Ancient DNA

George Poinar Jr. American Scientist. Volume 87, Issue 5. September/October 1999.

In 1991 a couple hiking in the Italian Alps made a startling and gruesome discovery: They found the frozen body of a man protruding from a glacier. The dead man’s clothing and shoes suggested that he was very unusual, and his discoverers wondered who he might be. The body was so unusual, in fact, that it aroused the interest of an international team of scientists. To determine the Iceman’s identity, a team led by Svante Paabo at the University of Munich consulted a remarkable source. They analyzed the man’s DNA.

Certainly, in the early 1990s, when this identification took place, DNA analysis had been used for forensic purposes hundreds of times. What made this case so unusual was the dead man’s age: He was some 5,000 years old.

As difficult as it is to believe, scientists have learned that the normally fragile DNA molecule can persist hundreds, thousands and even millions of years in certain extraordinarily preserved specimens. And it is a stroke of luck that the sophisticated tools of molecular biology, most often used to foretell an organism’s genetic future, can be employed in the service of deciphering an organism’s past. These days, scientists are exploiting the techniques of gene amplification and sequencing to decipher biological events that took place in the very distant past. They are reading some very ancient history encoded in DNA from people, animals and plants much older even than the so-called Tyrolean Iceman. These studies allow us to fill in missing links on phylogenetic trees or to identify the remains of organisms long extinct.

The notion that very old samples of nucleic acid might be preserved was considered too “far out” to be taken seriously just two decades ago. Since then, however, the field of ancient DNA research has come into its own, with an impressive range of research topics and even a journal, Ancient Biomolecules. A number of ancient specimens-a jaw fragment from a 50,000year-old Neanderthal and a tissue sample from a 30,000-year-old ground sloth among them have yielded DNA in quantities large enough for scientists to unravel their secrets.

Recent History

Two decades ago, molecular biologists were learning how to extract and amplify small stretches of DNA and determine the sequence of their nucleotide subunits. Comparisons of DNA sequences allow scientists to determine the relatedness of individuals and species. Species that are closely related share a great deal more of their DNA sequences than do ones that are more distantly related. Scientists now routinely amplify and compare genes to determine a person’s future risk of developing a disease. But the idea of applying these techniques to very old DNA was practically unthinkable.

Scientists anticipated that ancient DNA would be so badly degraded as to be useless in making sequence determinations. And even if it were not degraded, it would be available in such small quantities that it would be like having none at all. Scientists had so generally discounted the idea of extracting and sequencing DNA from a corpse long deceased that a 1980 account of the recovery of nucleic acids from an approximately 2,000-year-old corpse in China went virtually unnoticed. Nevertheless, in 1983 Montana physician Jack Tkach organized a small band of believers. The study group shared ideas and information, and it developed a newsletter describing a number of what then appeared to be very futuristic projects-including the possibility of recovering genes from extinct organisms.

The group’s optimism was rewarded the very next year. The first news of the recovery of DNA from a long-deceased organism struck like a bombshell. That year Russ Higuchi and his colleagues from Allan Wilson’s laboratory at the University of California at Berkeley cloned DNA from the quagga. This zebra-like animal once roamed the plains of southern Africa before it was hunted to extinction in 1878. The pelt that yielded its DNA was only 140 years old, but obtaining DNA from any extinct animal at all was a dream come true and made scientists seriously think about other possibilities.

The Higuchi paper appeared amid an ongoing controversy as to whether the quagga was more closely related to horses or zebras. Ultimately, comparison of the quagga DNA with horse and zebra DNA settled the question, proving the quagga to be more closely related to the zebra. The DNA evidence also provided considerable satisfaction to scientists who had already reached that conclusion via other, more traditional means.

The study amplified the small DNA sample into quantities large enough to be sequenced using the “template assay,” a forerunner of the polymerase chain reaction (PCR) technique. Following closely on the heels of the quagga study was a 1985 report from Paabo on the first successful recovery of DNA from an Egyptian mummy several thousand years old.

Of course, the development of PCR in the mid-1980s revolutionized DNA analysis and proved invaluable in the study of ancient DNA. Since PCR can fairly easily amplify the smallest quantities of DNA, it is an ideal technique for the study of ancient DNA, where only minute quantities of biological material can generally be recovered. Amplification supplies sufficient amounts of the ancient DNA sample for sequencing.

