrose Grange court tomb and also within the Isbister passage graves, although for the latter marginally significant (P=0.032 and P=0.054, respectively). This male excess is also noted when analyzing all individuals from British Isles megaliths together (P=0.0014). Outside the British Isles there is also a higher ratio of males to females (seen in the Ansarve Dolmen individuals), however the difference is not significant (P=0.25). If all genetically sexed individuals from megalithic contexts are analyzed together there is a significant over representation of males among these tombs (P=0.0031). Regarding non-megalithic burials, the Raschoille cave, (which has a sample size similar to the Isbister passage tomb) has more males than females but the difference is not significant (P=0.50). When analyzing all individuals buried in a non- megalithic context together (either in caves or as single inhumations, totaling a similar sample size as the individuals buried in the Primrose Grange tomb), slightly more males than females were observed but this difference was not significant (P=0.15 and P=0.37, respectively; see Table S4). P-values are not multi-test corrected.

Next, we tested whether the high ratio of males to females found among the megaliths of the British Isles was significantly higher than those that for the cave and single inhumation burials using a chi- square test with 1 degree of freedom (see Tables S3-S4 for samples sizes used for this comparison). The p-value of the chi-square statistic test was 0.40, and we cannot reject the possibility that the ratio 36

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of males to females buried in both megalithic tombs and burials from the other funerary traditions is similar. We further note a slight excess of males to females buried in caves and single inhumations in the British Isles, but this difference is not statistically significant with the current sample sizes analyzed.

One scenario that would fit our results is if there was an over representation of males buried within both megalithic and non-megalithic funerary traditions, since we observed a strong statistical support for more males than females buried in megalithic tombs in our first analysis. In summary, our results support the interpretation that patrilineal societies were buried in the megaliths, however given the current sample sizes available for individuals buried with other funerary practice (single inhumations and cave burials), it is currently not possible to ascertain whether this tradition was unique to megalithic societies. Larger samples sizes of genetic data per megalithic and non-megalithic burial grounds is need from both the British Isles and across the geographical range of the megalithic funerary tradition to further understand the burial dynamics that that we observe. Finally, we note that many more individuals have been determined within both the megalithic and non-megalithic funerary contexts than the individuals with genetic data used for this study. However, neither samples presented in this study, nor those from other publications (to the best of our knowledge), were selected a priori based on biological sex information, and thus our observations should represent a good proxy of the true male/ female ratio per site, and also when all individuals from each funerary tradition are analyzed together.

Table S3. Binomial probability of finding exactly (x) or more males per megalithic tomb (n>=6) or broad geographical region.

Megalith individuals Male/total ratio Prob. (X) or more males per site Ansarve dolmen 6/9 P=0.250 Primrose Grange court cairn 9/11 P=0.032 Isbister passage tomb 8/10 P=0.054 All megalith British Isles 31/42 P=0.0014 All megalith individuals 41/60 P=0.0031

Table S4. Binomial probability of finding exactly (x) or more males per non-megalithic burials (from caves or single inhumations) (n>=6) or broad geographical region. Bal4 was not included in the single inhumations, see S2.

Non megalith individuals Male/total ratio Prob. (X) or more males per site Raschoille cave 5/9 P=0.50 All British Isles caves 10/15 P=0.15 All British Isles single inhumations 6/10 P=0.37 All non-megalithic British Isles 16/25 P=0.11

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S9 Reference panels and group labels Genetic data from the ancient individuals from the present study and previous publications (Extended Dataset 1.3) were overlapped with four reference panels:

Panel 1) The Human Origins (103) genotype dataset comprised of 594,924 SNPs genotyped in 2,404 modern individuals from 203 populations, were used to perform the PCA and unsupervised ADMIXTURE v1.3 analyses (104). For the PCA analyses, only European populations were selected (see section S11.1).

Panel 2) 1,938,919 biallelic transversion SNPs with a minor allele frequency of at least 10% in Yorubans (YRI) from the 1000 genomes project (105) were used to maximize the genetic overlap between ancient samples, while reducing the potential effect of cytosine deaminations. A minimal allele frequency of 10% was selected in order to avoid the effect of Eurasian admixture into YRI, and thus to ensure that YRI acts as a true outgroup. aDNA data was overlapped with this panel to perform f3 shared drift and the kinship analyses performed with READ (69).

Panel 3) Simon Genome Diversity project (106) SNPs where at least two individuals from a European population harbour the minor allele (i.e. using the –mac 2 flag from PLINK v1.90b4.9 (107,108)

Panel 4) Autosomal SNPs from the 1.2M SNP captured array (43). Genetic data from all ancient individuals were screened for the 1,150,639 autosomal coordinates from this capture array. Genetic data overlapped with this panel were used for the demographic f4-statistic analyses. As described in section S11 both “all sites” and “only transversions” were used to perform the demographic f4- statistic analyses. Genetic data from the Ballito Bay A individual from Schlebush et al. (78) was also overlapped to these coordinates in order to be used as an outgroup for these analyses.

Panel 5) X-chromosome SNPs from the 1.2M SNP captured array (43). Genetic data from all ancient individuals were screened for the 49,704 X-chromosome coordinates from this capture array. Similarly, as for Panel 4, genetic data from all ancient individuals (including the Baillto Bay A sample) was overlapped with this panel to perform the sex-bias f4-statistics analyses, using both “all sites” and “only transversions” positions.

Genetic data from all ancient individuals was merged with these panels using the following approach: for each SNP site, a random read covering that site with minimum mapping and base quality of 30 was drawn (using Samtools 1.3v mpileup) and its allele was assumed to be homozygous in the ancient individual (79). Non-biallelic SNPs in the ancient individuals were excluded from the data. Genetic data from all ancient reference individuals was previously mapped and processed as described in section S4 in to order to homogenize data treatment.

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When performing demographic analyses, we refer to Neolithic individuals as a group (in general) using a geographical and temporal label (i.e. Spanish Early Neolithic individuals are labelled as “Spain_EN”), except for the Scandinavian and Irish groups which are labelled by archaeological site. Farmer capture data group labels also include a two-letter code (at the end of the label) to depict that this data was capture generated “_CP”, shotgun-generated data does not have any two-letter code. Labels at the individual level used for demographic analyses are described in detail in Extended Dataset 1.3. Use the notation “/” in French MN/LN_CP and Spain_LN/CA as an indication that the chronology at these sites extends through two time periods; however, the genetic profile of these individuals is broadly similar in qualitative terms (see Figures 2 and S18).

Given the close geographical proximity and genetic affinities of the shotgun-generated Scottish megalith individuals and Balintore (Fig. S19) these four individuals are referred as Scottish MN (Scottish_MN), and analysed as a group for the f-statistics analyses (Extended Dataset 1.3). This group should not be confused with the group of Scottish individuals from the 1.2M capture array in Olalde et al. (44) labelled Scotland_N_CP.

