Origin and Diffusion of mtDNA Haplogroup X

http://www.journals.uchicago.edu/AJHG/journal/issues/v73n5/40218/40218.text.html

Origin and Diffusion of mtDNA Haplogroup X 

Maere Reidla,1 Toomas Kivisild,1 Ene Metspalu,1 Katrin Kaldma,1
Kristiina Tambets,1 Helle-Viivi Tolk,1 Jüri Parik,1 Eva-Liis
Loogväli,1 Miroslava Derenko,2 Boris Malyarchuk,2 Marina
Bermisheva,1,3 Sergey Zhadanov,1,4 Erwan Pennarun,1,5 Marina
Gubina,1,4 Maria Golubenko,1,6 Larisa Damba,1,4 Sardana Fedorova,1,7
Vladislava Gusar,1,8 Elena Grechanina,8 Ilia Mikerezi,9 Jean-Paul
Moisan,5 André Chaventré,5 Elsa Khusnutdinova,3 Ludmila Osipova,4
Vadim Stepanov,6 Mikhail Voevoda,4 Alessandro Achilli,10 Chiara
Rengo,10 Olga Rickards,11 Gian Franco De Stefano,11 Surinder Papiha,12
Lars Beckman,13 Branka Janicijevic,14 Pavao Rudan,14 Nicholas
Anagnou,15 Emmanuel Michalodimitrakis,16 Slawomir Koziel,17 Esien
Usanga,18 Tarekegn Geberhiwot,19 Corinna Herrnstadt,20 Neil Howell,20
Antonio Torroni,10 and Richard Villems1

1Department of Evolutionary Biology, Institute of Molecular and Cell
Biology, Tartu University and Estonian Biocentre, Tartu, Estonia;
2Genetic Laboratory, Institute of Biological Problems of the North,
Magadan, Russia; 3Institute of Biochemistry and Genetics, Ufa Research
Center, Russian Academy of Sciences, Ufa, Russia; 4Institute of
Cytology and Genetics, Siberian Branch of the Russian Academy of
Sciences, Novosibirsk, Russia; 5Laboratoire d'Etude du Polymorphisme
de l'ADN, Faculté de Médecine, Nantes, France; 6Institute of Medical
Genetics, Siberian Branch of the Russian Academy of Medical Sciences,
Tomsk, Russia; 7Yakut Scientific Center, Russian Academy of Medical
Sciences, and Government of Republic Sakha (Yakutia), Yakutsk, Russia;
8Center of Clinical Genetics and Prenatal Diagnostics, Kharkov,
Ukraine; 9Department of Biology, Faculty of Natural Sciences, Tirana
University, Tirana, Albania; 10Dipartimento di Genetica e
Microbiologia, Università di Pavia, Pavia, Italy; 11Dipartimento di
Biologia, Università "Tor Vergata," Rome; 12Department of Human
Genetics, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne;
13Gotland University, Visby, Sweden; 14Institute for Anthropological
Research, Zagreb, Croatia; 15Institute of Molecular Biology and
Biotechnology and Department of Basic Sciences and 16Department of
Forensic Sciences and Toxicology, University of Crete School of
Medicine, Heraklion, Greece; 17Institute of Anthropology, Wroclaw,
Poland; 18Department of Haematology, University of Calabar, Calabar,
Nigeria; 19Birmingham and Solihull Teaching Hospital, Birmingham; and
20MitoKor, San Diego, CA

Received June 11, 2003; accepted for publication August 27, 2003;
electronically published October 20, 2003.

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA)
sequences belonging to haplogroup X and the survey of the
haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and
Africa revealed that haplogroup X is subdivided into two major
branches, here defined as "X1" and "X2." The first is restricted to
the populations of North and East Africa and the Near East, whereas X2
encompasses all X mtDNAs from Europe, western and Central Asia,
Siberia, and the great majority of the Near East, as well as some
North African samples. Subhaplogroup X1 diversity indicates an early
coalescence time, whereas X2 has apparently undergone a more recent
population expansion in Eurasia, most likely around or after the last
glacial maximum. It is notable that X2 includes the two complete
Native American X sequences that constitute the distinctive X2a clade,
a clade that lacks close relatives in the entire Old World, including
Siberia. The position of X2a in the phylogenetic tree suggests an
early split from the other X2 clades, likely at the very beginning of
their expansion and spread from the Near East.



