https://doi.org/10.1038/s41586-020-03053-2 Supplementary information A genetic history of the pre-contact Caribbean In the format provided by the authors and unedited Nature | www.nature.com/nature Supplementary Information A genetic history of the pre-contact Caribbean Daniel M. Fernandes, Kendra A. Sirak, Harald Ringbauer, Jakob Sedig, Nadin Rohland, Olivia Cheronet, Matthew Mah, Swapan Mallick, Iñigo Olalde, Brendan J. Culleton, Nicole Adamski, Rebecca Bernardos, Guillermo Bravo, Nasreen Broomandkhoshbacht, Kimberly Callan, Francesca Candilio, Lea Demetz, Kellie Sara Duffett Carlson, Laurie Eccles, Suzanne Freilich, Richard J. George, Ann Marie Lawson, Kirsten Mandl, Fabio Marzaioli, Weston C. McCool, Jonas Oppenheimer, Kadir T. Özdogan, Constanze Schattke, Ryan Schmidt, Kristin Stewardson, Filippo Terrasi, Fatma Zalzala, Carlos Arredondo Antúnez, Ercilio Vento Canosa, Roger Colten, Andrea Cucina, Francesco Genchi, Claudia Kraan, Francesco La Pastina, Michaela Lucci, Marcio Veloz Maggiolo, Beatriz Marcheco-Teurel, Clenis Tavarez Maria, Christian Martínez, Ingeborg París, Michael Pateman, Tanya M. Simms, Carlos Garcia Sivoli, Miguel Vilar, Douglas J. Kennett, William F. Keegan, Alfredo Coppa, Mark Lipson, Ron Pinhasi, David Reich 1 TABLE OF CONTENTS Ethics Statement 3 SI section 1. Overview of archaeological series and culture history 5 SI section 2. Archaeological site information 19 SI section 3. Newly-reported direct 14C dates and isotope data 48 SI section 4. Co-analysis of newly-reported data with the data reported in Nägele et al. 2020 57 SI section 5. Principal Component Analysis (PCA) 62 SI section 6. Unsupervised population structure analysis 66 SI section 7. Kinship and consanguinity analysis 68 SI section 8. Clade Grouping and Substructure Analysis with qpWave, TreeMix, and f4-statistics 76 SI section 9. Admixture modeling and estimates of ancestry proportions 91 SI section 10. Uniparental haplogroups 102 SI section 11. f-statistics and relatedness to modern language groups 108 SI section 12. qpGraph 109 SI section 13. Ability to detect a Carib migration into the Caribbean: simulations and qpWave 110 SI section 14. DATES 112 SI section 15. Relatedness of ancient individuals to present-day admixed Caribbean people 113 SI section 16. Analysis of phenotypic SNPs 114 SI section 17. Evaluation of the proposal of at least two sources of ancestry in Archaic Cuba 117 References 127 2 Ethics Statement Permissions for this study Permissions to carry out ancient DNA analysis of the human skeletal remains in this study were documented through authorization letters and Memorandums of Understanding (MOU) signed by a custodian who assumed responsibility for the skeletal remains collected from a specific geographic region or site. The authorization letter established permission to perform ancient DNA analysis as well as radiocarbon dating, isotopic analysis, and other bioarchaeological analyses on the ancient skeletal material and stated that 1) all skeletal material was recovered during past archaeological excavations and archived for scientific analysis; 2) the institution on behalf of which the custodian signed provided explicit permission to perform the analysis carried out in this study; and 3) the institution on behalf of which the custodian signed was supportive of publishing results of this study in a scientific journal. - Bahamas: Authorization letter signed by Michael Pateman (Director, Turks & Caicos National Museum); letter of permission for the export of skeletal material from The Bahamas and for ancient DNA, isotopic, and radiocarbon dating analyses signed by Keith L. Tinker (Director, The National Museum of The Bahamas); - Curaçao: Authorization letter signed by Dmitri Cloose (Director, National Archaeological Anthropological Memory Management, NAAM); - Dominican Republic: Authorization letter signed by Arq. Christian Martínez Villanueva (Director General. Museo del Hombre Dominicano); - Venezuela: Authorization letter signed by Prof. Carlos García Sívoli (on behalf of the Instituto de Investigaciones Bioantropológicas y Arqueológicas de la Universidad de Los Andes); - Haiti: Permission for the generation of full genome data using human skeletal remains was provided by the Peabody Museum of Natural History at Yale University; - Puerto Rico: Permission for the generation of full genome data using human skeletal remains was provided by the Peabody Museum of Natural History at Yale University. Collaboration and outreach In this study, we test hypotheses surrounding the geographic origins and genetic affinities of ancient peoples of the Caribbean islands using paleogenomic data as one line of evidence that is useful for studying the past. Anthropologists, archaeologists, and other scholars from each