In some of the demographic analyses in section S11.1 and S11.4 (i.e. the PCA and f4- statistics sections, respectively) we use the term “Atlantic farmers” to refer to individuals that have been excavated along the Atlantic coastal region. However, based on the results in this study as well as from observations from Martiniano et al. (22), suggesting a genetic signal for Neolithic individuals along the Atlantic coast of Europe, we also include in this category the Scandinavian Gökhem and Ansarve burials even though they are located in central Sweden and the Baltic Sea, respectively. As well as, Iberian individuals regardless of their geographical location within that peninsula, and also Middle- Late Neolithic individuals from southern France (see Extended Dataset 1.3).

The decision to include the Scandinavian burials as part of the “Atlantic farmers” group, was based on two observations. First, Martiniano et al. (22) suggested a genetic link between the Gökhem2 individual as well as other individuals found along the Atlantic coast of Europe (i.e. Iberian and British Isles Neolithic individuals) based on results from haplotype-based methods. Secondly, the PCA analyses from the present study (see section S11) display the Scandinavian individuals clustering within the genetic variation of the “Atlantic farmers” as well, thus supporting the previous observation reported by Martiniano et al. (22), and our broader demographic categorization of individuals.

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S10 Estimating kinship relationships among Megalith individuals The software READ (69) was used to identify bone elements that belonged to the same individual, and to infer kinship relationships among the individuals in the present study, and also the megalith reference data, per site (Table S2). READ is a heuristic method that uses the normalized proportion of non-matching alleles (P0) between pairs of individuals (using the median pairwise differences per group/population, or a defined value, as the normalizing factor), and compares it to the normalized value of theoretical expectations to infer familial relationships to a first or second degree. (READ is not implemented to detect more distant relationships). First-degree relationships are characterized by either parent-offspring or a full sibling relationship, whereas second-degree connections are represented by half-siblings, grandparent-grandchild, aunt/uncle-niece/nephew, or double cousins. READ constructs a distribution of normalized P0 scores per pairs of individuals (by computing the normalized P0 value in non-overlapping windows of 1 Mbps each), estimates an average and a standard error from such distribution, and assigns the value to one of the relatedness categories described above. The significance of READs predictions is displayed as the distance in multiples of the standard error (i.e. producing a Z-score value) from the observed normalized P0 assigned to a given relatedness category, to a “upper” (lesser related relationship) and “lower” (closer related relationship) relatedness threshold (see Fig. S8). Thus, if READ predicts that a pair of individuals are related to a second degree, it will display the certainty of such assignment by reporting the distance to both its upper (genetic signal of unrelated individuals; lower genetic related category than predicted), and lower (genetic signal of first-degree individuals) relatedness bound threshold values in Z-score units, which can be interpreted as X times the standard error observed. We use both concepts in an interchangeable way for reporting the analyses below. The higher the Z-score values the more certainty the reported normalized P0 value is away from either the “less related” or the “more related” categories, and well within the range of the category predicted by READ.

READ has been reported to be conservative when predicting familial relationships (even a low number of overlapping SNPs >1K), which makes it a good approximation to infer kin-related connections from ancient DNA data (69). We used two different approaches depending on how the data was generated. Shotgun-sequenced genome-wide data was overlapped with Panel 2 (1.9 million polymorphic transversion SNPs ascertained in 1KGP Yoruba individuals; see section S9). Such action allowed us to maximize the genetic overlap between pairs of ancient samples and reduced the potential effect of cytosine deamination. Genetic data from Olalde et al. (44) was overlapped with Panel 4 SNPs to maximize the genetic overlap among capture-generated data.

Given that this software uses a normalized pair-wise genetic distance between individuals to infer their relatedness, systematic differences in how the genetic data was generated could generate potentially spurious kinship relationships. Reference bias could also be a confounding factor if the 40

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average read-length of two given samples is shorter than to that of the rest of the individuals, since such samples would appear to be genetically closer to each other by also being more similar to the reference genome.

In order to infer kinship relationships following an unbiased strategy, we 1) use READ restricting to data generated with the same library-building strategy, labelling individuals by overlapping dates, 2) visually inspected the read-length size distribution for each individual using 1,000,000 random reads, 3) evaluated the affinity of each sample towards the reference human genome (using PLINK (107, 108) v1.90b4.9 –ibs-matrix flag), and 4) and contrasted READ predictions with the individual’s uniparental markers and biological sex assignation. It needs to be considered that only a few of the total number of individuals buried at the Primrose Grange and Ansarve sites harbored enough data to undertake nuclear analyses (see section S2). Thus, our estimate of the extent to which the groups were kin-related may be regarded as conservative.

In general, we ran READ for each archaeological site separately to obtain accurate normalization P0 values for the genetic baseline of each local farmer populations. However, given the close geographical proximity and the overlap in time between the Primrose Grange individuals and Carrowmore4 we analysed these data together as described below.

The Primrose Grange and Carrowmore tombs

Charactering genetic data from the Primrose Grange skeletal material per individual was challenging due to the fragmentary nature of the bone elements and also poor conservation of DNA in some of the remains. In order to account for such difficulties, genetic data was recovered employing four different library building strategies (BE, WGC, DR and SS) (see section S3). Kin-related analyses were performed only with BE and WGC data separately as explained below.

Most of the skeletal elements from the Primrose Grange individuals were not found in anatomical position or were found in different sections of the tombs (See Table S2). Thus, we ran a prior iteration of READ (once with BE and WGC data, respectively) and used these results in addition to a concordant mtDNA lineage assignation (i.e. by contrasting the mitochondrial haplotype call and the observance of private mutations) to identify bone elements that belonged to the same individual (see section S4 and Extended Dataset 1.1). After merging the sequencing reads by individual, data from 10 and 12 individuals was retained for the BE and WGC generated-libraries, respectively (see section S4).

The 95% confidence interval (C.I) dates from our Irish megaliths suggest that the tombs were used at least at two different time points (see Fig. 3). While Carrowmore4 was excavated at the Listoghil tomb at the Carrowmore site 2 km away from the Primrose Grange burial ground, the geographical 41

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proximity of these sites, their probable co-utilization of the tombs (see section S2), as well as a temporal overlap of Carrowmore4 with some of the Primrose Grange individuals inclined us to analyse the data from both tombs together.

The individuals Primrose2, Primrose17, Primrose18, Primrose12, and Primrose3 belonged to a first temporal usage of Tomb No. 1 at the Primrose Grange site (Group1, add time interwall). A latter funerary activity at this tomb is represented by Primrose16, Primrose10, Primrose6, Primrose7, Primrose13, and Primrose9 (Group2, add time interwall) (see Fig. 3). Primose16 overlap slightly with the former temporal group at the Primrose Grange site, however, since most of the C.I. overlap with the second group, we decided to incorporate this individual into the later funerary activity of the tomb.

