Some mammoth bones will have large proportions of mammoth DNA in them relative to the amount of microbial and other exogenous sources of DNA. These are the bones that we prefer to sequence. Unfortunately, it is very difficult to know the ratio of mammoth DNA to other DNA in a sample without going ahead and doing the experiment: extract DNA, sequence it, and see what you get.
Luckily, there are a few general rules about DNA preservation that can be used to guide sample selection. First, cold environments encourage DNA preservation. The chemical processes involved in DNA decay operate more slowly at lower temperatures. Good places to look for bones with well-preserved DNA include the frozen soils (permafrost) of the Arctic and high-altitude caves. Tropical islands are terrible places for DNA preservation, which is bad news for enthusiasts of resurrected dodos (although not all dodos died on Mauritius; some were transported alive to Europe and many of these remains can be found in existing museum collections). Second, ultraviolet light damages DNA. Ultraviolet light causes the same damage to DNA both during life and after death, only dead organisms don’t have the DNA repair mechanisms that keep us from getting terrible skin cancers every time we step into the sunshine. This again points to caves as ideal sources of well-preserved remains, and suggests that remains that are rapidly buried are likely to be better preserved than remains that sit exposed on the surface for many months or years. Third, water is particularly damaging to DNA. Rapid postmortem desiccation and preservation in dry or frozen conditions promotes long-term survival of DNA. Ancient DNA has been recovered from naturally mummified remains of humans, steppe bison, mammoths, and other species. Finally, different tissue types tend to be more or less susceptible to damage and decay. Bone, for example, appears to be a better source of intact DNA than is soft tissue, which perhaps has something to do with the structure of bone matrix or with the bone cells themselves. Hair is another excellent source of well-preserved DNA, as the hydrophobic exterior of the hair shaft limits the amount of water and microbes that might enter the hair and degrade the DNA.
THE TEMPORAL LIMITS OF DNA SURVIVAL
The laws of physics and biochemistry tell us that DNA does not survive forever, even in the best environments for preservation. With that in mind, knowing the age of a sample that we’re considering for a genome-sequencing project is useful in predicting how successful that project will be. While there is no strict rule that states a precise age beyond which DNA will not survive, biochemical modeling suggests an upper limit close to 100,000 years at moderate ambient temperatures. In practice, however, how old we can go varies considerably, and depends on where in the world the sample comes from, what element (hair, tooth, bone, mummified tissue, eggshell) is preserved, and what the preservation history of the sample has been. In warm environments where samples are immersed in water and exposed to UV light, every bit of useful DNA might be destroyed in less than a year. In the Arctic, if a sample is de-fleshed and immediately frozen and then remains underground and frozen from the time of burial to the time of excavation, the DNA within that sample may survive for many hundreds of thousands of years.
It is important to clarify what I mean by “useful” DNA. DNA does not exist as a beautifully preserved and informative molecule on one day and then dissolve into nonexistence overnight on its expiration date. The process of DNA decay includes both the accumulation of chemical damage and the gradual breaking down of the long strands of DNA into smaller and smaller fragments. Once the surviving fragments are shorter than around twenty-five or thirty base-pairs long, they are too short to map to a unique location in the genome and would therefore no longer be useful for genetic research. Fragments of DNA that are one or two base-pairs long may survive for an extremely long time even in very poor environments for preservation, but recovering these would not help us piece together the genome of an extinct species.
I was recently involved in a large international collaboration to sequence the complete genome of an ancient horse—the same kind of horse that runs the Kentucky Derby today, but a very old one. The bone we used was recovered from permafrost soil in the Canadian Arctic. When we found the bone, we knew that it was old. Very, very old. And we were very excited.
