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.

Archaeological Centre 779 00 Olomouc Czech Republic

Archeolodzy org Foundation 50316 Wrocław Poland

Begazy Tasmola Research Center of History and Archeology 050008 Almaty Kazakhstan

Biology and Biotechnology Faculty Al Farabi Kazakh National University 050040 Almaty Kazakhstan

BIOMICs Research Group University of the Basque Country Universidad del Pais Vasco Euskal Herriko Unibertsitatea 01006 Vitoria Gasteiz Spain

Centre for Egyptological Studies of the Russian Academy of Sciences Russian Academy of Sciences 119991 Moscow Russian Federation

Curt Engelhorn Center Archaeometry 68159 Mannheim Germany

Department of Anthropology Harvard University Cambridge MA 02138

Department of Anthropology National Museum of Natural History Smithsonian Institution Washington DC 20560

Department of Anthropology University of Auckland 01010 Auckland New Zealand

Department of Archaeogenetics Max Planck Institute for Evolutionary Anthropology 04103 Leipzig Germany

Department of Archaeogenetics Max Planck Institute for the Science of Human History 07745 Jena Germany

Department of Evolutionary Anthropology University of Vienna 1030 Vienna Austria

Department of Genetics Harvard Medical School Boston MA 02115

Department of Geography Prehistory and Archaeology University of the Basque Country Vitoria Gasteiz 01006 Spain

Department of Heritage Management Archaeological Heritage Office Saxony 01108 Dresden Germany

Department of Human Evolutionary Biology Harvard University Cambridge MA 02138

Department of Organismic and Evolutionary Biology Harvard University Cambridge MA 02138

Department of Prehistoric Archaeology Institute of Archaeology Czech Academy of Sciences 11801 Prague Czech Republic

Eurasia Department German Archaeological Institute 14195 Berlin Germany

Evolutionary Pathogenomics Max Planck Institute for Infection Biology 10117 Berlin Germany

Faculty of Biological Sciences Friedrich Schiller University 07743 Jena Germany

Faculty of Mathematics and Computer Science Friedrich Schiller University 07743 Jena Germany

History Department Al Farabi Kazakh National University 050040 Almaty Kazakhstan

Institute for Archaeological Sciences Eberhard Karls University of Tübingen 72074 Tübingen Germany

Institute for Pre and Protohistoric Archaeology and Archaeology of the Roman Provinces Ludwig Maximilian University Munich 80539 Munich Germany

Institute of Archaeology University of Wrocław 50139 Wrocław Poland

Institute of Ethnology and Anthropology Russian Academy of Science 119991 Moscow Russian Federation

Institute of Evolutionary Biology Consejo Superior de Investigaciones Cientificas Universitat Pompeu Fabra 08003 Barcelona Spain

Institute of Fundamental Medicine and Biology Kazan Federal University Kazan 420008 Russian Federation

Institute of Genetics and Physiology Al Farabi Kazakh National University Almaty 050060 Kazakhstan

Laboratory for Structural Analysis of Biomacromolecules Federal Research Center Kazan Scientific Center of the Russian Academy of Sciences 420111 Kazan Russian Federation

Nasledie Cultural Heritage Unit 355006 Stavropol Russian Federation

Research Institute and Museum of Anthropology Lomonosov Moscow State University 125009 Moscow Russian Federation

Research Laboratory of Paleoanthropological Study Institute of Archaeology named after A Kh Margulan Almaty 50010 Kazakhstan

Transmission Infection Diversification and Evolution Group Max Planck Institute for the Science of Human History 07745 Jena Germany

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Proc Natl Acad Sci U S A. 2022 May 24;119(21):e2204044119. doi: 10.1073/pnas.2204044119. PubMed

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Rasmussen S., et al. , Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. Cell 163, 571–582 (2015). PubMed PMC

Rascovan N., et al. , Emergence and spread of basal lineages of Yersinia pestis during the Neolithic decline. Cell 176, 295–305.e10 (2019). PubMed

Andrades Valtueña A., et al. , The stone age plague and its persistence in Eurasia. Curr. Biol. 27, 3683–3691.e8 (2017). PubMed

