Greek-Pathan E3b

ARTICLE
Y-chromosomal evidence for a limited Greek
contribution to the Pathan population of Pakistan
Sadaf Firasat1, Shagufta Khaliq1, Aisha Mohyuddin1, Myrto Papaioannou2,
Chris Tyler-Smith3, Peter A Underhill4 and Qasim Ayub*,1
1Biomedical and Genetic Engineering Division, Dr. AQ Khan Research Laboratories, Islamabad, Pakistan; 2Unit of
Prenatal Diagnosis, Center for Thalassemia, Laiko General Hospital, Athens, Greece; 3The Wellcome Trust Sanger
Institute, Hinxton, Cambridge, UK; 4Department of Genetics, Stanford University, Stanford, CA, USA
Three Pakistani populations residing in northern Pakistan, the Burusho, Kalash and Pathan claim descent
from Greek soldiers associated with Alexander’s invasion of southwest Asia. Earlier studies have excluded a
substantial Greek genetic input into these populations, but left open the question of a smaller
contribution. We have now typed 90 binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR)
loci mapping to the male-specific portion of the human Y chromosome in 952 males, including 77 Greeks in
order to re-investigate this question. In pairwise comparisons between the Greeks and the three Pakistani
populations using genetic distance measures sensitive to recent events, the lowest distances were
observed between the Greeks and the Pathans. Clade E3b1 lineages, which were frequent in the Greeks
but not in Pakistan, were nevertheless observed in two Pathan individuals, one of whom shared a 16 Y-STR
haplotype with the Greeks. The worldwide distribution of a shortened (9 Y-STR) version of this haplotype,
determined from database information, was concentrated in Macedonia and Greece, suggesting an origin
there. Although based on only a few unrelated descendants, this provides strong evidence for a European
origin for a small proportion of the Pathan Y chromosomes.
European Journal of Human Genetics (2007) 15, 121–126. doi:10.1038/sj.ejhg.5201726; published online 18 October 2006
Keywords: population genetics; Pakistan; Greek; Y chromosome polymorphism
Introduction
Pakistan lies within a region that was invaded by Alexander
the Great in 327–323 BC,1 although archeological evidence
in northern Pakistan suggests that the Greek
influence predates this invasion.2 Many ethnically and
linguistically distinct populations inhabit this region, three
of which (Burusho, Kalash and the Pathan) claim to be
descendents of Greek soldiers who invaded the Indian
subcontinent.3–5 A preliminary study using a limited
number of Y-chromosomal markers found no evidence
for admixture between the Greeks and Burusho or Pathan,
and provided ambiguous evidence of genetic admixture
between the Greek and Kalash populations.6 A subsequent
analysis of autosomal loci gave no indication that the
Kalash or other populations were genetically related to the
Greeks,7 and in another study the Kalash population were
shown as a genetic isolate8 who might therefore have
developed unusual genetic characteristics by drift.9
The genetic material comprising the great bulk of the
patrilinearally inherited human Y-chromosome is effectively
haploid and does not undergo inter-chromosomal
recombination, making it useful for evolutionary studies
and forensic investigations.10 In particular, it can provide
very high resolution haplotypes with known phylogenetic
relationships, and these can reveal low levels of admixture
that would not be detected by other methods. In the
Received 16 November 2005; revised 9 August 2006; accepted 1
September 2006; published online 18 October 2006
*Correspondence: Dr Q Ayub, Biomedical and Genetic Engineering
Division, Dr AQ Khan Research Laboratories, GPO Box 2891, Islamabad
44000, Pakistan. Tel: þ92 51 926 1142; Fax: þ92 51 926 1144;
E-mail:qayub@comsats.net.pk
European Journal of Human Genetics (2007) 15, 121–126
& 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00
www.nature.com/ejhg
present study, we have further investigated the origin and
the genetic relationship of these three Pakistani populations
with the extant Greek population by typing a large
set of markers from the male-specific region of the Y
chromosome in 77 Greeks and 875 Pakistani individuals,
and applying analytical methods that should be more
sensitive to low levels of admixture.
