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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 bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology.

[Significance] The bacterium Yersinia pestis has caused numerous historically documented outbreaks of plague and research using ancient DNA could demonstrate that it already affected human populations during the Neolithic. However, the pathogen’s genetic diversity, geographic spread, and transmission dynamics during this early period of Y. pestis evolution are largely unexplored. Here, we describe a set of ancient plague genomes up to 5,000 y old from across Eurasia. Our data demonstrate that two genetically distinct forms of Y. pestis evolved in parallel and were both distributed across vast geographic distances, potentially occupying different ecological niches. Interpreted within the archeological context, our results suggest that the spread of plague during this period was linked to increased human mobility and intensification of animal husbandry., The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology., This study was funded by the Max Planck Society, Max Planck Harvard Research Center for the Archaeoscience of the Ancient Mediterranean and the European Research Council under the European Union’s Horizon 2020 research and innovation program under Grant Agreement 771234 – PALEoRIDER (to W.H.), 856453 – HistoGenes (to J.K.), and 834616 – ARCHCAUCASUS (to S.H.). The Heidelberg Academy of Science financed the genetic and archeological research on human individuals from the Augsburg region within the project WIN Kolleg: “Times of Upheaval: Changes of Society and Landscape at the Beginning of the Bronze Age. M.E. was supported by the award “Praemium Academiae” of the Czech Academy of Sciences. M.D. was supported by the project RVO 67985912 of the Institute of Archaeology of the Czech Academy of Sciences, Prague. I.O. was supported by the Ramón y Cajal grant from Ministerio de Ciencia e Innovación, Spanish Government (RYC2019-027909-I). A. H€ubner was supported by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy (EXC 2051 – Project-ID 390713860). J.F.-E. and J.A.M.-A. were supported by the Diputación Foral de Alava, IT 1223-19, Gobierno Vasco. A. Buzhilova was supported by the Center of Information Technologies and Systems (CITIS), Moscow, Russia 121041500329-0. L. M., L.B.D., and E. Khussainova were supported by the Grant AP08856654, Ministry of Education and Science of the Republic of Kazakhstan. A. Beisenov was supported by the Grant AP08857177, Ministry of Education and Science of the Republic of Kazakhstan.

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

It has been hypothesized that the Neolithic transition towards an agricultural and pastoralist economy facilitated the emergence of human adapted pathogens. Here, we recovered eight Salmonella enterica subsp. enterica genomes from human skeletons of transitional foragers, pastoralists, and agro-pastoralists in western Eurasia that were up to 6,500 years old. Despite the high genetic diversity of S. enterica all ancient bacterial genomes clustered in a single previously uncharacterized branch that contains S. enterica adapted to multiple mammalian species. All ancient bacterial genomes from prehistoric (agro-)pastoralists fall within a part of this branch that also includes the human-specific S. enterica Paratyphi C, illustrating the evolution of a human pathogen over a period of five thousand years. Bacterial genomic comparisons suggest that the earlier ancient strains were not host specific, differed in pathogenic potential, and experienced convergent pseudogenization that accompanied their downstream host adaptation. These observations support the concept that the emergence of human adapted S. enterica is linked to human cultural transformations.

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

Archaeogenetic time transect in Europe unravels genetic and social changes before and after the arrival of “steppe” ancestry., Europe’s prehistory oversaw dynamic and complex interactions of diverse societies, hitherto unexplored at detailed regional scales. Studying 271 human genomes dated ~4900 to 1600 BCE from the European heartland, Bohemia, we reveal unprecedented genetic changes and social processes. Major migrations preceded the arrival of “steppe” ancestry, and at ~2800 BCE, three genetically and culturally differentiated groups coexisted. Corded Ware appeared by 2900 BCE, were initially genetically diverse, did not derive all steppe ancestry from known Yamnaya, and assimilated females of diverse backgrounds. Both Corded Ware and Bell Beaker groups underwent dynamic changes, involving sharp reductions and complete replacements of Y-chromosomal diversity at ~2600 and ~2400 BCE, respectively, the latter accompanied by increased Neolithic-like ancestry. The Bronze Age saw new social organization emerge amid a ≥40% population turnover.

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.).

This protocol describes how to obtain powder from the inner pulp chamber of teeth for the extraction of ancient DNA. It is ideal for the simultaneous isolation of both host endogenous and microbial DNA (e.g. for pathogens).

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing. This produces adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with initial USER enzyme treatment. This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream indexing protocols. The initial treatment with UDG (USER) removes molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448) and Briggs et al. (2010) Nucleic Acids Research 38 (6) (doi: 10.1093/nar/gkp1163)

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.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with initial USER enzyme treatment. This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. The initial treatment with UDG (USER) removes molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448) and Briggs et al. (2010) Nucleic Acids Research 38 (6) (doi: 10.1093/nar/gkp1163) This protocol has been optimised for reduced DNA input, to allow building of multiple libraries from a singe extract.

