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Southern East Asian Origin and Coexpansion of Mycobacterium Tuberculosis Beijing Family with Han Chinese

Southern East Asian Origin and Coexpansion of Mycobacterium Tuberculosis Beijing Family with Han Chinese

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis with

Tao Luoa,b,Iñaki 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 Medical Microbiology, School of Basic Medical Sciences, Fudan University, 200032, ; bLaboratory of Infection and Immunity, School of Basic Medical Science, West China Center of Medical Sciences, University, , Sichuan 610041, China; cGenomics and Health Unit, FISABIO Public Health, Valencia 46020, ; dCIBER (Centros de Investigación Biomédica en Red) in Epidemiology and Public Health, Instituto de Salud Carlos III, Madrid 28029, Spain; eDepartment of Tuberculosis Control, Zhuang Autonomous Region Center for Disease Control and Prevention, , Guangxi 530028, China; fState Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center 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 Disease Control and Prevention, Shanghai 200336, China; iMinistry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China; jDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel 4002, University of Basel, Basel CH-4003, Switzerland; and kCollaborative Innovation Center for Diagnosis and Treatment of Infectious 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 Mycobacterium great attentions due to its global emergence in recent decades tuberculosis and responsible for more than a quarter of the global (6, 7, 10–12), its tendency to cause disease outbreak (13–17), and tuberculosis epidemic. As the predominant genotype in , the its association with antibiotic resistance (12, 18). Experimental and Beijing family has been emerging in various areas of the world and is clinical evidences suggest a hypervirulent phenotype of Beijing often associated with disease outbreaks and antibiotic resistance. Re- strains (12, 19), and a higher mutation rate compared with other vealing the origin and historical dissemination of this strain family is strains (20). important for understanding its current global success. Here we char- According to genotyping data from previous molecular- acterized the global diversity of this family based on whole-genome epidemiology studies, most Beijing strains from widespread geo- sequences of 358 Beijing strains. We show that the Beijing strains graphic areas showed a remarkable degree of genetic similarity endemic in East Asia are genetically diverse, whereas the globally (6, 21), suggesting this strain family might have emerged from emerging strains mostly belong to a more homogenous subtype recent expansions. It was hypothesized that vaccination with “ ” known as modern Beijing. Phylogeographic and coalescent analy- Bacille Calmette Guerin (bacillus Calmette–Guérin) that has ses indicate that the Beijing family most likely emerged around been widely implemented in East Asian countries might be the 30,000 y ago in southern East Asia, and accompanied the early colo- force driving the dominance of this strain family in this area (21). nization by modern humans in this area. By combining the genomic Moreover, the global emergence of the Beijing family may have data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expan- sions in northern China during the era and subsequently Significance spread to other regions following the migration of . Our results support a parallel evolution of the Beijing family and Mycobacterium tuberculosis Beijing family is a group of glob- modern humans in East Asia. The dominance of the “modern” Beijing ally emerging bacterial strains that are responsible for more sublineage in East Asia and its recent global emergence are most than a quarter of the global tuberculosis epidemic. Here, we likely driven by its hypervirulence, which might reflect adaption to combine whole-genome sequencing and large-scale genotyp- increased human population densities linked to the agricultural tran- ing to map the temporal and spatial changes of the genetic sition in northern China. diversity within this strain family. We reveal a southern East Asia origin and a parallel evolution of this bacterial genotype MTBC Beijing family | origin | expansion | Han Chinese with modern humans in East Asia during the last 30,000 years. The recently globally emerged Beijing strains mainly belong to uberculosis has plagued human beings since ancient times and a hypervirulent subtype that most likely has initially been se- Tremains a leading cause of global morbidity and mortality. The lected for adaption to increased population densities during causative agent of human tuberculosis is the Mycobacterium tuber- the agricultural transition in northern China. culosis complex (MTBC), a group of organisms that harbor little Author contributions: T.L., F.L., S.G., J.M., R.L., K.W., and Q.G. designed research; T.L., D.L., genetic diversity compared with other bacteria (1). MTBC most B.L., J.W., C.Y., Q.L., M.G., G.S., and X.S. performed research; T.L., I.C., and L.W. analyzed likely originated in Africa, although its age is being debated (2–4). data; and T.L., I.C., L.W., S.G., and Q.G. wrote the paper. The human-adapted MTBC is highly clonal and is classified into The authors declare no conflict of interest. seven main phylogenetic groups, designated lineage 1 through This article is a PNAS Direct Submission. lineage 7 (2). These seven lineages show strong biogeographic as- Data deposition: The sequences in this paper have been deposited in the Sequence Read sociations that have been proposed to result from codiversification Archive database, www.ncbi.nlm.nih.gov/sra (accession no. SRP051093). with different human populations (2, 5). Lineage 2 that dominates 1I.C., D.L., B.L., J.W., and L.W. contributed equally to this work. in East Asia is one of the most successful MTBC variants; more 2To whom correspondence may be addressed. Email: [email protected], sebastien. than a quarter of the global tuberculosis epidemic is caused by this [email protected], [email protected], [email protected], or [email protected] lineage (6, 7). Lineage 2 contains strains that mostly belong to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. so-called Beijing family (8, 9). This strain family has attracted 1073/pnas.1424063112/-/DCSupplemental.