Materials and methods
DNA samples
DNA samples of 952 unrelated males were analyzed in this
study. The DNA was extracted directly from peripheral
blood mononuclear cells in case of the Greek samples
(n¼77) and from EBV-transformed lymphoblastoid cell
lines for the Pakistani samples (n¼875). The Pakistani
samples included Burusho, Kalash and Pathan individuals.
Informed consent was obtained from all participants in
this study.
Genotyping
All samples were typed for 90 binary markers11 following a
phylogenetic hierarchical approach to define evolutionary
Y lineages or haplogroups.10 The markers and the method
used for their genotyping are listed in Table 1. In addition,
16 multiallelic Y-STR (short-tandem-repeat) markers were
analyzed in these populations by three multiplex PCR
reactions12,13 to identify Y haplotypes.
Table 1 List of 90 Y-SNPs typed in this study
Y-SNP Typing method Polymorphism Y-SNP Typing method Polymorphism
M91 DHPLC del T M201 DHPLC G-T
M31 DHPLC G-C M147 DNA sequencing ins T
M6 DHPLC T-C M177 DNA sequencing C-T
PK1 AFLP C-A M143 DHPLC G-T
M32 DHPLC T-C M20 AFLP A-G
M60 DHPLC ins T M11 AFLP A-G
M150 DHPLC C-T M185 DHPLC C-T
M109 DHPLC C-T M27 ARMS-PCR C-G
M152 DHPLC C-T M76 DHPLC T-G
M218 DHPLC C-T M349 DHPLC G-T
RPS4Y AFLP C-T M357 DHPLC C-A
M8 DNA sequencing G-T PK3 ARMS-PCR T-C
M38 DNA sequencing T-G M214 ARMS-PCR A-G
M217 DNA sequencing A-C M175 DNA sequencing del TTCTC
M48 ARMS-PCR A-G M119 DHPLC A-C
PK2 ARMS-PCR T-C M101 DHPLC C-T
YAP PCR Alu ins M50 DHPLC T-C
SRY-8299 AFLP G-A M103 DHPLC C-T
sY81 AFLP A-G M110 DNA sequencing T-C
M35 ARMS-PCR G-C P31 DNA sequencing T-C
M78 ARMS-PCR C-T M88 DNA sequencing A-G
M148 DHPLC A-G M111 DNA sequencing del TT
M123 ARMS-PCR G-A M122 ARMS-PCR T-C
M136 DHPLC C-T L1Y PCR LINE1 ins
M89 ARMS-PCR C-T M134 DHPLC del G
M9 AFLP C-G M117 DHPLC del ATCT
92R7 AFLP C-T M133 DHPLC del T
M45 DHPLC G-A SRY+465 AFLP C-T
M74 DHPLC G-A PK4 DHPLC A-T
M207 ARMS-PCR A-G LLY22g AFLP C-A
M124 ARMS-PCR C-T M231 DHPLC G-A
M173 ARMS-PCR A-C TAT AFLP T-C
M73 DHPLC del GT M70 ARMS-PCR A-C
SRY-2627 AFLP C-T M193 DHPLC ins CAAA
SRY-1532 AFLP A-G-A 12f2 PCR del
M17 ARMS-PCR del G M172 ARMS-PCR T-G
M56 DHPLC A-T M12 DHPLC G-T
M157 DHPLC A-C M67 ARMS-PCR A-T
M87 DHPLC T-C M92 ARMS-PCR T-C
PK5 AFLP C-T M267 ARMS-PCR T-G
M242 ARMS-PCR C-T M62 ARMS-PCR T-C
M25 DHPLC G-C M52 ARMS-PCR A-C
M36 DHPLC T-G M69 DHPLC T-C
M97 DHPLC T-G Apt AFLP G-A
M170 ARMS-PCR A-C M82 DHPLC del AT
Greek ancestry of Pathan population of Pakistan
S Firasat et al
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European Journal of Human Genetics
Data analysis
Population pairwise FST values were estimated by using the
Arlequin package14 based on STR variation within haplogroups
weighted as described previously.6 Population
pairwise r genetic distances were calculated according to
Helgason et al.15 Median-joining networks were constructed
by Network 4.1.1.216 using the following five-fold
range weighting scheme. The weights assigned were
specific for the haplogroup and took into account the
Y-STR variation across the haplogroup in the Pakistani and
Greek populations: variance 0.00–0.14, weight 5; variance
0.15–0.29, weight 4; variance 0.30–0.44, weight 3;
variance 0.45–0.59, weight 2; variance 40.59, weight 1.