Set in Stone

Insects, animals and plants often become trapped in a tree’s resin, which hardens over long periods of time to form amber. Early on, my wife, Roberta, and I wondered whether the DNA of these specimens would be preserved. In 1982, while we were at the University of California at Berkeley, Roberta and I cut apart a small gnat trapped in Baltic amber that was 40 million years old. Under the electron microscope, we saw that the ancient cells of the fly were still completely intact. Inside the cell, preserved as though they had come from a more modern specimen, were the various compartments and components of the cell, including some lipid droplets, mitochondria and ribosomes. Most tantalizing was our observation of perfectly intact nuclei, the compartment that houses the cell’s DNA. Apparently the resin had acted as a preservative, drawing water out of the specimen while simultaneously fixing it.

Naturally, we asked ourselves whether it might be possible that the DNA had been preserved as well. Our Berkeley colleague Allan Wilson was also interested in the project, as was Russ Higuchi, who worked with us on it.

While we were trying to develop procedures to answer this question, another bombshell rocked the field. This time the report came in 1990 from Edward Golenberg and his team at the University of California at Riverside, who had managed to extract DNA from magnolia leaves thought to be about 15 million years old. It seemed another dream had come true. Could DNA persist for millions of years? How many millions, we wondered?

In 1992 Raul Cano and his colleagues at California Polytechnic State University extracted DNA from an insect in amber, a 20 million- to 40 million-year-old bee. The group followed that report with one in 1994, in which they retrieved DNA from a 120 million-year-old weevil in Lebanese amber. It is the oldest DNA recovered to date. Paleobiologists received this news enthusiastically. Amber deposits are widespread and contain a variety of biological inclusions. A doorway to the ancient world had definitely been opened.

Remarkable Preservation

Unfortunately, it is not always easy to look through that door. Not all attempts at DNA extraction are successful. As the field of ancient DNA matures, scientists are coming to learn just how difficult it can be to pass through at times. Some recent attempts to isolate DNA samples from insects trapped in amber have produced only contaminated products. The lesson from these results is that the procedure is by no means routine.

An important part of ancient-DNA research is learning how to identify specimens that are likely to yield intact DNA. Most DNA, of course, becomes degraded soon after an organism dies. But under certain circumstances, determined in large part by the microenvironment of the tissues at the time of death, some DNA can persist.

Animal specimens are best preserved under conditions that cause a corpse to desiccate quickly. For example, specimens buried in volcanic ash, preserved in resin that changes to amber, submerged in water high in tannins and low in oxygen, frozen in arctic ice or left in a dry, cool cave tend to remain well preserved. In contrast, conditions that expose the specimen to high temperatures and humidity tend to destroy it.

It is now apparent that nucleic acids such as DNA and RNA can survive longer when combined with other compounds in cells and tissues. After conducting a series of laboratory tests in 1993, Thomas Lindahl at Clare Hall Laboratories in England concluded that DNA, when exposed to the elements, would become completely degraded within 10,000 years at moderate temperatures. However, he noted that if the DNA were adsorbed to hydroxyapatite, a calcium-containing compound found in bones and teeth, its survival would be extended twofold. The DNA would last even longer if it were maintained in an environment of high ionic strength.

Hydroxyapatite is not the only compound that prolongs the life of a DNA molecule. George Sensabaugh at the University of California at Berkeley noted that endogenous DNA can be better preserved when it is combined with proteins called histones, which bind to DNA. My son Hendrik Poinar, then at the University of Munich, and an international team of scientists demonstrated that sugars also prolong the life of the DNA molecule to which they are bound.

From 1831 to 1836 the HMS Beagle sailed around the world, carrying with it British naturalist Charles Darwin. It was this trip that brought Darwin to the Galapagos Islands and shaped his thinking on natural selection. Along the way, Darwin made several other keen speculations about the relationships between the various species he observed. Several of these species are now extinct, and until recently his speculations have remained only that.

The questions Darwin attempted to answer using morphology, interbreeding and behavior are the same ones being asked today: namely, what are the true evolutionary relationships between organisms, and to what living groups are various extinct forms related? The difference is that today’s scientists have at their disposal the modem tools of molecular biology with which some are taking another look at Darwin’s conclusions.

For example, during his visit to New Zealand, Darwin studied two species of flightless bird: moas and kiwis. He proposed, quite surprisingly, the kiwi to be more closely related to the African ostrich than to its compatriot, the moa. In 1992, Alan Cooper at Victoria University in New Zealand and an international team extracted DNA from a fossil bone of the now-extinct moa and compared it with DNA from modem emus, rheas, cassowaries, ostriches and kiwis. They concluded that Darwin was right. It now seems likely that ancestors of New Zealand’s flightless birds invaded the islands at least two separate times, with one introduction leading to the moas and the other to the kiwis.

