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Growing reliance on animal and plant domestication in the Near East and beyond during the Pre-Pottery Neolithic B (PPNB) (the ninth to eighth millennium BC) has often been associated with a “revolutionary” social transformation from mobility toward more sedentary lifestyles. We are able to yield nuanced insights into the process of the Neolithization in the Near East based on a bioarchaeological approach integrating isotopic and archaeogenetic analyses on the bone remains recovered from Nevalı Çori, a site occupied from the early PPNB in Turkey where some of the earliest evidence of animal and plant domestication emerged, and from Ba'ja, a typical late PPNB site in Jordan. In addition, we present the archaeological sequence of Nevalı Çori together with newly generated radiocarbon dates. Our results are based on strontium ( 87 Sr/ 86 Sr), carbon, and oxygen (δ 18 O and δ 13 C carb ) isotopic analyses conducted on 28 human and 29 animal individuals from the site of Nevalı Çori. 87 Sr/ 86 Sr results indicate mobility and connection with the contemporaneous surrounding sites during the earlier PPNB prior to an apparent decline in this mobility at a time of growing reliance on domesticates. Genome-wide data from six human individuals from Nevalı Çori and Ba'ja demonstrate a diverse gene pool at Nevalı Çori that supports connectedness within the Fertile Crescent during the earlier phases of Neolithization and evidence of consanguineous union in the PPNB Ba'ja and the Iron Age Nevalı Çori.

The Neolithic and Bronze Ages were highly transformative periods for the genetic history of Europe but for the Aegean—a region fundamental to Europe’s prehistory—the biological dimensions of cultural transitions have been elucidated only to a limited extent so far. We have analysed newly generated genome-wide data from 102 ancient individuals from Crete, the Greek mainland and the Aegean Islands, spanning from the Neolithic to the Iron Age. We found that the early farmers from Crete shared the same ancestry as other contemporaneous Neolithic Aegeans. In contrast, the end of the Neolithic period and the following Early Bronze Age were marked by ‘eastern’ gene flow, which was predominantly of Anatolian origin in Crete. Confirming previous findings for additional Central/Eastern European ancestry in the Greek mainland by the Middle Bronze Age, we additionally show that such genetic signatures appeared in Crete gradually from the seventeenth to twelfth centuries bc, a period when the influence of the mainland over the island intensified. Biological and cultural connectedness within the Aegean is also supported by the finding of consanguineous endogamy practiced at high frequencies, unprecedented in the global ancient DNA record. Our results highlight the potential of archaeogenomic approaches in the Aegean for unravelling the interplay of genetic admixture, marital and other cultural practices. Results - The archaeogenetic dataset - Transregional genetic entanglements of Aegean populations - Neolithic to Early/Middle Bronze Age - Mobility in the Middle/Late Bronze Age Aegean - Insights into sex bias, biological kinship and marital practices Discussion Methods - Preparation of aDNA analysis - Analysis of population structure - Admixture analysis with ADMIXTOOLS - Admixture dating - Analysis of biological relatedness - Analysis of ROH - Direct AMS radiocarbon dating

The Middle and Late Bronze Age, a period roughly spanning the 2(nd) millennium BC (ca. 2000-1200 BC) in the Near East, is frequently referred to as the first 'international age', characterized by intense and far-reaching contacts between different entities from the eastern Mediterranean to the Near East and beyond. In a large-scale tandem study of stable isotopes and ancient DNA of individuals excavated at Tell Atchana (Alalakh, located in Hatay, Turkey), we explored the role of mobility at the capital of a regional kingdom, named Mukish during the Late Bronze Age, which spanned the Amuq Valley and some areas beyond. We generated strontium and oxygen isotope data from dental enamel for 53 individuals and 77 individuals, respectively, and added ancient DNA data of 10 newly sequenced individuals to a dataset of 27 individuals published in 2020. Additionally, we improved the DNA coverage of one individual from this 2020 dataset. The DNA data revealed a very homogeneous gene pool. This picture of an overwhelmingly local ancestry was consistent with the evidence of local upbringing in most of the individuals indicated by the isotopic data, where only five were found to be non-local. High levels of contact, trade, and exchange of ideas and goods in the Middle and Late Bronze Ages, therefore, seem not to have translated into high levels of individual mobility detectable at Tell Atchana., European Union (EU); Horizon 2020; European Research Council (ERC); Research İnnovation Programme; ERC-2015-StG; FoodTransforms