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

Genetic studies of Neolithic and Bronze Age skeletons from Europe have provided evidence for strong population genetic changes at the beginning and the end of the Neolithic period. To further understand the implications of these in Southern Central Europe, we analyze 96 ancient genomes from Switzerland, Southern Germany, and the Alsace region in France, covering the Middle/Late Neolithic to Early Bronze Age. Similar to previously described genetic changes in other parts of Europe from the early 3rd millennium BCE, we detect an arrival of ancestry related to Late Neolithic pastoralists from the Pontic-Caspian steppe in Switzerland as early as 2860–2460 calBCE. Our analyses suggest that this genetic turnover was a complex process lasting almost 1000 years and involved highly genetically structured populations in this region. European populations underwent strong genetic changes during the Neolithic. Here, Furtwangler et al. provide ancient nuclear and mitochondrial genomic data from the region of Switzerland during the end of the Neolithic and the Early Bronze Age that reveal a complex genetic turnover during the arrival of steppe ancestry.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

This protocol describes how to obtain powder from the inner pulp chamber of teeth for the extraction of ancient DNA. It is ideal for the simultaneous isolation of both host endogenous and microbial DNA (e.g. for pathogens).

The second plague pandemic, caused by Yersinia pestis, devastated Europe and the nearby regions between the 14th and 18th centuries AD. Here we analyse human remains from ten European archaeological sites spanning this period and reconstruct 34 ancient Y. pestis genomes. Our data support an initial entry of the bacterium through eastern Europe, the absence of genetic diversity during the Black Death, and low within-outbreak diversity thereafter. Analysis of post-Black Death genomes shows the diversification of a Y. pestis lineage into multiple genetically distinct clades that may have given rise to more than one disease reservoir in, or close to, Europe. In addition, we show the loss of a genomic region that includes virulence-related genes in strains associated with late stages of the pandemic. The deletion was also identified in genomes connected with the first plague pandemic (541–750 AD), suggesting a comparable evolutionary trajectory of Y. pestis during both events., The second plague pandemic, caused by Yersinia pestis, started in the 14th century and recurred in Europe until the 18th century. Here, the authors reconstruct ancient Y. pestis genomes from human remains spanning this period, shedding light into the phylogeography and population structure of the European strains.

The first historically documented pandemic caused by Yersinia pestis began as the Justinianic Plague in 541 within the Roman Empire and continued as the so-called First Pandemic until 750. Although paleogenomic studies have previously identified the causative agent as Y. pestis , little is known about the bacterium’s spread, diversity, and genetic history over the course of the pandemic. To elucidate the microevolution of the bacterium during this time period, we screened human remains from 21 sites in Austria, Britain, Germany, France, and Spain for Y. pestis DNA and reconstructed eight genomes. We present a methodological approach assessing single-nucleotide polymorphisms (SNPs) in ancient bacterial genomes, facilitating qualitative analyses of low coverage genomes from a metagenomic background. Phylogenetic analysis on the eight reconstructed genomes reveals the existence of previously undocumented Y. pestis diversity during the sixth to eighth centuries, and provides evidence for the presence of multiple distinct Y. pestis strains in Europe. We offer genetic evidence for the presence of the Justinianic Plague in the British Isles, previously only hypothesized from ambiguous documentary accounts, as well as the parallel occurrence of multiple derived strains in central and southern France, Spain, and southern Germany. Four of the reported strains form a polytomy similar to others seen across the Y. pestis phylogeny, associated with the Second and Third Pandemics. We identified a deletion of a 45-kb genomic region in the most recent First Pandemic strains affecting two virulence factors, intriguingly overlapping with a deletion found in 17th- to 18th-century genomes of the Second Pandemic.

Significance The first historically reported pandemic attributed to Yersinia pestis started with the Justinianic Plague (541–544) and continued for around 200 y as the so-called First Pandemic. To date, only one Y. pestis strain from this pandemic has been reconstructed using ancient DNA. In this study, we present eight genomes from Britain, France, Germany, and Spain, demonstrating the geographic range of plague during the First Pandemic and showing microdiversity in the Early Medieval Period. Moreover, we detect similar genome decay during the First and Second Pandemics (14th to 18th century) that includes the same two virulence factors, thus providing an example of potential convergent evolution of Y. pestis during large-scale epidemics., The first historically documented pandemic caused by Yersinia pestis began as the Justinianic Plague in 541 within the Roman Empire and continued as the so-called First Pandemic until 750. Although paleogenomic studies have previously identified the causative agent as Y. pestis, little is known about the bacterium’s spread, diversity, and genetic history over the course of the pandemic. To elucidate the microevolution of the bacterium during this time period, we screened human remains from 21 sites in Austria, Britain, Germany, France, and Spain for Y. pestis DNA and reconstructed eight genomes. We present a methodological approach assessing single-nucleotide polymorphisms (SNPs) in ancient bacterial genomes, facilitating qualitative analyses of low coverage genomes from a metagenomic background. Phylogenetic analysis on the eight reconstructed genomes reveals the existence of previously undocumented Y. pestis diversity during the sixth to eighth centuries, and provides evidence for the presence of multiple distinct Y. pestis strains in Europe. We offer genetic evidence for the presence of the Justinianic Plague in the British Isles, previously only hypothesized from ambiguous documentary accounts, as well as the parallel occurrence of multiple derived strains in central and southern France, Spain, and southern Germany. Four of the reported strains form a polytomy similar to others seen across the Y. pestis phylogeny, associated with the Second and Third Pandemics. We identified a deletion of a 45-kb genomic region in the most recent First Pandemic strains affecting two virulence factors, intriguingly overlapping with a deletion found in 17th- to 18th-century genomes of the Second Pandemic.