8136–8141 | PNAS | June 30, 2015 | vol. 112 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1424063112 Downloaded by guest on September 28, 2021 been due to its hypervirulence and association with drug re- A East sistance (7, 18). However, there were discrepant results regarding Europe 1 the relative protective effect of bacillus Calmette–Guérin vaccina-

tion against Beijing strains from animal infection experiments (19), East Europe 2 and many epidemiological studies failed to find any association between bacillus Calmette–Guérin vaccination and Beijing strains (22–25). The link between drug resistance and the Beijing family has Bj-MG3 primarily been observed in regions where this family has emerged Lineage 2.2 recently (e.g., Cuba, , countries of the former Soviet (Beijing) Union) but not in East Asian, where the Beijing family has been

endemic for a long time (18, 26). Furthermore, more recent studies 7.8 kya (7.1-8.6) indicate that the expansion of the Beijing family may have started NTF::IS6110 long before the introduction of vaccination and antibiotic treatment B (2, 3, 27). 11.3 kya (10.1-12.4) With the increased availability of genotyping data, the Beijing Bj-MG2 strains were proved more heterogeneous than initially estimated, RD181 18.5 kya – (16.4-20.6) and several Beijing sublineages have been identified (28 31). 12.2 kya 27.8 kya RD207 (10.7-13.6) Bj-MG1 (24.8-30.8) However, a full understanding of the genetic diversity of Beijing (Beijing) RD105 Extended RD105 Lineage 2.1 family is constrained by the low amount of nucleotide variation (Lineage 2) (Proto-Beijing) (8, 32). Whole-genome sequencing provides an ideal tool to 1.0E8

NDT Ne study the genetic diversity of MTBC, and new insights into the expansion 1.0E7 – (6.5 kya) per generation origin and evolution of MTBC have been gained (2, 4, 20, 33 C Pre-NDT 1.0E6 expansion 35). The genomic diversity of Beijing family was initially studied (10 kya) in a most recent study, in which a general East Asian origin and 1.0E5 recent expansions of this strain family were suggested (36). However, 1.0E4 1.0E3 the details about the origin and primary dissemination of Beijing 25 20 15 10 5 0 family remain unclear. Answering of these questions is important to Time (Kya) better understand the virulence of this lineage and its global success. Fig. 1. Genetic and geographic structure and population dynamics of Here, we combined whole-genome sequencing of key strains with global MTBC lineage 2 strains. (A) Places of origin for the 358 MTBC lineage detailed single nucleotide polymorphism (SNP) typing of a large 2 strains. The size of dot corresponds to the number of strains in each place. collection of clinical MTBC strains isolated from across China. Our The places were classified into four geographic areas marked in different results strongly support a southern East Asian origin of the MTBC colors, southern East Asia (SEAS, orange), northern East Asia (NEAS, blue), Beijing family and suggest a parallel evolution of this family with Southern Asia (SAS, green), and Northern Asia (NAS, cyan). (B) Maximum modern humans in East Asia during the last 30,000 y. clade credibility phylogeny inferred from genome-wide SNPs of the 358 strains, with estimated divergence date in thousand years. Highly congruent Results topology was obtained by maximum likelihood inference (SI Appendix,Fig. S3). Taxa were colored by the corresponding geographic areas. The 20 strains The Population Diversity of MTBC Lineage 2. We sequenced the with vague or unknown origin area were colored in white, and one strain whole genome of 79 clinical strains representative of the diversity of originating in the and two strains from South Africa were col- MTBC Beijing family in China in this study (40 strains) and a ored in black. The major nodes were colored to represent their most probable previous study (39 strains; ref. 2) (SI Appendix,Fig.S1). In addition, geographic origin as was indicated by phylogeographic analysis (SI Appendix, we genome sequenced 16 non-Beijing lineage 2 strains identified in Fig. S5). (C) Bayesian skyline plots showing changes in population diversity of Guangxi province. These 95 genomes were combined with 263 global MTBC lineage 2. Ne, effective population size in logarithmic scale. SI Appendix SI Appendix BIOLOGY