Susat J., et al. , A 5,000-year-old hunter-gatherer already plagued by Yersinia pestis. Cell Rep. 35, 109278 (2021). PubMed

Stenseth N. C., et al. , Plague dynamics are driven by climate variation. Proc. Natl. Acad. Sci. U.S.A. 103, 13110–13115 (2006). PubMed PMC

Gage K. L., Kosoy M. Y., Natural history of plague: Perspectives from more than a century of research. Annu. Rev. Entomol. 50, 505–528 (2005). PubMed

Hinnebusch B. J., Erickson D. L., Yersinia pestis biofilm in the flea vector and its role in the transmission of plague. Curr. Top. Microbiol. Immunol. 322, 229–248 (2008). PubMed PMC

Eisen R. J., et al. , Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. Proc. Natl. Acad. Sci. U.S.A. 103, 15380–15385 (2006). PubMed PMC

Vetter S. M., et al. , Biofilm formation is not required for early-phase transmission of Yersinia pestis. Microbiology (Reading) 156, 2216–2225 (2010). PubMed PMC

Spyrou M. A., et al. , Analysis of 3800-year-old Yersinia pestis genomes suggests Bronze Age origin for bubonic plague. Nat. Commun. 9, 2234 (2018). PubMed PMC

Yu H., et al. , Paleolithic to Bronze Age Siberians reveal connections with first Americans and across Eurasia. Cell 181, 1232–1245.e20 (2020). PubMed

Begier E. M., et al. , Pneumonic plague cluster, Uganda, 2004. Emerg. Infect. Dis. 12, 460–467 (2006). PubMed PMC

Bertherat E., et al. , Lessons learned about pneumonic plague diagnosis from two outbreaks, Democratic Republic of the Congo. Emerg. Infect. Dis. 17, 778–784 (2011). PubMed PMC

Lien-Teh W., Chun J. W. H., Pollitzer R., Wu C. Y., Plague: A Manual for Medical and Public Health Workers (Weishengshu, Shanghai Station, 1936).

Ratsitorahina M., Chanteau S., Rahalison L., Ratsifasoamanana L., Boisier P., Epidemiological and diagnostic aspects of the outbreak of pneumonic plague in Madagascar. Lancet 355, 111–113 (2000). PubMed

Richard V., et al. , Pneumonic plague outbreak, Northern Madagascar, 2011. Emerg. Infect. Dis. 21, 8–15 (2015). PubMed PMC

Gamsa M., The epidemic of pneumonic plague in Manchuria 1910–1911. Past Present 190, 147–183 (2006).

Arbaji A., et al. , A 12-case outbreak of pharyngeal plague following the consumption of camel meat, in north-eastern Jordan. Ann. Trop. Med. Parasitol. 99, 789–793 (2005). PubMed

Kehrmann J., et al. , Two fatal cases of plague after consumption of raw marmot organs. Emerg. Microbes Infect. 9, 1878–1880 (2020). PubMed PMC

Malek M. A., Bitam I., Drancourt M., Plague in Arab Maghreb, 1940-2015: A Review. Front. Public Health 4, 112 (2016). PubMed PMC

Christie A. B., Chen T. H., Elberg S. S., Plague in camels and goats: Their role in human epidemics. J. Infect. Dis. 141, 724–726 (1980). PubMed

Bin Saeed A. A., Al-Hamdan N. A., Fontaine R. E., Plague from eating raw camel liver. Emerg. Infect. Dis. 11, 1456–1457 (2005). PubMed PMC

Klimscha F., Transforming technical know-how in time and space. Using the digital atlas of innovations to understand the innovation process of animal traction and the wheel. eTopoi, Journal for Ancient Studies 6, 16–63 (2017).

Librado P., et al. , The origins and spread of domestic horses from the Western Eurasian steppes. Nature 598, 634–640 (2021). PubMed PMC

Hansen S., “The 4th millennium: A watershed in European prehistory” in Western Anatolia Before Troy. Proto-Urbanisation in the 4th Millennium BC? Horejs B., Mehofer M., Eds. (Academy of Science Press, 2014), pp. 243–259.

Anthony D. W., The Horse, the Wheel, and Language: How Bronze-Age Riders from Eurasian Steppes Shaped the Modern World (Princeton University Press, 2007).