Summary Modern humans have inhabited the Lake Baikal region since the Upper Paleolithic, though the precise history of its peoples over this long time span is still largely unknown. Here, we report genome-wide data from 19 Upper Paleolithic to Early Bronze Age individuals from this Siberian region. An Upper Paleolithic genome shows a direct link with the First Americans by sharing the admixed ancestry that gave rise to all non-Arctic Native Americans. We also demonstrate the formation of Early Neolithic and Bronze Age Baikal populations as the result of prolonged admixture throughout the eighth to sixth millennium BP. Moreover, we detect genetic interactions with western Eurasian steppe populations and reconstruct Yersinia pestis genomes from two Early Bronze Age individuals without western Eurasian ancestry. Overall, our study demonstrates the most deeply divergent connection between Upper Paleolithic Siberians and the First Americans and reveals human and pathogen mobility across Eurasia during the Bronze Age.

The first historically documented pandemic caused byYersinia pestisstarted as the Justinianic Plague in 541 within the Roman Empire and continued as the so-called First Pandemic until 750. Although palaeogenomic studies have previously identified the causative agent asY. pestis, little is known about the bacterium’s spread, diversity and genetic history over the course of the pandemic.To elucidate the microevolution of the bacterium during this time period, we screened human remains from 20 sites in Austria, Britain, Germany, France and Spain forY. pestisDNA and reconstructed six new genomes. We present a novel methodological approach assessing SNPs in ancient bacterial genomes, facilitating qualitative analyses of low coverage genomes from a metagenomic background. Phylogenetic analysis reveals the existence of previously undocumentedY. pestisdiversity during the 6th–7thcenturies, and provides evidence for the presence of multiple distinctY. pestisstrains in Europe. We offer genetic evidence for the presence of the Justinianic Plague in the British Isles, previously only hypothesized from ambiguous documentary accounts, as well as southern France and Spain, and that southern Germany seems to have been affected by at least two distinctY. pestisstrains. Four of the reported strains form a polytomy similar to others seen across theY. pestisphylogeny, associated with the Second and Third Pandemics. We identified a deletion of a 45 kb genomic region in the most recent First Pandemic strain affecting two virulence factors, intriguingly overlapping with a deletion found in 17th–18th-century genomes of the Second Pandemic.Significance StatementThe first historically reported pandemic attributed toYersinia pestisstarted with the Justinianic Plague (541–544) and continued for around 200 years as the so-called First Pandemic. To date, only oneY. pestisstrain from this pandemic has been reconstructed using ancient DNA. In this study, we present six new genomes from Britain, France, Germany and Spain, demonstrating the geographic range of plague during the First pandemic and showing microdiversity in the Early Medieval Period. Moreover, we detect similar genome decay during the First and Second Pandemic (17thto 18thcentury) that includes the same two virulence factors, thus providing an example of potential convergent evolution ofY. pestisduring large scale epidemics.

We study the impact of coil orientation on the motor threshold (MT) and present an optimal coil orientation for stimulation of the foot. The result can be compared to results of models that predict this orientation from electrodynamic properties of the media in the skull and from orientations of cells, respectively. We used a robotized TMS system for precise coil placement and recorded motor-evoked potentials with surface electrodes on the abductor hallucis muscle of the right foot in 8 healthy control subjects. First, we performed a hot-spot search in standard (lateral) orientation and then rotated the coil in steps of 10° or 20°. At each step we estimated the MT. For navigated stimulation and for correlation with the underlying anatomy a structural MRI scan was obtained. Optimal coil orientation was 33.1 ± 18.3° anteriorly in relation to the standard lateral orientation. In this orientation the threshold was 54 ± 18% in units of maximum stimulator output. There was a significant difference of 8.0 ± 5.9% between the MTs at optimal and at standard orientation. The optimal coil orientations were significantly correlated with the direction perpendicular to the postcentral gyrus ([Formula: see text]). Robotized TMS facilitates sufficiently precise coil positioning and orientation to study even small variations of the MT with coil orientation. The deviations from standard orientation are more closely matched by models based on field propagation in media than by models based on orientations of pyramidal cells.

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