unique genomes ( , Dataset S1 and ,Fig.S2) POPULATION of lineage 2 from 15 countries (Fig. 1A) published previously (ref- erences are appended in SI Appendix, SI References), which enabled other Beijing strains harbor a specific deletion in this region. The IS6110 us to analyze the diversity of MTBC lineage 2 at a global scale. strains of Bj-MG3 harbor an insertion of in the NTF region “ ” After excluding the repetitive, mobile elements and drug-resistance and correspond to the previously defined modern Beijing subtype associated genes, we identified 24,592 SNPs, which we used to (27). Consequently, the Bj-MG1, Bj-MG2, and other Beijing strains “ ” construct a phylogenetic tree of MTBC lineage 2 (Fig. 1B). We also correspond to the ancient Beijing subtype. The recently defined mapped the well characterized lineage 2 and Beijing family poly- East European subtype of Beijing family that is prevalent in morphic markers onto the phylogeny. Consistent with previous re- and other countries of the former Soviet Union (33) represented ports (8, 29), all lineage 2 strains harbored a genomic deletion in two monophyletic groups (East European 1 and 2) within Bj-MG3. RD105. The phylogeny is basally split into two sister clades, desig- The phylogenetically informative SNPs for defining MTBC lineage nated lineage 2.1 and 2.2 (Fig. 1B). Strains of lineage 2.2 all harbor 2 and major sublineages are summarized (SI Appendix, Dataset S2). a deletion in RD207 that is the Beijing-specific marker causing the characteristic spoligotypes of this family (37). In contrast, strains of The Southern East Asia Origin and Parallel Evolution of MTBC Beijing lineage 2.1 all have an intact RD207 and thus do not belong to the Family with Human Populations in East Asia. By mapping the patient Beijing family; we named these strains as “proto-Beijing,” indicating region of origin onto our MTBC lineage 2 phylogeny, we searched their close relationship with Beijing strains and the more ancestral for the most likely geographical origin of lineage 2 overall, and for state of them in RD207. Interestingly, except for one proto-Beijing the three major sublineages of the Beijing family. Our phylogeo- strain that exhibited the same deletion (3,466 bp) in RD105 as graphic analyses revealed southern East Asia as the most likely Beijing strains, all of the remaining 22 proto-Beijing strains har- location for the ancestor of lineage 2 as well as for the three Beijing bored a larger deletion (8,672 bp, Rv0068-Rv0075) in RD105 sublineages (SI Appendix,Fig.S5), which suggests a rapid radiation than Beijing strains; we named this deletion “extended RD105” of lineage 2 from this region to the rest of East Asia. (SI Appendix, Fig. S4). The Beijing strains included three major To further confirm the southern East Asian origin of lineage 2, monophyletic groups (MG), designated Bj-MG1 to Bj-MG3. Bj- we focused our attention on the geographical distribution of the MG1 represented the most early diverged branch within the family. proto-Beijing strains. Because the proto-Beijing branch repre- The strains of Bj-MG1 have an intact RD181, whereas all of the sent the most basal group of the lineage 2, the distribution of

