Southern East Asian origin and coexpansion ofMycobacterium tuberculosis Beijing family withHan ChineseTao Luoa,b, Inaki Comasc,d,1, Dan Luoe,1, Bing Luf,g,1, Jie Wuh,1, Lanhai Weii,1, Chongguang Yanga, Qingyun Liua,Mingyu Gana, Gang Suna, Xin Shenh, Feiying Liue, Sebastien Gagneuxj,2, Jian Meih,2, Rushu Lane,2, Kanglin Wanf,k,2,and Qian Gaoa,2

aKey Laboratory of Medical Molecular Virology of Ministries of Education and Health, Institutes of Biomedical Sciences and Institute of MedicalMicrobiology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; bLaboratory of Infection and Immunity, School of Basic MedicalScience, West China Center of Medical Sciences, Sichuan University, Chengdu, Sichuan 610041, China; cGenomics and Health Unit, FISABIO Public Health,Valencia 46020, Spain; dCIBER (Centros de Investigación Biomédica en Red) in Epidemiology and Public Health, Instituto de Salud Carlos III, Madrid 28029,Spain; eDepartment of Tuberculosis Control, Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention, Nanning, Guangxi 530028,China; fState Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, ChineseCenter for Disease Control and Prevention, Beijing 102206, China; gBeijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning,Beijing Center for Disease Control and Prevention, Beijing 100013, China; hDepartment of Tuberculosis Control, Shanghai Municipal Center for DiseaseControl and Prevention, Shanghai 200336, China; iMinistry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences andInstitutes of Biomedical Sciences, Fudan University, Shanghai 200433, China; jDepartment of Medical Parasitology and Infection Biology, Swiss Tropical andPublic Health Institute, Basel 4002, University of Basel, Basel CH-4003, Switzerland; and kCollaborative Innovation Center for Diagnosis and Treatment ofInfectious Diseases, Hangzhou 310003, China

Edited by William R. Jacobs Jr., Howard Hughes Medical Institute, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, and approved May 14,2015 (received for review December 23, 2014)

The Beijing family is the most successful genotype of Mycobacteriumtuberculosis and responsible for more than a quarter of the globaltuberculosis epidemic. As the predominant genotype in East Asia, theBeijing family has been emerging in various areas of the world and isoften associated with disease outbreaks and antibiotic resistance. Re-vealing the origin and historical dissemination of this strain family isimportant for understanding its current global success. Here we char-acterized the global diversity of this family based on whole-genomesequences of 358 Beijing strains. We show that the Beijing strainsendemic in East Asia are genetically diverse, whereas the globallyemerging strains mostly belong to a more homogenous subtypeknown as “modern” Beijing. Phylogeographic and coalescent analy-ses indicate that the Beijing family most likely emerged around30,000 y ago in southern East Asia, and accompanied the early colo-nization by modern humans in this area. By combining the genomicdata and genotyping result of 1,793 strains from across China, wefound the “modern” Beijing sublineage experienced massive expan-sions in northern China during the Neolithic era and subsequentlyspread to other regions following the migration of Han Chinese.Our results support a parallel evolution of the Beijing family andmodern humans in East Asia. The dominance of the “modern” Beijingsublineage in East Asia and its recent global emergence are mostlikely driven by its hypervirulence, which might reflect adaption toincreased human population densities linked to the agricultural tran-sition in northern China.

MTBC Beijing family | origin | expansion | Han Chinese

Tuberculosis has plagued human beings since ancient times andremains a leading cause of global morbidity and mortality. The

causative agent of human tuberculosis is the Mycobacterium tuber-culosis complex (MTBC), a group of organisms that harbor littlegenetic diversity compared with other bacteria (1). MTBC mostlikely originated in Africa, although its age is being debated (2–4).The human-adapted MTBC is highly clonal and is classified intoseven main phylogenetic groups, designated lineage 1 throughlineage 7 (2). These seven lineages show strong biogeographic as-sociations that have been proposed to result from codiversificationwith different human populations (2, 5). Lineage 2 that dominatesin East Asia is one of the most successful MTBC variants; morethan a quarter of the global tuberculosis epidemic is caused by thislineage (6, 7). Lineage 2 contains strains that mostly belong to theso-called Beijing family (8, 9). This strain family has attracted

