Yersinia pestis, the etiologic agent of plague, is a bacterium associated with wild rodents and their fleas. Historically it was responsible for three pandemics: the Plague of Justinian in the 6century AD, which persisted until the 8century []; the renowned Black Death of the 14century [], with recurrent outbreaks until the 18century []; and the most recent 19century pandemic, in which Y. pestis spread worldwide [] and became endemic in several regions []. The discovery of molecular signatures of Y. pestis in prehistoric Eurasian individuals and two genomes from Southern Siberia suggest that Y. pestis caused some form of disease in humans prior to the first historically documented pandemic []. Here, we present six new European Y. pestis genomes spanning the Late Neolithic to the Bronze Age (LNBA; 4,800 to 3,700 calibrated years before present). This time period is characterized by major transformative cultural and social changes that led to cross-European networks of contact and exchange []. We show that all known LNBA strains form a single putatively extinct clade in the Y. pestis phylogeny. Interpreting our data within the context of recent ancient human genomic evidence that suggests an increase in human mobility during the LNBA, we propose a possible scenario for the early spread of Y. pestis: the pathogen may have entered Europe from Central Eurasia following an expansion of people from the steppe, persisted within Europe until the mid-Bronze Age, and moved back toward Central Eurasia in parallel with human populations.

The history of the plague and the research on the causative agent Yersinia pestis.

Furthermore, Y. pseudotuberculosis-specific regions that have been lost in Y. pestis were still present in the LNBA strains ( Data S1 , sheet 5). We also observed genome decay in the LNBA clade mostly affecting flagellin genes and membrane proteins ( Figure S2 Data S1 , sheet 5).

Urease D (ureD) plays an important role in flea transmission. ureD expression causes a toxic oral reaction killing 30%–40% [] of infected fleas. ureD is a pseudogene in Y. pestis due to a frameshift mutation []. Close inspection of this gene revealed that the frameshift is not present, indicating that this gene was functional in the LNBA strains and possibly making them as toxic to fleas as their ancestor Y. pseudotuberculosis.

Silencing and reactivation of urease in Yersinia pestis is determined by one G residue at a specific position in the ureD gene.

The only plasmid virulence factor missing in the LNBA strains is ymt ( Figures 1 B and S2 ). ymt codes for the Yersinia murine toxin, an important virulence factor in flea transmission []. Expression of ymt protects against toxic blood digestion byproducts and permits colonization of the flea midgut []. Other plasmid virulence factors such as pla and caf1, absent in Y. pseudotuberculosis, were already present in the LNBA Y. pestis strains.

Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector.

Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector.

The percent coverage was calculated for genes related to virulence, flea transmission, colonization, and dissemination ( Figure 1 B). The YpfΦ prophage [], integrated only into the chromosomes of the 1.ORI strains responsible for the third pandemic [], was absent in all LNBA genomes. Additionally, yapC, possibly involved in the adhesion to mammalian cells, autoagglutination, and biofilm formation [], was lost in the three youngest LNBA strains (1343UnTal85, Post6, and RISE505).

We identified 423 single-nucleotide polymorphisms (SNPs) on the LNBA branch ( Data S1 , sheets 1–4), including strain-specific and shared SNPs, of which 114 are synonymous and 202 are non-synonymous (see STAR Methods ). The LNBA genomes share five SNPs ( Data S1 , sheet 2).

The branching point of the LNBA genomes and all other strains represents the most recent common ancestor (MRCA) of all currently available Y. pestis genomes, which was “tip-dated” using BEAST [] to 6,078 years (95% highest posterior density interval: 5,036–7,494 years), in agreement with previous estimates []. The time to the MRCA of Y. pestis and Y. pseudotuberculosis strain IP32953 was estimated at 28,258 years (95% highest posterior density interval: 13,200–44,631 years). The maximum clade credibility tree ( Figure S4 A) supports the same topology as the methods described above, with high statistical support of the LNBA branching point.

To assess the phylogenetic position of the reconstructed genomes in comparison to modern and ancient Y. pestis genomes (see STAR Methods ), we computed neighbor joining ( Figure S3 A), maximum parsimony ( Figure S3 B), and maximum likelihood ( Figures 1 and S3 C) trees. Our samples form a clade with the previously reported RISE509 and RISE505 strains [], with a bootstrap support > 95% for all methods.