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Address for correspondence and reprints: Dr. Maere Reidla, Department
of Evolutionary Biology, Tartu University, Estonian Biocentre, Riia
23, Tartu, 51010, Estonia. E-mail: [EMAIL PROTECTED]
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     mtDNA and the nonrecombining part of the Y chromosome are widely
used in archaeogenetic studies (Renfrew 2000; Cavalli-Sforza and
Feldman 2003) that aim to reveal the human past. The uniparental
inheritance and complete linkage of mutations in these two loci allow
an unambiguous determination of the phylogenetic relationships between
individual lineages. However, the putative genetic histories of the
lineages that are obtained do not fully reflect the complex dynamics
of ancient populations; thus, the data must be interpreted carefully.
Phylogenetic clustering of mtDNA haplogroups has been found to be
congruent with geographythere are haplogroups specific to African
(Chen et al. 1995; Watson et al. 1997), Asian (Ballinger et al. 1992;
Torroni et al. 1994; Kivisild et al. 2002), European/West Eurasian
(Torroni et al. 1996; Macaulay et al. 1999), and Native American
(Torroni et al. 1993) populations.

     Haplogroup X is an exception to this pattern of limited
geographical distribution. It is found, generally at low frequencies,
in both West Eurasians (Richards et al. 2000) and some northern groups
of Native Americans (Ward et al. 1991; Forster et al. 1996; Scozzari
et al. 1997; Brown et al. 1998; Smith et al. 1999; Malhi et al. 2001),
but, intriguingly, it is absent in modern north Siberian and East
Asian populations (Brown et al. 1998; Starikovskaya et al. 1998;
Schurr et al. 1999), which are genetically and geographically closest
to those of Native Americans. Among Siberians, haplogroup X mtDNAs
have only been detected in some Altaian populations of southwestern
Siberia (Derenko et al. 2001).

     When the sequence variation of the first hypervariable segment
(HVS-I) of the control region is analyzed, haplogroup X mtDNAs from
Europe and the Near East are found to yield similar coalescence times:
17,00030,000 years before present (YBP) and 13,70026,600 YBP,
respectively (Richards et al. 2000). These estimates are consistent
with a pre-Holocene origin and spread of this haplogroup into West
Eurasia. For Native Americans, the relatively old presence of
haplogroup X is confirmed by the analysis of ancient human remains
(Stone and Stoneking 1999; Malhi and Smith 2002). Moreover, Native
American haplogroup X mtDNAs form a clade distinct from that of West
Eurasians and with coalescence time estimates varying widely depending
on both the method of estimation and the number of assumed founders.
Thus, the coalescence times ranged from 12,00017,000 YBP to
23,00036,000 YBP, times that are consistent with both a pre- and a
postglacial population diffusion (Brown et al. 1998).

     To obtain further information about the extent of haplogroup X
diversity, 5 mtDNAs (from 1 Druze, 1 Estonian, 2 Georgians, and 1
Omani) were completely sequenced and were compared with 16 previously
published X sequences (fig. 1). These latter sequences included the 11
haplogroup X coding sequences published by Herrnstadt et al. (2002)
that have now been completed with the sequencing of the control
region.

A maximum parsimony tree of the 21 haplogroup X sequences revealed
that one nonsynonymous (13966) and three synonymous (6221, 6371, and
14470) substitutions in the coding region, as well as three
transitions in the control region (153, 16189, and 16278), distinguish
haplogroup X from the root of superhaplogroup N. Moreover, haplogroup
X is subdivided into two major subhaplogroups, designated "X1" and
"X2." Subhaplogroup X1, represented by a single Druze mtDNA in figure
1, differs from the root of haplogroup X by eight coding and three
control region transitions and lacks the two transitions (195 and
1719) that characterize X2. These two nucleotides are rather mutable
(Finnilä et al. 2001; Herrnstadt et al. 2002); thus, it cannot be
completely ruled out that X1 is indeed a subset of X2 that reverted at
both nucleotide positions. However, this possibility appears very
unlikely, especially when one considers the time depth and the
distinct geographic distribution of X1 (see below).

     In contrast with X1, X2 is well represented in the tree, and it
is further subdivided into at least six major clades (X2aX2f), which
include clades X1 and X2 as defined by Herrnstadt et al. (2002). All
20 X2 sequences, including the 2 Native American X2a sequences, share
transitions at nucleotide positions (nps) 195 and 1719. A recurrence
of the nonsynonymous substitution at 13708 was observed (clades X2b
and X2d). In addition, the nonsynonymous transition 13966 was found to
have reverted in the Moroccan X2b sequence. These were the only
recurrent mutations found among the 67 variable positions in the
coding region sequences. The ratio of nonsynonymous to synonymous
substitutions was 0.40 (18/45). Six mutations were located in RNA
genes.