great attentions due to its global emergence in recent decades(6, 7, 10–12), its tendency to cause disease outbreak (13–17), andits association with antibiotic resistance (12, 18). Experimental andclinical evidences suggest a hypervirulent phenotype of Beijingstrains (12, 19), and a higher mutation rate compared with otherstrains (20).According to genotyping data from previous molecular-

epidemiology studies, most Beijing strains from widespread geo-graphic areas showed a remarkable degree of genetic similarity(6, 21), suggesting this strain family might have emerged fromrecent expansions. It was hypothesized that vaccination withBacille Calmette Guerin (bacillus Calmette–Guérin) that hasbeen widely implemented in East Asian countries might be theforce driving the dominance of this strain family in this area (21).Moreover, the global emergence of the Beijing family may have

Significance

Mycobacterium tuberculosis Beijing family is a group of glob-ally emerging bacterial strains that are responsible for morethan a quarter of the global tuberculosis epidemic. Here, wecombine whole-genome sequencing and large-scale genotyp-ing to map the temporal and spatial changes of the geneticdiversity within this strain family. We reveal a southern EastAsia origin and a parallel evolution of this bacterial genotypewith modern humans in East Asia during the last 30,000 years.The recently globally emerged Beijing strains mainly belong toa hypervirulent subtype that most likely has initially been se-lected for adaption to increased population densities duringthe agricultural transition in northern China.

Author contributions: T.L., F.L., S.G., J.M., R.L., K.W., and Q.G. designed research; T.L., D.L.,B.L., J.W., C.Y., Q.L., M.G., G.S., and X.S. performed research; T.L., I.C., and L.W. analyzeddata; and T.L., I.C., L.W., S.G., and Q.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences in this paper have been deposited in the Sequence ReadArchive database, www.ncbi.nlm.nih.gov/sra (accession no. SRP051093).1I.C., D.L., B.L., J.W., and L.W. contributed equally to this work.2To whom correspondence may be addressed. Email: qiangao@shmu.edu.cn, sebastien.gagneux@unibas.ch, meijiansh@aliyun.com, gxlrshu@163.com, or wankanglin@icdc.cn

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1424063112/-/DCSupplemental.

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been due to its hypervirulence and association with drug re-sistance (7, 18). However, there were discrepant results regardingthe relative protective effect of bacillus Calmette–Guérin vaccina-tion against Beijing strains from animal infection experiments (19),and many epidemiological studies failed to find any associationbetween bacillus Calmette–Guérin vaccination and Beijing strains(22–25). The link between drug resistance and the Beijing family hasprimarily been observed in regions where this family has emergedrecently (e.g., Cuba, South Africa, countries of the former SovietUnion) but not in East Asian, where the Beijing family has beenendemic for a long time (18, 26). Furthermore, more recent studiesindicate that the expansion of the Beijing family may have startedlong before the introduction of vaccination and antibiotic treatment(2, 3, 27).With the increased availability of genotyping data, the Beijing

strains were proved more heterogeneous than initially estimated,and several Beijing sublineages have been identified (28–31).However, a full understanding of the genetic diversity of Beijingfamily is constrained by the low amount of nucleotide variation(8, 32). Whole-genome sequencing provides an ideal tool tostudy the genetic diversity of MTBC, and new insights into theorigin and evolution of MTBC have been gained (2, 4, 20, 33–35). The genomic diversity of Beijing family was initially studiedin a most recent study, in which a general East Asian origin andrecent expansions of this strain family were suggested (36). However,the details about the origin and primary dissemination of Beijingfamily remain unclear. Answering of these questions is important tobetter understand the virulence of this lineage and its global success.Here, we combined whole-genome sequencing of key strains withdetailed single nucleotide polymorphism (SNP) typing of a largecollection of clinical MTBC strains isolated from across China. Ourresults strongly support a southern East Asian origin of the MTBCBeijing family and suggest a parallel evolution of this family withmodern humans in East Asia during the last 30,000 y.

