Writers;
Daniel Falush,1 Thierry Wirth,1 Bodo
Linz
,1
Jonathan
K. Pritchard,2 Matthew Stephens,3 Mark Kidd,4
Martin
J. Blaser,5 David Y. Graham,6 Sylvie Vacher,7
Guillermo
I.
Perez-Perez,5 Yoshio Yamaoka,6 Francis Me´graud,7
Kristina
Otto,8 Ulrike Reichard,1 Elena Katzowitsch,8
Xiaoyan
Wang,1 Mark Achtman,1* Sebastian Suerbaum8
1Department of Molecular Biology, Max-Planck Institutfu¨r
Infektionsbiologie, 10117
Berlin
,
Germany
. 2Department of Human Genetics,
University
of
Chicago
,
Chicago
,
IL
60637
,
USA
. 3Department of Statistics,
University
of
Washington
,
Seattle
,
WA
98195–4322
,
USA
. 4Department of Surgery, Yale University School of Medicine,
New Haven, CT 06520–8062, USA. 5Department of Medicine,
New York
University
School
of Medicine,
New York
,
NY
10016–9196
,
USA
. 6VA
Medical
Center
,
Houston
,
TX
77030
,
USA
. 7Universite´ Victor Segalen Bordeaux 2, 33076
Bordeaux
,
France
. 8Institut fu¨r Hygiene und Mikrobiologie, Universita ¨t
Wu¨rzburg, Josef-Schneider Stra_e 2, 97080 Wu¨rzburg,
Germany
.
*To whom correspondence should be addressed.Email: achtman@mpiib-berlin.mpg.de
Helicobacter pylori,
a chronic gastric pathogen of human beings, can be divided into seven
populations and subpopulations with distinct geographical distributions. These
modern populations derive their gene pools from ancestral populations that arose
in
Africa
,
Central Asia
, and
East Asia
. Subsequent spread can be attributed to human migratory
fluxes such as the prehistoric colonization of
Polynesia
and the
Americas
, the neolithic introduction of farming to
Europe
, the Bantu expansion within
Africa
, and the slave trade.
Geographic subdivisions exist for a variety of human pathogens and commensals,
including JC virus (1), Mycobacterium tuberculosis (2), Haemophilus
influenzae (3), and Helicobacterpylori (4–8). H.
pylori, a Gram-negative bacterium
that colonizes the human gastric mucosa for decades and does not spread
epidemically (9), has the potential to be informative about human
migrations (10). Sequence diversity within H. pylori is greater
than that of most other bacteria (4) and about 50-fold greater than that
of human beings (11). Furthermore, frequent recombination between
different H. pylori strains (12–14) implies that only
partial linkage disequilibrium exists between polymorphic nucleotides within
genes (15), which increases the information content for population
genetic
analysis.
In this report, we use a population genetic tool that we have developed (16)
on a large, global sample of H. pylori isolates to define modern
populations and reconstruct their ancestral sources. Previous data with 20 H.
pylori isolates from
East Asia
,
Europe
, and
Africa
show that the sequences of fragments of seven housekeeping
genes and one virulence-associated gene (vacA) differ according to the
continent of origin (4). We sequenced the same fragments from 370 strains isolated from 27 geographical,
ethnic, and/or linguistic human groupings (Table 1). Of the 3850 nucleotides
sequenced for each isolate, 1418 were polymorphic and were used to define
bacterial populations (15).
Fig. 1. Relationships between modern populations (A), modern
subpopulations (B), and ancestral populations (C) of H. pylori.The
black lines show neighbor-joining population trees as measured
by _ˆ, the net nucleotide distance between
populations (15).The circle diameters indicate their genetic
diversity, measured as the average genetic distance between random pairs of
individuals.
The larger circles in (A) versus (C) reflect the
effects of admixture between ancestral populations.
Filled arcs reflect the number of isolates (A and B)
or nucleotides (C) in each population.Color coding is
consistent in different parts of the figure, except for modern hpEurope, which
is an
admixture between the ancestral AE1 and AE2
populations.Scales are at lower right.
The program STRUCTURE (16, 17) implements a Bayesian approach for
deducing population structure from multilocus data by a variety of models,
including the no-admixture model, which assumes that each individual has derived
all of its ancestry from only one population. We used this model to identify
four modern populations (15), designated
hpAfrica1, hpAfrica2, hpEastAsia, and hpEurope on the
basis of their current distributions (Table 1 and Fig. 1A).
