Ancient genomes in South Patagonia reveal population movements associated with technological shifts and geography Nakatsuka et al. 1 Supplementary Figures Supplementary Figure 1. Conditional heterozygosity analysis. All Middle Holocene Patagonians refers to the grouping of Chile_PuntaSantaAna_6600BP, Argentina_LaArcillosa2_5800BP, and Chile_Ayayema_4700BP. Narrow and thick grey segments: 1.96 and 3 standard errors, respectively. 2 Supplementary Figure 2. Unsupervised ADMIXTURE plot at different K values from 2-7. The run with the highest log likelihood score after 10 trials was taken for each K value. The cross-validation scores are given on the left. 3 PC2 (1.905%) VS PC1 (2.353%) 0.20 ● ● ●● 0.15 ●● ● ● ● ● ● 0.10 ● ● ● ●●● ● ● ● ● 0.05 Yámana; Modern (Beagle Channel); N PC2 = 20 Río Policarpo; ~700BP (Mitre Peninsula); N = 1 Hoste Island; ~1000BP (Beagle Channel); N = 1 ● Rousson & Willems Mission; ~100BP (Northern Tierra del Fuego); N = 1 Puerto Williams; ~800BP (Beagle Channel); N = 1 Margen Sur; ~700BP (Northern Tierra del Fuego); N = 2 Cape Horn Mission; ~100BP (Beagle Channel); N = 3 Pozo Tierra del Fuego 1; ~100BP (Northern Tierra del Fuego); N = 1 Río Pipo; ~1900BP (Beagle Channel); N = 1 Puesto Pescador● 1; ~400BP (Northern Tierra del Fuego); N = 1 ● ● ● ●● ● ●● ● ●● ● ● ● ●● ●● ●● 0.00 ● Almanza; ~200BP (Beagle Channel); N = 2 Río Grande;● ●● ~500BP (Northern Tierra del Fuego); N = 1 ● ● ● ● ● ● ● Acatushún; ~500BP (Beagle Channel); N = 1 Faro Méndez; ~100BP● (Northern Tierra del Fuego);● N = 1 ● Ayayema; ~4700BP (Middle Holocene Patagonia); N = 1 Cerro Johnny; ~400BP (Southern Continent); N = 1 ● ● La Arcillosa 2; ~5800BP (Middle Holocene Patagonia); N = 1 Kawéskar; Modern (Western Archipelago); N = 3 ● Punta Santa Ana; ~6600BP (Middle Holocene Patagonia); N = 1 ● Punta Santa María; ~900BP (Western Archipelago); N = 1 ● Caleta Falsa − S3; ~300BP (Mitre Peninsula); 0.05 N = 1 Yekchal; ~1100BP (Western Archipelago); N = 1 Caleta Falsa − S7; ~300BP (Mitre Peninsula); − N = 1 Hagenbeck Expedition; ~100BP (Western Archipelago); N = 2 ● ●● ● ●● Caleta Falsa − S8; ~500BP (Mitre Peninsula); N = 3 ● ●●● 0.10 − −0.15 −0.10 −0.05 0.00 0.05 0.10 PC1 ● Yámana; Modern (Beagle Channel); N = 20 Río Policarpo; ~700BP (Mitre Peninsula); N = 1 Hoste Island; ~1000BP (Beagle Channel); N = 1 ● Rousson & Willems Mission; ~100BP (Northern Tierra del Fuego); N = 1 Puerto Williams; ~800BP (Beagle Channel); N = 1 Margen Sur; ~700BP (Northern Tierra del Fuego); N = 2 Cape Horn Mission; ~100BP (Beagle Channel); N = 3 Pozo Tierra del Fuego 1; ~100BP (Northern Tierra del Fuego); N = 1 Río Pipo; ~1900BP (Beagle Channel); N = 1 Puesto Pescador 1; ~400BP (Northern Tierra del Fuego); N = 1 Almanza; ~200BP (Beagle Channel); N = 2 Río Grande; ~500BP (Northern Tierra del Fuego); N = 1 ● Acatushún; ~500BP (Beagle Channel); N = 1 ● Faro Méndez; ~100BP (Northern Tierra del Fuego); N = 1 Ayayema; ~4700BP (Middle Holocene Patagonia); N = 1 ● Cerro Johnny; ~400BP (Southern Continent); N = 1 ● La Arcillosa 2; ~5800BP (Middle Holocene Patagonia); N = 1 Kawéskar; Modern (Western Archipelago); N = 3 Punta Santa Ana; ~6600BP (Middle Holocene Patagonia); N = 1 ● Punta Santa