Using READ on WGC data

READ analysis of Group1 WGC data revealed two first-degree connections between Primrose2 & Primrose17, and Primrose17 & Primrose18, and with 1.87 and 2.01 SE below the threshold predicted for individuals related in second-degree, respectively (see Table S5).

Kin-analyses of Group2 predicted a first-degree relationship between Carrowmore4 & Primrose7, and two second-degree connections among Primrose6 & Primrose7, and Carrowmore4 & Primrose6. The significance of the first-degree relationship was 1.79 SE below the threshold for a second-degree relationship (Fig. S7a). However, it was 3.14 SE below the expected threshold for two unrelated individuals, suggesting a genetic connection between Carrowmore4 & Primrose7 at least in a second- degree. The second-degree relationships from Group2 consisted of connections between Primrose6 & Primrose7 and Carrowmore4 & Primrose6, which were predicted to be 1.04 and 0.50 SE below their upper bound (Fig. S7b).

Using READ on BE data

READ analysis of Group1 BE data again predicted a first-degree connection between Primrose2 & Primrose17, and a second-degree relationship between Primrose17 & Primrose18. The former prediction was statistically significant with a normalized P0 value 6.97 SE below the signal for second-degree and 27.54 SE above that of identical twins/same individual, while the latter was 1.83 SE below, and 5.43 SE above the genetic signal of two unrelated individuals and a first-degree connection, respectively (Fig. S8a). No kinship connections were predicted for Group2 BE (Fig. S8b).

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a) b)

Figure S7. READ’s average pairwise P0 values for a) Primrose group1 and b) group2 WGC data

a) b)

Figure S8. READ’s average pairwise P0 values for a) Primrose group1and b) group2 BE data

Table S5. READ results Primrose and Carrowmore4 using WGC (A) and BE (B) data.

(A) (B) Grp1 relatioship Z_upper Z_lower Grp1 relatioship Z_upper Z_lower prs017WGCprs018WGC First_Degree 2.01 -0.07 prs017BEprs018BE Second_Degree 1.83 -5.43 prs02WGCprs017WGC First_Degree 1.87 -3.37 prs02BEprs017BE First_Degree 6.97 -27.54

Grp2 relatioship Z_upper Z_lower car4WGCprs06WGC Second_Degree 1.37 -0.41 car4WGCprs07WGC First_Degree 1.73 -0.94 43 prs06WGCprs07WGC Second_Degree 0.01 -4.04

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Addressing reference bias as confounding factor potentially influencing kinship predictions

ADNA is characterized on one hand by being highly fragmented, which is reflected in a sequencing read-length distribution shifted towards small fragments (typically 50-80 nt long). On the other hand, aDNA has been shown to harbor cytosine deaminations at the ends of its fragments (83) a factor that need to be accounted for during read mapping (88). If the reads from two samples on average are shorter than to that of the rest of the individuals analyzed in the same temporal group in the READ analyses, thus having on average a higher mapping quality score (i.e. enriching for reads with less changes to the reference), such individuals would appear to be genetically closer to each other due to a shared high similarity to the reference genome without reflecting a real biological connection.

In order to discard such a confounding effect from our kin-related inferences, we undertook a twofold action.

1) We visually inspected the read-length distribution for each individual (Fig. S9)

2) Evaluated the affinity of each sample towards the human reference genome (hg19) using PLINK v1.90b4.9 –ibs-matrix flag (107,108) (see Table S6).

Figure S9. Read length distribution for both temporal Irish Megalith groups for either BE (a-b) or WGC (c-d) data. 44

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Table S6. (A) Read length average and standard deviation from the read length distribution per each sample in Fig. S9. (B) Reference bias represented as the identity to the human reference for each individual produced with either WGC or BE data. (A) (B) Group 1 Average S.D. Group 2 Average S.D. Sample Ref identity Sample Ref identity prs002WGC 105.07 40.21 car004WGC 101.84 42.26 Ref 100% Ref 100% prs003WGC 115.58 45.23 prs006WGC 102.7 43.36 prs013WGC 98.34% prs018BE 98.35% prs012WGC 113.72 46.91 prs007WGC 116.78 46.66 prs016WGC 98.23% prs017BE 98.33% prs017WGC 90.59 36.52 prs010WGC 115.85 43.34 prs018WGC 98.21% prs010BE 98.33% prs018WGC 92.43 41.04 prs0134WGC 74.83 28.9 car004WGC 98.20% prs012BE 98.33% prs016WGC 102.62 45.7 prs010WGC 98.18% prs006BE 98.32% prs004WGC 116.67 44.35 prs017WGC 98.18% prs003BE 98.31% prs006WGC 98.17% prs013BE 98.30% Group 1 Average S.D. Group 2 Average S.D. prs012WGC 98.16% prs016BE 98.28% prs002BE 86.07 35.53 prs006BE 84.26 36.7 prs002WGC 98.14% prs002BE 98.28% prs003BE 97.09 40.46 prs010BE 102.08 40.26 prs009WGC 98.14% prs009BE 98.27% prs012BE 99.6 43.57 prs013BE 64.25 23.24 prs007WGC 98.11% prs017BE 87.22 39.58 prs016BE 94.16 43.00 prs003WGC 98.09% prs018BE 79.98 37.08 prs009BE 100.37 43.06

Fig. S9 and Table S6 display how kinship relationships inferred from READ are in general not affected by a shared higher reference bias. For instance, for either of the first-degree relationships predicted by READ (Primrose2 & Primrose17 and Carrowmore4 & Primrose7), neither pair of individuals has a smaller average read-length size than that of other individuals from the same group (true for both WGC and BE data). In both cases (for both types of data) it usually involves data reads that are slightly longer in one individual than in the other, which is reflected as different affinity to the human reference genome (Table S6). Therefore, we concluded that reference bias could not be a confounding factor to explain the higher similarity between Primrose2 & Primrose17 and Carrowmore4 & Primrose7.

The same scenario described above is also observed for Primrose6 & Primrose7 (predicted to be second-degree related). In the case of the second-degree connection between Carrowmore4 & Primrose6, the read-length size is similar between the samples, and smaller than that of the other individuals from Group2 WGC. However, Primrose16 has a similar average read-length to that of both individuals and no kin-connections with this sample were predicted by READ. Moreover, Primrose13 has an even smaller average read-length size than Carrowmore4, Primrose6, and Primrose16. While read-length size distributions of Primrose17 and Primrose18 WGC data could be affected by reference bias (due to a shorter average read-length than the rest of the Group1 WGC), the Primrose17 & Primrose18 kin-connection is statistically significant when using BE data, which also show different average read-length sizes for these two individuals.

