Supplementary Information The genetic structure of the world’s first farmers Table of Contents 1 SI 1 – Description of ancient samples and archaeological context 2-11 SI 2 – Human Origins dataset 12-13 SI 3 – Population groupings in the ancient Near East 14-16 SI 4 – Pervasive Basal Eurasian ancestry in the ancient Near East 17-44 SI 5 – Ancient Near Easterners had less Neanderthal ancestry than ancient Europeans 45-50 SI 6 – Y-chromosome haplogroup variation in the ancient Near East 51-58 SI 7 – Admixture history of ancient West Eurasians 59-112 SI 8 – Population admixture into Eastern Africans from the Levantine Neolithic 113-120 SI 9 – Constraints on the origins of Ancestral North Indians 121-128 SI 10 – Modeling admixture from ghost Populations 129-138 SI 11 – Admixture in East Asians and Eastern European hunter-gatherers 139-152 Supplementary Information 1 Description of ancient samples and archaeological context This note presents information on the archaeological context of 45 individuals from the ancient Near East for whom we report genome-wide data in this study. We give uncalibrated dates in radiocarbon years (“bp”). We give calibrated dates in years before the common era, converting from uncalibrated to calibrated dates using IntCal131 and OxCal4.22 (https://c14.arch.ox.ac.uk/oxcal/OxCal.html). We indicate dates obtained directly on the skeleton for which we obtained ancient DNA by the suffix “calBCE”. We indicate indirect dates obtained based on archaeological context as “BCE”. Iran Hotu Cave (Iran) Hotu Cave is located in the foothills of the Alborz Mountains, near the modern town of Behshahr on the southern shore of the Caspian Sea in northern Iran. The site was excavated to a depth of 12 meters by the American physical anthropologist Carleton Coon in the spring of 1951, and provides evidence of human occupation at various times from the early Mesolithic to the post-Achaemenid periods3-5. Skeletal remains of three sets of individuals (I, II, III) were recovered from Pleistocene Gravel 4 of Trench D, the deepest sounding undertaken (Section 2)3. In Coon’s preliminary excavation report, this gravel was initially identified as an aceramic Neolithic level, but was subsequently reclassified as a Mesolithic horizon.1,2,3 In 1955, two charcoal samples recovered in association with the skeletal remains from depths of 9.5 and 10.15 meters yielded uncalibrated radiocarbon ages of 9190±590 bp and 9270±570 bp respectively6. In 2013, AMS analyses of collagen extracted from a tooth from Mesolithic skeleton I yielded an uncalibrated radiocarbon age of 9480±40 bp (9119-8637 calBCE)7. The set III of individuals includes one adult partially complete skeleton (HotuIIIa) and the remains of a young child estimated to be 1.5-2 years old (HotuIIIb). We sampled the right petrous bone of HotuIIIb for DNA analysis. Subsequent AMS analysis of this specimen yielded an uncalibrated radiocarbon age of 7250 ± 40 bp (6218-6034 calBCE). The discrepancy in the direct dates of skeletal remains (Hotu I and IIIb) recovered from such close proximity to one another in the same archaeological horizon causes us to doubt the age of the Hotu IIIb specimen. There are no stratigraphic unconformities indicating that this specimen may have been introduced from overlying Neolithic cave deposits, and we suspect that the later than anticipated date may result from the use of modern-day organic materials such as the shellac or glue in post-excavation reconstruction of the Hotu IIIb crania. Genetic similarities between the CHG and Hotu IIIb specimens demonstrate that there are continuities between Iranian Neolithic 2 populations and local hunter-gatherer groups in northern Iran and the Caucasus, rather than with Epipalaeolithic or Neolithic populations originating in Anatolia or the Levant. The earlier date (9119- 8637 BCE)8 obtained from the Hotu I skeleton is not essential to support this observation, as the continuity of Iran Neolithic with hunter-gatherers of the southern Caucasus/Iran highlands rather than with those of the Levant is evident when either the HotuIIIb specimen or the securely dated pre- Neolithic Caucasus hunter-gatherers8 from Georgia are used (main text). We show a conservatively wide range that includes both the earliest date from Hotu I and the later direct date from HotuIIIb in Fig. 1a, although based on the arguments made here we strongly favor the earlier date and Mesolithic attribution of the HotuIIIb individual. • HotuIIIb (I1293): 1.5-2 year old individual. Ganj Dareh (Iran) Tepe Ganj Dareh is a mound on the floor of the Gamas-Ab Valley, and is situated at an altitude of ~1,400 meters in the High Zagros region of Kermanshah Province in western Iran. It is one of several mounds discovered during