Further analyses split hpEastAsia into the
hspAmerind, hspEAsia, and hspMaori subpopulations, and
hpAfrica1 into hspWAfrica and hspSAfrica (Fig. 1B). These results confirm and
extend previous data showing geographical subdivisions
(4, 7, 8). Almost all H. pylori strains isolated
from various countries in
East Asia
were assigned to the
hspEAsia subpopulation. The hspMaori subpopulation was isolated exclusively from
Maoris and other Polynesians in
New Zealand
, whereas the hspAmerind strains were isolated from Inuits
and from Amerinds in North and
South America
. The hspSAfrica and hpAfrica2 populations were found only in
South Africa
, where they made up a majority of the strains isolated. The
hspWAfrica strains were found at low frequency in
South Africa
but at high frequency in
West Africa
and also in the
Americas
, particularly among African Americans in
Louisiana
and
Tennessee
. The hpEurope population contained almost all H. pylori from
Europeans as well as from Turks, Israelis, Bangladeshis, Ladakhis, and Sudanese.
These bacteria were also isolated from the
Americas
and
Australia
, and from whites, blacks, and Cape Coloured in
South Africa
, where they were predominantly associated with whites.
The current global sample is still incomplete, and additional isolates from
large parts of
Asia
and
Africa
and from aboriginal groups around the world will be needed
to determine whether additional populations exist. However, our definition of
seven modern populations and subpopulations provides a solid basis for deducing
the global patterns of spread of H. pylori with their human hosts.
Our attempts to define subpopulations by the same method among the 200 hpEurope
isolates were not successful because of inconsistent clustering (15). We
hypothesized that this inconsistency reflected the complex history of
Europe
, which was populated in several independent waves of
migration (18) of unknown genetic composition (19). We have
therefore developed an approach, the linkage model in STRUCTURE, that can
reconstruct ancestral populations even after substantial genetic hybridization (16).
This approach uses the mosaic ancestry of genomes within breeding species,
assigning individual nucleotides to ancestral populations on the basis of
their linkage to neighboring nucleotides.
Analysis of the global H. pylori sample with the linkage model defined
five ancestral populations (15), which we named ancestral Africa1,
Africa2, EastAsia, Europe1 (AE1), and Europe2 (AE2) (Fig. 1C). H. pylori strains
within modern hpEurope are recombinants between AE1 and AE2 bacteria. No single
isolate possesses more than 80% estimated ancestry from either of these
populations (fig. S1); instead, each genome is a mosaic of multiple small
chromosomal chunks (Fig. 2, F and G; fig. S2). In contrast, the other
populations are more homogeneous.
Despite clear evidence for occasional import (Fig. 2,
C and D), many isolates have derived 85 to 98% of their nucleotides from the
ancestral population (Fig. 2, A and B; fig. S2).
Recombination between populations alters their genetic distances and blurs the
branching order of trees (20). The ability to infer nucleotide pools in
ancestral populations now allows more accurate estimates of ancestral
relationships and evolutionary history. The ancestral population tree (Fig. 1C)
suggests that Africa2 evolved before the other populations split and that AE1
and ancestral
East Asia
diverged from each other most recently. Additional detailed
analyses (15) support these inferences.
Knowledge of ancestral gene pools also allows inferences about gene flow between
populations. The high diversity in hpEurope (Fig. 1A) is due to fusion between
AE1 and AE2. Within our sample, the proportion of AE1 nucleotides is highest in
Finland
,
Estonia
, and Ladakh (Fig. 3A). However, all European isolates also
possess AE2 nucleotides, but only 3 of 17 isolates from Ladakh do so (fig. S1).
