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Genetic Study Points to Ancient Split for Isolated Population in Pakistan

NEW YORK (GenomeWeb) – Pakistan's Kalash population appears to have been isolated from other populations for thousands of years, according to work led by researchers from the Wellcome Trust Sanger Institute and the University of Trieste.

As they reported today in the American Journal of Human Genetics, the researchers did array-based genotyping on nearly two-dozen Kalash men, along with whole-genome sequencing on one of the individuals. Through comparisons with other populations, they found evidence of ancient divergence between the Kalash and populations in South Asia and other parts of the world.

"The Kalash were actually an ancient genetic isolate that split more than 8,000 to 12,000 years ago — depending on what analysis you do — from the other South Asian and European populations," Sanger researcher Qasim Ayub, co-first author on the study, told GenomeWeb.

"Since the split, we found that they've been pretty isolated and have experienced no admixture from the neighboring populations in Pakistan or from any other Eurasian populations," he added.

The Kalash population resides in isolated mountain valley communities in the Hindu Kush mountain range in what is now northwestern Pakistan. The group has long been recognized for its unique ethnic, linguistic, and cultural features, Ayub explained, and some scholars have claimed the group is descended from Alexander III's Greek army.

A prior analysis of microsatellite makers by Human Genome Diversity Panel project members suggested that the Kalash clustered independently from African, East Asian, Oceanic, Native American, and Eurasian/Middle Eastern populations.

Such differentiation was initially attributed to isolation and genetic drift, Ayub noted, though subsequent analyses indicated that the Kalash also carry Y chromosome lineages not found in other populations in Pakistan.

Using the Illumina HumanOmni2.5M-8 BeadChip arrays, the researchers genotyped 23 male individuals residing in three valleys in the Hindu Kush mountain area. These included 10 samples collected for the HGDP, along with another 13 samples obtained by the study's authors between 1994 and 2000.

In addition, they did genome sequencing on one of these Kalash men with the Illumina HiSeq 2000, producing sequences that cover the human genome to an average depth of 30-fold.

The team then compared genetic patterns in the Kalash with those found in individuals from 35 present-day populations in Africa, Asia, Europe, and the Caucasus.

It also looked at their relationship to three ancient individuals sequenced for other studies: the Tyrolean Iceman (Ötzi), who was a European Neolithic farmer; a Mesolithic European hunter-gatherer known as the La Braña 1 individual; and a Paleolithic hunter-gatherer from Siberia, called the Mal'ta boy.

Both the full genome sequence and the multiple Kalash SNP profiles pointed to a long separation from other populations, diverging at least 6,000-years-ago and perhaps as far back as 12,000 years.

The latter finding contradicts results from an earlier study that claimed to see relatively recent genetic evidence for admixture with Eurasians — patterns that appeared to support the possibility of Kalash ancestry from Alexander III's Greek army, which invaded the Indian subcontinent some 326 years before the Common Era.

The researchers have not yet attempted to model the early human migrations that may have brought the Kalash ancestors to present-day Pakistan.

It's generally believed that the same populations that moved from Central Asia to Europe also migrated into the Indian subcontinent and replaced indigenous populations there, Ayub said. But he and his colleagues are hesitant to draw too many conclusions based on a single complete Kalash genome.

Sequences from that first genome sequence suggest the Kalash are related to the Mal'ta boy, a Paleolithic hunter-gatherer from a population believed to be among the European ancestral groups.

A study published in Nature earlier this year indicated that Siberia's Mal'ta boy also shared ancestry with a group of Eastern European hunter-gatherers that was ancestral to a Yamnaya steppe herder population, which in turn contributed substantially to Europe's current population structure.

Given such clues, Ayub noted, "we assume that the same populations that migrated westward into Europe may have migrated down into Afghanistan and down into present-day Pakistan."

Researchers at the Sanger Institute are gearing up to sequence 25 additional Kalash individuals using samples collected for the HGDP.

Those sequences are expected provide new details about Kalash demography and population history, Ayub said, as well as information on rare variants and adaptations that are present in the population.

For example, results from the current study indicated that the Kalash carry ancestral alleles at previously described lactase persistence variants, meaning they should not be expected to digest milk.

But those genetic patterns belie the pastoral nature of the population, which practice goat herding and celebrate an annual "milk day" celebration, suggesting future studies of the population might find new lactose tolerance alleles in the population.