By piecing together the genetic information locked inside a frozen, fossilized bone, scientists have deciphered the complete genome of an extinct prehistoric horse that roamed the Yukon more than 700,000 years ago. The work rewrites the evolutionary history of the horse and smashes the previous record for the oldest complete genome ever sequenced. In doing so, it redefines how far back in time scientists can travel using DNA sequences as their guide.

Every time a cowboy throws a leg over the saddle and gallops off on his horse, he's riding on top of 4 million years of evolutionary history. But this history is mostly a mystery. We know surprisingly little about how natural selection and thousands of years of selective breeding by humans have shaped these animals on the genetic scale.

Horses were once considered a textbook example for the smooth transition of one species into another, a perfect illustration of Darwin’s theories. Ancient equine species – dog-sized animals with five toes – gradually evolved into towering, hooved thoroughbreds. Or so the story went. But with every fossil that was unearthed, a more tangled picture emerged.

Then DNA sequencing came along, allowing scientists to reconstruct how organisms change over time down to the resolution of single letters in the DNA code.

In the new study, a multinational team of scientists led by Ludovic Orlando and Eske Willerslev at the University of Copenhagen used what's become a common approach: comparing the DNA of modern species to DNA recovered from fossil remains, in this case a fossil bone fragment found near Thistle Creek, Canada. By pushing DNA sequencing technology to its limits, they were able to rewind the evolutionary clock back further than ever before.

, from the Klondike region, Yukon. Credit: D.G. Froese

The previous record for oldest genome was an 80,000-year-old ancient cousin of humans whose genome was sequenced from a single finger bone found in Siberia. The Thistle Creek horse appears to be nearly ten times as old, which provided new challenges for the scientists. DNA sequencing technology is constantly improving, but the information that researchers get in the end is only as good as the DNA that they start with. And that’s where scientists like Orlando are fighting a losing battle against nature.

Recent technological advances, several developed solely for this work, allowed the horse genome wranglers to read their DNA sequences with as little as a single molecule of starting material. And beefed up computing power meant they could rebuild genomes stretching billions of bases from chunks as small as 25 individual letters. “It is a 12.2 billion-piece jigsaw puzzle,” said Mike Bunce, a paleogeneticist at Murdoch University, who was not involved in the study.

Not only was the DNA heavily degraded, the bone itself had adopted a host of microbial residents, the tiny engines of decomposition, each full of their own DNA. The team again turned to powerful computer programs to pick out which sequences belonged to the horse and which belonged to the bacteria.

The final product of all this work was a complete rough draft sequence of the Thistle Creek horse's genome.

In order to place the Thistle Creek Horse on the evolutionary timeline, the researchers compared its genome to those of a younger extinct species, several modern domestic horses, a donkey, and a wild Asian horse. The results of this comparison, reported today in Nature, push back the origin of the Equus lineage, which includes all living horses, zebras and donkeys, to a common ancestor living 4 million years ago.

As part of their analysis, the team sequenced the genome of the Przewalski’s horse, an endangered species native to the Mongolian steppes. Their results confirm that the Przewalski’s horse is Earth’s last remaining truly wild horse population, highlighting a critical need for species conservation. Finally, along the way, the researchers assembled the first complete genome of the donkey, a creature that seems forever doomed to life in the horse’s shadow.

The team also uncovered other chemical secrets locked within the Thistle Creek bone. Using machines designed to smash proteins into their amino acid building blocks, the researchers decoded the sequence of 73 prehistoric horse proteins. Orlando says they originally looked at the proteins as a way to gauge how well the sample had been preserved, but were surprised to find so many untouched. Studying the proteins that flowed through the bloodstream of this horse provides a snapshot of molecules in action taken three quarters of a million years ago.

But the most fascinating question raised by the work is this: How damaged and scarce can ancient DNA be before scientists will be unable to weave a genome from its frayed strands?

Last year, Bunce and colleagues squashed the dreams of Jurassic Park fans when they demonstrated the frustratingly short half-life of DNA. Every 521 years or so, about half the DNA in any particular sample will break down into its chemical components. Even when buried in permafrost, Earth's cold-storage freezer, long DNA molecules become short ones and individual DNA bases are erased forever. While this spells a huge setback for scientists aiming clone extinct species, it doesn't rule out sequencing extinct genomes.

Bunce predicts that in the coming years, there will be a race to sequence even more degraded prehistoric genomes using less DNA.

Przewalski horses, the last remaining species of wild horse, in Khomyntal, Western Mongolia.

Eddy Rubin, director of the Department of Energy’s Joint Genome Institute, predicts a shift in how researchers study these ancient species. “DNA is really a very accurate predictor of what happened," he says, "much more so than bony structures.” Rubin suggests that a warming climate and thawing permafrost means “there may be other samples that reside in friendly environments out there that could push back what we know about the origin of species.”

That includes our own. The ability to reconstruct fossil genomes is already revolutionizing the study of human origins.

Until recently, scientists trying to retrace our evolution have focused primarily on bones uncovered in tropical environments, such as the famous Australopithecus skeleton known as Lucy. But important human relatives like Neanderthals and Denisovans lived alongside our ancestors as far north as Siberia.

This new research provides a scaffold to build upon as genome detectives push the one million year threshold. And as DNA sequencing technology marches forward, geneticists are able to reach further back in time. But the Thistle Creek horse reminds us that decoding a DNA sequence only tells part of the story. Researchers around the world continue to analyze the genetics of horses past and present using this data as their guide, perhaps one day identifying the changes that molded modern horses.

As scientists amass more and more sequencing data from less and less starting material at an ever faster pace, they promise to keep their colleagues busy figuring out what it all means for many years to come.