Uniparentally-inherited markers on mitochondrial DNA (mtDNA) and the non-recombining regions of the Y chromosome (NRY), have been used for the past 30 years to investigate the history of humans from a maternal and paternal perspective. Researchers have preferred mtDNA due to its abundance in the cells, and comparatively high substitution rate. Conversely, the NRY is less susceptible to back mutations and saturation, and is potentially more informative than mtDNA owing to its longer sequence length. However, due to comparatively poor NRY coverage via shotgun sequencing, and the relatively low and biased representation of Y-chromosome variants on capture assays such as the 1240 k, ancient DNA studies often fail to utilize the unique perspective that the NRY can yield. Here we introduce a new DNA enrichment assay, coined YMCA (Y-mappable capture assay), that targets the "mappable" regions of the NRY. We show that compared to low-coverage shotgun sequencing and 1240 k capture, YMCA significantly improves the mean coverage and number of sites covered on the NRY, increasing the number of Y-haplogroup informative SNPs, and allowing for the identification of previously undiscovered variants. To illustrate the power of YMCA, we show that the analysis of ancient Y-chromosome lineages can help to resolve Y-chromosomal haplogroups. As a case study, we focus on H2, a haplogroup associated with a critical event in European human history: the Neolithic transition. By disentangling the evolutionary history of this haplogroup, we further elucidate the two separate paths by which early farmers expanded from Anatolia and the Near East to western Europe., Open Access funding enabled and organized by Projekt DEAL. This study was funded by the Max Planck Society, the French (ANR) and German (DFG) Research Foundations under the INTERACT project (ANR-17-FRAL-0010, DFG-HA-5407/4-1, 2018-2021) to M.R. and W.H., the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant agreement no. 771234-PALEoRIDER to W.H., the award Praemium Academiae of the Czech Academy of Sciences to M.E. and the project RVO 67985912 of the Institute of Archaeology of the Czech Academy of Sciences, Prague to M.S.

This collection contains established ancient DNA (aDNA) specific protocols for Illumina next generation sequencing. It covers sampling of skeletal remains for regions preserving higher amounts of aDNA, DNA extraction methods designed for retrieval of very short DNA fragments, and various library construction methods (including treatments for the 'removal' of typical aDNA damage). All protocols have been adapted after published protocols (see each for the corresponding original publication), and are used primarily in the Department of Archaeogenetics at the MPI-SHH (Jena, Germany). All protocols in this collection can be used together, and in some cases swapped out with others. All protocols prior indexing are mostly performed in a dedicated ancient DNA laboratory - please see the guidelines section of each protocol for more information. The order of protocols is as follows: 1. Sampling for aDNA Sampling of Pars petrosa (os temporale, ''petrous bones') for human aDNA extraction Sampling of teeth for human and microbial (e.g. pathogen) aDNA extraction Sampling of dental calculus for oral microbiome aDNA extraction 2. Retrieval of DNA Ancient DNA Extraction from Skeletal Material (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A protocol) Ancient DNA extraction from dental calculus (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A with extra washing steps from Ozga et al. 2016 Am J Phys Anthropol) 3. Library Preparation Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (requires 3.1, based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with partial damage removal treatment from Rohland et al. 2015 Philos Trans R Soc Lond B Biol Sci) Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with complete damage removal treatment from Briggs et al. 2010 Nucleic Acids Res) Dual-Indexing of aDNA libraries for Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) 4. Preparation for Sequencing Amplification and Pooling for dual-indexed Illumina library for sequencing