Luo et al. PNAS | June 30, 2015 | vol. 112 | no. 26 | 8137 Downloaded by guest on September 28, 2021 proto-Beijing strains could provide important clues for the geo- expansion before NDT (9–11 kya). This scenario of MTBC graphical origin of lineage 2. Firstly, we searched for proto- lineage 2 is consistent with the latest anthropological findings Beijing strains among 2,346 MTBC strains isolated from 13 that suggested the population expansion in East Asia started provinces across China (38). We identified 11 (<0.5%; 95% CI before the agriculture transition (45). Taken together, our data 0.2–0.8) proto-Beijing strains (SI Appendix, Table S1), all of support a southern origin and a parallel evolution between MTBC which were isolated from four areas in southern China, including lineage 2 and modern humans in East Asia. seven strains from Guangxi province. Considering the relative over- representation of proto-Beijing strains in Guangxi, we then typed A “South-To-North-And-Back-To-South” Scenario and Coexpansion 1,404 strains collected in that province and identified 36 (2.6%; 95% of MTBC Beijing Family with Han Chinese. The human migration – CI, 1.8–3.5) additional proto-Beijing strains (including the 16 strains in East Asia included two main stages: In the first stage (10 we genome sequenced). We further identified 11 proto-Beijing 40 kya), modern humans underwent a great northward migration strains from previous studies and we found most of them isolated that extended to northern China and Siberia (46); in a more from patients born in southern China or South East Asia countries recent second stage (recent 8,000 y), the population in northern (Table 1). Taken together, a total of 58 proto-Beijing strains were China (Han Chinese) underwent massive (Neolithic) expansions detected in this and previous studies. Among the 55 strains with and subsequent migrations to southern China (47). To explore available geographical information, 54 (98%) were from southern potential correlations between these events and the genetic popu- China or Southeast Asian areas (Table 1). This aggregating dis- lation structure of MTBC in China, we genotyped 1,385 Beijing tribution of proto-Beijing and its absence in northern China fur- strains collected from 11 regions throughout China, and combined ther support the southern origin of MTBC lineage 2. these data with published data of 408 Beijing strains from two ad- The southern origin of MTBC lineage 2 is coincident with the ditional regions (48, 49). Similar to our genome-based analysis, we found the three major Beijing sublineages widely distribute in anthropological evidence for the initial arrival of modern hu- all areas, with the dominance of “modern” Beijing (Bj-MG3) in all mans in East Asia (39, 40). To test this notion, we estimated the areas except (Fig. 2A). Additionally, we found that the prev- age of lineage 2 and compared with the dates of East Asian alence of “modern” Beijing strains in Han group was significantly humans (Fig. 1B and SI Appendix, Table S2). The dates were higher than that in the Minority group (t test, P = 0.03) (Fig. 2B, calculated using the MTBC-70 model published earlier (2) that for group classifications, see SI Appendix, SI Materials and Methods). supports an African origin of MTBC around 70,000 y ago and We further divided the seven Han populations into subsequent dispersals of MTBC following the two waves of “ ” and Southern Han groups separated by the Yangzi River (39, 47). Out-Of-Africa migrations of modern humans. According to We found the prevalence of “modern” Beijing significantly higher that model, the ancestor of the TbD1-deleted MTBC lineages in the Northern Han group (t test, P = 0.03). (lineage 2, 3, and 4) was most likely dispersed along with the By reconstructing the changes in population diversity of Bei- second wave of Out-of-Africa migration of modern human into jing sublineages over time using Bayesian skylines, we found that Eurasia (2). The dominant human Y-chromosome haplogroup the “modern” Beijing sublineage mainly accounted for the NDT (HG) NO in East Asia is believed to be associated with this expansion of MTBC lineage 2 (Fig. 2C). This sublineage has migration (41, 42). According to the MTBC-70 model, we esti- experienced an approximately 300-fold increase in population – mated the age of lineage 2 at 25 32 kya, which is consistent with size during the expansion (SI Appendix, Fig. S6), making it the the date of human HG NO (41, 43). We also used Bayesian dominant genotype by the end of the NDT (Fig. 2C). According skylines to estimate the changes in the effective population size to our phylogeographic analysis, most ancestral nodes of “mod- of global lineage 2 over time, and tested the concordance with ern” Beijing strains during the initial stage of NDT exhibit mixed demographic changes of modern human (Fig. 1C). Similar to our population pools of southern and northern East Asia (SI Ap- previous study (2), we detected a strong population expansion pendix, Fig. S5), indicating rapid expansion in one area and during the period of Neolithic Demographic Transition (NDT) subsequent extensive transmission to the other. This evidence, (6–7 kya), which is concordant with the start of the agriculture together with the massive North–South migration history of Han transition in China (44). Furthermore, we detected an earlier Chinese and the highest prevalence of “modern” Beijing strains in Northern Han population, strongly suggests that the initial Neolithic expansion of “modern” Beijing strains has taken place Table 1. Distribution of proto-Beijing strains identified in this in northern China. This scenario is further supported by the high and previous studies concordance between the timing of the NDT expansion of this Number of sublineage and the date of intensive agriculture transition in Origin proto-Beijing strains Typing method Reference North China around 6,000 y ago (41). Taken together, our data support a hypothesized “South- China To-North-And-Back-To-South” scenario for the phylogeography Guangxi 43 LSP and SNP/WGS This study of MTBC Beijing strains in China (Fig. 3 A and B). Specifically, 2 LSP and SNP This study the southern East Asia origin of Beijing sublineages and the wide 1 WGS 62 distribution of these sublineages in both southern and northern 1 LSP and SNP This study China indicate that the Beijing family experienced initial - Sichuan 1 LSP and SNP This study vergences in the original population and then dispersed to other 1 LSP and SNP 63 areas of China along with the early northward migrations of modern Unknown region 3* WGS 2 humans. After the successful expansion in northern China, the Other countries “modern” Beijing transmitted to southern China along with the 1 WGS 2 massive North–South migration of Han Chinese in recent 2,000 y. 2* LSP 34 The “modern” Beijing underwent successful expansions along 1* LSP 34 with the Han Chinese in South China and became the dominant 1 WGS 64 MTBC strains. South 1* WGS 2 Discussion LSP, large sequence polymorphism; SNP, single nucleotide polymorphism; WGS, whole-genome sequencing. Based on population genomics analyses and extensive genotyp- *Isolates from American immigrants born in corresponding areas. ing data, we propose a southern origin and a parallel evolution of

8138 | www.pnas.org/cgi/doi/10.1073/pnas.1424063112 Luo et al. Downloaded by guest on September 28, 2021 ABC

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-MG3 sublineage in Han and Minority areas. Histogram bars represent the mean rate of prevalence and SE. (C) Changes of effective population size of Beijing sublineages during the past 10,000 y. The dashed line indicates the end of Neolithic expansion around 2,000 y ago.