Network was also used to estimate the time to the most
recent common ancestor (TMRCA). The r genetic distance
matrix was used to construct phylogenetic tree by
neighbor-joining method17,18 using the MEGA software
package.19
Results
The Y-chromosomal lineages and their frequencies in the
Greeks, Burusho, Kalash, Pathan and the rest of the
Pakistani population are shown in Figure 1. The combination
of biallelic markers identified 12 Y-chromosomal
haplogroups or lineages in the Greeks, 17 in the Burusho
and 15 in the Pathan populations. Only eight Y lineages
were found in the Kalash population. Principal component
analysis of Y haplogroup frequencies incorporating published
data from European20 and West Asian21,22 populations
(Figure 2) revealed that the Pakistani populations
cluster together, separately from the Europeans, consistent
with the previous conclusion that none of the Pakistani
populations had a large male contribution from Greece,
Figure 1 A rooted maximum-parsimony tree of Y lineages found in the Greek, Burusho, Kalash, Pathan and Pakistan. The lineages were defined by
binary markers whose designations and population frequencies are given below each branch. The YCC lineage names10 are shown below the
frequencies. Branch lengths are arbitrary.
Figure 2 A plot of the first two principal coordinates based upon
the analysis of Y haplogroup frequencies in (a) Pakistani and Greek
populations using 33 biallelic markers. (b) Pakistani, Greek and
selected populations from Europe, Africa, Middle East and West Asia.
Greek ancestry of Pathan population of Pakistan
S Firasat et al
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European Journal of Human Genetics
and demonstrating that this conclusion was not an artifact
of the low phylogenetic resolution used before.
The genetic distances between the populations were then
calculated using measures that are more sensitive to recent
events (Table 2). Pakistani–Greek population pairwise FST
values based on the variation of STRs within haplogroups6
ranged from 0.131 to 0.213, with the lowest value between
the Pathan and the Greeks. Pairwise r genetic distances
(the number of steps between a haplotype in one population
and the closest haplotype in the second population,
averaged over all comparisons)15 ranged from 4.3 to 8.1,
with the lowest value again between the Pathan and the
Greeks. Phylogenetic analysis using the matrix of r genetic
distances between populations with tree validation carried
out by bootstrap resampling (10 000 replicates) also
demonstrated that of the three Pakistani populations, the
Pathans were closest to the Greek (Figure 3). These results
therefore suggest that there might have been a low degree
of recent Pathan–Greek admixture. To investigate this
possibility further, we have examined individual lineages.
Clade E lineages were more frequent in the Greeks (21%)
as compared to Pakistan (4%). The majority of haplogroup
E chromosomes belonged to clade E3b and all Greek and
Pakistani samples were resolved into the branches E3b1
(M78) and E3b3 (M123). Among the three Pakistani
populations claiming Greek descent, this clade was observed
only in the Pathans. The Pathan samples belonged
to clade E3b1 that constituted 17% of the Greek samples.
A median-joining network of clade E Y chromosomes
was constructed in order to examine the genetic relationship
between these Greek and Pathan samples. A duplication
of 10 and 13 repeat units was observed in the
clade-E-derived Y chromosomes for the trinucleotide
repeat DYS425 and this locus was, therefore, excluded
from the network. The most striking feature of this
network was the sharing of haplotypes between the Pathan
and Greek samples (Figure 4). One Pathan individual
shared the same Y-STR haplotype with three Greek
individuals, and the other Pathan sample was separated
from this cluster by a single mutation at the DYS436 locus.
This demonstrates a very close relationship between the
Pathan and Greek E lineages, but how surprising is this?
