Ancient Horse Bone Yields Oldest Sequence of DNA Known to Science

A team of international researchers have been able to sequence a substantial portion of the genome of an ancestral horse from a leg bone of a horse that roamed about in Canada some 700,000 years ago.  This research sheds light on the evolution of the Equine family and dramatically extends the known limit of DNA survival.  DNA material has been recovered from other Pleistocene aged fossils, those of Cave Bears (Ursus spelaeus), Mastodons and Mammoths but this DNA recovery is from a sample more than half a million years older than other material successfully studied to date.

The study has just been published in the academic journal “Nature” the fossilised leg bone was part of a collection of horse fossils preserved in the Canadian permafrost of the Yukon.  The fossils were found at a location known as Thistle Creek (west-central Yukon Territory)

The fossils are estimated to be around 735,000 years old (Mid Pleistocene), and the fifteen centimetre long leg bone which was used in the DNA study, is part of a collection excavated out of the cold, hard permafrost back in 2003.  Leg bones, being relatively thick and robust have a greater potential for preserving organic material within them than smaller more fragile bone material.

Ancient Genetic Material Recovered from a Horse Fossil

700,000 year old genetic material recovered from a fossil bone.

700,000 year old genetic material recovered from a fossil bone.

Picture Credit: D.G. Froese/Nature Journal

In a recent study, the leg bones of 158 Moas (extinct, giant flightless birds), were used to develop a framework for estimating the half-life of DNA.  This controversial research from New Zealand suggested a half-life of DNA at just 521 years.  The scientists involved with this study programme calculated that fragments of DNA could survive in perfect preservation conditions for perhaps as long as 6.8 million years, but analysis of this highly unstable, ancient material would probably prove impossible.  This new study of DNA related to Equus (horses), may not be 6.8 million years old but the ancient horse leg bone has yielded the most ancient DNA sequence know to science thus far.

To read more about the study of genetic material in Moa bones: Controversial Research Proposes a Half-Life for DNA

Ludovic Orlando, an evolutionary biologist at the University of Copenhagen and one of the co-authors of the scientific paper, explained that the team wanted to determine whether there might be any biological molecules left in this sample.  The first step was to try to identify any evidence of amino acids that would have constituted any collagen (protein found in bone), within the specimens studied.  Once the team had established that there were these proteins present, the researchers moved on in their analysis with the aim of trying to extract DNA from the ancient horse’s leg bone.
Recently, a team of scientists from Manchester University were able to identify an animal from fragments of tibia (leg bone) found in the Yukon.  The fossil bone fragments, much older than the horse fossils (dated to around 3.5 million years ago), were found to belong to a type of ancient camel.  The Manchester-based team were able to make this conclusion by tracing minute amounts of collagen in the fossilised bone and then de-coding it to see how closely it resembled the collagen found in living species.  The technique is referred to as “collagen fingerprinting”.  However, Ludovic Orlando and his colleagues were able to go further and to identify a quantity of genetic material within their sample, allowing them to sequence about seventy percent of this ancient horse’s genome.
To read more about the study of ancient camels from the Yukon: Camels of the Arctic
Although for the majority of their 700,000 years of incarceration the horse fossils were protected from liquid water (ground water present in a fossil matrix can speed up the breakdown of organic material) and biological contaminants, the scientists were able to identify from climate change studies that the area in which the fossils had been found had thawed out on several occasions.  However, some of the Equus genetic material had still been preserved.  Of the twelve billion or so DNA molecules identified most were from bacteria that had populated the bone after the horse had died.  However, using DNA from the modern horse species as reference markers, the researchers were able to identify “endogenous DNA”, genetic material from the ancient horse itself.
Fragments of Fossil Bone Analysed for Their Genetic Content
"Flogging a dead horse" to extract DNA.
Picture Credit: Dr. Orlando
From the detailed analysis of the organic material, the scientists were able to identify about 3% of the total “pool of organic data” as being horse DNA, that’s about 40 million DNA molecules.
Orlando described this finding as:
“A bit of horse DNA in an ocean of microbial DNA.”
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 ancient horse, which team members at Everything Dinosaur have nick-named “Old Ned”, a reference to the theme to the television series “Steptoe and Son”, father and son rag and bone men who owned a horse.

The first approach the team attempted resulted in relatively poor yields of horse-derived sequences, so they turned to a technology that could directly analyse single molecules of DNA.  Using very powerful computers the scientists were able to distinguish the horse DNA material from the contaminating bacterial DNA, eventually revealing about seventy percent of the entire genome.

Although far from complete, the resulting genetic data has led to some interesting conclusions.  For example, the presence of Y chromosome markers demonstrate that the fossil material came from a stallion (male).

The recovered DNA material was then compared to the genome of a modern donkey, zebras, the domestic horse and the Mongolian wild horse (Przewalski’s horse), this enabled the scientists to build up a comprehensive map outlining the evolution of Equus.  The “family tree” that was developed suggests that the Thistle Creek equine was a basal member of the horse family although the very first horses, as we know them today, the forerunner of the donkey, zebra and horse first evolved some 4 to 4.5 million years ago.  This new genetic dating evidence indicates that the horse family is much more ancient than previously thought.

In addition, when it comes to looking at the Przewalski’s horse, the last truly wild horse left on the planet, this new study suggests that the horse family tree that led to today’s wild horses split from the line leading to the domestic horse some fifty thousand years ago and not as previously thought some one hundred and sixty thousand years ago.

New Insight into the Evolution of Modern Horses

Przewalski's horse DNA studied as part of this research.

Picture Credit: AFP/JIJI

A survey of the recovered DNA sequence looking for specific material in the genes known as single nucleotide polymorphisms (SNPs) has enabled the scientists to estimate past population sizes.  It seems that over the last two million years or so, the horse family has experienced a number of significant population declines and then population explosions.  Analysis of climate change data shows that there is a close match between rising horse numbers and improving (warmer) climate.

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, Dr. Orlando went onto explain:

“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 is the alleles that prime to the racing performance in domestics were not present at that time.”

Commenting on the wider implications of the study, co-author Eske Willerslev of the University of Copenhagen stated:

“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.”

A spokesperson from Everything Dinosaur stated:

“Ten years ago, such developments in extracting viable genetic material from such fossil material was simply not possible, next generation sequencing technology has completely revised our expectations.  If DNA can be identified from fossil material more than 700,000 years old, then this widens the net for scientists as they seek to retrieve more genetic data from fossils.”

This branch of genetics is certainly leading to a whole range of new and exciting developments, not least of which is a more complete understanding of the evolution of extant types of horse – all 4 million years of it.  The fossil material from the Yukon is an example of a “rocking horse” that could well have “rocked” our understanding of the ability of organic material to survive fossilisation.

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