Similarly, AE2 nucleotides are most frequent in
Spain
,
Sudan
, and
Israel
, but the isolates from
Sudan
and
Israel
possess lower levels of AE1 than do European isolates. Thus,
AE1 and AE2 probably reached
Europe
from different sources, AE1 primarily from the direction of
central
Asia
and AE2 primarily from the Near East and
North Africa
.
|
Code
|
|
|
Source
|
|
|
|
No.of
isolates assigned to
|
|
|
|
|
|
|
|
|
|
hpAfrica1
|
hpEastAsia
|
|
|
|
Region
|
Country
|
Ethnic
|
Linguistic
|
hpEurope
|
hpAfrica2
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
hspWAfrica hspSAfrica hspMaori
|
hspAmerind
|
hspEAsia
|
|
1
|
East Asia
|
Korea
|
|
Korean
|
|
|
|
|
|
|
11
|
|
2
|
East Asia
|
Singapore
|
|
Sino-Tibetan
|
2
|
|
|
|
|
|
9
|
|
3
|
India
|
Ladakh
|
North Indian
|
Sino-Tibetan
|
17
|
|
|
|
|
|
|
|
4
|
India
|
|
Bangladeshi*
|
Indo-European
|
9
|
|
|
|
|
|
|
|
5
|
Africa
|
South
Africa
|
Black
|
Niger-Congo
|
2
|
7
|
|
17
|
|
|
|
|
6
|
Africa
|
South
Africa
|
White
|
Indo-European
|
10
|
3
|
|
9
|
|
|
|
|
7
|
Africa
|
South
Africa
|
Cape Coloured†
|
Indo-European
|
4
|
|
6
|
25
|
|
|
|
|
8
|
Africa
|
Burkina
Faso
|
|
Niger-Congo
|
|
|
12
|
|
|
|
|
|
9
|
Africa
|
Senegal
|
|
Niger-Congo
|
|
|
5
|
|
|
|
|
|
10
|
Africa
|
Sudan
|
|
Semitic
|
2
|
|
|
|
|
|
|
|
11
|
N.America
|
USA
|
African
American
|
Indo-European
|
3
|
|
10
|
|
|
|
|
|
12
|
N.America
|
USA
|
White
|
Indo-European
|
2
|
|
3
|
|
|
|
|
|
13
|
N.America
|
Canada/USA
|
Inuit
|
Eskimo-Aleut
|
4
|
|
|
|
|
8
|
|
|
14
|
N.America
|
Canada
|
Athabaskan†
|
Na-Dene
|
|
|
|
|
|
4
|
|
|
15
|
S.America
|
Colombia
|
Mestizo
|
Indo-European
|
11
|
|
1
|
|
|
|
|
|
16
|
S.America
|
Colombia
|
Huitoto†
|
Witotoan
|
12
|
|
|
|
|
4
|
|
|
17
|
S.America
|
Venezuela
|
Piaroa†
|
Salivan
|
2
|
|
1
|
|
|
1
|
|
|
18
|
Australasia
|
New
Zealand
|
Polynesian§
|
Austronesian
|
3
|
|
2
|
|
23
|
|
|
|
19
|
Australasia
|
Australia
|
White
|
Indo-European
|
3
|
|
|
|
|
|
|
|
20
|
Europe
|
UK
|
|
Indo-European
|
19
|
|
1
|
|
|
|
1
|
|
21
|
Europe
|
Estonia
|
|
Uralic
|
11
|
|
|
|
|
|
|
|
22
|
Europe
|
Finland
|
|
Uralic
|
9
|
|
|
|
|
|
|
|
23
|
Europe
|
Germany
|
|
Indo-European
|
12
|
|
|
|
|
|
|
|
24
|
Europe
|
Italy
|
|
Indo-European
|
6
|
|
|
|
|
|
|
|
25
|
Europe
|
Spain�
|
|
Indo-European
|
37
|
|
|
|
|
|
|
|
26
|
Europe
|
Germany
|
Turkish
|
Altaic
|
10
|
|
|
|
|
|
|
|
27
|
Near East
|
Israel
|
|
Semitic
|
5
|
|
|
|
|
|
|
|
|
Other¶
|
|
|
|
5
|
|
2
|
|
|
|
5
|
|
Total
|
|
|
|
|
200
|
10
|
43
|
51
|
23
|
17
|
26
|
|
*Isolates from Bangladeshis resident in the
UK
are listed here as being from
India
.
†Speak English but with elements of Khoisan. †Collectively referred to
as Amerinds in the
|
|
|
text. §Polynesian isolates were from 18 Maoris, 8 Samoans, and
2 Tongans in
New
Zealand
.