In ancient DNA research, it is crucial to know how old the bones are. Knowing the age of each bone provides a way to correlate changes in population size and genetic diversity with environmental changes. For example, horses went locally extinct in North America around 12,000 years ago. As I discussed in chapter 1, the two competing hypotheses to explain horse extinction are that horses fared poorly through the peak of the last ice age around 20,000 years ago or that they were overhunted by humans that arrived in North America after around 14,000 years ago. Knowing that horses were gone by 12,000 years ago is not the same as knowing why horses disappeared. To distinguish between these hypotheses, we need to know when horse populations began to decline. And, to learn this, we have to know the age of each bone.
There are several ways to learn the age of a bone, fossil, or archaeological artifact. In some environmental contexts, such as caves or archaeological sites, these items might be found in a clearly defined layer or stratum within which other items whose age is known are also found. There might be a concentration of fossils that are only found together during a particular time interval, or examples of a prehistoric technology that is only used during a specific period of prehistory. Unfortunately, such layers are not common in permafrost, where most of our horse bones are found.
The age of most permafrost-preserved bones is estimated using a process called radiocarbon dating. Radiocarbon dating measures the ratio of two isotopes of carbon—carbon-14 and carbon-12—in organismal remains and uses this ratio to estimate how long ago the organism died. Carbon-14 is a radioactive isotope of carbon that is created in the atmosphere when cosmic rays collide with nitrogen. Carbon-12 is the normal carbon isotope. Both forms of carbon combine with oxygen to form carbon dioxide, which plants absorb through photosynthesis. Animals then eat the plants, and the carbon from those plants is incorporated into their bones. At any point in time, the ratio of the two forms of carbon is the same in the atmosphere as it is in the organisms living in that atmosphere. Carbon-14 is radioactive and decays at a predictable rate with a half-life of 5,700 years. Because organisms stop taking up carbon after death, we can calculate how long it has been since the organism died based on how much carbon-14 is left in its remains.
Radiocarbon dating is a powerful and pleasantly precise way to estimate the age of permafrost bones. However, the amount of carbon-14 in the atmosphere is very, very small relative to the amount of carbon-12—only about one part per trillion of the carbon in the atmosphere is carbon-14—and the half life of carbon-14 is very short. After around 40,000 years or so, too little carbon-14 will remain in an organism to measure accurately. Radiocarbon dating is useful, therefore, only over this very brief, recent time interval.
Fortunately, there is another way to estimate the age of a permafrost-preserved bone. When volcanoes erupt, they send out a wide fan of very fine dust, which is often referred to as volcanic ash or tephra. The tephra produced by each eruption is unique in its geochemical composition. And, as it turns out, geochemists have developed several ways to learn the ages of these volcanic eruptions. These methods are based on the premise that high heat “resets” the age of the minerals, so that different properties of the minerals can then be measured to estimate when the eruption occurred.
Volcanic tephra is deposited across wide swaths of Alaska and the Yukon Territory and marks eruptions of volcanoes as far to the west as the Aleutian Islands and Alaskan peninsula. When the dust settles, a blanket of white forms on top of the permafrost. As time goes forward, permafrost sediments pile up on top of the layer of volcanic ash, which now clearly delineates fossils buried before the eruption, beneath the tephra, from those buried after the eruption, in permafrost above the tephra. This method is not as precise as radiocarbon dating, but it does provide a means to approximate the ages of bo
nes that are too old to be dated using radiocarbon. This is the method we used to learn the age of our very old horse bone.
HOW OLD IS TOO OLD?
My favorite place to do fieldwork is the Klondike gold-mining region just outside of Dawson City, in Canada’s Yukon Territory. Gold mining, it turns out, is great for ice age paleontology. Most gold miners in the Klondike use a process called placer mining (plate 6). In placer mining, water from the spring snowmelt is collected into holding ponds. After the sun thaws any exposed permafrost, the water is pumped to the active mining site and blasted against the thawed mud. This washes away anything that is not solid ice. The mining then stops for a little while as the warm sun melts away the next layer of frozen mud. Then, the water is turned back on and the freshly melted mud is washed away. This process is repeated until the permafrost is gone and only the gold-bearing gravels remain.