ResultsThe Population Diversity of MTBC Lineage 2. We sequenced thewhole genome of 79 clinical strains representative of the diversity ofMTBC Beijing family in China in this study (40 strains) and aprevious study (39 strains; ref. 2) (SI Appendix, Fig. S1). In addition,we genome sequenced 16 non-Beijing lineage 2 strains identified inGuangxi province. These 95 genomes were combined with 263unique genomes (SI Appendix, Dataset S1 and SI Appendix, Fig. S2)of lineage 2 from 15 countries (Fig. 1A) published previously (ref-erences are appended in SI Appendix, SI References), which enabledus to analyze the diversity of MTBC lineage 2 at a global scale.After excluding the repetitive, mobile elements and drug-resistanceassociated genes, we identified 24,592 SNPs, which we used toconstruct a phylogenetic tree of MTBC lineage 2 (Fig. 1B). We alsomapped the well characterized lineage 2 and Beijing family poly-morphic markers onto the phylogeny. Consistent with previous re-ports (8, 29), all lineage 2 strains harbored a genomic deletion inRD105. The phylogeny is basally split into two sister clades, desig-nated lineage 2.1 and 2.2 (Fig. 1B). Strains of lineage 2.2 all harbora deletion in RD207 that is the Beijing-specific marker causing thecharacteristic spoligotypes of this family (37). In contrast, strains oflineage 2.1 all have an intact RD207 and thus do not belong to theBeijing family; we named these strains as “proto-Beijing,” indicatingtheir close relationship with Beijing strains and the more ancestralstate of them in RD207. Interestingly, except for one proto-Beijingstrain that exhibited the same deletion (3,466 bp) in RD105 asBeijing strains, all of the remaining 22 proto-Beijing strains har-bored a larger deletion (8,672 bp, Rv0068-Rv0075) in RD105than Beijing strains; we named this deletion “extended RD105”(SI Appendix, Fig. S4). The Beijing strains included three majormonophyletic groups (MG), designated Bj-MG1 to Bj-MG3. Bj-MG1 represented the most early diverged branch within the family.The strains of Bj-MG1 have an intact RD181, whereas all of the

other Beijing strains harbor a specific deletion in this region. Thestrains of Bj-MG3 harbor an insertion of IS6110 in the NTF regionand correspond to the previously defined “modern” Beijing subtype(27). Consequently, the Bj-MG1, Bj-MG2, and other Beijing strainscorrespond to the “ancient” Beijing subtype. The recently definedEast European subtype of Beijing family that is prevalent in Russiaand other countries of the former Soviet Union (33) representedtwo monophyletic groups (East European 1 and 2) within Bj-MG3.The phylogenetically informative SNPs for defining MTBC lineage2 and major sublineages are summarized (SI Appendix, Dataset S2).

The Southern East Asia Origin and Parallel Evolution of MTBC BeijingFamily with Human Populations in East Asia. By mapping the patientregion of origin onto our MTBC lineage 2 phylogeny, we searchedfor the most likely geographical origin of lineage 2 overall, and forthe three major sublineages of the Beijing family. Our phylogeo-graphic analyses revealed southern East Asia as the most likelylocation for the ancestor of lineage 2 as well as for the three Beijingsublineages (SI Appendix, Fig. S5), which suggests a rapid radiationof lineage 2 from this region to the rest of East Asia.To further confirm the southern East Asian origin of lineage 2,

we focused our attention on the geographical distribution of theproto-Beijing strains. Because the proto-Beijing branch repre-sent the most basal group of the lineage 2, the distribution of

Ne per generation

Pre-NDT expansion (10 kya)

Time (Kya)

NDT expansion (6.5 kya)

1.0E7

1.0E5

1.0E4

1.0E6

05 10 15 20 25 1.0E3

1.0E8

RD105 (Lineage 2)

RD207 (Beijing)

Extended RD105

RD181

27.8 kya (24.8-30.8)