_Includes two Basque speakers. ¶ “Other” summarizes unique isolates
from
|
|
|
the following sources: hspEastAsia:
Japan
,
China
,
Hong
Kong
,
Thailand
,
and a Japanese from
Peru
;
hpEurope:
France
,
Lithuania
,
Holland
,
Thailand
,
and an Asian in
Cape Town
,
South
|
|
|
Africa
; hspWAfrica:
Gambia
and
Guatemala
.
|
|
| |
|
|
|
|
|
|
|
|
|
|
|
|
Further reconstruction of the history of H. pylori is best done in
the context of current knowledge about human migration. As with a human
population tree (21), hpEurope derives from a short central branch
between hpEastAsia and hpAfrica1 (Fig. 1A), hinting at a parallel history of
intercontinental gene flow to
Europe
for humans and bacteria.
Furthermore, the relative contribution of AE2 versus AE1 correlates
significantly with the first principle component of European human variation
(table S1), which is thought to reflect the entry of neolithic farmers into
Europe
from the
Near East
(20). The second principle component has been
tentatively attributed to the migratory fluxes that brought Uralic languages to
Europe, and indeed correlated weakly with AE1 versus AE2 (r _ 0.6, P _.13)
(table S1). It seems that neither AE1 nor AE2 was harbored by the original
Paleolithic hunter-gatherers in
Europe
, because considerable AE1 or AE2 ancestry is found outside
Europe
, whereas Paleolithic Y-chromosome haplotypes are largely
restricted
to
Europe
(18).
Fig. 2. Ancestral
sources of individual nucleotides in eight selected isolates.The origin of each
polymorphic nucleotide (colors as in Fig. 1C) is shown for each of the eight
gene fragments. The geographical sources of each isolate are shown above each
graph.
Known human migrations can also explain the spread of hpEastAsia and hpAfrica1
populations (Fig. 3B). Current models (22, 23) agree that speakers
of Austronesian languages (Maoris and other Polynesians) arrived in
New Zealand
after sequential island-hopping that is likely to have
resulted in repeated human population bottlenecks. Indeed, consistent with
population bottlenecks, the genetic diversity within the hspMaori sample is
extremely low (Fig. 1), and the pattern of nucleotide polymorphisms
within subpopulations implies that there has been strong drift in the evolution
of the hsp-Maori population (15) (fig. S3). The isolation of hpEastAsia
from Native Americans (7, 8) can be similarly explained by
hpEastAsia’s being carried during the colonization of the
Americas
that began at least 12,000 years ago. Unlike hspMaori,
hspAmerind did not show signs of strong drift, implying that H. pylori accompanied
the ancestors of modern Amerinds and Inuits in large
numbers of individuals and/or was introduced on multiple occasions.
The high degree of similarity between hspWAfrica and hspSAfrica (Fig. 1B, fig.
S3) is concordant with the low genetic distances (20) observed between
speakers of the Niger-Congo family of languages and is consistent with
hspSAfrica’s being carried to Southern Africa during the rapid expansion of
Bantu farmers from central West Africa (24). Given this scenario, one
possibility to account for the extremely distinct hpAfrica2 population is that
they colonized the Khoisan hunter-gatherer inhabitants of
Southern Africa
, who fall on one of the deepest branches of an African human
population tree (20) and are very distinct from Bantu.
Modern migrations of slaves from
West Africa
to the
Americas
and of Europeans to
South Africa
, the
Americas
, and
Australasia
are probably responsible for the current existence of
hspWAfrica and hpEurope in these and other locations (Table 1). According to
this interpretation, the past few centuries since modern human migrations were
too short for the distinctions between multiple bacterial populations to become
blurred. The assignments of particular human migrations
to migrations of H. pylori populations can
allow dating of the bacterial population tree by archaeological events.
The five ancestral populations existed before the separation of hspAmerind from
the other hpEastAsia populations (Fig. 1, B and C), which is estimated to have
occurred at least 12,000 years ago. Accordingly, H.pylori has probably
accompanied anatomically modern humans since their origins.
Fig. 3. Putative
modern and ancient migrations of H. pylori. (A) Average proportion
of ancestral nucleotides
by source.Numbers correspond to the codes in Table 1 and colors are as in
Fig.1C.
( B) Interpretation.Arrows
indicate specific migrations of humans and H. pylori populations.BP,
years before present.
The high sequence diversity in H. pylori allows the recognition of
distinct populations after centuries of coexistence in individual geographic
locations, as demonstrated in the
Americas
and
South Africa
. Even after thousands of years of contact in
Europe
between bacteria introduced by distinct waves of migration,
residual shortrange linkage disequilibrium
has allowed us to identify ancestral chunks of chromosome. Thus, analysis of H.
pylori from
human populations could also help resolve details of
human migrations.