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Uniparental marker calling and biological sex assignation concordance

In order to confirm READ’s kin-predictions and explore the nature of their kin-relationships, we contrasted READ results with our uniparental lineage and biological sex assignations (see Fig. 3).

Primrose2 & Primrose17

READ predicts a statistically significant first-degree relationship between Primrose2 (male) and Primrose17 (female), by placing their normalized P0 value of 6.97 SE below the signal for second- degree, and 27.54 SE above that of identical twins/same-individual, when using BE data. Moreover, the cal 95% CI dating overlaps largely in time (Fig. 3). Given these observations in addition that both individuals present different mitochondrial lineages and biological sex assignations, a mother and son or sibling relationship can be excluded, thus the only possibility for the Primrose17 & Primrose2 connection is a father-daughter relationship (see Fig. S10).

Figure S10. Pedigree structures predicted for Primrose2, Primrose17, and Primrose18 individuals based on uniparental markers, biological sex, and READ prediction. Males are displayed with green color and females in orange.

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Primrose17 & Primrose18

Both individuals are males, their cal 95% CI dating overlaps largely in time (Table 1), and they share the same mitochondrial lineage (including private mutations; see Extended Dataset 1.1). Their Y- chromosome haplotypes might belong to different major haplogroup lineages: I2a2 (Primrose17) and I2a1 (Primrose18), however such level of resolution is only supported by a couple of SNPs overlapped with 1 read each (see section S7). READ predicts a significant second-degree connection where the normalized P0 lays 1.83 SE below, and 5.43 SE above, the genetic signals of two unrelated individuals and a first-degree connection, respectively.

Thus, the data suggest a half-sibling or double-cousin connection where the same maternal lineage will be shared (Fig. S10), over a grandfather-grandson or uncle-nephew relationship given that the Y- chromosome major lineage might not be shared. However, due to low coverage the latter two scenarios cannot be fully discarded.

Carrowmore4 & Primrose7

Both individuals were genetically predicted to be males and have different maternal lineages. Interestingly, Carrowmore4 had a mitochondrial haplotype not seen in any of the Primrose Grange individuals. Due to the low average genome-wide coverage of both individuals, a full Y-chromosome haplotype call was not possible to obtain. However, both males harbour derived and ancestral reads suggesting that both might belong to haplogroup I2a2 with an unresolved haplotype. Again, such calls were predicted with a lesser degree of certainty due to low-coverage (see section S7). Carrowmore4 seems to be slightly older than Primrose7 when comparing the cal 95% CI dating (Table S1; see Fig. 3). READ predicts that Primrose7 and Carrowmore4 are related to a first-degree relationship with a normalized P0 value of 1.79 SE below the threshold for second-degree, and 0.89 SE above such for the prediction of identical twins/same individual. While such genetic signal might not be statistically significantly different from that of a second-degree relationship, such normalized P0 value lays 3.14 SE below the signal of a pair of unrelated individuals thus, Primrose7 and Carrowmore4 seem to be related at least to second degree.

Contamination could be a factor accounting for the closer genetic distance between Primrose7 and Carrowmore4 than that among the other Primrose Grange individuals that we sequenced. We notice that Pirmorse7 presents a wider contamination C.I. than Carrowmore4, where ContaMix predicts 1.44% (0.18-14.26%), and 0.03% (0-0.72%) contamination, respectively (Table1 and Extended Dataset 1.4). However, the wider contamination C.I. in the former individual might reflect nosier estimates than in the later given that Primrose7 mtDNA coverage is ~ 43.4x whereas Carrowmore4 is ~ 451.69x. Moreover, we provide two observations that argue against our results being impacted by contamination: First both individuals had a different mtDNA haplotype call prediction from Haplogrep2 (98-99), K1a4a1 and T2c1d1, respectively (see Table1) which were supported by a high

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confidence prediction (> 0.9). Secondly, for neither of the individuals did Haplogrep2 (98, 99) predict an alternative mtDNA haplotype call which matched the other maternal lineage.

Index jumping could also account for this degree of high genetic similarity between these two samples. However, while WGC data for Primrose7 and Carrowmore4 were generated on the same day, and sequenced on the same lane, such was the case for some of the other Primrose Grange individuals (i.e. Primrose3, Primrose9, and Primrose10), with higher levels of DNA preservation, were not predicted to be related to Primrose7 or Carrowmore4, neither were they related among themselves. Moreover, jumping index events have been reported occur at a low rate on the Illumina HiseqX platform (which we use to generate our data), and events that do occur seem to be more common between libraries with a higher DNA content and those that have lesser preservations (109), probably reflecting the fact that it is easier to detect index jumping in such cases. If index jumping would have occurred in those lanes sequenced on the same machine on the same day, it should be easier to detect index jumping between libraries with higher endogenous DNA content (i.e. Primrose3, Primrose9, and Primrose10) to those with lower amounts of endogenous DNA, where the index jumping flow would be from the former individuals to the latter. READ does not predict a familial relationship between Primrose3, Primrose9, and Primrose10 to Primrose7 or Carrowmore4, and again the alternative mtDNA calls predicted from Haplogrep2 (98, 99) does not suggest the presence of a lineages belonging to the former individuals. Thus, since we fail to detect a putative genetic connection due to index jumping in the most likely scenario for detecting it, we assumed that if it occurred, it would have happened at a very low rate between the Primrose7 and Carrowmore4 samples which have the same endogenous DNA content. Finally, any index jumping would be seen as contamination of a particular sample, and the contamination estimates are overall low.

In summary, after accounting for reference bias, contamination and index jumping, relying on READs first-degree relationship prediction between Carrowmore4 and Primrose7, and given that both males present different maternal lineages, and that the former individual might be slightly younger than the latter, a parent-offspring where Carrowmore4 is the father of Primrose7 is the most plausible kinship relationship.

Carrowmore4 & Primrose6

Carrowmore4 and Primrose6 presented the least significant second-degree relationship predicted by READ, where the normalized P0 values laid 0.50SE below the signal of two unrelated individuals, and 1.8SE above that of pair of first-degree individuals. However, as mentioned before READ has been reported to have low false positive rates (<0.1% for 1KGP sites) (69). Primrose6 is genetically characterized as female and Carrowmore4 as male; both individuals have different mitochondrial haplotypes. Such observations, in addition to that the cal 95% CI dating of both individuals overlap almost entirely in time (Fig. 3), would favour a scenario in which the second-degree relationship is represented by either half-siblings or double-cousins, over a grandmother-grandson or aunt-nephew

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relationship. As discuss in the main text, a scenario that will be consistent with the Carrowmore4 & Primrose7 and Primrose6 & Primrose7 READ predictions as well, would be if Carrowmore4 and Primrose6 are related as double-cousins.