Frachetti M. D., Multiregional emergence of mobile pastoralism and nonuniform institutional complexity across Eurasia. Curr. Anthropol. 53, 2–38 (2012).

Hübler R., et al. , HOPS: Automated detection and authentication of pathogen DNA in archaeological remains. Genome Biol. 20, 280 (2019). PubMed PMC

Huson D. H., et al. , MEGAN community edition–Interactive exploration and analysis of large-scale microbiome sequencing data. PLOS Comput. Biol. 12, e1004957 (2016). PubMed PMC

Feldman M., et al. , A high-coverage Yersinia pestis genome from a sixth-century Justinianic Plague victim. Mol. Biol. Evol. 33, 2911–2923 (2016). PubMed PMC

Keller M., et al. , Ancient Yersinia pestis genomes from across Western Europe reveal early diversification during the First Pandemic (541–750). Proc. Natl. Acad. Sci. U.S.A. 116, 12363–12372 (2019). PubMed PMC

Bos K. I., et al. , A draft genome of Yersinia pestis from victims of the Black Death. Nature 478, 506–510 (2011). PubMed PMC

Bos K. I., et al. , Eighteenth century Yersinia pestis genomes reveal the long-term persistence of an historical plague focus. eLife 5, e12994 (2016). PubMed PMC

Spyrou M. A., et al. , Historical Y. pestis genomes reveal the European Black Death as the source of ancient and modern plague pandemics. Cell Host Microbe 19, 874–881 (2016). PubMed

Spyrou M. A., et al. , Phylogeography of the second plague pandemic revealed through analysis of historical Yersinia pestis genomes. Nat. Commun. 10, 4470 (2019). PubMed PMC

Bouckaert R., et al. , BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLOS Comput. Biol. 15, e1006650 (2019). PubMed PMC

Lillie M., Budd C., Potekhina I., Hedges R., The radiocarbon reservoir effect: New evidence from the cemeteries of the middle and lower Dnieper basin, Ukraine. J. Archaeol. Sci. 36, 256–264 (2009).

Lillie M., Budd C., Potekhina I., Stable isotope analysis of prehistoric populations from the cemeteries of the Middle and Lower Dnieper Basin, Ukraine. J. Archaeol. Sci. 38, 57–68 (2010).

Wickham H., ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, NY, 2009).

Kahle D., Wickham H., ggmap: Spatial visualization with ggplot2. R J. 5, 144–161 (2013).

Rudis B., Bolker B., Schulz J., ggalt: Extra Coordinate Systems, “Geoms”, Statistical Transformations, Scales and Fonts for “ggplot2” (2017). https://CRAN.R-project.org/package=ggalt. Accessed 15 February 2017.

Kassambara A., ggpubr: “ggplot2” Based Publication Ready Plots (2020). https://github.com/kassambara/ggpubr. Accessed 27 June 2020.

R Development Core Team, R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2008).

Inkscape Project, Inkscape (2020). https://inkscape.org/news/2020/05/04/introducing-inkscape-10/. Accessed 23 November 2020.

Bos K. I., et al. , Paleomicrobiology: Diagnosis and evolution of ancient pathogens. Annu. Rev. Microbiol. 73, 639–666 (2019). PubMed

Derbise A., Carniel E., YpfΦ: A filamentous phage acquired by Yersinia pestis. Front. Microbiol. 5, 701 (2014). PubMed PMC

Hinnebusch B. J., et al. , Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector. Science 296, 733–735 (2002). PubMed

Pradel E., et al. , New insights into how Yersinia pestis adapts to its mammalian host during bubonic plague. PLoS Pathog. 10, e1004029 (2014). PubMed PMC

Yang X., Pan J., Wang Y., Shen X., Type VI secretion systems present new insights on pathogenic Yersinia. Front. Cell. Infect. Microbiol. 8, 260 (2018). PubMed PMC

Ponnusamy D., et al. , High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection. Infect. Immun. 83, 2065–2081 (2015). PubMed PMC

Liang Y., et al. , Chromosomal rearrangement features of Yersinia pestis strains from natural plague foci in China. Am. J. Trop. Med. Hyg. 91, 722–728 (2014). PubMed PMC

