https://doi.org/10.1038/s41586-021-03335-3 Supplementary information Initial Upper Palaeolithic humans in Europe had recent Neanderthal ancestry In the format provided by the authors and unedited Nature | www.nature.com/nature 1 Supplementary Information 2 Contents: 3 4 Supplementary Information 1 ................................................................................................. 2 5 Bacho Kiro Cave and human remains .................................................................................... 2 6 Supplementary Information 2 ............................................................................................... 11 7 Ancient DNA processing, quality controls and contamination estimates ............................ 11 8 Supplementary Information 3 ............................................................................................... 32 9 Datasets for downstream analyses ........................................................................................ 32 10 Supplementary Information 4 ............................................................................................... 36 11 Relatedness, sex determination and Y chromosome analyses ............................................. 36 12 Supplementary Information 5 ............................................................................................... 41 13 Population relationships ....................................................................................................... 41 14 Supplementary Information 6 ............................................................................................... 77 15 Admixture Graph Modelling ................................................................................................ 77 16 Supplementary Information 7 ............................................................................................... 86 17 Neandertal ancestry in Bacho Kiro individuals .................................................................... 86 18 Supplementary Information 8 ............................................................................................. 108 19 Neandertal introgressed segments in Bacho Kiro individuals and dating of Neandertal 20 admixture ............................................................................................................................ 108 21 Supplementary information 9 ............................................................................................. 123 22 Sharing of Neandertal introgressed segments between Bacho Kiro and present-day 23 individuals .......................................................................................................................... 123 24 Supplementary Information 10 ........................................................................................... 130 25 Neandertal deserts .............................................................................................................. 130 26 27 1 28 Supplementary Information 1 29 Bacho Kiro Cave and human remains 30 31 Bacho Kiro Cave is located on the northern slope of the Balkan Mountains and 5 km away from 32 the city of Dryanovo in Bulgaria. First excavated in 1938 by D. Garrod1 and later in 1970s by 33 a team led by J. Kozłowski2, Bacho Kiro Cave was recently re-opened and excavated by a 34 research team led by the National Archaeological Institute with Museum in Sofia and the 35 Department of Human Evolution at the Max Planck Institute for Evolutionary Anthropology in 36 Leipzig3,4. New excavations focused on two sectors adjacent to the area excavated in 1970s, the 37 Main Sector and the Niche 1 as detailed previously3,4. The archaeological sequence of the cave 38 spans the late Middle Palaeolithic (MP) and the early Upper Palaeolithic (UP), with an 39 archaeologically rich assemblage in Layer I3,4 recognized as a variant of the Initial Upper 40 Palaeolithic (IUP)5. 41 Five human specimens from the new excavations were found in a direct association with 42 an IUP assemblage in Bacho Kiro Cave (Fig. 1)3. A human lower molar (F6-620) was found in 43 the upper part of the Layer J in the Main Sector of the cave and four bone fragments (AA7-738, 44 BB7-240, CC7-2289 and CC7-335) were found in the Layer I in the Niche 1 sector. These 45 specimens were directly radiocarbon dated to between 45,930 and 42,580 cal. BP3,4 (re- 46 calibrated with OxCal 4.4.26 and IntCal207, Tab. S1.1). Their mitochondrial genomes (mtDNA) 47 are attributed to modern humans, suggesting that they are the oldest modern humans in Europe 48 recovered to date3. 49 Specimens from Kent’s Cavern in United Kingdom8 and Grotta del Cavallo in Italy9 50 have been claimed to represent the earliest evidence of modern humans in western Europe (Fig. 51 1). However their dates are based on the archaeological context and the exact provenance of 52 these specimens has been extensively debated10,11. Modern human remains older than 40,000 53 cal. BP in Europe are extremely sparse (Fig. 1)12-14. Moreover, genome-wide data has been 54 retrieved for only three modern humans from Eurasia that are older than this15-17 and each of 55 these individuals was found outside of any archaeological context. 56 The archaeological sites with IUP assemblages span an area from North Africa, 57 southwest Asia, eastern and central Europe to north China18,19 (Fig. 1, Tab. S1.2), and have 58 been suggested to be associated with the first wave of modern human dispersals out-of-Africa19. 59 However, human remains found in the IUP context to date are relatively few12,20-22, highlighting 60 the relevance of Bacho Kiro Cave IUP human finds3,4. 2 61 Two additional bone fragments were found in Layer B in the Main Sector of Bacho Kiro 62 Cave (F6-597) and in the collections of the 1970s excavations in a position corresponding to 63 the interface of Layers B/C (BK1653). These two specimens were directly dated to 36,320- 64 35,600 cal. BP and 35,290-34,610 cal. BP3,4, respectively (re-calibrated with OxCal 4.4.26 and 65 IntCal207, Tab. S1.1). Although the lithic assemblages from these layers are sparse, they are 66 likely to be Aurignacian3,4. Thus, there is ~10,000 years between the two groups of human 67 remains found in the Bacho Kiro Cave. Even though there are more individuals that are older 68 than 30,000 cal. BP and from whom genome-wide data has been retrieved (Fig. 1, Tab. S1.1)23- 69 26, this genetic record is still quite fragmentary compared to more recent timeperiods 70 (https://reich.hms.harvard.edu/downloadable-genotypes-present-day-and-ancient-dna-data- 71 compiled-published-papers/). 72 73 References SI1: 74 1 Garrod, D. A., Howe, B. & Gaul, J. Excavations in the cave of Bacho Kiro, north-east 75 Bulgaria. Bulletin of the American School of Prehistoric research 15, 46-76 (1939). 76 2 Kozłowski, J. K. & Ginter, B. Excavation in the Bacho Kiro Cave (Bulgaria): Final 77 Report. 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