Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague

[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.Peer reviewe

Femur (anterior view) showing drilling sites for the collection of both cortical and cancellous material.

Femur (anterior view) showing drilling sites for the collection of both cortical and cancellous material.</p

Representative comparison of deamination patterns in reads from the KRA004 dental pulp chamber libraries mapping to both the HG19 human reference and <i>Yersinia pestis</i> CO92 reference genomes.

Representative comparison of deamination patterns in reads from the KRA004 dental pulp chamber libraries mapping to both the HG19 human reference and Yersinia pestis CO92 reference genomes.</p

S6 Fig -

Screenshots showing SNPs at positions (A) 699494, (B) 2262577, and (C) 1009185 for the low coverage genomes KRA008, KRA028, and KRA032. Reads shown here are untrimmed. (TIF)</p

Fig 2 -

(A) Historical Y. pestis diversity including the Second, and Third Pandemics. New high-coverage genomes from Krakauer Berg are denoted in blue. Strain and lineage acronyms follow conventions established in the published literature [5,14,65]. Bootstrap values above 90% are denoted with an asterisk, and numeric values of all lower percentages are shown. (B) Zoomed view of the Y. pestis diversity within pestis secunda (grey area in A). High coverage genomes are denoted in blue, whereas the tentative, manual placements of low-coverage genomes are denoted in orange. Lineage-characteristic SNPs (Table 1) are indicated in circles at their respective branches.</p

EAGER mapping to <i>Y</i>. <i>pestis</i> post capture.

Pestis secunda (1356–1366 CE) is the first of a series of plague outbreaks in Europe that followed the Black Death (1346–1353 CE). Collectively this period is called the Second Pandemic. From a genomic perspective, the majority of post-Black Death strains of Yersinia pestis thus far identified in Europe display diversity accumulated over a period of centuries that form a terminal sub-branch of the Y. pestis phylogeny. It has been debated if these strains arose from local evolution of Y. pestis or if the disease was repeatedly reintroduced from an external source. Plague lineages descended from the pestis secunda, however, are thought to have persisted in non-human reservoirs outside Europe, where they eventually gave rise to the Third Pandemic (19th and 20th centuries). Resolution of competing hypotheses on the origins of the many post-Black Death outbreaks has been hindered in part by the low representation of Y. pestis genomes in archaeological specimens, especially for the pestis secunda. Here we report on five individuals from Germany that were infected with lineages of plague associated with the pestis secunda. For the two genomes of high coverage, one groups within the known diversity of genotypes associated with the pestis secunda, while the second carries an ancestral genotype that places it earlier. Through consideration of historical sources that explore first documentation of the pandemic in today’s Central Germany, we argue that these data provide robust evidence to support a post-Black Death evolution of the pathogen within Europe rather than a re-introduction from outside. Additionally, we demonstrate retrievability of Y. pestis DNA in post-cranial remains and highlight the importance of hypothesis-free pathogen screening approaches in evaluations of archaeological samples.</div

SNPs for ancient branch 1 genomes, limited to those that define diversity within the pestis secunda.

SNPs for ancient branch 1 genomes, limited to those that define diversity within the pestis secunda.</p

Gene annotation and effects of SNPs that define diversity in the pestis secunda.

Gene annotation and effects of SNPs that define diversity in the pestis secunda.</p

EAGER mapping results for shotgun sequencing prior to <i>Y</i>. <i>pestis</i> capture.

EAGER mapping results for shotgun sequencing prior to Y. pestis capture.</p

SNP table showing the seven diagnostic Second Pandemic <i>Y</i>. <i>pestis</i> SNPs (p1-p7 [25]) and additional <i>pestis secunda</i> diversity comparing profiles of the Black Death genotype, all Krakauer Berg genomes (KRA0—), known examples of <i>pestis secunda</i> (Bergen op Zoom, London St. Mary’s Graces, Bolgar), and the modern CO92 reference (accession number: AL590842).

Amongst the two high-coverage genomes (blue), a minimum of 3 reads covering each position were required for SNP identification, while SNPs in the low-coverage genomes (orange) were evaluated at minimum 1-fold coverage, with number of reads supporting the SNP shown in parentheses. Sites where this coverage was not achieved (for both high or low-coverage genomes) are denoted with Ns. Nucleotides diagnostic of pestis secunda/Branch 1 strains are shown in bold.</p