Cingolani P., et al. , A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012). PubMed PMC

Minnich S. A., Rohde H. N., “A rationale for repression and/or loss of motility by pathogenic Yersinia in the mammalian host” in The Genus Yersinia: From Genomics to Function, Perry R. D., Fetherston J. D., Eds. (Advances In Experimental Medicine And Biology, Springer, 2007), pp. 298–311. PubMed

Key F. M., et al. , Emergence of human-adapted Salmonella enterica is linked to the Neolithization process. Nat. Ecol. Evol. 4, 324–333 (2020). PubMed PMC

Zhou Z., et al. , Pan-genome analysis of ancient and modern Salmonella enterica demonstrates genomic stability of the invasive para C lineage for millennia. Curr. Biol. 28, 2420–2428.e10 (2018). PubMed PMC

Vågene Å. J., et al. , Salmonella enterica genomes from victims of a major sixteenth-century epidemic in Mexico. Nat. Ecol. Evol. 2, 520–528 (2018). PubMed

Schuenemann V. J., et al. , Genome-wide comparison of medieval and modern Mycobacterium leprae. Science 341, 179–183 (2013). PubMed

Mendum T. A., et al. , Mycobacterium leprae genomes from a British medieval leprosy hospital: Towards understanding an ancient epidemic. BMC Genomics 15, 270 (2014). PubMed PMC

Schuenemann V. J., et al. , Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe. PLoS Pathog. 14, e1006997 (2018). PubMed PMC

Guellil M., et al. , A genomic and historical synthesis of plague in 18th century Eurasia. Proc. Natl. Acad. Sci. U.S.A. 117, 28328–28335 (2020). PubMed PMC

Sun Y.-C., Hinnebusch B. J., Darby C., Experimental evidence for negative selection in the evolution of a Yersinia pestis pseudogene. Proc. Natl. Acad. Sci. U.S.A. 105, 8097–8101 (2008). PubMed PMC

Chouikha I., Hinnebusch B. J., Silencing urease: A key evolutionary step that facilitated the adaptation of Yersinia pestis to the flea-borne transmission route. Proc. Natl. Acad. Sci. U.S.A. 111, 18709–18714 (2014). PubMed PMC

Stenseth N. C., et al. , Plague: Past, present, and future. PLoS Med. 5, e3 (2008). PubMed PMC

Gage K. L., Montenieri J. A., Thomas R. E., “The role of predators in the ecology, epidemiology, and surveillance of plague in the United States” in Proceedings of the Vertebrate Pest Conference 1994 (University of California, Davis, CA, 1994), pp. 200–206.

Mahmoudi A., et al. , Plague reservoir species throughout the world. Integr. Zool. 16, 820–833 (2021). PubMed

Allentoft M. E., et al. , Population genomics of Bronze Age Eurasia. Nature 522, 167–172 (2015). PubMed

Haak W., et al. , Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522, 207–211 (2015). PubMed PMC

Scott A., et al. , Emergence and intensification of dairying in the Caucasus and Eurasian steppes. Nat. Ecol. Evol. 10.1038/s41559-022-01701-6. PubMed DOI PMC

Wilkin S., et al. , Dairy pastoralism sustained eastern Eurasian steppe populations for 5,000 years. Nat. Ecol. Evol. 4, 346–355 (2020). PubMed PMC

Nyirenda S. S., et al. , Potential roles of pigs, small ruminants, rodents, and their flea vectors in plague epidemiology in Sinda District, eastern Zambia. J. Med. Entomol. 54, 719–725 (2017). PubMed

Dai R., et al. , Human plague associated with Tibetan sheep originates in marmots. PLoS Negl. Trop. Dis. 12, e0006635 (2018). PubMed PMC

Koskiniemi S., Sun S., Berg O. G., Andersson D. I., Selection-driven gene loss in bacteria. PLoS Genet. 8, e1002787 (2012). PubMed PMC

Ochman H., Moran N. A., Genes lost and genes found: Evolution of bacterial pathogenesis and symbiosis. Science 292, 1096–1099 (2001). PubMed

Sheppard S. K., Guttman D. S., Fitzgerald J. R., Population genomics of bacterial host adaptation. Nat. Rev. Genet. 19, 549–565 (2018). PubMed

