1
Out-of-Africa migration and Neolithic co-expansion of Mycobacterium
tuberculosis with modern humans
Iñaki Comas1,2@, Mireia Coscolla3,4*, Tao Luo5*, Sonia Borrell3,4, Kathryn E. Holt6,
Midori Kato-Maeda7, Julian Parkhill8, Bijaya Malla3,4, Stefan Berg9, Guy Thwaites10,
Dorothy Yeboah-Manu11, Graham Bothamley12, Jian Mei13, Lanhai Wei14, Stephen
Bentley8, Simon R. Harris8, Stefan Niemann15, Roland Diel16, Abraham Aseffa17, Qian
Gao5@, Douglas Young18,19#, Sebastien Gagneux3,4#@
1 Genomics and Health Unit, Centre for Public Health Research (CSISP-FISABIO),
46020 Valencia, Spain
2 CIBER in Epidemiology and Public Health, Spain
3 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public
Health Institute, 4002 Basel, Switzerland
4 University of Basel, 4002 Basel, Switzerland
5 Key Laboratory of Medical Molecular Virology, Institutes of Biomedical Sciences and
Institute of Medical Microbiology, Shanghai Medical College, Fudan University,
Shanghai 200032, China
6 Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, 3010 Victoria, Australia
7 Division of Pulmonary and Critical Care Medicine, University of California San
Francisco, 94143 San Francisco, USA
8 Pathogen Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, CB10 1SA, UK
9 TB Research Group, Veterinary Laboratories Agency, Weybridge, New Haw,
Addlestone, Surrey KT15 3NB, UK
10 Department of Infectious Disease/Centre for Clinical Infection and Diagnostics
Research, King's College London, SE1 1UL, United Kingdom
11 Noguchi Memorial Institute for Medical Research, University of Ghana, LG 581
Legon, Ghana
12 Department of Respiratory Medicine, Homerton University Hospital, London E9
6SR, UK
13 Department of Tuberculosis Control, Shanghai Municipal Center for Disease Control
and Prevention, 1380 W Zhongshan Road, Shanghai, 200336, China
14 Ministry of Education Key Laboratory of Contemporary Anthropology, School of
Life Sciences and Institutes of Biomedical Sciences, Fudan University, 200433
Shanghai, China.
15 Molecular Mycobacteriology, Research Center Borstel, 23845 Borstel, Germany
16 Institute for Epidemiology, Schleswig-Holstein University Hospital, Niemannsweg
11, 24105 Kiel, Germany
17 Armauer Hansen Research Institute, P.O. Box 1005 Addis Ababa, Ethiopia
18 MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK
19 Division of Medicine and Centre for Molecular Microbiology and Infection, Imperial
College London, London SW7 2AZ, UK
*=these authors contributed equally to this work
#=these authors jointly supervised this work
@=corresponding authors
2
Abstract
Tuberculosis caused 20% of all human deaths in the Western world between the 17th
and 19th centuries, and remains a cause of high mortality in developing countries. In
analogy to other crowd diseases, the origin of human tuberculosis has been associated
with the Neolithic Demographic Transition, but recent studies point to a much earlier
origin. Here we used 259 whole-genome sequences to reconstruct the evolutionary
history of the Mycobacterium tuberculosis complex (MTBC). Coalescent analyses
indicate that MTBC emerged about 70 thousand years ago, accompanied migrations of
anatomically modern humans out of Africa, and expanded as a consequence of increases
in human population density during the Neolithic. This long co-evolutionary history is
consistent with MTBC displaying characteristics indicative of adaptation to both low-
and high host densities.
Tuberculosis killed one in five adults in Europe and North-America between the 17th
and 19th centuries1, and remains today a cause of high morbidity and mortality in much
of the developing world2. Infectious diseases of humans can be divided into two broad
categories3. Crowd diseases are generally highly virulent and depend on high host
population densities to maximize pathogen transmission and reduce the risk of pathogen
extinction through exhaustion of susceptible hosts4. Many crowd diseases emerged
during the Neolithic Demographic Transition (NDT) starting around ten thousand years
ago (kya), as the development of animal domestication increased the likelihood of
zoonotic transfer of novel pathogens to humans, and agricultural innovations supported
increased population densities that helped sustain the infectious cycle3. In contrast, older
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human infections are often characterized by slow progression to disease, sometimes
involving reactivation after many years of latent or asymptomatic infection; these
characteristics have been proposed to reflect adaptation to low host population densities
by allowing repletion of the reservoir of susceptible individuals5. Tuberculosis is
reminiscent of a typical crowd disease in killing up to 50% of individuals when left
untreated3,6, and having evolved a mode of aerosol transmission that is promoted by
high host densities. However, tuberculosis also displays a pattern of chronic
progression, latency and reactivation that is characteristic of a pre-NDT disease7.