Although the extended survival of DNA is good news to those who want to study ancient DNA, it can pose a problem when attempting to liberate these DNA molecules from the compounds to which they are bound. For example, while attempting to recover endogenous DNA from fecal samples of extinct ground sloths, Hendrik Poinar and his colleagues showed that chemical bonds linking DNA to sugars had to be severed before the DNA could be amplified and sequenced.

In addition to the normal problems inherent in molecular biology, scientists in the field of ancient DNA must contend with maddeningly small amounts of DNA, which is inevitably degraded. Much time is spent in trying to recover DNA from samples that turn out to have no remaining nucleic acids. Some researchers have actually devised tests to predict whether any DNA remains in ancient tissue.

Jeffrey Bada at the Scripps Institution of Oceanography in La Jolla, California, looked at the disposition of certain amino acids, the subunits from which proteins are made, to determine the likelihood of finding intact DNA in a specimen. In particular, Bada focused on aspartate. Like many amino acids, aspartate can exist in one of two configurations, mirror images of each other known as the D- or L- forms.

Living beings on earth overwhelmingly employ the L-form of amino acids, but these gradually become converted to the D-form, producing a mixture of equal amounts of D- and L-amino acids. The degree of conversion depends on the conditions surrounding the organism at the time of death. Moisture, temperature and the attachment of metal ions to proteins-the same factors that affect the degradation of DNA-also promote the conversion of L- to rr amino acids. Bada and his colleagues predicted they would find a correlation between the degree of amino acid conversion in a specimen and the amount of DNA that could be recovered from it.

In 1996, a study by Hendrik Poinar, Matthias Hoss, Bada and Paabo showed that when the ratio between D- and L-aspartate exceeds 0.08, there is little chance that DNA is still present; otherwise chances are pretty good that some DNA still remains. Since this ratio can be determined with only a few milligrams of the sample, and the results can be obtained within a few days and at little expense, this has proved to be a useful and fast technique to assess the quality of DNA in an ancient specimen.

In addition to the problem of extracting DNA, there is the almost universal problem of contamination. Microorganisms can remain on a specimen for long periods as spores. In 1995, Raul Cano and Monica Borucki reported that they could actually culture bacterial spores tens of millions of years old removed from the body of an insect in amber. Ancient microbial DNA can confound the analysis of a specimen’s DNA, as can modern microbial DNA.

Icemen and Other Mysteries

Scientists tolerate the frustrations and disappointments of working with ancient DNA because the rewards are so great. One of the most intriguing mysteries solved by applying modern analytical techniques was the identity of the Tyrolean Iceman. DNA analysis indicated that this 30-to-40-year-old man was most closely related to modern Europeans, specifically those from alpine and northern Europe.

Also associated with the Iceman was a braided grass coat, which he was wearing at the time of his death, and a pair of oval leather shoes that contained grass insulation. A 1994 analysis of the DNA from the grass of the coat by Franco Rollo and his colleagues at the University of Camerino, Italy revealed its similarity to modern-day species belonging to the Festuceae and the Hordeae families, both of which are cereal crops that have been under cultivation for some time. That same year, German scientists K. Haselwandter and M. R. Edner isolated and cultivated fungi from the grass inside the Iceman’s shoes. The team identified the fungi as belonging to the genera Septoria, Leptoshpaeria and Sclerotinia, varieties that cause leaf-spot disease to this day. Most likely the fungi infected the grass before the Iceman gathered it.

Another of the great curiosities of our time is the way in which our hominid ancestors interacted with one another. A particularly compelling question revolves around the possible relations between Homo sapiens and Neanderthals. Anthropologists would like to know whether Neanderthals were eliminated by Homo sapiens or interbred with them. In 1997 Matthias Krings of the University of Munich and colleagues in Germany and the United States reported the findings from their analysis of mitochondrial DNA extracted from the remains of a Neanderthal man. This corpse, estimated to be between 30,000 and 300,000 years old, had been discovered in 1856 by a schoolteacher in the Neander Valley, near Dusseldorf, Germany The 1997 analysis of this man’s DNA concluded that Neanderthals contributed no DNA to modem human beings and therefore could not be ancestors to Homo sapiens. The study led Krings’s group to believe that modem humans had instead replaced Neanderthals. They also revised uoward the age of the common ancestor of the two species. The fossil record indicates that Neanderthals and modern humans diverged between 250,000 and 300,000 years ago. The Krings group interpreted the DNA evidence to mean that the split took place between 550,000 and 690,000 years ago. The authors of this study caution, however, that their findings are based on the only specimen from which DNA extraction has been possible so far.