Johnson T. L., et al. , Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae). Microbiology (Reading) 160, 2517–2525 (2014). PubMed PMC

Eisen R. J., Dennis D. T., Gage K. L., The role of early-phase transmission in the spread of Yersinia pestis. J. Med. Entomol. 52, 1183–1192 (2015). PubMed PMC

Bland D. M., Miarinjara A., Bosio C. F., Calarco J., Hinnebusch B. J., Acquisition of yersinia murine toxin enabled Yersinia pestis to expand the range of mammalian hosts that sustain flea-borne plague. PLoS Pathog. 17, e1009995 (2021). PubMed PMC

Sebbane F., Jarrett C. O., Gardner D., Long D., Hinnebusch B. J., Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proc. Natl. Acad. Sci. U.S.A. 103, 5526–5530 (2006). PubMed PMC

Zimbler D. L., Schroeder J. A., Eddy J. L., Lathem W. W., Early emergence of Yersinia pestis as a severe respiratory pathogen. Nat. Commun. 6, 7487 (2015). PubMed PMC

Wong D., et al. , Primary pneumonic plague contracted from a mountain lion carcass. Clin. Infect. Dis. 49, e33–e38 (2009). PubMed

Mathieson I., et al. , The genomic history of southeastern Europe. Nature 555, 197–203 (2018). PubMed PMC

Neumann G. U., Andrades Valtueña A., Fellows Yates J. A., Stahl R., Brandt G., Tooth sampling from the inner pulp chamber for ancient DNA extraction (protocols.io, 2020). 10.17504/protocols.io.bqebmtan. Accessed 24 March 2021. DOI

Velsko I., Skourtanioti E., Brandt G., Ancient DNA extraction from skeletal material (protocols.io, 2020). 10.17504/protocols.io.baksicwe. Accessed 30 October 2020. DOI

Dabney J., et al. , Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl. Acad. Sci. U.S.A. 110, 15758–15763 (2013). PubMed PMC

Meyer M., Kircher M., Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. 2010, pdb.prot5448 (2010). PubMed

Rohland N., Harney E., Mallick S., Nordenfelt S., Reich D., Partial uracil-DNA-glycosylase treatment for screening of ancient DNA. Philos. Trans. R. Soc. Lond. B Biol. Sci. 370, 20130624 (2015). PubMed PMC

Aron F., Neumann G. U., Brandt G., Half-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (protocols.io, 2020). 10.17504/protocols.io.bmh6k39e. Accessed 24 March 2021. DOI

Olalde I., et al. , The genomic history of the Iberian Peninsula over the past 8000 years. Science 363, 1230–1234 (2019). PubMed PMC

Gansauge M.-T., Aximu-Petri A., Nagel S., Meyer M., Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nat. Protoc. 15, 2279–2300 (2020). PubMed

Fellows Yates J. A., et al. , Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager. PeerJ 9, e10947 (2021). PubMed PMC

Schubert M., Lindgreen S., Orlando L., AdapterRemoval v2: Rapid adapter trimming, identification, and read merging. BMC Res. Notes 9, 88 (2016). PubMed PMC

Li H., Durbin R., Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009). PubMed PMC

Broad Institute, Picard Tools (March 12, 2020). https://broadinstitute.github.io/picard/. Accessed 12 March 2020.

McKenna A., et al. , The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010). PubMed PMC

Bos K. I., et al. , Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis. Nature 514, 494–497 (2014). PubMed PMC

Quinlan A. R., Hall I. M., BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). PubMed PMC

Paradis E., Schliep K., ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics 35, 526–528 (2019). PubMed

Hijmans R. J., geosphere: Spherical trigonometry (2019). https://CRAN.R-project.org/package=geosphere 2019. Accessed 26 May 2019.

Oksanen J., et al. , vegan: Community ecology package (2020). https://cran.r-project.org/web/packages/vegan/index.html. Accessed 28 November 2020.

A. A. Valtueña, M. A. Spyrou, G. U. Neumann, LNBAplague (2022). GitHub. https://github.com/aidaanva/LNBAplague. Deposited 21 October 2020.

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