MTBC Beijing family with modern humans in East Asia during route along the migrations and expansions of human (for ad- the last 30,000 y. Previously, a general East Asian/Chinese origin ditional discussion, see SI Appendix, SI Discussion). of this strain family was suggested based on its highest preva- As we summarized, the prevalence of Beijing family in Eurasia is lence rate and/or highest level of diversity in those regions (18, dominated by the “modern” sublineage (Fig. 3C). Although 27, 36, 50). In the current study, we provided further evidence “modern” Beijing strains are less prevalent in some regions (e.g., and proposed a southern East Asia origin of Beijing family, and Korea), there is evidence indicating they are now which is consistent with the latest anthropological evidence emerging in those areas (51, 52). Besides, recent genotyping data about the colonization and migrations of modern human in East from other regions increasingly show that the globally emerging Asia (39, 40). The current distribution of Beijing sublineages in Beijing strains are also dominated by the “modern” sublineage (23, China and surrounding regions could be explained with our 53–55). For example, the well-known hypervirulent strains including proposed “South-To-North-And-Back-To-South” transmission W strains, strain 210 and HN878 that are responsible for multiple

A C 40-10 Kya BIOLOGY POPULATION

Bj

B Recent 8 Kyr

MG3

East European Beijing

Proto-Beijing 100% 50% Beijing (unknown) 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 the Neolithic transition. (B) The Neolithic expansion of “modern” Beijing strains (Bj-MG3 sublineage) in Northern China and coexpansion with Han Chinese in recent 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.

Luo et al. PNAS | June 30, 2015 | vol. 112 | no. 26 | 8139 Downloaded by guest on September 28, 2021 disease outbreaks in the United States all belong to the “modern” suggests the population expansions of Beijing family were corre- Beijing genotype. The dominance of “modern” Beijing strains in lated with the Industrial Revolution and the first World War in both endemic and epidemic areas strongly suggest they are hyper- Europe. However, this scenario is less concordant with the de- virulent compared with the “ancient” genotype, which has been mography of human population in East Asian, where the Beijing supported by several experimental studies using infection models. family is endemic (for additional discussion, see SI Appendix, For example, a recent study showed “modern” Beijing caused more SI Discussion). histopathological changes and mortality in mice (56). Most of all, In summary, by systematically studying the global diversity of three more recent studies all found that “modern” Beijing strains the MTBC Beijing family, we generated strong support for a induced lower and/or delayed proinflammatory that may enable the southern East Asia origin and a parallel evolution of this lineage bacteria to escape from host immune response (57–59). Epidemi- with modern humans in East Asia. The endemic Beijing strains ologically, numerous studies from various geographical settings in East Asia are diverse, whereas the globally emerging strains have suggested a higher transmissibility of “modern” Beijing ge- mostly belong to the highly homogenous “modern” Beijing sub- notype, as indicated by increased prevalence in young people (i.e., a type. Our results suggest that the hypervirulent phenotype ob- proxy for ongoing transmission) (12, 23, 51, 60), or a higher geno- served in the “modern” sublineage may have been selected by the typic clustering proportion than “ancient” genotype (26, 52, 55). increase human densities during the Neolithic and later human One study also showed an increased prevalence of “modern” Bei- expansions. The hypervirulence and effectiveness of “modern” jing genotype in bacillus Calmette–Guérin-vaccinated patients (23). Beijing strains to resist bacillus Calmette–Guérin vaccinations and Taken together, both epidemical and experimental evidence anti-TB drugs may have further promoted their global emergence support the hypervirulence of “modern” Beijing sublineage. in the past decades. Further experimental and epidemiological How, then, did this hypervirulent phenotype evolve? We pre- research is needed to study the hypervirulence of “modern” viously hypothesized that increases in human populations might Beijing subtype, the molecular determinants and the selection have selected for increased virulence (2). The increased and more forces that have contributed to its global success. dense-populations in northern China during the NDT might have been the initial driving force for selecting the “modern” Beijing Materials and Methods strains; this notion is supported by the extensive expansions of Genome Sequencing and Evolutionary Analyses. Briefly, we selected 95 rep- “modern” Beijing we observed during that period. The establish- resentative lineage 2 strains based on composite analyses of SNP and VNTR ment of permanent human settlements and continuous availability genotypes of 908 Beijing strains (26) and 36 proto-Beijing strains collected of susceptible hosts provided ideal conditions for the spread of from six regions of China. We performed paired-end genome sequencing on the Illumina HisEq 2000 with an expected coverage of 100. We identified hypervirulent strains (2). additional 263 unique genomes (each of them is different from any other All of the above conclusions were based on the MTBC-70 model genome by more than 10 SNPs) of MTBC lineage 2 strains from previous that proposes MTBC originally dispersed out of Africa around 70 kya studies (SI Appendix, Dataset S1). We applied Bayesian evolutionary analyses along with modern humans (2). Recently, a Holocene model by running BEAST (v1.8.0) (65) based on both MTBC-70 and MTBC-Holocene for the origin and initial dispersal of MTBC was proposed that model. We used RASP (66) that implements both Bayesian and parsimony supports a much younger age for the MTBC (less than 6,000 y) approaches to analyze the ancestral geographic ranges of MTBC lineage 2 (4). Indeed, tuberculosis has been recently detected from human and sublineages. remains older than 8 kya by both paleopathological and molecular evidence (61), indicating that MTBC may be older than the Ho- Genotyping. A real-time PCR melting curve assay (67) was used for typing of locene model suggests. By rerunning our data with the Holocene six SNPs in 3R (DNA replication, recombination, and repair) genes (SI Ap- pendix, Fig. S1). Multiplex PCRs targeting RD105 and RD207 genomic regions model, we estimated the age of lineage 2 as 2.0 kya (1.3–3.0, 95% SI Appendix were applied to identify non-Beijing lineage 2 (proto-Beijing) strains. HPD) ( ,TableS2). Although some human migration Details of materials and methods are included in SI Appendix, SI Materials routes during the past several millennia may have contributed to and Methods. the spread of MTBC lineage 2, they were not concordant with the C current distribution of Beijing sublineages (Fig. 3 , for additional ACKNOWLEDGMENTS. This work was supported by the Natural Science discussion, see SI Appendix, SI Discussion). The Holocene model Foundation of China (91231115 and 31301033), the Key Project of Chinese suggests a similar short- and long-term substitution rate of MTBC National Programs, China (2013ZX10003004-001), China Postdoctoral Science (4), which is more than 10-fold faster than the MTBC-70 suggests Foundation (2012M52082), Ramón y Cajal Spanish Research Grant RYC-2012- SI Appendix SI Discussion 10627, the MINECO Research Grant SAF2013-43521-R, the Swiss National (ref. 2, for additional discussion, see , ). By Science Foundation (PP00P3_150750), the European Research Council (309540- applying the short-term rate of MTBC, Merker et al. (36) recently EVODRTB), and SystemsX.ch.