(B) Percent coverage of virulence factors located on the Yersinia pestis chromosome and plasmids, plotted in R using the ggplot2 package. The numbers represent specific genes: (1) ymt gene, (2) pla gene, (3) filamentous prophage YpfΦ, (4) Y. pestis-specific genes. Related to Figure S2 . See also Data S1

(A) Maximum-likelihood tree of all Yersinia pestis genomes, including 1,265 SNP positions with complete deletion. Nodes with support ≥ 95% are marked with an asterisk. The colors represent different branches in the Y. pestis phylogeny: branch 0 (black), branch 1 (red), branch 2 (green), branch 3 (blue), branch 4 (orange), and LNBA Y. pestis branch (purple). Y. pseudotuberculosis-specific SNPs were excluded from the tree for clarity of representation. In the light-colored boxes, discussed losses and gains of genomic regions and genes are indicated. Related to Figure S3

To authenticate the ancient origin of the bacterial genomes, we evaluated terminal deamination damage common to ancient DNA []. Our samples presented typical damage profiles similar to the corresponding associated human DNA ( Figure S1 ), and multi-strain infection was not observed ( Figure S1 ).

“Strong” positive individuals were shotgun sequenced to a depth of 379,155,741–1,529,935,532 reads. RK1001 and GEN72 were further enriched for Y. pestis DNA using in-solution capture (see STAR Methods ). After mapping to the reference genome (Y. pestis CO92, NC_003143.1 ), we reconstructed genomes for all six candidates with a mean coverage between 3- and 12-fold, with 86%–94% of the reference covered 1-fold ( Table 2 Figure S2 ). The reads were independently mapped to the three Y. pestis CO92 plasmids yielding mean coverages of 7- to 24-fold (pCD1), 3- to 14-fold (pMT1), and 18- to 43-fold (pPCP1; Table S1 Figure S2 ).

To evaluate whether an individual was potentially Y. pestis-positive, we calculated a score based on the number of specific reads mapping to Y. pestis compared to other Yersinia (see STAR Methods ). Individuals with a positive score were deemed potential candidates. Those with scores > 0.005 and reads mapping to all three Y. pestis plasmids were considered “strong” positives. We identified five “strong” candidates: one individual from Rasshevatskiy (RK1001; North Caucasus, Russia), one from Gyvakarai (Gyvakarai1; Lithuania), one from Kunila (Kunila II; Estonia), and two from Augsburg, Germany (Haunstetten, Unterer Talweg 85 Feature 1343 [1343UnTal85]; Haunstetten, Postillionstrasse Feature 6 [6Post]). One individual from Beli Manastir-Popova zemlja (GEN72; Croatia) did not pass the “strong” candidate threshold but was included by virtue of having the highest number of reads mapping to the Y. pestis chromosome and plasmids (chromosome = 993, pCD1 = 243, pMT1 = 111, pPCP1 = 22). For additional archaeological information, see Table 2 and STAR Methods

The radiocarbon dates were calibrated with Calib 7.1. calBP = calibrated years Before Present; cal BC = calibrated years Before Christ. All individuals were directly radiocarbon dated. See also Table S1

A total of 563 tooth and bone samples dating from the Late Neolithic to the Bronze Age (LNBA) from Russia (n = 122), Hungary and Croatia (n = 139), Lithuania (n = 27), Estonia (n = 45), Latvia (n = 10), and Germany (n = 220) were screened for Y. pestis by mapping reads ranging from 700,000 to 21,000,000 against a multi-fasta reference of 12 different Yersinia ( Table 1 ).

Genomes from the NCBI RefSeq/Nucleotide Database, Used in the Multi-species Reference Panel for Screening for Y. pestis aDNA

Table 1 Genomes from the NCBI RefSeq/Nucleotide Database, Used in the Multi-species Reference Panel for Screening for Y. pestis aDNA

Discussion

7 Rasmussen S.

Allentoft M.E.

Nielsen K.

Orlando L.

Sikora M.

Sjögren K.-G.

Pedersen A.G.

Schubert M.

Van Dam A.

Kapel C.M.O.

et al. Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. 7 Rasmussen S.

Allentoft M.E.

Nielsen K.

Orlando L.

Sikora M.

Sjögren K.-G.

Pedersen A.G.

Schubert M.

Van Dam A.

Kapel C.M.O.

et al. Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. 7 Rasmussen S.