Analysis of ancient DNA can also resolve issues regarding the evolution of animals. In particular, scientists have noted that unrelated animals may share similar features, a condition known as convergent evolution. For example, insects and birds both have wings, even though they are not closely related. Scientists have puzzled over this phenomenon for decades. Even Charles Darwin, in his famous work on the origin of species in 1859, dealt with this problem under the heading of “Analogical Resemblances” and defined it as “resemblances in quite distinct beings, which have been adapted for the same function.”

Scientists looking at ancient DNA confronted the issue in a study of the thylacine, an extinct, carnivorous, dog-like marsupial. Thylacines were the largest carnivorous marsupials in Australia and were last seen in the wild in Tasmania in the 1930s. Farmers hoping to protect their sheep from thylacine attacks trapped them. In fact, the Tasmanian government offered a bounty for their destruction from 1888 until 1909. The jaws of the thylacine superficially resemble those of the wolf, a placental mammal, but on closer inspection one sees differences in the structure and number of premolars and molars. Some scientists saw a resemblance between the thylacine and the Borhyaenidae, an extinct group of marsupial carnivores from South America. They noted some characteristics of the teeth and pelvis that were shared exclusively between the two groups of animals. However, a study conducted in 1989 by Richard Thomas, who was then at the University of California at Berkeley, and an international group of colleagues compared thylacine DNA with that of living South American carnivorous marsupials and concluded that they were not related, the similarities being, like wings, an example of convergent evolution.

Ground sloths, which are all extinct today, make a challenging topic for ancient DNA studies. One of the controversies surrounding these creatures has to do with whether they were directly related to the tree sloths, and if so, to which of the two groups. Matthias Hoss from the University of Munich and colleagues attempted to answer this question using the DNA from 13,000-year-old remains of the ground sloth Mylodon darwin, which came from the Mylodon cave in Chile. DNA analysis demonstrated this animal to be more closely related to the two-toed tree sloth than to the three-toed sloth. But ancient DNA need not only be extracted directly from the specimen in question. Answers can also come from material that passed through the animal. For example, Hendrik Poinar and colleagues in Munich looked at dung samples from North America thought to belong to a ground sloth. The study helped the scientists determine that among the variety of plants eaten by the ground sloth was the yucca plant. The analysis also showed that the plants eaten by the ground sloth are now found at altitudes considerably higher than they were in the past. This provided evidence of environmental change over the past 10,000 or so years.

Modern Mysteries

The mysteries that can be solved by DNA analysis are by no means limited to events that took place in the very distant past. Events that happened in more recent times can also be illuminated by DNA analysis. Among these are the positive identification of the family of Tsar Nicholas II, whose bodies had been recovered from a hastily dug grave not far from Ekaterinburg, Russia.

Although the facial areas of the skulls of these bodies had been destroyed, samples of mitochondrial DNA could still be extracted from some of the bones. In their analysis, Peter Gill and his colleagues at the Forensic Science Service of the Central Research and Support Establishment in Reading, England, positively matched the extracted DNA with that of living relatives of the Tsar.

In a second well-publicized case, DNA analysis revealed the identity of remains found in a cemetery in Southern Brazil. In 1979, after a man named Wolfgang Gerhard drowned, rumors quickly spread that the dead man was none other than the infamous Josef Mengele, the “Angel of Death” who performed medical experiments on inmates at the Nazi death camps during the Second World War. Authorities, however, dismissed these claims when an examination of certain skeletal features failed to correspond to features described in Mengele’s medical records.

A DNA analysis conducted in the early 1990s by Alec Jeffreys and his colleagues at the University of Leicester in England indicated otherwise. Specifically, a match between DNA extracted from the dead man’s femur and the DNA of Mengele’s surviving son strongly indicated that the exhumed remains of Wolfgang Gerhard were in actuality those of Josef Mengele. On the basis of this DNA evidence, the German authorities closed the case concerning the whereabouts of the elusive Dr. Mengele.

The molecular biology revolution of the last several decades has reached into every conceivable corner of biological investigation. The study of ancient DNA will continue to grow as the technology improves and, in elucidating mysteries of the past, ensures itself a robust future.