1. Achtman M (2008) Evolution, population structure, and phylogeography of geneti- 11. Glynn JR, et al. (2005) Mycobacterium tuberculosis Beijing genotype, northern Ma- cally monomorphic bacterial pathogens. Annu Rev Microbiol 62:53–70. lawi. Emerg Infect Dis 11(1):150–153. 2. Comas I, et al. (2013) Out-of-Africa migration and Neolithic coexpansion of Myco- 12. Parwati I, van Crevel R, van Soolingen D (2010) Possible underlying mechanisms for bacterium tuberculosis with modern humans. Nat Genet 45(10):1176–1182. successful emergence of the Mycobacterium tuberculosis Beijing genotype strains. 3. Wirth T, et al. (2008) Origin, spread and demography of the Mycobacterium tuber- Lancet Infect Dis 10(2):103–111. culosis complex. PLoS Pathog 4(9):e1000160. 13. Caminero JA, et al. (2001) Epidemiological evidence of the spread of a Mycobacte- 4. Bos KI, et al. (2014) Pre-Columbian mycobacterial genomes reveal seals as a source of rium tuberculosis strain of the Beijing genotype on Gran Canaria Island. Am J Respir New World human tuberculosis. Nature 514(7523):494–497. Crit Care Med 164(7):1165–1170. 5. Gagneux S, et al. (2006) Variable host-pathogen compatibility in Mycobacterium tu- 14. Agerton TB, et al. (1999) Spread of strain W, a highly drug-resistant strain of Myco- berculosis. Proc Natl Acad Sci USA 103(8):2869–2873. bacterium tuberculosis, across the United States. Clin Infect Dis 29(1):85–92. discussion 6. Bifani PJ, Mathema B, Kurepina NE, Kreiswirth BN (2002) Global dissemination of the 93–85. Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 10(1):45–52. 15. Affolabi D, et al. (2009) Possible outbreak of streptomycin-resistant Mycobacterium 7. Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D (2002) Worldwide occur- tuberculosis Beijing in Benin. Emerg Infect Dis 15(7):1123–1125. rence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg 16. Narvskaya O, et al. (2002) Nosocomial outbreak of multidrug-resistant tuberculosis Infect Dis 8(8):843–849. caused by a strain of Mycobacterium tuberculosis W-Beijing family in St. Petersburg, 8. Hershberg R, et al. (2008) High functional diversity in Mycobacterium tuberculosis Russia. Eur J Clin Microbiol Infect Dis 21(8):596–602. driven by genetic drift and human demography. PLoS Biol 6(12):e311. 17. Frieden TR, et al. (1996) A multi-institutional outbreak of highly drug-resistant tu- 9. Comas I, Homolka S, Niemann S, Gagneux S (2009) Genotyping of genetically mono- berculosis: Epidemiology and clinical outcomes. JAMA 276(15):1229–1235. morphic bacteria: DNA sequencing in Mycobacterium tuberculosis highlights the limi- 18. Anonymous; European Concerted Action on New Generation Genetic Markers and tations of current methodologies. PLoS ONE 4(11):e7815. Techniques for the Epidemiology and Control of Tuberculosis (2006) Beijing/W ge- 10. Cowley D, et al. (2008) Recent and rapid emergence of W-Beijing strains of Myco- notype Mycobacterium tuberculosis and drug resistance. Emerg Infect Dis 12(5): bacterium tuberculosis in Cape Town, South Africa. Clin Infect Dis 47(10):1252–1259. 736–743.