Worldwide data for the 16-element haplotype are not
available, but a subset of nine of the STRs are included in
by the Y-STR Haplotype Reference Database (YHRD)23 and
were used to search this. The haplotype DYS19¼13;
389I¼13; 389II¼30; 390¼24; 391¼10; 392¼11;
393¼13; 438¼10; 439¼12 was found in 53 individuals
in a worldwide population sample of 7897 haplotypes and
was highly specific for the Balkans (Figure 5). The contour
map shows a major concentration around Macedonia and
Greece, with a low scattering in other European countries,
Table 2 Weighted population pairwise r genetic distances
(below diagonal) and FST values (above diagonal)
based on STR variation within haplogroups
Greek Burusho Kalash Pathan
Greek 0.000 0.188 0.213 0.131
Burusho 5.659 0.000 0.214 0.196
Kalash 8.066 3.882 0.000 0.219
Pathan 4.277 2.451 3.254 0.000
Figure 3 Neighbor-joining tree showing the relationship between
the Greek and three Pakistani ethnic groups. The tree is based on r
genetic distances. Bootstrap values from 10 000 replicates are shown.
Figure 4 Median-joining network of clade E lineages in Pakistan
(open circles) and Greece (hatched circles). Circles represent haplotypes
and have an area proportional to frequency. The Pathan
individuals are shown in black.
Figure 5 Contour map showing the 9 Y-STR haplotype frequency
distribution in Eurasia and northern Africa. This haplotype was shared
between three Greeks and a Pathan individual belonging to clade
E3b1.
Greek ancestry of Pathan population of Pakistan
S Firasat et al
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European Journal of Human Genetics
Tunisia, West Africa and the Pathans. This gives a strong
indication of a European, possibly Greek, origin of these
Pathan Y chromosomes.
Discussion
An extensive analysis of Y diversity within Greeks and
three Pakistani populations – the Burusho, Kalash and
Pathan – who claim descent from Greek soldiers allowed
us to compare Y lineages within these populations and
re-evaluate their suggested Greek origins. This study as a
whole seems to exclude a large Greek contribution to any
Pakistani population, confirming previous observations.7
However, it provides strong evidence in support of the
Greek origins for a small proportion of Pathans, as
demonstrated by the clade E network (Figure 4) and
the low pairwise genetic distances between these two
populations.
The Pathans were the only population among the three
that claim Greek ancestry in which clade E was present.
This branch is observed in Europe, Middle East, North and
East Africa with a suggested origin in East Africa.24 Subclade
E3b is common in Europe and probably originated in
Africa.25 Compelling evidence in support of the genetic
relationship between the Pathan and Greek E3b1 Y
chromosomes was provided by the median-joining network
(Figure 4). One Pathan shared a Y-STR haplotype, that
included a duplication of 10 and 13 repeat units for the
DYS425 locus, with three Greek individuals and the other
was separated from this cluster by a single mutation, which
enabled us to estimate the TMRCA (mean7SD) using the
Network software as between 20007400 and 500071200
YBP depending upon the observed26 or inferred mutation
rates,27 respectively. This coincides with the period of
Alexander’s invasion during 327–323 BC. This haplotype
was not observed in any other E3b1-derived Pakistani Y
chromosome but was highly specific for the Balkans – the
highest frequency being in Macedonia.
It is worth emphasizing here that the chance of picking
up rare events largely amplified by drift affecting a limited
portion of the population cannot be discounted, and
Cruciani et al28 also recommend caution when using
microsatellite alleles as surrogates of unique event polymorphisms.
The genetic data alone do not tell us when the
Balkan chromosomes arrived in Pakistan: it is necessary to
turn to the historical record for this. There has been no
known Greek admixture within the last few generations,
but in addition to Alexander’s armies, the possibility of
admixture between the Greek slaves who were brought
to this region by Xerxes around one hundred and fifty
years before Alexander’s arrival, and the local population,
cannot be discounted. At that time Afghanistan
and present day Pakistan were part of the Persian
Empire.1 Nevertheless, Alexander’s army of 25 000–30 000
mercenary foot soldiers from Persia and West Asia and
5000–7000 Macedonian cavalry29 perhaps provides a more
likely explanation because of their elite status and
substantial political impact on the region.
Acknowledgements
We thank the donors for the samples used in this study. This work was
supported by aWellcome Trust Collaborative Research Initiative Grant
to S Qasim Mehdi. CT-S was supported by the Wellcome Trust. We
would like to thank S Siddiqi and A Mansoor for technical assistance
and Professors LL Cavalli-Sforza, SQM and anonymous reviewers for
their helpful comments.
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