Primrose6 & Primrose7

Primrose6 (female) and Primrose7 (male) represent the second-best second-degree connection inferred by READ using WGC data 1.04 SE below the genetic signal of an unrelated pair of individuals, and 1.53 above that of a first-degree relationship. As before, the low significant output from READ, is most likely due to the low number of SNPs used to perform the READ analysis since these individuals share more drift with each-other than with other Primrose Grange individuals. However, both individuals share different, although closely-related, mitochondrial lineages (K1a+195 and K1a4a1). Mitochondrial contamination in both individuals was predicted to be rather low (Extended Dataset 1.4). The cal 95% CI dating range of Primrose6 shows a slightly older mean than that of Primrose7 as in the previous case (Fig. 3). Given their different mtDNA lineages, a grandmother-grandson relationship can be excluded, thus, this relationship might be represented by half-siblings (although unlikely given their shorter temporal overlap), aunt-nephew or double cousins.

As described in the main text, one possibility that will be consistent with the Carrowmore4 & Primrose7, Primrose6 & Primrose7, and Carrowmore4 & Primrose6 READ predictions is if Primrose6 and Primrose7 reflect a third-degree relationship where there is higher than expected background inbreeding which would could mimic the signal of second-degree relationship.

The Ansarve Dolmen

We look for kin-connections among the Ansarve individuals by analysing genetic data from Ansarve3, Ansarve5, Ansarve8, Ansarve14, and Ansarve17 that overlap broadly in time. Ansarve16 was excluded from this analysis since there is at least a 100 years gap between the former and the latter individuals (see Fig. 2). READ predicts a statistically significant second-degree relationship between Ansarve14 and Ansarve17, 13.01 SE below the signal of two unrelated individuals, and 4.61 SE above the observed threshold for a pair of first-degree individuals (see Fig. S11 below).

Both male individuals date to a very similar time-range and have different mitochondrial lineages. Their >2.5x genome coverage allow us to infer that they share the same Y chromosome haplotype (see and Table 1). Thus, any type of a paternally-connected second-degree relationship among them is section S7 possible (half-siblings, double cousins, grandparent-grandchild and uncle-nephew) given the combination of their uniparental markers (see Fig. S12). Even though READ predicted no kin- related connection between Ansarve3 and Ansarve5, this pair of individuals seem to be more related to each other than the Ansarve genetic baseline, which could indicate they were related in a third or fourth degree.

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Figure S11. READ’s average pairwise P0 values for the Ansarve individuals except for Ansarve16

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Figure S12. Pedigree structures predicted for the Ansarve14 and Ansarve17 individuals based on uniparental markers, biological sex, and READ prediction. Males are displayed with green color and females in orange.

The Midhowe, Lairo and Balintore burials

The Midhowe1 and Midhowe2 individuals were excavated from the Midhowe chambered cairn located on the island of Rousay (Orkney Islands). READs performance improves substantially when at least four individuals represent the genetic structure of a group. For this analysis we included the Lairo1 and Balintore4 individuals as belonging to the same group as the Midhowe samples. Lairo1 was also excavated from a long horned stalled chambered cairn, different from the Midhowe burial, but also located on the island of Rousay, suggesting that Lairo1 might be representing the same underlying genetic structure as the Midhowe samples since there is some overlap in time. Balintore4 was excavated in mainland Scotland, and thus as priori it should not be included to identify kin- related connections among the individuals buried in the Rousay Island. However, since Balintore4 (as Lairo1) displays a similar genetic profile to the Midhowe individuals (see PCA and admixture plot),

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and date to a similar time we decide to include him in this group. Finally, Midhowe1 might be as much as 100-400 years older than the other three individuals (which overlap more in time, see Table 1). Neither the addition of Balintore4 from mainland Scotland, nor the incorporation of Midhowe1, potentially older that the others, resulted in any kinship relationships inferred by READ (Fig. S13), suggesting that Midhowe1 and Midhowe2 individuals were probably not related to a first or second degree.

Figure S13. READ’s average pairwise P0 values for the Scottish individuals.

The Gökhem passage grave

We look for kinship relationships among the individuals buried in the passage grave (Frälsegården) at Gökhem (16, 17). We overlapped genetic data from these individuals to Panel 3, as with the previously described shotgun-sequenced data to maximize the genetic inferences among pairs of Gökhem individuals. READ detects a possible second-degree relationship between Gökhem5 and Gökhem7. However, the normalized mean genetic distance between these individuals is just 0.23 SE above the genetic signal of two unrelated individuals (Fig. S14), and thus is difficult to ascertain if these individuals were indeed kin-related due to their low coverage data and wide C.I. overlap.

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Figure S14. READ’s average pairwise P0 values for the Gökhem individuals.

The Isbister, Holm of Papa Westrey, and La Mina burials

We used genetic data from other megalithic sites previously reported in other studies (43, 44), where genome-wide data was captured and was available from at least four individuals at each site, to investigate if kinship relationships might have also been present in the passage graves of La Mina (Spain) and the chambered cairns of Holm of Papa Westrey and Isbister (Orkney Isles). We overlapped all available data per each tomb, for which the individual C.I. dating overlapped temporally (see below), with the 1.2M SNPs coordinates used to capture the data initially (Panel 4), and ran READ on each dataset independently. No kinship relationships were predicted within neither of these reference megalith datasets (Fig. S15). The following individuals were used for the kinship analyses. La Mina: I0405, I0406, I0407, I0408; Holm of Papa Westray: I2636, I2637, I2650, I2651; Isbister: I2934, I2935, I2978, I2979, I3085.

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a) b)

Figure S15. READ’s average pairwise P0 values for the individuals from a) Isbister, b) Holm of Papa Westray, c) La Mina.

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S11 Population genetic analyses S11.1 Principal component analysis

We performed principal component analysis (PCA) to characterize the genetic affinities of the individuals from the present study to reference ancient Neolithic “farmers” and Mesolithic hunter- gatherers, as well as present-day European populations (Fig. S16-17). The PCA was conducted on 429 individuals from 50 European populations extracted from the Human Origins panel (Panel 1, see section S9) (103). We used PLINK v1.90b4.9 (107, 108) to remove SNPs with a lot of missing data using --geno 0.1 flag and SNPs with minor allele frequencies < 1% using --maf 0.01 flag. This was done to obtain a genetic structure resolution of present-day Europeans unaffected by drift (particularly in Eastern Europeans). To finalize the processing in the present-day individuals, a random allele was selected at SNPs where modern individuals were heterozygous, which made the data set completely haploid.