Allentoft M.E.

Nielsen K.

Orlando L.

Sikora M.

Sjögren K.-G.

Pedersen A.G.

Schubert M.

Van Dam A.

Kapel C.M.O.

et al. Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. 23 Shimodaira H. Multiple comparisons of log-likelihoods and combining nonnested models with applications to phylogenetic tree selection. Figure 2 Map of Proposed Yersinia pestis Circulation throughout Eurasia Show full caption (A) Entrance of Y. pestis into Europe from Central Eurasia with the expansion of Yamnaya pastoralists around 4,800 years ago. (B) Circulation of Y. pestis to Southern Siberia from Europe. Only complete genomes are shown. The prehistoric genomes presented here are the first to reveal Y. pestis diversity in the European LNBA. This complements contemporary Y. pestis genomes from Bronze Age individuals recovered from Southern Siberia [] and offers higher resolution to evaluate the evolution and dissemination of prehistoric plague strains. All LNBA genomes, including those previously reconstructed from Southern Siberia [], form a distinct clade. The strains RISE509 [] and RK1001 occupy the most basal position of all Y. pestis genomes sequenced to date, formally tested with CONSEL [] ( Figure S4 B). These data are compatible with two scenarios. In scenario 1, plague was introduced multiple times to Europe from a common reservoir between 5,000 and 3,000 BP. The bacterium spread to Europe from a source most likely located in Central Eurasia at least four times during a period of over 1,000 years: once to Lithuania and Croatia, once to Estonia, and twice to Germany. In this model, the phylogeny of the LNBA lineages results exclusively from their temporal relationship. In scenario 2, plague entered Europe from Central Eurasia once during the Neolithic. A reservoir was established within or close to Europe from which it circulated, and ultimately it moved back to Central Eurasia during the Bronze Age ( Figure 2 ).

24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 25 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. With few genomes available, it is difficult to disentangle the two hypotheses; however, interpreting our data in the context of human genetics and archaeological data can offer some resolution. Ancient human genomic data point to a change in mobility and large-scale expansion of people from the Caspian-Pontic Steppe associated with the Yamnaya complex, both east and west starting around 4,800 BP. These people carried a distinct genetic component that is also seen in highly mobile groups associated with the Southern Siberian Afanasievo complex, the Yamnaya complex, and the Central and Eastern European Corded Ware complex []. In Central European individuals, it is first observed in the Corded Ware complex and then becomes part of the genetic composition of most subsequent and all modern-day European populations [].

24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 25 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 10 Mathieson I.

Roodenberg S.A.

Posth C.

Szécsényi-Nagy A.

Rohland N.

Mallick S.

Olade I.

Broomandkhoshbacht N.

Cheronet O.

Fernandes D.

et al. The Genomic History Of Southeastern Europe. 11 Mittnik A.

Wang C.-C.

Pfrengle S.

Daubaras M.

Zariņa G.

Hallgren F.

Allmäe R.

Khartanovich V.

Moiseyev V.

Furtwängler A.

et al. The Genetic History of Northern Europe. 24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 26 Tebelškis P.

Jankauskas R. The Late Neolithic grave at Gyvakarai in Lithuania in the context of current archaeological and anthropological knowledge. Our earliest indication of plague in Europe is found in Croatia and the Baltic, coinciding with the arrival of “steppe ancestry” [] in human populations. The Baltic Late Neolithic Y. pestis genomes (Gyvakarai1 and KunilaII) were reconstructed from individuals associated with the Corded Ware complex. Along with the Croatian Y. pestis genome (Vučedol complex), these are derived from a common ancestor shared with the Yamnaya-derived RK1001 and Afanasievo-derived RISE509. This supports the notion of the pathogen spreading in the context of the large-scale expansion of steppe peoples from Central Eurasia to Eastern and Central Europe. Furthermore, human genomic analyses indicate that RISE509, Gyvakarai1, KunilaII, and GEN72 carry “steppe ancestry” []. Evidence for such long-distance contact is also present in the archaeological record. For example, the Gyvakarai1 burial is characterized by a specific inventory of grave items (e.g. hammer-headed pins) and distinct skeletal morphology that have no analogs in earlier local populations [].

24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 25 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 27 Olalde I.

Brace S.

Allentoft M.E.

Armit I.

Kristiansen K.

Rohland N.