Human tuberculosis was traditionally believed to have originated from animals4, but
more recent phylogenetic analyses of MTBC have suggested that strains adapted to
cause tuberculosis in animals diverged from the major human strains before NDT8–13.
Moreover, human-associated MTBC is an obligate human pathogen with no known
animal or environmental reservoir, suggesting that changes in human demography are
likely to affect the evolution of MTBC. Here we used a population genomics approach
to explore the evolutionary history of human MTBC, with a particular focus on the
impact of changing host population sizes over time. Our results suggest a model that
allows reconciliation of the apparent discrepancy between MTBC features characteristic
of crowd diseases versus those indicative of adaptation to low host densities.
The global diversity of human-adapted MTBC
We generated the genome sequences of 220 strains representative of the global diversity
of MTBC (Supplementary Table 1) and 39 additional strains corresponding to the
lineage 2 "Beijing" family. In the global dataset, after excluding repetitive and mobile
elements, we identified 34,167 polymorphic sites (SNPs) (Supplementary Table 2),
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which we used to reconstruct the phylogenetic relationships between these strains (Fig.
1A). This genome-based phylogeny was congruent with previous phylogenies based on
other markers and resolved seven major lineages, with animal-adapted strains clustering
together with the strains from Lineage 68. The phylogeny includes the recently
described Lineage 7, which to date has only been observed in Ethiopia or recent
Ethiopian emigrants14. Principal component analysis confirmed all main MTBC
lineages, and highlighted the close phylogenetic relationship between the Eurasian
Lineages 2, 3 and 4. These three lineages have collectively been referred to as
evolutionarily “modern” (Fig. 1B) in the past, because of their comparably more derived
position on the MTBC phylogeny and because they are thought to have spread more
recently8,11. The maximum genetic distance between any two strains was 2,188 SNPs
and involved a human and an animal strain, and 1,856 SNPs when only human clinical
isolates were considered. Only 387 (1.1%) of the SNPs were homoplastic. Homoplasy
can arise as a consequence of false-positive SNP calls, because of positive selection,
recurrent mutations or because of recombination as recently suggested15. However, the
fact that only 1.1% of the sites are homoplastic supports the view that the population
structure of MTBC is largely clonal with little ongoing recombination occurring
between strains16,17.
African origin and co-divergence of MTBC with modern humans
Several studies have proposed an African origin for MTBC8,10,12. We decided to
formally test this hypothesis using our new whole-genome data. We used three
independent phylogeographical analyses to determine the likely geographic origin of the
most recent common ancestor of MTBC. Two different Bayesian analyses identified
5
Africa as the most likely origin of MTBC, with East- and West Africa showing a
combined posterior probability of 90% and 67%, respectively (Supplementary Figs. 1,
2, and 3). Similarly, a Maximum Parsimony approach predicted 100% probability of an
African origin. Taken together, these data support an African origin for MTBC.
Next we sought to determine the putative age of the association between MTBC and its
human host. Given that human-adapted MTBC is limited to humans, and both
anatomically modern humans and MTBC originated in Africa, we tested whether
MTBC and humans might have diverged in parallel; this would be particularly likely if
the association between the two predates the NDT, as previously postulated8,10,12. To
explore this possibility, we first compared our new MTBC phylogeny to a
corresponding tree constructed from 4,955 mitochondrial genomes representative of the
main human haplogroups (Supplementary Table 3)18. We observed striking similarities
(Figs. 1C and 1D). In both cases, the early branching clades are found exclusively in
Africa. Moreover, the trichotomy formed by branching of the Out-of-Africa M and N
mitochondrial macro-haplogroups from the L3 African source population is mirrored in
the MTBC phylogeny by a similar relationship between Lineage 1, Eurasian Lineages
2/3/4, and the African Lineages 5 and 6. In addition to this qualitative similarity,
comparison of the most common mitochondrial haplogroups with the most frequent
MTBC lineages in the same country revealed a strong quantitative association
(Parsimony Score and Association Index tests; P < 0.01 in all cases) (Supplementary
Fig. 4, Supplementary Table 5 and Supplementary Table 6). Taken together, these data
are consistent with MTBC evolving in parallel with its human host.