Next, we conducted PCAs for each ancient individual presented in table Extended Dataset 1.3 separately, with only overlapping SNPs, and setting as missing potential deaminated variants from the processed panel discussed above. PCAs were performed using smartpca from the EIGENSOFT package, with the “numoutlieriter: 0 and “r2thresh: 0.2” parameters. Procrustes analysis (16) was then used to transform each individual's PC1 and PC2 loadings to the coordinate system of the PCA using all SNPs as described in (105). The result was plotted using an in-house R script from the vegan library. As previously reported, Middle Neolithic individuals fall in between the genetic variation of the so called Western European hunter-gatherers (WHGs) and that of the Early Neolithic individuals in the PCA, reflecting their higher HG admixture levels (17, 43, 110); both Neolithic temporal groups cluster around the genetic variation of present-day Sardinians (Figs. 2 and S16, and (111)).

In order to assess if differences in funerary cultural practices could explain some of the genetic variation observed in the Neolithic individuals, we labeled them as either “megalith farmers” or “non- megalith farmers”, in addition to plotting the individuals from the present study (Fig. S17). Rather than a separation by different burial traditions, megalith farmers fall within the variation from Iberian, British Isles, and Scandinavian Neolithic individuals, as previously described by Martiniano et al. (22). Next we labelled the Neolithic individuals as either “Atlantic farmers” or “Central European farmers” based on geography and archaeology as explained above (section S9).

While temporal differences in the Neolithic individuals to some extent drive their genetic affinities (Figs. 2a and S16), the largest source of variation seems to be derived from their geographical place of origin.

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Figure S16. PCA of 429 present-day European individuals (grey dots) with published Neolithic, and WHG samples (filled symbols), and ancient samples from this study (non-filled symbols) plotted onto the first two principal components. The Neolithic individuals are labeled in a tempo-graphical manner except for the Scandinavian and Irish individuals which are named after the archaeological site. To ease visualization and reduce saturation in this plot, farmer groups from which both capture and shotgun data was generated were given the same symbol and group label (without the “_CP” subfix) i.e. Anatolian_EN, Germany_EN, Hungary_EN, Spain_EN, Spain_MN and Spain_CA (see Extended Dataset 1.3 for details)

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Figure S17. PCA of 429 present-day European individuals (grey dots) with published Neolithic individuals, labeled as “megalith and non-megalith farmers”, Western European Mesolithic hunter-gatherer (WHG) samples (filled symbols), and ancient samples from this study (non-filled symbols) plotted onto the first two principal components.

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S11.2 Unsupervised Admixture

A model-based clustering algorithm implemented in ADMIXTURE v1.3 (104) was used to estimate ancestry components and to cluster individuals in order to characterize the genetic structure in the Neolithic individuals from this study (Fig. S18). ADMIXTURE v1.3 was conducted on 1718 individuals from 179 populations from the Human Origins data set (103), which was merged with the ancient individuals as described in Extended Dataset 1.3. Data was pseudo-haploidized by randomly selecting one allele at each heterozygous site of present-day individuals. Finally, the dataset was filtered for linkage disequilibrium using PLINK v1.90b4.9 (107, 108) with parameters (--indep- pairwise 200 25 0.4), this retained 77,934 SNPs. ADMIXTURE v1.3 was run in 20 replicates with different random seeds for ancestral clusters from K=2 to K=14. Common signals between independent runs for each K were identified using the LargeKGreedy algorithm of CLUMPP (112). CLUMPP predicted that K = 7 was the highest K at which >80% of the runs were consistent in their ancestral component prediction (Fig. 2b). Clustering was visualized using pong (113). Starting from K=7 the Western-European Mesolithic genetic component is first defined. From that K onwards, the Middle Neolithic individuals are modelled as a mixture of Mesolithic HG ancestry and the Early Neolithic ancestry component maximized in ancient Anatolian agriculturalist. The results for all K’s are shown in Fig. S18.

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Figure S18. ADMIXTURE v1.3 results for the present study and ancient reference individuals (Extended dataset1.3) in addition to 179 present-day populations from the Human Origins Panel (103).

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S11.3 Shared drift, measured as f3, between individuals from megaliths and other Neolithic sites

Shared drift, quantified as f3, was computed using ADMIXTOOLS v5.0 (103) at a group level as a first approximation to investigate the Neolithic demographic affinities of the individuals from this study. Individuals were clustered geographically and temporally as displayed in Extended Dataset 1.3. Data was overlapped with Panel 2, and we used 1KGP Yoruba (YRI) as an outgroup to compute f3 of the form (x, y; YRI), for pairs of ancient Neolithic groups. Since our data was generated mainly by direct shotgun sequencing after library-building and some of the individual’s temporal and geographical reference data have been SNP-captured, we restricted the f3 shared drift analyses to use only samples with shotgun-sequence data (Extended Dataset 1.3) in order to avoid a methodological bias. Therefore, the AnatolianNW_EN individuals (Barcins) from Hofmanová et al. (114) were used as a proxy for Anatolian farmers (see Extended Dataset 1.3). We restricted to only use reference individuals with >10% genome coverage. Results are displayed in the form a heatmap where red indicates more shared drift between pairs of Neolithic groups (Fig. S19). The shared drift analyses suggest that the Neolithic individuals relate to other Neolithic individuals from similar geographies and chronologies. However, some f3 pairwise group comparisons suggest a demic connection among Atlantic European Middle Neolithic individuals, more so than to Central European Neolithic individuals (Fig. S19), for instance, between the British Isles and Iberia (Scottish_MN and Primrose to Spain_CA), and between Scandinavia (Gökhem2) and the British Isles. As seen in Figs. S16-17, we did not observe connections exclusively among ”megalith, or non-megalith farmers”, again providing strong indications that the Neolithic individuals related to each other by geographic and chronological similarities rather than funerary traditions (Extended Dataset 1.3).

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Figure S19. f3 shared drift per group analysis of the form x, y; YRI.

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S11.4 Paired f4-tests to infer demography

The PCA clustering patterns (Fig. 2a and S16) and greater shared drift values observed among some Atlantic European Middle Neolithic individuals, than to Central European Neolithic individuals (Fig. S19), could denote the genetic fingerprint of a Neolithic migration along the Atlantic facade. Alternatively, such observation could also be explained by a shared HG admixing demography, given that notable HG-admixture proportions have been reported in some Middle Neolithic individuals (15,44,109).

To address such scenario, we designed a f4-statistics paired tests in which we examine the genetic affinity between a test Neolithic group to two “Neolithic farmer sources” f4(outgroup, x, Central European EN, Atlantic farmer source), while accounting for a shared admixture history with HGs f4(outgroup, HG, Anatolian_EN_CP, x) using aDNA data overlapped with Panel 4 (Figs. S20-S22). Error bars show two block-jackknife standard errors estimated by ADMIXTOOLS v5.0 (103). As HG sources we used the 12 WHG employed for the demographic paired f4-statistics analyses depicted in Extended Dataset 1.3. Furthermore, we did not filter for transitions in order to extract as much information as possible from these analyses following the strategy as in section S9. We rationalized that cytosine deaminations should not covariate among samples, particularly not between different groups or populations, and thus, DNA damage (if anything) should instead introduce “noise” to the f4- statistic signals. Extended Dataset 1.5 displays f4-statistic results of addressing a Neolithic migration along the Atlantic coast, controlling for HG admixture using both “all sites” and “only transversions”.