18.5 kya (16.4-20.6)

11.3 kya (10.1-12.4)

12.2 kya (10.7-13.6)

7.8 kya (7.1-8.6)

NTF::IS6110

Lineage 2.1 (Proto-Beijing)

Bj-MG1

Bj-MG2

Bj-MG3

East Europe 1

East Europe 2

Lineage 2.2 (Beijing)

A

B

C

Fig. 1. Genetic and geographic structure and population dynamics ofglobal MTBC lineage 2 strains. (A) Places of origin for the 358 MTBC lineage2 strains. The size of dot corresponds to the number of strains in each place.The places were classified into four geographic areas marked in differentcolors, southern East Asia (SEAS, orange), northern East Asia (NEAS, blue),Southern Asia (SAS, green), and Northern Asia (NAS, cyan). (B) Maximumclade credibility phylogeny inferred from genome-wide SNPs of the 358strains, with estimated divergence date in thousand years. Highly congruenttopology was obtained by maximum likelihood inference (SI Appendix, Fig.S3). Taxa were colored by the corresponding geographic areas. The 20 strainswith vague or unknown origin area were colored in white, and one strainoriginating in the United States and two strains from South Africa were col-ored in black. The major nodes were colored to represent their most probablegeographic origin as was indicated by phylogeographic analysis (SI Appendix,Fig. S5). (C) Bayesian skyline plots showing changes in population diversity ofglobal MTBC lineage 2. Ne, effective population size in logarithmic scale.

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proto-Beijing strains could provide important clues for the geo-graphical origin of lineage 2. Firstly, we searched for proto-Beijing strains among 2,346 MTBC strains isolated from 13provinces across China (38). We identified 11 (<0.5%; 95% CI0.2–0.8) proto-Beijing strains (SI Appendix, Table S1), all ofwhich were isolated from four areas in southern China, includingseven strains from Guangxi province. Considering the relative over-representation of proto-Beijing strains in Guangxi, we then typed1,404 strains collected in that province and identified 36 (2.6%; 95%CI, 1.8–3.5) additional proto-Beijing strains (including the 16 strainswe genome sequenced). We further identified 11 proto-Beijingstrains from previous studies and we found most of them isolatedfrom patients born in southern China or South East Asia countries(Table 1). Taken together, a total of 58 proto-Beijing strains weredetected in this and previous studies. Among the 55 strains withavailable geographical information, 54 (98%) were from southernChina or Southeast Asian areas (Table 1). This aggregating dis-tribution of proto-Beijing and its absence in northern China fur-ther support the southern origin of MTBC lineage 2.The southern origin of MTBC lineage 2 is coincident with the

anthropological evidence for the initial arrival of modern hu-mans in East Asia (39, 40). To test this notion, we estimated theage of lineage 2 and compared with the dates of East Asianhumans (Fig. 1B and SI Appendix, Table S2). The dates werecalculated using the MTBC-70 model published earlier (2) thatsupports an African origin of MTBC around 70,000 y ago andsubsequent dispersals of MTBC following the two waves of“Out-Of-Africa” migrations of modern humans. According tothat model, the ancestor of the TbD1-deleted MTBC lineages(lineage 2, 3, and 4) was most likely dispersed along with thesecond wave of Out-of-Africa migration of modern human intoEurasia (2). The dominant human Y-chromosome haplogroup(HG) NO in East Asia is believed to be associated with thismigration (41, 42). According to the MTBC-70 model, we esti-mated the age of lineage 2 at 25–32 kya, which is consistent withthe date of human HG NO (41, 43). We also used Bayesianskylines to estimate the changes in the effective population sizeof global lineage 2 over time, and tested the concordance withdemographic changes of modern human (Fig. 1C). Similar to ourprevious study (2), we detected a strong population expansionduring the period of Neolithic Demographic Transition (NDT)(6–7 kya), which is concordant with the start of the agriculturetransition in China (44). Furthermore, we detected an earlier

expansion before NDT (9–11 kya). This scenario of MTBClineage 2 is consistent with the latest anthropological findingsthat suggested the population expansion in East Asia startedbefore the agriculture transition (45). Taken together, our datasupport a southern origin and a parallel evolution between MTBClineage 2 and modern humans in East Asia.