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Age of the association of MTBC and humans
The similarities in tree topology and phylogeographic distribution suggest that MTBC
already infected the early human populations of Africa. To further explore the
association between MTBC and its human host, we tested for possible imprints of
ancient human divergence times on the main phylogenetic lineages of MTBC using a
Bayesian approach19. Several approaches have been used to date bacterial phylogenies
(see refs. 20–22 for some examples). Unfortunately, none of these were applicable here
because of the following reasons. First, although ancient DNA has been used to study
the evolutionary history of other bacteria20, and similar studies have been performed in
tuberculosis in the past23, no relevant whole-genome data are currently available from
ancient DNA of MTBC strains. Second, although a mutation rate for MTBC has
recently been estimated based on a macaque infection model and molecular
epidemiological data24,25, it is well known that such short-term mutation rates cannot
easily be extrapolated to long-term substitution rates relevant for the time-scale
discussed here26,27. Third, and related to the previous point, although the isolation dates
of some of the strains included in our analysis were known, at best they would allow
calculation only of a short-term mutation rate. Moreover, when performing a tip-to-date
analysis of those strains (N = 49), we found that in contrast to several other bacterial
species21,28–30, no significant correlation between isolation time and phylogenetic
divergence exists in MTBC (correlation coefficient = 0.047).
Because of these limitations, we used an alternative approach to date our MTBC
phylogeny. Specifically, we used as initial calibration points several key dates in human
evolution. We tested three alternative models in which the coalescent time of the most
basal MTBC Lineages 5 and 6 was calibrated against: i) the emergence of anatomically
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modern humans 185 +/-20 kya (MTBC-185)31, ii) the coalescent time of the L3
mitochondrial haplogroup 70 +/-10 kya (MTBC-70)32, and iii) the beginning of the
NDT 10 +/-2 kya (MTBC-10)3 (Table 1). We compared the timing of the branching
points predicted by each of the models with estimated dates of known events in human
history. A recent model based on human whole-genome analyses suggests that the
global dispersal of modern humans occurred through two major waves; an initial eastern
dispersal around the Indian Ocean starting 62-75 kya, and a later dispersal into Eurasia
25-38 kya33. Our MTBC-70 model showed a striking correlation with these human
migration events by dating a first split of Lineage 1 at 67 kya (95% highest probability
density (HPD): 48-88 kya) coinciding with the first wave of human migration31, and a
second split at 46 kya (95% HPD: 31-61 kya) matching the later dispersal throughout
Eurasia (Fig. 2a, Supplementary Fig 5)34,35. Coalescent dates for the branch leading to
Lineages 4 and 2 in the MTBC-70 model (30-46 kya and 32-42 kya, respectively) show
a good correlation with archaeological evidence of presence of modern humans in
Europe35 and East Asia36. In contrast, our alternate model MTBC-185 postulates initial
branching of Out-of-Africa lineages as early as 126-174 kya when focusing on the
branch leading to 'modern' strains (Supplementary Fig. 6), which would suggest the
global dispersal of MTBC preceded that of anatomically modern humans. MTBC-10, by
definition, implies global dispersal within the last 10 ky (Supplementary Fig. 7). While
MTBC has been spread by trade and conquest in recent centuries8, the pattern of this
dispersal does not match the phylogeographic distribution discussed above. Finally, a
fourth model (MTBC-65) using the coalescent time of mitochondrial haplogroup M as a
calibration time-point for MTBC Lineage 1 generated very similar results to MTBC-70
(Table 1). In summary, our phylogenetic analysis based on a 70 ky timeframe shows
that MTBC has been infecting humans at least for the last 70 ky.
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Neolithic co-expansion of MTBC and humans
All the data presented so far strongly support the notion that human tuberculosis indeed
predates NDT. How then could the features of tuberculosis typical of crowd diseases
have arisen? To address this question, we used Bayesian skyline plots to estimate the
changes in effective population size over time of the pathogen and human populations19.