As observed, both versions denote trends in which Atlantic Neolithic individuals are genetically closest to each other than to Central European farmers (Fig. S20-S22), however this trend only become significant when more data is used by using the “all sites” dataset. We use this strategy to formally investigate a long-range Neolithic migration along the Atlantic coast to the exclusion of the Central European Neolithic individuals/groups. Source Neolithic farmer groups were restricted to only include capture data to avoid dataset bias.

As described in section S9 the Neolithic groups are broadly defined geographically and temporally; ascription to specific groups is described in Extended Dataset 1.3. Figures S20-S22 display the affinities of the test Neolithic groups to either a Central European, an Iberian, French or British Isles Neolithic source (Spain EN_CP, Spain MN_CP, France MN_LN_CP, England N_CP, Scotland N_CP, Wales N_CP; see below and Extended Dataset 1.3). We replicate previous reports of a demic connection between the British Isles and Iberia to the exclusion of Central Europe using Germany Early Neolithic individuals (Germany_EN_CP, LBK captured data, Extended Dataset 1.3) as a proxy (19, 44, 115), while accounting for the shared HG admixture level. The observation that the Central Middle Neolithic group based on capture data (CE_MN_CP; represented by the Baalberge, Esperstedt and Salzmünde individuals, Extended Dataset 1.3) has similar levels of HG admixture as other Atlantic Neolithic individuals, but they are not significantly closer to any Atlantic individual than to

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Central European Early Neolithic, further supports the observation of a demic connection among the Atlantic individuals to the exclusion of Central European Early Neolithic.

We found a Neolithic-specific genetic affinity between the British Isles (Scottish and English capture data) and Scandinavia to the exclusion of the Central European Middle Neolithic individuals (Figs. S21 right and S22 left). The signal is maximized in for comparisons involving Gökhem2, or when the Scandinavian Neolithic proxy is represented by the Ansarve individuals and the British Neolithic proxy by Scotland_N_CP (Extended Dataset 1.3). The Scotland_N_CP group was used preferentially as a British Isles Neolithic proxy, due to the higher sample size (N=35) than the other British Isles groups (England_N_CP, N=14 and Wales_N_CP, N=2 Extended Dataset 1.3). This observation could be compatible with the genetic fingerprint of a continuous Neolithic farmer-specific migration along the European Atlantic coast as it has been suggested from the archaeological record (8, 9, 14). The different genetic affinities of the two Scandinavian groups to British Isles farmers could be explained by structure among Scandinavian Neolithic farmers. While this newly reported genetic link among Atlantic coast farmers is interesting, we notice that it is weaker than the greater affinity observed between the Iberian Peninsula and British Isles (19, 44, 115) (Extended Dataset 1.3). Migrations between the British Isles and Scandinavia along the Atlantic coast might have been less frequent than the migrations between Iberia and the British Isles. Figs. S20-S22 display the contrast of the genetic affinities of Atlantic and Central European farmers.

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Figure S20. Paired f4-tests to test the genetic affinity of x Neolithic test populations to either an Atlantic or Central European (Germany_EN_CP) Neolithic source group i.e. f4(outgroup, x, Central European EN, Atlantic farmer source), as a function of its HG admixture when compared to an Early Neolithic Anatolian group f4(outgroup, HG, Anatolian_EN_CP, x). Atlantic sources: Spain-_EN_CP (left) and Spain_MN_CP (right) (Extended Dataset 1.3). Top row: test populations are plotted using a temporal geographical label following the legend above. Bottom row: populations are labeled as Atlantic, Central European or Anatolian Farmers; symbol shape same as top row. Error bars show two block-jackknife standard errors. The type of error bar line represents whether a given test had a Z-score >= 3 (solid), >= 2 (two dash), >= 1 (dash), > 0 (dotted).

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Figure S21. Paired f4-tests for genetic affinity of the Neolithic test populations to either a Atlantic (see below) or Central European (Germany_EN_CP) Neolithic source group f4(outgroup, x, Central European EN, Atlantic farmer source), as a function its HG admixture when compared to an Early Neolithic Anatolian group f4(outgroup, HG, Anatolian_EN_CP, x). Atlantic sources: France_MN/LN_CP (left) and England_N_CP (right) (Extended Dataset 1.3). Top row: test populations are plotted using a temporal geographical label following the legend above. Bottom row: populations are labeled as Atlantic, Central European or Anatolian Farmers; symbol shape same as top row. Error bars show two block-jackknife standard errors. The type of error bar line represents whether a given test had a Z-score >= 3 (solid), >= 2 (two dash), >= 1 (dash), > 0 (dotted).

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Figure S22. Paired f4-tests for genetic affinity of the Neolithic test populations to either a Atlantic (see below) or Central European (Germany_EN_CP) Neolithic source group f4(outgroup, x, Central European EN, Atlantic farmer source), as a function its HG admixture when compared to an Early Neolithic Anatolian group f4(outgroup, HG, Anatolian_EN_CP, x). Atlantic sources: Scotland_N_CP (left) and Wales_N_CP (right) (Extended Dataset 1.3). Top row: test populations are plotted using a temporal geographical label following the legend above. Bottom rows: populations are labeled as Atlantic, Central European or Anatolian Farmers; symbol shape same as top row. Error bars show two block-jackknife standard errors. The type of error bar line represents whether a given test had a Z-score >= 3 (solid), >= 2 (two dash), >= 1 (dash), > 0 (dotted).

11.5 Sex-bias admixture analyses

The autosomal and Y-chromosome genetic data from the Megalith individuals demonstrate (previous) encounters with HG groups. However, is not clear if such an admixture process might have happened in the recent or distant past. We use f4-statistics to assess if the genetic affinities of these individuals to either a Mesolithic HG or Neolithic farmer source population differed between the autosomes and the X-chromosome (see below). This is used as a proxy to investigate the extent to which a sex-biased 66

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admixture scenario might have occurred between Mesolithic HGs and Neolithic farmers among the ancestors of the individuals analyzed here.