A “South-To-North-And-Back-To-South” Scenario and Coexpansionof MTBC Beijing Family with Han Chinese. The human migrationin East Asia included two main stages: In the first stage (10–40 kya), modern humans underwent a great northward migrationthat extended to northern China and Siberia (46); in a morerecent second stage (recent 8,000 y), the population in northernChina (Han Chinese) underwent massive (Neolithic) expansionsand subsequent migrations to southern China (47). To explorepotential correlations between these events and the genetic popu-lation structure of MTBC in China, we genotyped 1,385 Beijingstrains collected from 11 regions throughout China, and combinedthese data with published data of 408 Beijing strains from two ad-ditional regions (48, 49). Similar to our genome-based analysis, wefound the three major Beijing sublineages widely distribute inall areas, with the dominance of “modern” Beijing (Bj-MG3) in allareas except Tibet (Fig. 2A). Additionally, we found that the prev-alence of “modern” Beijing strains in Han group was significantlyhigher than that in the Minority group (t test, P = 0.03) (Fig. 2B,for group classifications, see SI Appendix, SI Materials and Methods).We further divided the seven Han populations into Northern Hanand Southern Han groups separated by the Yangzi River (39, 47).We found the prevalence of “modern” Beijing significantly higherin the Northern Han group (t test, P = 0.03).By reconstructing the changes in population diversity of Bei-

jing sublineages over time using Bayesian skylines, we found thatthe “modern” Beijing sublineage mainly accounted for the NDTexpansion of MTBC lineage 2 (Fig. 2C). This sublineage hasexperienced an approximately 300-fold increase in populationsize during the expansion (SI Appendix, Fig. S6), making it thedominant genotype by the end of the NDT (Fig. 2C). Accordingto our phylogeographic analysis, most ancestral nodes of “mod-ern” Beijing strains during the initial stage of NDT exhibit mixedpopulation pools of southern and northern East Asia (SI Ap-pendix, Fig. S5), indicating rapid expansion in one area andsubsequent extensive transmission to the other. This evidence,together with the massive North–South migration history of HanChinese and the highest prevalence of “modern” Beijing strainsin Northern Han population, strongly suggests that the initialNeolithic expansion of “modern” Beijing strains has taken placein northern China. This scenario is further supported by the highconcordance between the timing of the NDT expansion of thissublineage and the date of intensive agriculture transition inNorth China around 6,000 y ago (41).Taken together, our data support a hypothesized “South-

To-North-And-Back-To-South” scenario for the phylogeographyof MTBC Beijing strains in China (Fig. 3 A and B). Specifically,the southern East Asia origin of Beijing sublineages and the widedistribution of these sublineages in both southern and northernChina indicate that the Beijing family experienced initial di-vergences in the original population and then dispersed to otherareas of China along with the early northward migrations of modernhumans. After the successful expansion in northern China, the“modern” Beijing transmitted to southern China along with themassive North–South migration of Han Chinese in recent 2,000 y.The “modern” Beijing underwent successful expansions alongwith the Han Chinese in South China and became the dominantMTBC strains.

DiscussionBased on population genomics analyses and extensive genotyp-ing data, we propose a southern origin and a parallel evolution of

Table 1. Distribution of proto-Beijing strains identified in thisand previous studies

OriginNumber of

proto-Beijing strains Typing method Reference

ChinaGuangxi 43 LSP and SNP/WGS This studyFujian 2 LSP and SNP This study

1 WGS 62Zhejiang 1 LSP and SNP This studySichuan 1 LSP and SNP This studyTaiwan 1 LSP and SNP 63Unknown region 3* WGS 2

Other countriesLaos 1 WGS 2

2* LSP 34Vietnam 1* LSP 34Malaysia 1 WGS 64South Korea 1* WGS 2

LSP, large sequence polymorphism; SNP, single nucleotide polymorphism;WGS, whole-genome sequencing.*Isolates from American immigrants born in corresponding areas.