Mallick S.

Booth T.

Szécsényi-Nagy A.

Mittnik A.

et al. The Beaker Phenomenon and the Genomic Transformation of Northwest Europe. 24 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. 28 Kuzmina E.E. The Prehistory of the Silk Road. 29 Koryakova L.

Epimakhov A.V. The Urals and Western Siberia in the Bronze and Iron Ages. 8 Vandkilde H. Bronzization: the Bronze Age as pre-modern globalization. 9 Hansen S. The 4th Millennium: a watershed in European prehistory. The younger Late Neolithic Y. pestis genomes from southern Germany are derived from the Baltic strains, and one of these is found in an individual associated with the Bell Beaker complex. Previous analyses have shown that Bell Beaker individuals from Germany also carry “steppe ancestry” []. This suggests that Y. pestis may have spread further southwest, analogous to the human “steppe” component. The youngest of the LNBA Y. pestis genomes (RISE505, Southern Siberia) associated with the Central Eurasian Andronovo complex, descends from the Central European strains, suggesting a spread into Southern Siberia. Interestingly, genome-wide human data show that individuals associated with the Sintashta, Srubnaya, and Andronovo cultural complexes in the Eurasian steppes (dating around 3,700–3,300 calibrated years before present) carried mixed ancestry of middle Neolithic European farmers and Bronze Age steppe people, suggesting a backflow of human genes from Europe to Central Eurasia []. Archaeologically, there seems to be a close connection between the Eastern European Abashevo cultural complex and Sintashta that might have included population shifts from west to east. In particular, the post-Sintashta Andronovo complex is an epoch of population shifts affecting all areas east of the Urals to the western borders of China, including populations with European origin []. The steppe, a natural corridor connecting people and their livestock throughout Central and Western Eurasia, might have facilitated the spread of strains related to the European Early Bronze Age Y. pestis to Southern Siberia, where RISE505 was found. In our view, human genetic ancestry and admixture, in combination with the temporal series within the LNBA Y. pestis branch, supports scenario 2. Y. pestis was possibly introduced to Europe from the steppe around 4,800 BP. Thereafter, a local reservoir was established within or in close proximity to Europe. The European Y. pestis strain was disseminated to Southern Siberia potentially through anthropogenic processes connected to the backflow of human genetic ancestry from Western Eurasia into Southern Siberia. The pathogen diversity mirrors the archaeological evidence, which indicates intensification of Eurasian trade networks from the beginning of the Bronze Age [].

30 Hinnebusch B.J. Biofilm-dependent and biofilm-independent mechanisms of transmission of Yersinia pestis by fleas. 31 Hinnebusch B.J.

Fischer E.R.

Schwan T.G. Evaluation of the role of the Yersinia pestis plasminogen activator and other plasmid-encoded factors in temperature-dependent blockage of the flea. 32 Eisen R.J.

Bearden S.W.

Wilder A.P.

Montenieri J.A.

Antolin M.F.

Gage K.L. Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. 31 Hinnebusch B.J.

Fischer E.R.

Schwan T.G. Evaluation of the role of the Yersinia pestis plasminogen activator and other plasmid-encoded factors in temperature-dependent blockage of the flea. 33 Chouikha I.

Hinnebusch B.J. Yersinia--flea interactions and the evolution of the arthropod-borne transmission route of plague. 34 Sun Y.-C.

Jarrett C.O.

Bosio C.F.

Hinnebusch B.J. Retracing the evolutionary path that led to flea-borne transmission of Yersinia pestis. 32 Eisen R.J.

Bearden S.W.

Wilder A.P.

Montenieri J.A.

Antolin M.F.

Gage K.L. Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. 35 Eisen R.J.

Dennis D.T.

Gage K.L. The Role of Early-Phase Transmission in the Spread of Yersinia pestis. 36 Vetter S.M.

Eisen R.J.

Schotthoefer A.M.

Montenieri J.A.

Holmes J.L.

Bobrov A.G.

Bearden S.W.

Perry R.D.

Gage K.L. Biofilm formation is not required for early-phase transmission of Yersinia pestis. 37 Johnson T.L.

Hinnebusch B.J.

Boegler K.A.

Graham C.B.

MacMillan K.

Montenieri J.A.

Bearden S.W.

Gage K.L.