María; ~900BP (Western Archipelago); N = 1 ● Caleta Falsa − S3; ~300BP (Mitre Peninsula); N = 1 Yekchal; ~1100BP (Western Archipelago); N = 1 Caleta Falsa − S7; ~300BP (Mitre Peninsula); N = 1 Hagenbeck Expedition; ~100BP (Western Archipelago); N = 2 Caleta Falsa − S8; ~500BP (Mitre Peninsula); N = 3 Lapa Do Santo; ~9600BP (Ancient Brazil); N = 5 ● Chane (Equatorial−Tucanoan); N = 1 Laranjal; ~6600BP (Ancient Brazil); N = 2 Karitiana (Equatorial−Tucanoan); N = 4 ● Moraes; ~5800BP (Ancient Brazil); N = 1 Piapoco (Equatorial−Tucanoan); N = 2 Conchalí; ~700BP (Ancient Central Chile); N = 2 Surui (Equatorial−Tucanoan); N = 2 Los Rieles; ~12000BP (Ancient Central Chile); N = 1 Yukpa (Equatorial−Tucanoan); N = 1 Los Rieles; ~5100BP (Ancient Central Chile); N = 1 ● Aconcagua; ~500B (Late Central Andes); N = 1 ● Aymara (Andes); N = 11 Cuncaicha; ~3300BP (Late Central Andes); N = 1 Quechua (Andes); N = 3 Cuncaicha; ~4000BP (Late Central Andes); N = 1 ● Huilliche (Central Chile); N = 27 Kaillachuro; ~3800BP (Late Central Andes); N = 1 Pehuenche (Central Chile); N = 27 Laramate; ~900BP (Late Central Andes); N = 6 ● Cuncaicha; ~9000BP (Early Andes); N = 1 Pica Ocho; ~700BP (Late Central Andes); N = 1 LaGalgada; ~4100BP (Early Andes); N = 1 Río Uncallane; ~1800BP (Late Central Andes); N = 5 Lauricocha; ~3500BP (Early Andes); N = 1 Arroyo Seco II; ~7700BP (Pampas Region); N = 5 Lauricocha; ~5800BP (Early Andes); N = 1 Laguna Chica; ~6800BP (Pampas Region); N = 3 Lauricocha; ~8600BP (Early Andes); N = 2 ● Laguna Toro; ~2400BP (Pampas Region); N = 1 Supplementary Figure 3. Principal Components Analysis (PCA). Ancient individuals were projected onto principal components inferred from the modern individuals after masking modern individuals to remove non-Native American ancestry (see Online Methods). Lapa Do Santo; ~9600BP (Ancient Brazil); N = 5 ● Chane (Equatorial−Tucanoan); N = 1 Laranjal; ~6600BP (Ancient Brazil); N = 2 Karitiana (Equatorial−Tucanoan); N = 4 4 ● Moraes; ~5800BP (Ancient Brazil); N = 1 Piapoco (Equatorial−Tucanoan); N = 2 Conchalí; ~700BP (Ancient Central Chile); N = 2 Surui (Equatorial−Tucanoan); N = 2 Los Rieles; ~12000BP (Ancient Central Chile); N = 1 Yukpa (Equatorial−Tucanoan); N = 1 Los Rieles; ~5100BP (Ancient Central Chile); N = 1 ● Aconcagua; ~500B (Late Central Andes); N = 1 ● Aymara (Andes); N = 11 Cuncaicha; ~3300BP (Late Central Andes); N = 1 Quechua (Andes); N = 3 Cuncaicha; ~4000BP (Late Central Andes); N = 1 ● Huilliche (Central Chile); N = 27 Kaillachuro; ~3800BP (Late Central Andes); N = 1 Pehuenche (Central Chile); N = 27 Laramate; ~900BP (Late Central Andes); N = 6 ● Cuncaicha; ~9000BP (Early Andes); N = 1 Pica Ocho; ~700BP (Late Central Andes); N = 1 LaGalgada; ~4100BP (Early Andes); N = 1 Río Uncallane; ~1800BP (Late Central Andes); N = 5 Lauricocha; ~3500BP (Early Andes); N = 1 Arroyo Seco II; ~7700BP (Pampas Region); N = 5 Lauricocha; ~5800BP (Early Andes); N = 1 Laguna Chica; ~6800BP (Pampas Region); N = 3 Lauricocha; ~8600BP (Early Andes); N = 2 ● Laguna Toro; ~2400BP (Pampas Region); N = 1 Color Key 0 0.05 0.1 0.15 0.2 Value ● ● ● ● ● ● Laguna Chica ~6800BP Arroyo Seco II ~7700BP Lapa Do Santo ~9600BP Ancient Brazil