Much to the bemusement of the miners, we are not particularly interested in the gold. We are, however, very interested in the thousands of bones that are unearthed as the permafrost is washed away (plates 7–9). In the Klondike, around 80 percent of these are bones from the extinct steppe bison, about 10 percent are horses, and the rest are mainly mammoths, bears, lions, caribou, wolves, and muskoxen. Crucially, placer mining is slow and methodical, which means that many of these bones can be picked out of the permafrost while they’re still frozen. These bones are impeccably preserved.
We found the really old horse bone in a gold mine near Thistle Creek. The site was special, even among Klondike gold mines. A few years earlier, a team of geologists led by Duane Froese from the University of Alberta discovered that the permafrost near Thistle Creek was very old. In fact, it was the oldest permafrost ever discovered. They knew this because they found a volcanic ash layer called the Gold Run tephra associated with the permafrost mud. The Gold Run tephra was deposited across the central Yukon around 700,000 years ago. So, when we found out that horse bones were preserved within 700,000-year-old permafrost, we could not wait to see whether they contained any horse DNA.
Duane recovered seven bones, all of them larger than those of present-day domestic horses, from the layer of permafrost that was associated with the Gold Run tephra. He made sure that the bones were kept frozen rather than allowed to thaw as they were transported out of the field and into storage. We took subsamples from two of these horse bones for DNA analysis and, to our surprise and delight, were able to recover DNA from both. I repeat: we were able to recover authentic, ancient horse DNA from two 700,000-year-old bones.
The fragments of DNA recovered from these horse bones are the oldest ancient DNA sequences that have been isolated from a specimen whose age is well constrained. However—extraordinary claims require extraordinary proof. Were our results real? We think so. We were extremely careful to make sure the sample was kept frozen and kept away from other samples or other sources of contaminating DNA. The fragments of DNA that we recovered from the bones were short and very badly damaged, as is expected when working with ancient DNA. Our analyses of the sequence data indicated that the old horse was evolutionarily more ancient than living horses. And the results were repeatable. We extracted DNA from these horses in my lab at Oxford and my lab at Penn State, and my colleague Ludovic Orlando and his team at the University of Copenhagen extracted DNA from one of these horse bones several times. The results from all of these extractions were consistent with each other, in terms of both the actual sequences recovered and the damage profile of the recovered DNA. Together, these observations support the authenticity of the very ancient horse DNA.
By the time we finished sequencing ancient horse DNA from this bone, we had generated nearly 12 billion fragments of DNA. We took each of these fragments and tried to match them to the genome sequence of a domestic horse, which had been assembled and published a few years earlier. Around 1 percent of our 12 billion fragments matched different parts of the domestic horse genome, indicating that this tiny component of the DNA recovered from this bone was horse DNA. The other 11.9 billion fragments matched sequences of plant DNA, fungal DNA, bacterial DNA, and other environmental DNA. That is a terrible ratio of horse-to-environmental DNA, and yet we still sequenced the genome of this very ancient horse.
Why did DNA survive in this bone for such an exceptionally long time? We can’t say for certain. The bone was found in the oldest permafrost soil that is known to exist, and the bone probably never thawed between the time of burial and 700,000 years later, when we pulled it out of the frozen ground. Unless older, permanently frozen soils are discovered or fossils are recovered from older ice cores, this may be the age limit of DNA survival in bones.
Exceptional preservation is not limited to the Arctic. Caves have also been found to preserve DNA for a remarkably long time. Most of the Neandertal bones that have been sequenced, for example, were recovered from caves. Recently, DNA was recovered from 300,000-year-old cave bears and a 400,000-year-old hominin from bones preserved in Spanish caves. Environmental stability is known to promote DNA preservation, and caves tend to be consistent in both ambient temperature and humidity, which perhaps explains these examples of exceptionally long-term preservation.
