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Ancient Genome Prompts Rethink of Timing of Evolutionary Events in Horse Lineage

NEW YORK (GenomeWeb News) – Horses, donkeys, and zebras may have split from a shared common ancestor much earlier than once suspected, a new genome sequencing study suggests.

As they reported online today in Nature, investigators at the University of Copenhagen, BGI-Shenzhen, and elsewhere sequenced genomic DNA from the fossilized remains of a horse believed to have lived in what's now the Yukon Territory of Canada as far back as 700,000 years ago or more.

Together with new genome sequences for a 43,000-year-old Pleistocene horse, a present-day Przewalski's horse, five modern-day domestic horses, and a donkey, the ancient genome prompted researchers to propose divergence within the equid lineage going back four million years or more — about double the time estimated previously.

"This pretty large dataset of horse genomes we can then use to address a number of quite fundamental questions to horse evolution," co-senior author Eske Willerslev, director of the University of Copenhagen's Centre for GeoGenetics, said during a press briefing at the World Conference of Science Journalists in Helsinki this week.

"One is the time of split of the so-called equids, all the horse line animals — the zebras, the donkey, the horses," he said. "And this is something that has been heavily discussed and we actually could recalibrate that to about 4 to 4.5 million years ago, which is about twice as much as was previously thought."

The team's scrutiny of the new equine sequences at hand also verified the notion that the so-called Przewalski's horse belongs to wild horse lineage that split from domestic horses some 38,000 to 72,000 years ago. Moreover, it suggested that there is still a fair bit of genetic variation within that wild cousin of domestic horses, which doesn't show signs of recent mixing with members of the domestic lineage.

"We find similar levels of genetic variation among Przewalski's and domestic populations," Willerslev and co-authors wrote, "indicating that the former are genetically viable and worthy of conservation efforts."

The genome for the domestic horse, Equus ferus caballus, was described in a 2009 Science study published by members of the Horse Genome Project, following an announcement regarding the availability of a draft sequence a couple years earlier.

Such genomic data has made it possible to explore the genetic basis for a range of modern domestic horse traits. But much of what's known about the evolutionary history of the horses and their equine relatives has come from fossils collected over the years.

The horse has long been a "classical, simple example of morphological evolution — how we go from four toes, into three toes, into two toes, into one toe," Willerslev noted. "And now, suddenly, we can actually follow parts of that evolution through genomics."

For their study of horse evolution, Willerslev and his team decided to try their hand at sequencing an extremely old horse fossil found at Thistle Creek, in Canada's Yukon Territory, about a decade ago.

The fossil — believed to belong to a horse from the Middle Pleistocene era that lived some 560,000 to 780,000 years ago — was first subjected to mass spectrometry-based analysis on collagen peptides and peptides from dozens of other proteins.

After those tests showed that the sample was surprisingly well preserved, the researchers used a combination of Illumina and Helicos sequencing to try sequencing genomic DNA from the sample, which is hundreds of thousands of years older than any of the ancient samples previously sequenced.

In the process, they generated enough sequence data from the ancient horse to cover the existing horse reference genome to a depth of just more than 1-fold.

On top of that, the group did genome sequencing on a donkey and horses from the Arabian, Icelandic, Norwegian fjord, Standardbred, and Thoroughbred domestic horse breeds, generating around 8- to 21-fold coverage for each representative, on average.

The sequenced the genomes of a 43,000-year-old (Late Pleistocene era), pre-domesticated horse and a Przewalski's horse from present day to 1.8-fold and nearly 10-fold average coverage, respectively.

Once extinct in the wild, study authors noted, the Przewalski's horse was successfully reintroduced to its native Mongolia and is now classified as an endangered species.

Using this data, the team put the time to the most recent shared common ancestor of wild and domestic horses, donkeys, and zebras at between four and four-and-a-half million years ago. Since then, genetic data suggests horse populations have increased and waned in ways that coincide with environmental conditions over time.

"The whole population history of the horse is heavily driven by climactic changes: basically, when it's fairly cold, it's good to be a horse," Willerslev said. "When it's warm, it's pretty bad."

The data offered a peek at more recent splits in the equine lineage as well. For instance, the researchers estimated that the lineages leading to Przewalski's horse and the domestic horse diverged some 50,000 years ago.

And given the genetic patterns found in Przewalski's horse, the team verified that it is "truly wild," Willerslev said. "There are not domestic genetics present in that horse."

The collection of horse sequences also made it possible to take a look at the genes that have been subject to selection during horse evolution. In the Thistle Creek horse samples, for instance, the researchers saw signs of selection on genes contributing to the ability to smell and immune system function, Willerslev noted, while genes related to muscle compactness seem to have shifted during horse domestication.

The Przewalski's horse genome houses its own genetic variants, study authors found, which fall in sequences that may have been subject to selection in that lineage since it split from the equine branch leading to domestic horses.