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MTBC Beijing family with modern humans in East Asia duringthe last 30,000 y. Previously, a general East Asian/Chinese originof this strain family was suggested based on its highest preva-lence rate and/or highest level of diversity in those regions (18,27, 36, 50). In the current study, we provided further evidenceand proposed a southern East Asia origin of Beijing family,which is consistent with the latest anthropological evidenceabout the colonization and migrations of modern human in EastAsia (39, 40). The current distribution of Beijing sublineages inChina and surrounding regions could be explained with ourproposed “South-To-North-And-Back-To-South” transmission

route along the migrations and expansions of human (for ad-ditional discussion, see SI Appendix, SI Discussion).As we summarized, the prevalence of Beijing family in Eurasia is

dominated by the “modern” sublineage (Fig. 3C). Although“modern” Beijing strains are less prevalent in some regions (e.g.,Japan and Korea), there is evidence indicating they are nowemerging in those areas (51, 52). Besides, recent genotyping datafrom other regions increasingly show that the globally emergingBeijing strains are also dominated by the “modern” sublineage (23,53–55). For example, the well-known hypervirulent strains includingW strains, strain 210 and HN878 that are responsible for multiple

A B C

Fig. 2. Association of MTBC “modern” Beijing sublineage (Bj-MG3) with Han Chinese. (A) Relative prevalence of Beijing sublineages in 13 provinces of China.The “ancient” Beijing strains from previous studies with unclear sublineage attribution were colored black. (B) Comparison of the prevalence of Bj-MG3sublineage in Han and Minority areas. Histogram bars represent the mean rate of prevalence and SE. (C) Changes of effective population size of Beijingsublineages during the past 10,000 y. The dashed line indicates the end of Neolithic expansion around 2,000 y ago.

C

East European Beijing

Beijing (unknown) Proto-Beijing

MG3

Bj

A40-10 Kya

B

Recent 8 Kyr

100% 50% 10%

Fig. 3. “South-To-North-And-Back-To-South” of MTBC lineage 2. (A) Hypothesized south-to-north transmission of “ancient” Beijing strains before theNeolithic transition. (B) The Neolithic expansion of “modern” Beijing strains (Bj-MG3 sublineage) in Northern China and coexpansion with Han Chinese inrecent 2,000 y. (C) Current distribution of MTBC Beijing family and sublineages in Eurasia. Detailed information about the prevalence of MTBC Beijing family/Beijing sublineages in each area was summarized in SI Appendix, Tables S3 and S4.

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disease outbreaks in the United States all belong to the “modern”Beijing genotype. The dominance of “modern” Beijing strains inboth endemic and epidemic areas strongly suggest they are hyper-virulent compared with the “ancient” genotype, which has beensupported by several experimental studies using infection models.For example, a recent study showed “modern” Beijing caused morehistopathological changes and mortality in mice (56). Most of all,three more recent studies all found that “modern” Beijing strainsinduced lower and/or delayed proinflammatory that may enable thebacteria to escape from host immune response (57–59). Epidemi-ologically, numerous studies from various geographical settingshave suggested a higher transmissibility of “modern” Beijing ge-notype, as indicated by increased prevalence in young people (i.e., aproxy for ongoing transmission) (12, 23, 51, 60), or a higher geno-typic clustering proportion than “ancient” genotype (26, 52, 55).One study also showed an increased prevalence of “modern” Bei-jing genotype in bacillus Calmette–Guérin-vaccinated patients (23).Taken together, both epidemical and experimental evidence

support the hypervirulence of “modern” Beijing sublineage.How, then, did this hypervirulent phenotype evolve? We pre-viously hypothesized that increases in human populations mighthave selected for increased virulence (2). The increased and moredense-populations in northern China during the NDT might havebeen the initial driving force for selecting the “modern” Beijingstrains; this notion is supported by the extensive expansions of“modern” Beijing we observed during that period. The establish-ment of permanent human settlements and continuous availabilityof susceptible hosts provided ideal conditions for the spread ofhypervirulent strains (2).All of the above conclusions were based on the MTBC-70 model