Our MTBC dataset revealed a main signal of population size increase starting 10 kya to
2.5 kya (Fig. 2B), suggesting that the expansion of MTBC occurred as a consequence of
the increase in population densities that followed the establishment of first human
settlements during the NDT37, and not just because of a general increase in the total
number of humans peopling the planet at the time. To test if the human population
dynamics around that period coincide with that of the MTBC we used a dataset
previously described to maximize the information on human demographics during the
Neolithic (Supplementary Table 6)38. The resulting skyline plot shows a Neolithic
expansion of humans around 4-8 kya (Supplementary Fig. 8) coinciding with that of
MTBC (Spearman R = 0.99, p <0.00001; Fig. 2B, Supplementary Fig. 8). Taken
together, these findings indicate that the Neolithic contributed to the success of MTBC,
not by enhancing the likelihood of zoonotic transfer to humans as previously proposed,
but because of a combined increase in host population size and density.
The evolutionary history of MTBC at a regional scale
To analyze MTBC evolution at a regional level, we focused on Lineage 2, which
includes the “Beijing” family of strains. These strains have received particular attention
because of their hyper-virulence in laboratory models, their recent dissemination in
9
human populations, and their association with drug resistance39. Supplementing our
global diversity set with an additional 39 Lineage 2 genomes from China, we observed a
strong correlation between skyline plots derived from the Lineage 2 genomes and a set
of human mitochondrial genomes enriched with haplogroups from East Asia of likely
origin just before, during or after the Neolithic (Spearman R = 0.97, p <0.001; Fig. 3A,
Supplementary Fig. 9). MTBC-70 dating for Lineage 2 is consistent with an initial
arrival coincident with archeological evidence of anatomically modern humans in East
Asia36 (32-42 kya, Supplementary Fig. 5), a first expansion (6-11 kya, Figs. 3B and 3C)
alongside the emergence of agriculture in China 8 kya40, and a subsequent main
expansion of the "Beijing" strains (3-5 kya, Supplementary Fig. 9) coinciding with the
spread of agriculture to neighbouring regions (Figs. 3B and 3C)37.
In summary, our data on the global and regional expansion of MTBC during the NDT
supports the view that while NDT was not the only period leading to strong increases in
human population sizes, it was the period where in addition to human population
growth, the densities of human populations increased following the first establishment
of permanent human settlements. Hence, in addition to providing a springboard for
global domination by modern humans, NDT was also central to the success of MTBC
by generating growing numbers of susceptible hosts living in increasingly crowded
conditions.
Concluding remarks
The common origin in Africa, the congruence in phylogeography, and the dating of
major branching events, lead us to conclude that MTBC has been co-evolving with
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anatomically modern humans for tens of thousands of years. The marked expansion of
MTBC during the NTD, but not during earlier human expansion events41,42, suggests
that the success of this pathogen was primarily driven by increases in human host
density, which is typical of crowd diseases. However, the striking match between the
MTBC and human mitochondrial phylogenies supports a much older association
between MTBC and its host, and suggest that carriage of MTBC was ubiquitous in
hunter-gatherer populations migrating out of Africa well before NDT. The fidelity of
this match is surprising. Considering their vulnerability and small numbers (some of
today’s hunter-gatherers live in groups of 20 or less43), it might have been anticipated
that tuberculosis disease would have had a significant detrimental impact on these
groups, and might therefore have precipitated its own extinction. In fact, the
correspondence between MTBC phylogeny with early human migration is strikingly
similar to that observed with low virulence Helicobacter pylori44. Perhaps latent
infection with MTBC imparted some degree of immunity against more lethal pathogens
encountered in the new environment or in contact with archaic human populations? The
ongoing analyses of the human microbiota highlight the fuzzy boundaries between
commensalism and pathogenecity during health and disease45. A recent study has
suggested that co-infection with H. pylori might protect against active tuberculosis
disease46. Conversely, whether latent tuberculosis infection protects against gastric
ulcers or stomach cancer caused by H. pylori in individuals infected with both bacteria
is unknown but an intriguing possibility. In such a case, a positive feedback between
both infections would result in an asymptomatic individual benefiting from being
infected by both bacterial species.
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Alternatively, one could think of a model in which early populations carried the
infection in a less virulent form, with transmis
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