8140 | www.pnas.org/cgi/doi/10.1073/pnas.1424063112 Luo et al. Downloaded by guest on September 28, 2021 19. Hanekom M, et al. (2011) Mycobacterium tuberculosis Beijing genotype: A template 43. Rootsi S, et al. (2007) A counter-clockwise northern route of the Y-chromosome for success. Tuberculosis (Edinb) 91(6):510–523. haplogroup N from towards Europe. Eur J Hum Genet 15(2):204–211. 20. Ford CB, et al. (2013) Mycobacterium tuberculosis mutation rate estimates from dif- 44. Barton L, et al. (2009) Agricultural origins and the isotopic identity of domestication ferent lineages predict substantial differences in the emergence of drug-resistant in northern China. Proc Natl Acad Sci USA 106(14):5523–5528. tuberculosis. Nat Genet 45(7):784–790. 45. Zheng HX, et al. (2011) Major population expansion of East Asians began before 21. van Soolingen D, et al. (1995) Predominance of a single genotype of Mycobacterium neolithic time: Evidence of mtDNA genomes. PLoS ONE 6(10):e25835. tuberculosis in countries of east Asia. J Clin Microbiol 33(12):3234–3238. 46. Su B, et al. (1999) Y-Chromosome evidence for a northward migration of modern 22. Shi C, et al. (2007) Immune responses and protective efficacy induced by 85B antigen humans into Eastern Asia during the last Ice Age. Am J Hum Genet 65(6):1718–1724. and early secreted antigenic target-6 kDa antigen fusion protein secreted by 47. Wen B, et al. (2004) Genetic evidence supports demic diffusion of Han culture. Nature recombinant bacille Calmette-Guérin. Acta Biochim Biophys Sin (Shanghai) 39(4): 431(7006):302–305. 290–296. 48. Yu Q, et al. (2013) Genetic diversity of Mycobacterium tuberculosis isolates from Inner 23. Kremer K, et al. (2009) Vaccine-induced immunity circumvented by typical Myco- , China. PLoS ONE 8(5):e57660. bacterium tuberculosis Beijing strains. Emerg Infect Dis 15(2):335–339. 49. XP, et al. (2013) Genotyping and drug resistance analysis of Mycobacterium 24. van Crevel R, et al. (2001) Mycobacterium tuberculosis Beijing genotype strains as- tuberculosis in . Chin J Antitub 35(9):655–659. sociated with febrile response to treatment. Emerg Infect Dis 7(5):880–883. 50. Mokrousov I (2008) Genetic geography of Mycobacterium tuberculosis Beijing ge- 25. Ahn HC, Yang JH, Lee HB, Rhee YK, Lee YC (2000) Effect of combined therapy of oral notype: A multifacet mirror of human history? Infect Genet Evol 8(6):777–785. anti-tubercular agents on theophylline pharmacokinetics. Int J Tuberc Lung Dis 4(8): 51. Iwamoto T, et al. (2009) Population structure dynamics of Mycobacterium tuberculosis 784–787. Beijing strains during past decades in Japan. J Clin Microbiol 47(10):3340–3343. 26. Yang C, et al. (2012) Mycobacterium tuberculosis Beijing strains favor transmission 52. Wada T, et al. (2009) High transmissibility of the modern Beijing Mycobacterium but not drug resistance in China. Clin Infect Dis 55(9):1179–1187. tuberculosis in homeless patients of Japan. Tuberculosis (Edinb) 89(4):252–255. 27. Mokrousov I, et al. (2005) Origin and primary dispersal of the Mycobacterium tu- 53. Kato-Maeda M, et al. (2010) Differences among sublineages of the East-Asian lineage berculosis Beijing genotype: Clues from human phylogeography. Genome Res 15(10): of Mycobacterium tuberculosis in genotypic clustering. Int J Tuberc Lung Dis 14(5): 1357–1364. 538–544. 28. Filliol I, et al. (2006) Global phylogeny of Mycobacterium tuberculosis based on single 54. Iwamoto T, et al. (2012) Genetic diversity and transmission characteristics of Beijing nucleotide polymorphism (SNP) analysis: Insights into tuberculosis evolution, phylo- family strains of Mycobacterium tuberculosis in . PLoS ONE 7(11):e49651. genetic accuracy of other DNA fingerprinting systems, and recommendations for a 55. Hanekom M, et al. (2007) A recently evolved sublineage of the Mycobacterium tu- minimal standard SNP set. J Bacteriol 188(2):759–772. berculosis Beijing strain family is associated with an increased ability to spread and 29. Tsolaki AG, et al. (2005) Genomic deletions classify the Beijing/W strains as a distinct cause disease. J Clin Microbiol 45(5):1483–1490. genetic lineage of Mycobacterium tuberculosis. J Clin Microbiol 43(7):3185–3191. 