Eisen R.J. Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae). Even though Y. pestis seems to have spread in patterns strikingly similar to human movements ( Figure 2 ), the mode of transmission during this early phase of its evolution cannot be easily determined. Most contemporary cases of Y. pestis infection occur via a flea vector and stem from sylvatic rodent populations with resistance to the bacterium. Flea transmission is accomplished by one of two mechanisms []: blockage-dependent transmission [] or early-phase transmission (EPT) []. In the former, Y. pestis obstructs the flea digestive system by producing a biofilm that blocks the flea’s foregut within 1–2 weeks post-infection. This blockage prevents a blood meal from reaching the flea’s midgut, and blood regurgitation during failed feeding sheds live bacteria into the host []. The blockage-dependent transmission requires a functional ymt gene and hms locus, and non-functional rcsA, pde2, and pde3 genes []. ymt protects Y. pestis within the digestive system of the flea, allowing colonization of the flea midgut. The hms locus is involved in biofilm formation, and rcsA, pde2, and pde3 are biofilm downregulators. However, Y. pestis can be transmitted within the first 1–4 days after entering the flea prior to colonization of the midgut and biofilm formation [] (the EPT model). This model is currently less understood than blockage-dependent transmission but has been shown to be both biofilm [] and ymt independent [].

7 Rasmussen S.

Allentoft M.E.

Nielsen K.

Orlando L.

Sikora M.

Sjögren K.-G.

Pedersen A.G.

Schubert M.

Van Dam A.

Kapel C.M.O.

et al. Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago. The genetic characteristics of the LNBA genomes (i.e., lack of ymt, functional pde2, and rcsA) were previously interpreted as evidence that early forms of Y. pestis were unable to cause blockage in the flea gut, thus suggesting that the bubonic form of the disease evolved later []. However, as none of these genes seem to be required for EPT, one cannot exclude that LNBA Y. pestis infections could have been acquired from fleas via this transmission mode. Under this model, transmission would have been less efficient since a functional UreD would have reduced the number of flea vectors by 30%–40%.

38 Lathem W.W.

Price P.A.

Miller V.L.

Goldman W.E. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. 39 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. 40 Zimbler D.L.

Schroeder J.A.

Eddy J.L.

Lathem W.W. Early emergence of Yersinia pestis as a severe respiratory pathogen. 38 Lathem W.W.

Price P.A.

Miller V.L.

Goldman W.E. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. 41 Ochman H.

Moran N.A. Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis. The presence of mammalian virulence-related genes such as pla and caf1 indicates that LNBA Y. pestis was to some extent adapted to these hosts. The LNBA pla presents the ancestral I259 variant, shown to be less efficient in infiltrating the host [] than the derived T259 form []. Strains carrying the ancestral variant can cause pneumonic disease but are less efficient in colonizing other tissues []. This indicates that LNBA Y. pestis could have caused a pneumonic or less severe bubonic form. The genome decay we detected, affecting membrane and flagellar proteins possibly involved in interactions with the host’s immune system, could indicate adaptation to new hosts or pathogenic lifestyles [].

42 Perry R.D.

Fetherston J.D. Yersinia pestis--etiologic agent of plague. 43 Barrett Ronald

Kuzawa Christopher W.

McDade Thomas

Armelagos G.J. Emerging and re-emerging infectious diseases: the third epidemiologic transition. 44 Ryabogina N.E.

Ivanov S.N. Ancient agriculture in Western Siberia: problems of argumentation, paleoethnobotanic methods, and analysis of data. Modern plague is a rodent-adapted disease, in which commensal species such as Rattus rattus and their fleas play a central role as disease vectors for humans []. Although rodent-flea transmission is compatible with the genomic makeup of the LNBA strains, disease dynamics may have differed in the past. The Neolithic is considered to be a time period in which new diseases were introduced into human groups during transition from a mobile to sedentary lifestyle. Adoption of agriculture and increased population density are thought to have acted synergistically to change the disease landscape []. Whether commensal rodent populations were large enough to function as a plague reservoir during human migrations at this time is unknown. In Central Eurasian Bronze Age cultures, agriculture (i.e., large-scale food storage) was mostly absent []. However, contact between sylvatic rodents in the steppe, pastoralists, and their herds might have been frequent in these environments. Alternative models of transmission involving different host species, perhaps even humans or their livestock, might carry some traction, as the ancient disease may have behaved differently from what we know today.