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The terrestrial subsurface houses a significant proportion of the Earth’s microbial biomass. Our understanding about terrestrial subsurface microbiomes is almost exclusively derived from groundwater and porous sediments. To obtain more insights about endolithic microbiomes and their metabolic status, we investigated rock samples from the vadose zone, fractured aquifers, and deep aquitards. Using methods from paleogenomics, we recovered sufficient DNA for metagenomics from rock specimens independent of porosity, lithology, and depth. We estimated between 2.81 and 4.25 × 105 cells × g−1 rock. DNA damage patterns revealed paleome signatures (genetic records of past microbial communities) for three rock specimens from the vadose zone. The taxonomy and functional potential of paleome communities revealed increased abundances of chemolithoautotrophs, and a broader metabolic potential for aromatic hydrocarbon breakdown. Our study suggests that limestones represent ideal archives for genetic records of past microbial communities, due to their specific conditions facilitating long-term DNA preservation.

Industrialization - including urbanization, participation in the global food chain, and consumption of heavily processed foods - is thought to drive substantial shifts in the human microbiome. While diet strongly influences stool microbiome composition, the influence of diet on the oral microbiome, and particularly dental plaque, is largely speculative. Here we investigated whether dental plaque microbial communities are distinctly different across populations with dissimilar subsistence strategies and degree of industrialized market integration. Using a metagenomic approach, we compared the dental plaque microbiomes of Baka foragers and Nzime subsistence agriculturalists in Cameroon (n = 46) with the dental plaque and calculus microbiomes of highly industrialized populations in North America and Europe. We found that differences in microbial taxonomic composition between populations were minimal, with high conservation of abundant microbial taxa and no significant differences in microbial diversity related to dietary practices. Instead, we find that the major source of variation in dental plaque microbial species composition is related to tooth location and oxygen availability, and may be influenced by toothbrushing or other dental hygiene measures. Our results support that dental plaque, in contrast to the stool microbiome, maintains an inherent stability against ecological perturbations in the oral environment.

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.

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

This protocol converts partially completed double-stranded DNA libraries e.g. from: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx.doi.org/10.17504/protocols.io.bakricv6) into dual-indexed Illumina libraries ready for sequencing on an Illumina platform. Other variants of the above protocol can also be used. This protocol includes steps for indexing PCR, clean-up, and post-indexing qPCR. This protocol is modified after Kircher, M., Sawyer, S. & Meyer, M., 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic acids research, 40(1), p.e3. http://dx.doi.org/10.1093/nar/gkr771.

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized. Results - Site excavation. - Archaeofaunal remains. - Cementum analysis and dental eruption/wear. - Animal DNA. Discussion Methods - Excavation. - Radiocarbon dating. - Thermoluminescence dating. - Zooarchaeology and ZooMS. - Cementum analysis. - DNA analysis. -- PCR amplification. -- Library construction and sequencing. -- PCA of genome-wide sequences. -- Mitochondrial genome analysis.

Present-day Tibetans have adapted both genetically and culturally to the high altitude environment of the Tibetan Plateau, but fundamental questions about their origins remain unanswered. Recent archaeological and genetic research suggests the presence of an early population on the Plateau within the past 40 thousand years, followed by the arrival of subsequent groups within the past 10 thousand years. Here, we obtain new genome-wide data for 33 ancient individuals from high elevation sites on the southern fringe of the Tibetan Plateau in Nepal, who we show are most closely related to present-day Tibetans. They derive most of their ancestry from groups related to Late Neolithic populations at the northeastern edge of the Tibetan Plateau but also harbor a minor genetic component from a distinct and deep Paleolithic Eurasian ancestry. In contrast to their Tibetan neighbors, present-day non-Tibetan Tibeto-Burman speakers living at mid-elevations along the southern and eastern margins of the Plateau form a genetic cline that reflects a distinct genetic history. Finally, a comparison between ancient and present-day highlanders confirms ongoing positive selection of high altitude adaptive alleles.

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).

SummaryThe Eastern Eurasian Steppe was home to historic empires of nomadic pastoralists, including the Xiongnu and the Mongols. However, little is known about the region’s population history. Here we reveal its dynamic genetic history by analyzing new genome-wide data for 214 ancient individuals spanning 6,000 years. We identify a pastoralist expansion into Mongolia ca. 3000 BCE, and by the Late Bronze Age, Mongolian populations were biogeographically structured into three distinct groups, all practicing dairy pastoralism regardless of ancestry. The Xiongnu emerged from the mixing of these populations and those from surrounding regions. By comparison, the Mongols exhibit much higher Eastern Eurasian ancestry, resembling present-day Mongolic-speaking populations. Our results illuminate the complex interplay between genetic, sociopolitical, and cultural changes on the Eastern Steppe.

Southern Italy is a key region for understanding the agricultural transition in the Mediterranean due to its central position. We present a genomic transect for 19 prehistoric Sicilians that covers the Early Mesolithic to Early Neolithic period. We find that the Early Mesolithic hunter-gatherers (HGs) are a highly drifted sister lineage to Early Holocene western European HGs, whereas a quarter of the Late Mesolithic HGs ancestry is related to HGs from eastern Europe and the Near East. This indicates substantial gene flow from (south-)eastern Europe between the Early and Late Mesolithic. The Early Neolithic farmers are genetically most similar to those from the Balkan and Greece, and carry only a maximum of ∼7% ancestry from Sicilian Mesolithic HGs. Ancestry changes match changes in dietary profile and material culture, except for two individuals who may provide tentative initial evidence that HGs adopted elements of farming in Sicily.One-sentence summaryGenome-wide and isotopic data from prehistoric Sicilians reveal a pre-farming connection to (south-) eastern Europe, and tentative initial evidence that hunter-gatherers adopted some Neolithic aspects prior to near-total replacement by early farmers.

Summary The Eastern Eurasian Steppe was home to historic empires of nomadic pastoralists, including the Xiongnu and the Mongols. However, little is known about the region’s population history. Here, we reveal its dynamic genetic history by analyzing new genome-wide data for 214 ancient individuals spanning 6,000 years. We identify a pastoralist expansion into Mongolia ca. 3000 BCE, and by the Late Bronze Age, Mongolian populations were biogeographically structured into three distinct groups, all practicing dairy pastoralism regardless of ancestry. The Xiongnu emerged from the mixing of these populations and those from surrounding regions. By comparison, the Mongols exhibit much higher eastern Eurasian ancestry, resembling present-day Mongolic-speaking populations. Our results illuminate the complex interplay between genetic, sociopolitical, and cultural changes on the Eastern Steppe., Graphical Abstract, Highlights • Genome-wide analysis of 214 ancient individuals from Mongolia and the Baikal region • Three genetically distinct dairy pastoralist groups in Late Bronze Age Mongolia • Xiongnu nomadic empire formed through mixing of distinct local and distant groups • No selection on the lactase persistence alleles despite 5,000 years of dairy culture, Ancient DNA from over 200 individuals inhabiting the Eastern Eurasian Steppe during the Bronze Age provides insights into the population history of the Mongols and Xiongnu.

This protocol converts partially completed double-stranded DNA libraries e.g. from: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx.doi.org/10.17504/protocols.io.bakricv6) into dual-indexed Illumina libraries ready for sequencing on an Illumina platform. Other variants of the above protocol can also be used. This protocol includes steps for indexing PCR, clean-up, and post-indexing qPCR. This protocol is modified after Kircher, M., Sawyer, S. & Meyer, M., 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic acids research, 40(1), p.e3. http://dx.doi.org/10.1093/nar/gkr771.

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).