56. Ribeiro SC, et al. (2014) Mycobacterium tuberculosis strains of the modern sublineage 30. Mestre O, et al. (2011) Phylogeny of Mycobacterium tuberculosis Beijing strains of the Beijing family are more likely to display increased virulence than strains of the constructed from polymorphisms in genes involved in DNA replication, recombination ancient sublineage. J Clin Microbiol 52(7):2615–2624. and repair. PLoS ONE 6(1):e16020. 57. Faksri K, et al. (2014) Heterogeneity of phenotypic characteristics of the modern and 31. Mokrousov I, et al. (2002) Phylogenetic reconstruction within Mycobacterium tuber- ancestral Beijing strains of Mycobacterium tuberculosis. Asian Pac J Allergy Immunol culosis Beijing genotype in northwestern Russia. Res Microbiol 153(10):629–637. 32(2):124–132. 32. Smith NH, Gordon SV, de la Rua-Domenech R, Clifton-Hadley RS, Hewinson RG (2006) 58. van Laarhoven A, et al. (2013) Low induction of proinflammatory cytokines parallels Bottlenecks and broomsticks: The molecular evolution of Mycobacterium bovis. Nat evolutionary success of modern strains within the Mycobacterium tuberculosis Beijing Rev Microbiol 4(9):670–681. genotype. Infect Immun 81(10):3750–3756. 33. Casali N, et al. (2014) Evolution and transmission of drug-resistant tuberculosis in a 59. Chen YY, et al. (2014) The pattern of cytokine production in vitro induced by ancient Russian population. Nat Genet 46(3):279–286. and modern Beijing Mycobacterium tuberculosis strains. PLoS ONE 9(4):e94296. 34. Comas I, et al. (2010) Human T cell epitopes of Mycobacterium tuberculosis are 60. Buu TN, et al. (2009) The Beijing genotype is associated with young age and multi- evolutionarily hyperconserved. Nat Genet 42(6):498–503. drug-resistant tuberculosis in rural Vietnam. Int J Tuberc Lung Dis 13(7):900–906. 35. Ford CB, et al. (2011) Use of to estimate the mutation rate 61. Hershkovitz I, et al. (February 13, 2015) Tuberculosis origin: The Neolithic scenario. of Mycobacterium tuberculosis during latent infection. Nat Genet 43(5):482–486. Tuberculosis (Edinb), 10.1016/j.tube.2015.02.021. 36. Merker M, et al. (2015) Evolutionary history and global spread of the Mycobacterium 62. Lin N, Liu Z, Zhou J, Wang S, Fleming J (2013) Draft genome sequences of two super- tuberculosis Beijing lineage. Nat Genet 47(3):242–249. extensively drug-resistant isolates of Mycobacterium tuberculosis from China. FEMS 37. Flores L, et al. (2007) Large sequence polymorphisms classify Mycobacterium Microbiol Lett 347(2):93–96. tuberculosis strains with ancestral spoligotyping patterns. J Clin Microbiol 45(10): 63. Chuang PC, Chen HY, Jou R (2010) Single-nucleotide polymorphism in the fadD28 3393–3395. gene as a genetic marker for East Asia Lineage Mycobacterium tuberculosis. J Clin 38. Dong H, et al. (2010) Spoligotypes of Mycobacterium tuberculosis from different Microbiol 48(11):4245–4247. Provinces of China. J Clin Microbiol 48(11):4102–4106. 64. Ng KP, et al. (2013) Draft genome sequence of the first isolate of extensively drug- 39. Shi H, et al. (2005) Y-chromosome evidence of southern origin of the East Asian- resistant (XDR) Mycobacterium tuberculosis in Malaysia. Genome Announcements specific haplogroup O3-M122. Am J Hum Genet 77(3):408–419. 1(1):e00056-12. 40. Stoneking M, Delfin F (2010) The human genetic : Weaving a 65. Drummond AJ, Ho SY, Phillips MJ, Rambaut A (2006) Relaxed phylogenetics and BIOLOGY

complex tapestry. Curr Biol 20(4):R188–R193. dating with confidence. PLoS Biol 4(5):e88. POPULATION 41. Yan S, et al. (2014) Y chromosomes of 40% Chinese descend from three Neolithic 66. Yu Y, Harris AJ, Blair C, He X (2015) RASP (Reconstruct Ancestral State in Phylogenies): super-grandfathers. PLoS ONE 9(8):e105691. A tool for historical biogeography. Mol Phylogenet Evol 87:46–49. 42. Rasmussen M, et al. (2011) An Aboriginal Australian genome reveals separate human 67. Luo T, et al. (2011) Multiplex real-time PCR melting curve assay to detect drug- dispersals into Asia. Science 334(6052):94–98. resistant mutations of Mycobacterium tuberculosis. J Clin Microbiol 49(9):3132–3138.

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