     The data obtained from the analyses of complete mtDNA sequences
belonging to haplogroup X were then used to survey 13,589 mtDNAs
(21,682 when mtDNA data from the literature are included) from 66
populations of Eurasia and North Africa (table 1). A total of 175
mtDNAs were found to harbor the four coding region transitionsat 6221,
6371, 13966, and 14470that define haplogroup X. The four markers were
always found in association and, by combination of the control and
coding variation, all 175 mtDNAs could be apportioned to
subhaplogroups X1 and X2 (table 2). No other main branches occur, and
the root haplotype was not present among the sequences. The adenine at
np 153 appears relatively conserved in the phylogenetic context of 376
complete human mtDNA sequences taken worldwide, exhibiting a change to
guanine only in haplogroups A and X (Ingman et al. 2000; Finnilä et
al. 2001; Maca-Meyer et al. 2001; Derbeneva et al. 2002; Mishmar et
al. 2003). In contrast, this position shows a high level of variation
in the background of haplogroup X as both the 153A and 153G alleles
are present in its different subclades. It is possible that the AG
transition at 153 arose only once in the haplogroup X ancestor and the
recurrent reverse mutations in 11 branches in figure 2 bear witness to
the process of favored fixation of the more stable A allele.

 Subhaplogroup X1 was found to be largely restricted to the
Afro-Asiaticspeaking populations of North Africa and neighboring
areas, including Ethiopia, suggesting a possible geographic diffusion
of X1 alongside the Mediterranean Sea and the Red Sea (table 1). This
subhaplogroup is subdivided into the two clades X1a and X1b, which are
defined by two and five coding region mutations, respectively (fig.
2). Both clades share a recurrent transition at 146 in HVS-II. The
coalescence time of the entire X1 subhaplogroup using HVS-I variation
is 42,900 ± 18,100 YBP, whereas the coalescence time of the X1a clade
is 17,900 ± 11,900 YBP.

     Virtually all (97.2%) haplogroup X mtDNAs from the Near East, the
South Caucasus, and Europe were found to belong to subhaplogroup X2,
as did all (100%) of those from Siberia and Central Asia and some
(36.8%) of those from North Africa (table 2). Thus, subhaplogroup X2
is characterized by a very wide geographic range but also by an
infrequent occurrence. Indeed, it generally comprises <5% of the
mtDNAs in West Eurasian and North African populations (table 1). Three
exceptions include the Druze, the Georgians, and the Orkney Islanders,
among whom the frequency of X2 reaches 11%, 8%, and 7%, respectively.
The high frequencies of X2 in the Druze and the Orkney Islanders are
combined with a low haplotype diversity (0.400 and 0.473,
respectively), and the relatively high frequency in these populations
is most likely due to genetic drift and founder events. Overall, it
appears that the populations of the Near East, the Caucasus, and
Mediterranean Europe harbor subhaplogroup X2 at higher frequencies
than those of northern and northeastern Europe (P < .05) and that X2
is rare in Eastern European as well as Central Asian, Siberian, and
Indian populations and is virtually absent in the Finno-Ugric and
Turkic-speaking people of the Volga-Ural region. Coalescence time
estimates based on HVS-I and coding region variation17,900 ± 2,900 YBP
and 21,600 ± 4,000 YBP, respectively (figs. 1 and 2)are consistent
with the range expansion of X2 around or after the last glacial
maximum (LGM). It is intriguing that the estimated coalescence time
for X2 alone is very close to that obtained from HVS-I data for the
entire haplogroup X (20,200 ± 3,100 YBP) (fig. 2). However, the latter
is probably an underestimate due to both the higher proportion (>90%)
of X2 mtDNAs included in the calculations and the fact that the HVS-I
consensus sequence of X2 is completely identical to that of the
overall haplogroup X.

     Two-thirds of the subhaplogroup X2 sequences fall into the five
clades X2bX2f (fig. 2). Two sequencesone from Lebanon and one from
Georgialacked the transition at np 1719, suggesting either the
presence of an early X2 branch or a reversion at that nucleotide
position. The sister groups X2b and X2c (X1 and X2, respectively, in
the work of Herrnstadt et al. 2002) encompass one-third of the
European sequences (excluding the samples from the North Caucasus). It
is of interest that some North African sequences (from Morocco and
Algeria) belong to X2b as well. Subhaplogroup X2b shows a diversity
that is consistent with a postglacial population expansion in both
West Eurasia and North Africa. Clades X2e and X2f encompass the
majority (87.1%) of the sequences from the South Caucasus area and
show coalescence times (12,000 ± 4,000 YBP and 10,800 ± 5,000 YBP,
respectively) consistent with a Late Upper Paleolithic (LUP) origin
and a subsequent spread in the region. We found significant
differences between the haplogroup distribution between the North and
the South Caucasian samples, a result that indicates a major
geographical barrier between the two regions. The South Caucasian
sample is enriched in mtDNAs belonging to clades X2e and X2f (P <
.01), whereas the North Caucasian sample shows a higher proportion of
sequences derived at nps 225 and 16248 (P < .01).

