Researchers have excluded a large Greek contribution to any Pakistani population but provide strong evidence in support of the Greek origins for a small proportion of Pathans.

The study was conducted by researchers from the Biomedical and Genetic Engineering Division of the Dr AQ Khan Research Laboratories, Pakistan, Unit of Prenatal Diagnosis at the Laiko General Hospital, Greece, The Wellcome Trust Sanger Institute, UK and the Department of Genetics at Stanford University, US, and was published in the European Journal of Human Genetics (2007) as ‘Y-chromosomal evidence for a limited Greek contribution to the Pathan population of Pakistan’.

It investigated the origin and the genetic relationship of these three Pakistani populations with the extant Greek population. The research was done by typing a large set of markers from the male-specific region of the Y-chromosome in 77 Greeks and 875 Pakistani individuals.

The DNA samples of 952 unrelated males were analysed, extracted directly from peripheral blood mononuclear cells in the case of the Greek samples and from the EBV-transformed lymphoblastoid cell lines for the Pakistani samples. The Pakistani samples included Burusho, Kalash and Pathan individuals whose informed consent was obtained.

Values were estimated based on STR variation within haplogroups. Population pairwise genetic distances (the number of steps between a haplotype in one population and the closest haplotype in the second population, averaged over all comparisons) were calculated and median-joining networks were constructed using a five-fold range weighting scheme whereby weights assigned were specific for the haplogroup and took into account the Y-STR variation across the haplogroup in the Pakistani and Greek populations.

The network was also used to estimate the time to the most recent common ancestor (TMRCA) and a genetic distance matrix was used to construct phylogenetic tree by neighbor-joining method.

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, while only eight Y lineages were found in the Kalash population. Principal component analysis of Y haplogroup frequencies incorporating published data from European and West Asian populations revealed that the Pakistani populations cluster together and separately from the Europeans, which is consistent with the study’s earlier conclusion.

The genetic distances between the populations were calculated using measures that were more sensitive to recent events. Pakistani-Greek population pairwise values based on the variation of STRs within haplogroups ranged from 0.131 to 0.213, with the lowest value between the Pathan and the Greeks. Pairwise genetic distances ranged from 4.3 to 8.1, with the lowest value again between the Pathan and the Greeks. Phylogenetic analysis using the matrix of genetic distances between populations with tree validation also demonstrated that of the three Pakistani populations, the Pathans were closest to the Greek. These results therefore suggest that there might have been a low degree of recent Pathan-Greek admixture.

To investigate this possibility further, the researchers 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. 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 demonstrated a very close relationship between the Pathan and Greek E lineages.

What gives a strong indication of a European, possibly Greek, origin of these Pathan Y-chromosomes is the map shown in the figure, which shows a major concentration around Macedonia and Greece, with a low scattering in other European countries, Tunisia, West Africa and the Pathans.

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, the Middle East, North and East Africa with a suggested origin in East Africa. Sub-clade E3b is common in Europe and probably originated in Africa. Compelling evidence in support of the genetic relationship between the Pathan and Greek E3b1 Y chromosomes was provided by the median-joining network. 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.

According to the analysis in the study, none of the Pakistani populations, except Pathans, had a large male contribution from Greece. It is worth emphasising that the chances of picking up rare events largely amplified by drift affecting a limited portion of the population cannot be discounted. The genetic data alone do not tell us when the Balkan chromosomes arrived in Pakistan and 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 must also be taken into account as well as the fact that at that time, Afghanistan and present day Pakistan were part of the Persian Empire.

Therefore, keeping in mind the analysis and observations done by the researchers and the results that they arrived at, it can be concluded that of the three nations - Burusho, Kalash and Pathan - only Pathans can be concluded to be descenders of the Greek.