Environmental stability does not, however, seem to be an absolute requirement. We recently pieced together the complete 16,000-base-pair mitochondrial genome of a 100,000-year-old bison bone that was recovered from an ancient lake site in Colorado. The bone belonged to an extinct species of bison called Bison latifrons whose horns spanned an astonishing 2.5 meters in width—five times the width of today’s American bison. The bison bone and the DNA within it somehow survived despite undergoing thousands of seasonal shifts between cold winters and hot summers. The DNA that we recovered from the bone was in terrible condition but, remarkably, remained usable. Would we want to use that particular bison bone as the source of genetic material from which to begin the process of resurrecting Bison latifrons? Not unless we absolutely had to. Less than 0.1 percent of the DNA in the bone was bison DNA, the average fragment length was in the range of thirty base-pairs long, and the sequences were badly damaged. But if this bone was the only bone that we had access to and we really wanted to bring the giant bison back to life, we could use this bone to sequence its genome. We would only get a little bit of bison DNA out at a time, and it would be very expensive. But we would probably, eventually, get the sequence mostly correct.
Fortunately for the mammoth and the passenger pigeon, we don’t have to rely on badly preserved bones with tiny amounts of DNA. Passenger pigeons died out only a century ago, and hundreds of birds are preserved in museum collections around the globe. Well-preserved mammoth remains are even more abundant. If we limit ourselves to the last 40,000 years—which puts us in the range of radiocarbon dating and allows us to know how old the bones we’re working with actually are—there are probably thousands, if not hundreds of thousands, of mammoth remains already in museum and university collections across the world. Most of these are from permafrost deposits, including from the Klondike. Many of these have already been subjects of ancient DNA research, even genome-sequencing projects. We need not be limited, however, to samples sitting on a shelf somewhere at room temperature, subject to faster rates of DNA decay. All we need to do to find an extremely well preserved mammoth bone is get on an airplane, and then a helicopter, and then a boat perhaps, and make our way to the Arctic.
CHAPTER 4
CREATE A CLONE
When you are working in the tundra, nobody cares if you sing loudly and out- of-tune as you walk along a meandering river. Nobody laughs at the five layers of clothing you’re wearing or mocks the variety of nets you’ve donned in your latest, ill-fated attempt to limit mosquito access to your flesh. And nobody bats an eye when your battered Mi-8 helicopter makes an unexpected stop in the middle of the Siberian tundra and picks up a French-speaking couple, their five-year-old child, and a large, red cooler.
These are lessons I learned in the summer of 2008, during what I fondly remember as my strang
est and least successful bone-hunting season. That summer, we spent several weeks living in a small encampment surrounded by lakes on the low-lying tundra of the Taimyr Peninsula. We were hunting mammoths.
The Taimyr expedition was led by Bernard Buigues, a seasoned and pleasantly eccentric arctic explorer, and there was no reason to suspect it would not go well. For decades, Bernard, as president of CERPOLEX (CERcles POLaires EXpédition), led overland expeditions into Siberia and to the North Pole. These expeditions began from his well-appointed base in Khatanga, a small Russian town situated on the Khatanga River in Krasnoyarsk Krai. By the early 2000s, Bernard’s interests had shifted to expeditions of a more scientific variety, and he had formed Mammuthus, an organization attached to CERPOLEX with the stated goal of exploring and celebrating the Arctic and its many treasures. As the name implies, however, Mammuthus was particularly focused on recovering and facilitating scientific investigations of mummified remains of mammoths. The formation of Mammuthus was either opportunistic or timely, as mummies of mammoths and other ice age giants have been popping out of the Siberian permafrost at a surprising rate since the turn of this century.
Upon meeting Bernard, it was impossible not to have complete faith in both his leadership and the success of the expedition. By 2008, Bernard had decades of experience working in the Siberian tundra. He had seemingly endless energy and enthusiasm, a deep knowledge of the logistical challenges of working in Siberia (and how to circumvent these challenges), and a large collection of warm coats. Most importantly, he had a long history of collaboration with the people living in the region, which goes some way to explain why he is so often the first to get access to newly discovered mammoth mummies. By all reasoning, the expedition should have gone well.
How to Clone a Mammoth Page 8