Elucidation of the pattern of population subdivision is also of medical
relevance (25). Geographically variable results regarding the association
of putative virulence factors with disease (26) might well reflect
differences in the local prevalence of the individual H. pylori populations.
Similarly, the development of diagnostic tests, antibiotics,
and vaccines needs to account for global diversity and
will be aided by the availability of representative isolates.
References
and Notes
1.
H.T.Agostini, R.Yanagihara, V.Davis, C.F.Ryschkewitsch, G.L.Stoner, Proc.
Natl. Acad. Sci. U.S.A. 94, 14542
(1997). 2.K. Kremer et al., J. Clin. Microbiol. 37, 2607
(1999). 3. J.M.Musser et al., Rev. Infect. Dis. 12, 75
(1990). 4.M. Achtman et al., Mol. Microbiol.
32, 459 (1999).
5.D.
Kersulyte et al., J. Bacteriol. 182, 3210 (2000). 6.
A.K.Mukhopadhyay et al., J. Bacteriol. 182,
3219 (2000). 7.Y. Yamaoka et al., FEBS Lett. 517, 180
(2002). 8.C. Ghose et al., Proc. Natl.
Acad. Sci. U.S.A. 99,
15107 (2002). 9. R.A.Feldman, in Helicobacter pylori: Molecular and Cellular
Biology, M.Achtman, S.Suerbaum, Eds.(Horizon Scientific,
Wymondham
,
UK
, 2001), pp.29 –51. 10. A.Covacci, J.L.Telford, G.Del
Giudice, J.Parsonnet, R.Rappuoli, Science 284, 1328 (1999). 11.
W.H.Li, L.A.Sadler, Genetics 129, 513 (1991). 12. D.Kersulyte,
H.Chalkauskas, D.E.Berg, Mol. Microbiol. 31, 31 (1999). 13.D.
Falush et al., Proc. Natl. Acad. Sci. U.S.A. 98, 15056
(2001). 14.S. Suerbaum et al., Proc. Natl. Acad. Sci. U.S.A. 95,
12619 (1998). 15.Materials and methods, details of the STRUCTURE analysis, and
analysis of the pattern of divergence between populations are available on Science
Online. 16. D.Falush, M.Stephens, J.K.Pritchard, in preparation; available
at www.mpiib-berlin.mpg.de/str2.pdf.
17. J.K.Pritchard, M.Stephens, P.Donnelly, Genetics 155, 945
(2000). 18.O. Semino et al., Science 290, 1155 (2000). 19.
L.Chikhi, R.A.Nichols, G.Barbujani, M.A.Beaumont, Proc. Natl. Acad. Sci.
U.S.A. 99, 11008 2002). 20. L.L.Cavalli-Sforza, P.Menozzi, A.Piazza, The
History and Geography of Human Genes (Princeton Univ. Press, Princeton, NJ,
1994). 21. E.S.Poloni, L.Excoffier, J.L.Mountain, A.Langaney,
L.L.Cavalli-Sforza, Ann. Hum. Genet. 59, 43 (1995). 22.
J.M.Diamond, Nature 403, 709 (2000). 23. S.Oppenheimer,
M.Richards, Sci. Prog. 84, 157 (2001). 24.C. Ehret, Int. J.
Afr. Hist. Stud. 34, 5 (2001). 25. J.F.Wilson et al., Nature
Genet. 29, 265 (2001). 26. D.Y.Graham, Y.Yamaoka, Helicobacter 5
(suppl.1), S3 (2000). 27.We thank all the colleagues listed in the
supporting online text who have supplied bacterial isolates, DNA, and
information and C.Josenhans for critical reading of the manuscript.Expert
technical assistance was provided by S.Friedrich, A.Wirsing, and E.Bernard.
Supported by grants from the Deutsche Forschungsgemeinschaft (Ac 39/10-3,
SFB479/TP A5), the Bundesministerium fu¨r Bildung und Forschung Pathogenomics
Network, and NIH (RO2GM63270).
Supporting
Online Material
www.sciencemag.org/cgi/content/full/299/5612/1582/DC1
Materials
and Methods
Supporting
Text
Figs.S1
to S3
Tables
S1 and S2
References
26 November 2002
; accepted
16 January 2003
Traces of Human Migrations in Helicobacter pylori Populations