A fragment of a fossilised bone thought to be more than 700,000 years old has yielded the genome of an ancient relative of modern-day horses. This predates all previous ancient DNA sequences by more than 500,000 years. The bone was found preserved in Canadian permafrost following the animal's demise. The ancestor of all equines existed around four million years ago. A remnant of the long bone of an ancient horse was recovered from the Thistle Creek site, located in the west-central Yukon Territory of Canada.
Palaeontologists estimated that the horse had last roamed the region sometime between a half to three-quarters of a million years ago. An initial analysis of the bone showed that despite previous periods of thawing during inter-glacial warm periods, it still harboured biological materials, connective tissue and blood-clotting proteins, which are normally absent from this type of ancient material. "You would be amazed how much material of this kind is actually out there... museums are full of fossil material from all over the planet”, says Keith Dobney from University of Aberdeen. "We were really excited because it meant that the preservation was really good," he said. "So at that stage we thought, let's try a DNA extraction to see how much of the genome we could characterise."
The multi-national team of researchers pulverised a fragment of the bone to recover its DNA, then subjected it to high-throughput, next-generation gene sequencing to unravel the blueprint of this antediluvian mount. The first approach they tried resulted in relatively poor yields of horse-derived sequences, so they turned to a technology that could directly analyse single molecules of DNA. This proved far more successful, but they still had an abundance of data to plough through.
Using high-powered computers and an existing horse genome sequence as a reference, the scientists sifted through the 12 billion sequencing reads to distinguish between DNA motifs belonging to the ancient horse and those from contaminating organisms, such as bacteria accumulated from the environment. Przewalski's horse, once extinct in the wild, is viewed as the only remaining truly wild horse. From the resulting equine DNA fragments, they reconstructed a draft of its genome. Although the derived sequence data only covered around 70% of the entire genome, this was sufficient foundation for some revealing analyses.
The tell-tale presence of Y chromosome markers showed that the Thistle Creek bone had belonged to a male. But the DNA also enabled them to reconstruct the evolutionary history of the larger Equus genus, which includes modern-day horses and zebras. The scientists also determined the DNA sequence of a donkey, an ancient pre-domestication horse dating back around 43,000 years, five modern horses and a Przewalski's horse, which possibly represents the last surviving truly wild horse population.
Family trees, based on similarity of the DNA sequences, revealed the relationships between these equine stable-mates and their longer evolutionary history. The Thistle Creek genome was reassuringly ancestral to the modern horses, positioned as it was at the base of the tree. Geological dating evidence meant that the researchers could calibrate the rate of evolution in the different branches, and from this look back into the depths of the tree to approximate the age of the Equus genus ancestor, the forerunner to the donkey, zebra and horse.
DNA was extracted from pieces of the ancient bone. The results suggested it grazed the grasslands between 4 and 4.5 million years ago, twice as long ago as most previous estimates. Through surveying sequence diversity in a larger number of domestic and Przewalki's horse samples, by looking in the genes for what are known as single nucleotide polymorphisms or SNPs, past population sizes could be modelled. Over the last two million years horses had experienced significant population expansions and collapses associated with climatic changes, and one collapse coincided with the date when the Thistle Creek and modern horses diverged.
The location of the genetic differences between the ancient and modern horses also provided tantalising clues into some of the possible consequences of these genetic differences, as Dr Orlando explained. "Once you have the genome, one thing you can do is to actually look at different genes that we know today are important for different traits. What we've learned for example the alleles that prime to the racing performance in domestics were not present at that time, for example."
Commenting on the wider implications, Eske Willerslev of the University of Copenhagen said, "Pushing back the time barrier is important because it has implications for our evolutionary understanding of anything from hominins to other animals, because we can look further back in time than people have done previously." Palaeoecologist Keith Dobney from the University of Aberdeen echoed the sentiment. "There were many things we said wouldn't be possible in ancient DNA [studies] not that long ago, until next generation sequencing came along and all of a sudden everything has changed, and I mean everything," he said.
Modern sequencing approaches and better fossil specimens will allow scientists to gaze further and further back into the mists of ancient evolution. Prof Dobney said that procuring samples for future studies should not be a problem. "You would be amazed how much material of this kind is actually out there. Museums are full of fossil material from all over the planet, caves are fantastic stable environments for preservation and some of the best preserved DNA has come out of cave deposits," he said.
"Even if you look at the Przewalski horse, which has a divergence time of only about 50,000 years ago... and compare it to the domestic horse, you can already see differences," observed Prof Willerslev. "I would definitely say it would not look like a horse as we know it… but we would expect it to be a one-toed horse."