that proposes MTBC originally dispersed out of Africa around 70 kyaalong with modern humans (2). Recently, a Holocene modelfor the origin and initial dispersal of MTBC was proposed thatsupports a much younger age for the MTBC (less than 6,000 y)(4). Indeed, tuberculosis has been recently detected from humanremains older than 8 kya by both paleopathological and molecularevidence (61), indicating that MTBC may be older than the Ho-locene model suggests. By rerunning our data with the Holocenemodel, we estimated the age of lineage 2 as 2.0 kya (1.3–3.0, 95%HPD) (SI Appendix, Table S2). Although some human migrationroutes during the past several millennia may have contributed tothe spread of MTBC lineage 2, they were not concordant with thecurrent distribution of Beijing sublineages (Fig. 3C, for additionaldiscussion, see SI Appendix, SI Discussion). The Holocene modelsuggests a similar short- and long-term substitution rate of MTBC(4), which is more than 10-fold faster than the MTBC-70 suggests(ref. 2, for additional discussion, see SI Appendix, SI Discussion). Byapplying the short-term rate of MTBC, Merker et al. (36) recently

suggests the population expansions of Beijing family were corre-lated with the Industrial Revolution and the first World War inEurope. However, this scenario is less concordant with the de-mography of human population in East Asian, where the Beijingfamily is endemic (for additional discussion, see SI Appendix,SI Discussion).In summary, by systematically studying the global diversity of

the MTBC Beijing family, we generated strong support for asouthern East Asia origin and a parallel evolution of this lineagewith modern humans in East Asia. The endemic Beijing strainsin East Asia are diverse, whereas the globally emerging strainsmostly belong to the highly homogenous “modern” Beijing sub-type. Our results suggest that the hypervirulent phenotype ob-served in the “modern” sublineage may have been selected by theincrease human densities during the Neolithic and later humanexpansions. The hypervirulence and effectiveness of “modern”Beijing strains to resist bacillus Calmette–Guérin vaccinations andanti-TB drugs may have further promoted their global emergencein the past decades. Further experimental and epidemiologicalresearch is needed to study the hypervirulence of “modern”Beijing subtype, the molecular determinants and the selectionforces that have contributed to its global success.

Materials and MethodsGenome Sequencing and Evolutionary Analyses. Briefly, we selected 95 rep-resentative lineage 2 strains based on composite analyses of SNP and VNTRgenotypes of 908 Beijing strains (26) and 36 proto-Beijing strains collectedfrom six regions of China. We performed paired-end genome sequencing onthe Illumina HisEq 2000 with an expected coverage of 100. We identifiedadditional 263 unique genomes (each of them is different from any othergenome by more than 10 SNPs) of MTBC lineage 2 strains from previousstudies (SI Appendix, Dataset S1). We applied Bayesian evolutionary analysesby running BEAST (v1.8.0) (65) based on both MTBC-70 and MTBC-Holocenemodel. We used RASP (66) that implements both Bayesian and parsimonyapproaches to analyze the ancestral geographic ranges of MTBC lineage 2and sublineages.

Genotyping. A real-time PCR melting curve assay (67) was used for typing ofsix SNPs in 3R (DNA replication, recombination, and repair) genes (SI Ap-pendix, Fig. S1). Multiplex PCRs targeting RD105 and RD207 genomic regionswere applied to identify non-Beijing lineage 2 (proto-Beijing) strains.

Details of materials and methods are included in SI Appendix, SI Materialsand Methods.

ACKNOWLEDGMENTS. This work was supported by the Natural ScienceFoundation of China (91231115 and 31301033), the Key Project of ChineseNational Programs, China (2013ZX10003004-001), China Postdoctoral ScienceFoundation (2012M52082), Ramón y Cajal Spanish Research Grant RYC-2012-10627, the MINECO Research Grant SAF2013-43521-R, the Swiss NationalScience Foundation (PP00P3_150750), the European Research Council (309540-EVODRTB), and SystemsX.ch.

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