survey work in the area9. It measures about 40 meters in diameter and 7 to 8 meters in height and has five identified levels (A to E), with the lowest level E being the oldest. Permanent architecture is seen earliest at level D. Philip E. L. Smith led an excavation of roughly 20% of the mound in four seasons between 1967 and 1974. Zooarchaeological and archaeobotanical evidence show a population exploiting ovicaprids, with goats dominant, as well as evidence for use of wild barley but no plant domesticates. There is evidence of herding but no evidence for decreased size or changes in horn core morphology10,11. Current evidence places the occupation of the site at approximately 8000-7700 calBCE11. None of the human remains have been directly dated. The human remains are highly fragmentary. The current minimum number of individual is 116, of which 56 are catalogued as skeletons each represented by more than 4 elements12. The following six Ganj Dareh individuals (all petrous bone samples) are included in this study. None has a direct radiocarbon date: • GD13A (I1290): 30-50 year-old male from level C. • GD14B (I1944): 3-4 year-old child from level B1. • GD16 (I1945): 5.5-7.5 year-old child from level D. • GD37 (I1949): 30-50 year-old male from levels D/E. • GD39 (I1951): 1.5-2.5 year-old child from level D crypt. • GD1150 (I1955): 18-30 year-old male from levels A/D. This individual is a clear genetic outlier and was analyzed separately from the other individuals pending radiocarbon dating, which confirmed that it was not from the Neolithic period but of recent origin (1430-1485 calCE (330±30 BP, Beta- 432801); it is thus labeled Iran_recent. 3 Seh Gabi (Iran) The site of Seh Gabi is a series of seven small mounds, identified as A through G, on a branch of the Gamas Ab River in the Kangavar Valley of the High Zagros region of Kermanshah Province, western Iran, about 6 kilometers from the much larger Chalcolithic and Bronze Age site of Godin Tepe. The mounds were scattered over an area of ~15 hectares with four excavated by Louis D. Levine and the Royal Ontario Museum between 1971 and 1973. Occupation lasted from the Late Neolithic through the Chalcolithic periods13. The human material, recovered in 1971 and 1973, comprises a minimum of 31 individuals that are almost entirely subadults: fetuses, infants and children <4 years of age; many with evidence of pathology. We report ancient DNA data on six samples (all petrous bones) from Mound A (n=2), Mound B (n=3), and Mound C (n=1). No radiocarbon dates have been obtained on Mound A, and so we use archaeological dates for the Chalcolithic of 4500-3500 BCE. Radiocarbon dates have been obtained from Mound B although not on the skeletons for which we report DNA. The dates obtained range from 5630±80 bp (SI-4915) to 5020±70 bp (SI-4910), and hence we represent the dates for samples from this bound by the union of the 95.4% calibrated confidence intervals (4680-3662 calBCEP14,15). We directly dated the Mound C individual who is Late Neolithic: • SG19 (I1665): fetal remains, ~26 weeks old from Mound A. • SG21 (I1674): fetal remains, ~35 weeks old from Mound A. • SG7 (I1662): fetal remains, ~34 weeks old from Mound B. • SG11 (I1670): ~6 month-old infant from Mound B. • SG16 (I1661): ~9 months old infant from Mound B. • SG2 (I1671): 5837-5659 calBCE (OxA-33168, 6850±40 bp). ~6 month-old infant from Mound C. Levant Raqefet Cave (Israel) Raqefet Cave is located within a southern extension of Mt. Carmel. It has a long prehistoric sequence spanning the Middle Palaeolithic through to the Neolithic periods16. The first excavation was led by Noy and Higgs (1970-1972), and the most recent excavation was led by Nadel and Lengyel (2004- 2011). The first chamber was used extensively by the Natufians, who left on the terrace 100 bedrock mortars, cupmarks and cupules17. Some of the mortars are among the largest ever recorded for Natufian sites18, and one of the deepest has a grid-like pattern incised inside the shaft19. The samples we analyze for ancient DNA (all of which are petrous bones) were found in single or double primary burials lying on their back or on their side in flexed positions. These are part of a cluster of Natufian graves created within a short time span in a dedicated area of the first chamber of the cave. The area 4 contains approximately 30 human skeletons, all but one of which are in an area of about 15 meters squared. Faunal analysis indicates feasting by the open graves and the burial of food remains within the graves20. Phytolith analysis indicates the use of plant materials in the graves and in the mortars21. In four graves, the pit was lined with greens and flowers prior to inhumation, as reflected by dozens of plant impressions in each22.