geographic region included in this study played an integral role in the interpretation of data and formulation of results and were included as co-authors. We also consulted multiple other Caribbean-based stakeholders throughout the course of the project to solicit critical feedback and local perspectives. A major goal was to ensure that local narratives were considered during the process of contextualizing these data, and thus the involvement and feedback of local scholars and stakeholders was crucial. The inferences that we make as part of this project are intended to provide new information about the genetic ancestry of the people who lived in the Caribbean prior to the colonization by Europeans 3 in the late 15th century and to explore the genetic contribution from the ancient peoples of this region to the genomes of present-day Caribbean people. We note that our study of genetic ancestry of people in this region should not be conflated with individual and/or community perceptions of identity, as identity cannot be defined by genetics alone. Genetic data are one form of knowledge that contributes to understanding the past, and oral traditions and other forms of Indigenous knowledge can coexist with scientific data. Protection of archaeological sites Latitude and longitude coordinates for the archaeological sites from which the skeletal remains of individuals examined were excavated are reported in Supplementary Data 1. The Society for American Archaeology’s Ethical Principle #6 (https://www.saa.org/career-practice/ethics-in-professional- archaeology) states that, “An interest in preserving and protecting in situ archaeological sites must be taken into account when publishing and distributing information about their nature and location”; thus, we have provided latitude and longitude coordinates limited to two decimal degree digits in an attempt to balance protecting archaeological site integrity against publishing the geographic data needed to test genetic correlations with distance measures and meet open science replicability standards. 4 SI1 - Overview of archaeological series and culture history in the Caribbean and northern South America The Caribbean Similarities and differences in material culture are arranged in classification systems to identify patterns in the archaeological record of a region. Two taxonomic systems are employed for the Caribbean islands. The first and most general is an “Age” system based on the introduction of significant changes in technology. The major weakness of the Age system is its emphasis on broad categories of technological change (i.e., flaked stone, ground stone, pottery). In this regard, Ages are not time sensitive and each spans millennia. In addition, Ages do not reflect local differences in the ways these technologies were expressed. For these reasons a second system, based on the time- space periodization of internal developments, was created to capture localized changes in style (“Series”). These systems are hierarchical in that “internal” stylistic changes are subsumed under “external” technological introductions. The “Age” system is divided into Lithic, Archaic, Ceramic, and Historic Ages (Rouse 1992). Irving Rouse, a foundational figure in Caribbean archaeology, developed the system to recognize the most significant changes in technology observed in the region. Technological change occurs in three main ways: independent invention, diffusion of ideas, or the movement of people who carry the new technology. The latter (the arrival of new immigrants from the mainland bearing new technology) was assumed when developing this nomenclature. Working within this frame of reference, the pattern of human movement(s) into the islands was reconstructed primarily through comparisons of technologies between Caribbean archaeological sites and mainland material assemblages (Wilson 2007). In this approach, technological change is viewed as the product of external influences, and the introduction of new technologies supposedly leads to the physical and/or cultural displacement of previously established practices (Rouse 1992). In reality, even if cultural practices changed, existing technology(s) usually continued in use, albeit often in different ways (Rodríguez Ramos 2010). We begin by providing a brief description of three distinct Ages of Caribbean occupation (we do not focus in detail on the Lithic or Historic Ages in this work as we study no individuals dating to either of these time periods). These Ages began and ended at different times on different islands; therefore, we provide the earliest date for each Age. For ease of interpretation, we discuss archaeological periods and corresponding ceramic typological styles in this section and the following section using BCE/CE dates.