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic., The research was funded by the Max Planck Society, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (771234–PALEoRIDER, to W.H.; 805268–CoDisEASe to K. Bos; 834616–ARCHCAUCASUS to S.H.), the Slovak Academy of Sciences and the European Union’s Seventh Framework Programme and Marie Curie Actions under the Programme SASPRO (1340/03/03 to P.C.R.), the ERA.NET RUS Plus–S&T programm of the European Union’s Seventh Framework Programme (277–BIOARCCAUCASUS to S.Re. and S.H.), the Werner Siemens Stiftung (“Paleobiochemistry”, to CW), the Award Praemium Academiae of the Czech Academy of Sciences (to M.E.), the Institute of Archaeology of the Czech Academy of Sciences (RVO 67985912, to M.Dobe.), the Russian Foundation for Basic Research (19-09-00354a, to M.K.K. and V.V.K.; 19-78-10053 to SSh), the German Research Foundation (DFG-HA-5407/4-1–INTERACT to W.H. and RE2688/2 to S.Re.), the French National Research Agency (ANR-17-FRAL-0010–INTERACT, to M.F.D., M.Ri., S.Ro., S.Sai., D.Bi., and P.Le.), the Wenner-Gren Dissertation Fieldwork Grant (9558 to S.Sab.), and the Ministry of Education and Science of the Republic of Kazakhstan (AP08856654 to L.B.D., L.M., and E.Kh. and AP08857177 to A.Z.B.).

The Middle and Late Bronze Age Near East, a period roughly spanning the second millennium BC (ca. 2000-1200 BC), is frequently referred to as the first ‘international age’, characterized by intense and far-reaching contacts between different entities from the eastern Mediterranean to the Near East and beyond. In a large-scale tandem study of stable isotopes and ancient DNA of individuals excavated at Tell Atchana (Alalakh), situated in the northern Levant, we explore the role of mobility at the capital of a regional kingdom. We generated strontium isotope data for 53 individuals, oxygen isotope data for 77 individuals, and added ancient DNA data from 9 new individuals to a recently published dataset of 28 individuals. A dataset like this, from a single site in the Near East, is thus far unparalleled in terms of both its breadth and depth, providing the opportunity to simultaneously obtain an in-depth view of individual mobility and also broader demographic insights into the resident population. The DNA data reveals a very homogeneous gene pool, with only one outlier. This picture of an overwhelmingly local ancestry is consistent with the evidence of local upbringing in most of the individuals indicated by the isotopic data, where only five were found to be ‘non-local’. High levels of contact, trade, and exchange of ideas and goods in the Middle and Late Bronze Ages, therefore, seem not to have translated into high levels of individual mobility detectable at Tell Atchana.

Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from archaeological dental calculus, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110) and adapted for dental calculus by Mann et al. (2018) Scientific Reports (doi: https://doi.org/10.1038/s41598-018-28091-9).

Here, we report genome-wide data analyses from 110 ancient Near Eastern individuals spanning the Late Neolithic to Late Bronze Age, a period characterized by intense interregional interactions for the Near East. We find that 6th millennium BCE populations of North/Central Anatolia and the Southern Caucasus shared mixed ancestry on a genetic cline that formed during the Neolithic between Western Anatolia and regions in today’s Southern Caucasus/Zagros. During the Late Chalcolithic and/or the Early Bronze Age, more than half of the Northern Levantine gene pool was replaced, while in the rest of Anatolia and the Southern Caucasus, we document genetic continuity with only transient gene flow. Additionally, we reveal a genetically distinct individual within the Late Bronze Age Northern Levant. Overall, our study uncovers multiple scales of population dynamics through time, from extensive admixture during the Neolithic period to long-distance mobility within the globalized societies of the Late Bronze Age. Video Abstract - Introduction - Results -- Sample Corpus and Data Compilation -- The Late Neolithic/Early Chalcolithic Genetic Structure in Anatolia, Northern Levant, and Caucasian Lowlands -- Neolithic Admixture and a Common Genetic Profile of Chalcolithic and Bronze Age Groups -- Admixture Modeling of the Chalcolithic and Bronze Age Groups -- Genetic Turnover in the Bronze Age Northern Levant -- Evidence for Individual Mobility in Alalakh - Discussion -- Genetic Homogenization across Anatolia and the Southern Caucasus prior to the Bronze Age -- Population and Territorial State Dynamics in the Northern Levant -- Conclusions STAR★Methods

Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from skeletal material powder (e.g. bone, teeth), modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).

The colonization patterns of oceanic islands are often interpreted through transmarine dispersal. However, in islands with intense human activities and unclear geological history, this inference may be inappropriate. Cyprus is such an island, whose geotectonic evolution has not been clarified yet to the desired level for biogeographical reconstructions, leaving the questions of ‘how the Cypriote biota arrived’ and ‘does the dispersal have the formative role in patterns of its diversification’ unanswered. Here, we address these issues through a reconstruction of the evolutionary history of six herptiles (Ablepharus budaki, Ophisops elegans, Acanthodactylus schreiberi, Telescopus fallax, Pelophylax cf. bedriagae, and Hyla savignyi) by means of mitochondrial DNA (cytochrome b and 16S rRNA), applying a Bayesian phylogenetic, biogeographical, and chronophylogenetic analyses. The phylogeographical analyses show that the colonization history of those species in Cyprus started in the late Miocene and extended into the Pliocene and Pleistocene, with geodispersal, transmarine dispersal, and human-mediated dispersal having their share in shaping the diversification of Cypriote herptiles. The revealed patterns could be divided into three biogeographical categories: old colonizers that arrived in Cyprus during the late Miocene or early Pliocene either by a land bridge (geodispersal) which connected Cyprus with the mainland or by transmarine dispersal, younger colonizers that reached the island through transmarine dispersal from the Middle East, and new settlers that arrived through human-induced (voluntary or not) introductions. This work advances our knowledge of the biogeography of Cyprus and highlights the need to consider both geo- and transmarine dispersal when dealing with islands whose associations do not have a straightforward interpretation. © 2013 The Linnean Society of London

This collection contains established ancient DNA (aDNA) specific protocols for Illumina next generation sequencing. It covers sampling of skeletal remains for regions preserving higher amounts of aDNA, DNA extraction methods designed for retrieval of very short DNA fragments, and various library construction methods (including treatments for the 'removal' of typical aDNA damage). All protocols have been adapted after published protocols (see each for the corresponding original publication), and are used primarily in the Department of Archaeogenetics at the MPI-SHH (Jena, Germany). All protocols in this collection can be used together, and in some cases swapped out with others. All protocols prior indexing are mostly performed in a dedicated ancient DNA laboratory - please see the guidelines section of each protocol for more information. The order of protocols is as follows: 1. Sampling for aDNA Sampling of Pars petrosa (os temporale, ''petrous bones') for human aDNA extraction Sampling of teeth for human and microbial (e.g. pathogen) aDNA extraction Sampling of dental calculus for oral microbiome aDNA extraction 2. Retrieval of DNA Ancient DNA Extraction from Skeletal Material (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A protocol) Ancient DNA extraction from dental calculus (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A with extra washing steps from Ozga et al. 2016 Am J Phys Anthropol) 3. Library Preparation Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (requires 3.1, based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with partial damage removal treatment from Rohland et al. 2015 Philos Trans R Soc Lond B Biol Sci) Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with complete damage removal treatment from Briggs et al. 2010 Nucleic Acids Res) Dual-Indexing of aDNA libraries for Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) 4. Preparation for Sequencing Amplification and Pooling for dual-indexed Illumina library for sequencing