Inferring demographic events from patterns of genetic variation on the X-chromosome using ancient DNA data is challenging. Low coverage (typical of shotgun-based aDNA data) further constrains the maximum number of X-chromosome SNPs (which are already limited by the size of the X- chromosome), particularly when restricting to transversions to avoid confounding biases of cytosine deaminations. In order to maximize our probabilities to overlap aDNA data at X-chromosome informative sites and taking into account that some of the megalith data we use as reference have been SNP-captured, we use the autosomal and X-chromosome SNPs coordinates from the 1.2M capture SNP array panel and performed analyses on all sites as described in section 11.4. However, prior to the f4-statistics analyses the X-chromosome was also pruned, using PLINK v1.90b4.9 (107,108) (-- indep-pairwise 200 25 0.4), in order to account the strong effect of LD on this chromosome. We used ADMIXTOOLS v5.0 (103) to run f4-statistics analyses with default parameters. Standard errors and Z scores for f4-statistics were estimated using a weighted block jackknife.

Differences in the affinities to a Mesolithic HG or a Neolithic farmer source were investigated testing the robustness of the topology (outgroup, x; HG, F) on the autosomes or the X-chromosome, respectively. We used the Ballito Bay A genome (78) as an outgroup. Once we obtained results from the f4-statistics, we normalized the f4-statistics point estimate (Z = f4X − f4A / √σ2A + σ2X), to detect sex-bias admixture as previously described by Mathieson et al. (80). We interpreted a significantly different from zero positive Z score, as that such farmer had more HG affinity in its autosomes than in the X-chromosome (or more farmer affinity in the X-chromosome than in the autosomes). Conversely, a significantly different from zero negative Z score will imply that a farmer has more HG affinity in the in the X-chromosome than in the autosomes (or more farmer affinity in the autosomes than in the X-chromosome).

Therefore, the former scenario will suggest that more male than female HGs admixed with farmers i.e. admixture occurred in a sex-biased manner mostly from male HGs to female farmers. On the other hand, the later scenario would suggest that more male than female farmers admixed with HGs, thus admixture occurred in a sex-biased manner mostly from male farmers to female HGs. Previous studies have shown that early farmer males typically carry J , G, H, and T Y-chromosome macro-lineages, whereas Mesolithic and sub-Neolithic HGs have been noted to mostly present I2 lineages (15, 43, 44, 79, 116). The very high frequency of I2a male-lineages among the farmer groups of the 4th millennium BCE (particularly individuals from a megalithic context), suggest substantial mixing of HG males with these groups. We performed this test on megalith farmers at a group level across all geographical range, as well as non-megalith farmers from the British Isles following the same strategy as in section S8 (see Extended Dataset 1.3 for labels of such farmers at a group level). We required a minimum nuclear coverage of 0.05X for shotgun-generated data in order to undertake this analysis. All individuals that had ≥0.05X were included in the analysis. Only one individual (Gökhem2) from

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the Frälsegården, Gökhem site was kept for this analysis. All ancient individuals that were captured (panel 4) were kept for the particular group.

As HG sources we used the 12 WHG employed for the demographic paired f4-statistics analyses. As the “farmer” source we used eight Early Neolithic individuals mur1, CB13, ne1, ne5, ne6, Bar31, Bar8 and Stuttgart (see Extended Dataset 1.3) with an autosomal average coverage > 1x to maximize information harvested from the Early Neolithic source.

Table S7 summarizes the results of our sex-bias admixture test for the megalith groups investigated. As observed in this table, none of the results are significantly different from zero. However, the Scottish_MN group (shotgun generated) was the group which displayed the highest positive f4 Z score, suggesting a tendency towards HG male-sex biased admixture in their recent past. In contrast, the Ansarve and Gökhem individuals displayed admixture with HG both for the autosomes and the X- chromosome, which may be indicative of a scenario of recent admixture with HGs in Scandinavia (see Extended Dataset 1.6).

Table S7. f4-statistic Z-score of the difference between hunter-gatherer-related affinity on the autosomes and on the X chromosome for each megalith and non-megalith population investigated.

2 2 Population f4 X − f4 A √σ A + σ X f4Zscore Ansarve -0,004912 0,003024052 -1,6243109 Gökhem2 -0,00458 0,003691321 -1,2407482 Primrose 0,001388 0,00263866 0,5260245 Ballynahatty 0,001291 0,003441831 0,375091 Scottish_MN 0,004558 0,003057551 1,4907353 Non-Megalith_farmer_CP 0,000308 0,002369784 0,1299697 Megalith_farmer_CP 0,00005 0,002467281 0,0202652 All_Brittish_Isles_farmers_CP 0,000572 0,002331486 0,2453371 Spain_MN_CP (La Mina) 0,002713 0,002961535 0,916079 PortugalDol_LN 0,000101 0,003499301 0,0288629

S11.6 The European Y-chromosome haplogroup distribution in time and space

In order to assess the temporal and geographical distribution of Y-chromosome lineages in the prehistoric HGs, and Neolithic farmers we screened data from previous publications (15, 43, 44, 79, 116). In brief, we obtained the lineage calls from previous studies (assuming such calls were correct) and co-analysed these with the Y-chromosome lineage calls we present in this study (described in section S7). We assessed the frequency of “major” haplogroups reported up to date (in the above- mentioned studies) for the HG and Neolithic farmer groups defined temporally, and by the major geographical (European) areas where Y-chromosome data has been produced (Fig. S25). We defined

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those major haplogroups by looking at lower resolution branches that were common to several individuals. As observed from Fig. S25, Northern, Northwestern European, and Scandinavian HGs are characterized mostly by I2 lineages, except for the Baltic HGs, which also display the Q1, and R1b macro-lineages. The major lineages I2a and R1b are observed in Iron Gates HGs. As previously described (15, 43, 44, 79, 116). Early Neolithic central European and Anatolian males are mainly represented by the G2a, H2, J2a, and C1a lineages. However, all macro lineages (as here defined) are observed in in Central European and Anatolian Neolithic males. Early Neolithic males from Spain are also mainly represented by G2a lineages, but also with some presence of the H2, E1b, and I2 lineages. Middle Neolithic males from Spain are a mix of the I2a, G2a, and H2 lineages, and interestingly, Neolithic British males are exclusively classified as belonging to the I2a lineage as previously reported (44).

Figure S23. Frequencies of Y-chromosome major haplogroups in northwestern European and Scandinavian HGs (NWE Scand HG) n=11, Iron Gates HG n=22, Zvejnieki HG (E Baltic HG) n=14, Early Neolithic Balkan (EN Balkan) n=18, LBK Austria & Germany (EN Central Europe) n=15, Early Neolithic Spain (EN Spain) n=9, Middle and Late Neolithic Southern France (MN/LN France) n=24, Middle Neolithic Spain (MN Spain) n=16, and Neolithic Britain (N British Isles) n=35) (from 15,43,44,79,116).

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S12. Primrose Grange, Tomb. Original field documentation.

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Primrose Grange, Tomb. Original field documentation.

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Primrose Grange, Tomb. Original field documentation.

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Primrose Grange, Tomb. Original field documentation.

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Primrose Grange, Tomb. Original field documentation.

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