Piapoco Equatorial−Tucanoan Andes ● Huilliche Early Andes Conchalí; ~700BP Late Central Andes Pehuenche Pampas Region ● Aymara Central Chile Quechua Ancient Central Chile Laramate ~900BP Western Archipelago Río Uncallane; ~1800BP Beagle Channel Lauricocha ~8600BP Mitre Peninsula Laranjal ~6600BP Kawéskar Modern Beagle Channel ~800BP ● Beagle Channel ~1500−100BP Cape Horn Mission ~100BP ● Mitre Peninsula ~400BP ● Western Archipelago ~800BP ● Yámana Modern Northern Tierra del Fuego ~500BP Surui Karitiana Hagenbeck Expedition ~100BP Supplementary Figure 4. Heatmap of pairwise FST. FST was estimated among South American modern and ancient populations. The dendrogram was estimated with hierarchical clustering using FST as distances (see also Supplementary Data File 3K). For point legend, refer to Supplementary Figure 3. 5 A) B) Supplementary Figure 5. Carbon and Nitrogen isotopes analyses (A) Carbon and nitrogen isotope values for all ancient individuals. When different measurements were available they were averaged (Supplementary Data File 1 contains all values). (B) Maritime reservoir-corrected radiocarbon date distributions of all individuals with radiocarbon dates. Bracket below each probability distribution: 2σ confidence interval. 6 A) 242 242 60 Mbuti 105 USA-AK_USR1_11400BP 18 12 32 Argentina_LagunaToro_2400BP 18 4 25 38 53 351 9% 285 91% 4 1 LaArcillosa2_5800BP PuntaSantaAna_6600BP 96 0 53 Ayayema_4700BP Chile_Conchali_700BP 35 269 5 1 50% 50% 32% 68% 18 3 69% 6 163 12 41% 31% 59% 14 7% 68 93% 36 63 51% 49% Kawéskar_800BP Yámana_1500-100BP Haush_400BP Selk’nam_500BP Aónikenk_400BP 7 B) 209 209 155 Mbuti 121 USA-AK_USR1_11400BP 72 4 50 Argentina_LagunaToro_2400BP 23 18 27 41 3 68 0 391 8% 92% 252 5 0 LaArcillosa2_5800BP PuntaSantaAna_6600BP 250 105 0 0 67 Ayayema_4700BP Chile_Conchali_700BP 50 293 5 0 53% 34% 40% 47% 66% 18 0 41 68% 5 180 10 1 42% 20 32% 58% 60% 4% 54 96% 29 96% 4% 89 44% 56% Kawéskar_800BP Yámana_1500-100BP Haush_400BP Selk’nam_500BP Aónikenk_400BP Yámana_modern 8 C) 200 200 173 Mbuti 121 USA-AK_USR1_11400BP 72 4 49 Argentina_LagunaToro_2400BP 23 18 28 43 3 67 0 391 8% 92% 269 4 0 LaArcillosa2_5800BP PuntaSantaAna_6600BP 69 103 0 0 68 Ayayema_4700BP Chile_Conchali_700BP 51 291 3 0 53% 34% 59% 47% 66% 15 0 67% 21 13 8 33% 172 0 10 56% 44% 26 4% 42 96% 41% 40 21% 103 79% 39% 61% Kawéskar_800BP Yámana_1500-100BP Haush_400BP Selk’nam_500BP Aónikenk_400BP Kawéskar_modern 9 D) 237 237 98 Mbuti 122 USA-AK_USR1_11400BP 107 4 50 Argentina_LagunaToro_2400BP 22 113 29 45 6 73 0 386 11% 89% 295 5 0 LaArcillosa2_5800BP PuntaSantaAna_6600BP 35 97 7 0 63 Ayayema_4700BP Chile_Conchali_700BP 49 298 4 0 52% 33% 49% 48% 67% 17 0 69% 37 12 8 31% 164 0 10 56% 44% 25 5% 51% 43 95% 39 2 66% 101 34% 40% 60% Kawéskar_800BP Yámana_1500-100BP Haush_400BP Selk’nam_500BP Aónikenk_400BP Huilliche_modern 10 E) 248 248 76 Mbuti 122 USA-AK_USR1_11400BP 79 4 50 Argentina_LagunaToro_2400BP 24 72 30 46 7 75 0 384 12% 88% 286 4 0 LaArcillosa2_5800BP PuntaSantaAna_6600BP 35 97 7 0 65 Ayayema_4700BP Chile_Conchali_700BP 49 295 4 0 52% 33% 78% 48% 67% 17 0 12 68% 140 8 32% 118 0 10 55% 45% 26 5% 22% 39 42 95% 0 86% 103 14% 39% 61% Kawéskar_800BP Yámana_1500-100BP