     Clade X2e, defined by the synonymous substitution at 15310,
encompasses all haplogroup X sequences in the Altaians (fig. 2). Among
the nine Altaian X sequences, eight harbor the founder HVS-I motif of
X2e, and seven of these eight also carry the HVS-II founder motif. As
a result, a very low haplotype diversity of haplogroup X (0) in the
Altaian region was obtained, making it significantly different from
the haplotype diversities for haplogroups C and D (0.835 and 0.943,
respectively) in the same region. Moreover, the nine Altaian mtDNAs do
not harbor any nucleotide difference between nps 16090 and 16365.
Therefore, under the assumption that these sequences are a random
sample of the Altaian haplogroup X, an estimated  value <0.33 (P <
.05) was obtained. This value corresponds to a time depth of <6,700
years (Forster et al. 1996), and it would suggest that Altaians have
acquired haplogroup X2 only relatively recently. This scenario is
supported by the concomitant presence in the Altaians of a wide range
of other West Eurasian haplogroups (H, J, I, T, U1, U4, and U5) that
comprise 27% of their mtDNAs. Indeed, any recent migration (for
example, from the [southern] Caucasus region) that might have carried
X2e mtDNA sequences to the Altai region would also explain the
presence of the other West Eurasian mtDNA haplogroups in modern
Altaians.

     Further northeast of the Altai area, haplogroup X sequences were
detected in the Tungusic-speaking Evenks, of the Podkamennaya Tunguska
basin (Central Siberia). In contrast to the Altaians, the Evenks did
not harbor any West Eurasian mtDNA haplogroups other than X. However,
neither of the two Evenk X haplotypes showed mutations characteristic
of the Native American clade X2a. Instead, one sequence was a member
of X2b and the other of X2* (fig. 2). Thus, one possible scenario is
that several X haplotypes arrived in Siberia from western Asia during
the Palaeolithic, but only X2a crossed Beringia and survived in modern
Native Americans. Alternatively, the presence of two phylogenetically
different haplogroup X mtDNA sequences in this particular
subpopulation of Evenks might be due to recent gene flow.

     The Native Americanspecific clade X2a appears to be defined by
five mutations, three in the coding region (8913, 12397, and 14502)
and two in the control region (200 and 16213) (fig. 1). The transition
at np 200 was seen in virtually all previously analyzed Native
American haplogroup X mtDNAs, whereas the transition at np 16213 was
absent in some of the Ojibwa described by Brown et al. (1998). We
surveyed our Old World haplogroup X mtDNAs for the five diagnostic X2a
mutations (table 2) and found a match only for the transition at np
12397 in a single X2* sequence from Iran. In a parsimony tree, this
Iranian mtDNA would share a common ancestor with the Native American
clade (fig. 2). Yet, the nonsynonymous substitution at np 12397
converting threonine to alanine cannot be regarded a conservative
marker, as it has also been observed in two different phylogenetic
contextsin haplogroups J1 and L3eamong 794 complete mtDNA sequences
(Finnilä et al. 2001; Maca-Meyer et al. 2001; Herrnstadt et al. 2002).
Therefore, the scenario that the threonine to alanine change in the
haplogroup X background is indeed due to recurrence appears most
plausible.

     These findings leave unanswered the question of the geographic
source of Native American X2a in the Old World, although our analysis
provides new clues about the time of the arrival of haplogroup X in
the Americas. Indeed, if we assume that the two complete Native
American X sequences (from one Navajo and one Ojibwa) began to diverge
while their common ancestor was already in the Americas, we obtain a
coalescence time of 18,000 ± 6,800 YBP, implying an arrival time not
later than 11,000 YBP.

     The results of this study point to the following conclusions.
First, haplogroup X variation is completely captured by two ancient
clades that display distinctive phylogeographic patternsX1 is largely
restricted to North and East Africa, whereas X2 is spread widely
throughout West Eurasia. Second, it is apparent that the Native
American haplogroup X mtDNAs derive from X2 by a unique combination of
five mutations. Third, the few Altaian (Derenko et al. 2001) and
Siberian haplogroup X lineages are not related to the Native American
cluster, and they are more likely explained by recent gene flow from
Europe or from West Asia. Fourth, the split between "African" X1 and
"Eurasian" X2 subhaplogroups of X is phylogenetically as deep as that
within the branches of haplogroup U that also differ profoundly in
their phylogeography. Thus, subhaplogroup U6 is largely restricted to
North Africa (as X1), whereas subhaplogroup U5 is widespread in West
Eurasia (as X2). The phylogeographic patterns and the coalescence
times that we obtained here suggest that the basic phylogenetic
structures of the mtDNA haplogroups in West Eurasia and North Africa
are as ancient as the beginning of the spread of anatomically modern
humans in this region. Finally, phylogeography of the subclades of
haplogroup X suggests that the Near East is the likely geographical
source for the spread of subhaplogroup X2, and the associated
population dispersal occurred around, or after, the LGM when the
climate ameliorated. The presence of a daughter clade in northern
Native Americans testifies to the range of this population expansion.