Ancient DNA

SIGNIFICANCE: Scientists from the Max Planck Institute for Evolutionary Anthropology announced during the 2009 Annual Meeting of the American Association for the Advancement of Science (AAAS) that they had generated a first-draft sequence equivalent to more than 60 percent of the complete Neanderthal genome and were comparing it to that of their closest relatives, human beings, which might shed light on the origin of humankind as well as the evolutionary process. What makes this composite sequence of three billion bases remarkable is that it is derived from thirty-thousand-year-old samples of ancient DNA. In 2013, the Max Planck Institute announced it had verified an approximately four-hundred-thousand-year-old mitochondrial genome sequence of a relative of the Neanderthals in Spain, marking the first time that such DNA was extracted from somewhere other than permafrost. There is no doubt that the sequencing technology developed for the Human Genome Project in 2000, which provided the biochemical code for all the genes in the human genome, and subsequent refinements in sequencing technology, has played a crucial role in helping to decipher the Neanderthal genome. Moreover, advances in polymerase chain reaction (PCR) technology have made possible the amplification of small, fragile samples of ancient Neanderthal DNA so they can be sequenced and analyzed. These and other advances have allowed for detailed genetic analyses of ancient DNA, helping researchers determine the origins of both novel and ancient strains of microorganisms; establish evolutionary paths of plant, fish, and animal species, including human beings; document new species; and classify those that are endangered.

Researchers in the 2020s were working to use ancient DNA to reconstruct the history of human health, including past epidemics. They hoped their findings would lead to a better understanding of genomic diversity and disease.

Introduction: Dinosaurs and Jurassic Park

Ancient DNA research is carried out by retrieving DNA sequences from museum specimens, archaeological finds, fossilized and mummified remains, and ancient microorganisms embedded in ice, rock, or amber. Only a miniscule amount of the original matter is present in these sources. For years, researchers struggled with their tiny and often degraded samples of ancient DNA, and the fruits of their labor were often equivocal.

In 1993, filmmaker Stephen Spielberg produced the highly popular motion picture Jurassic Park, based on Michael Crichton’s 1990 novel of the same name, in which scientists are able to bring dinosaurs to life using polymerase chain reaction (PCR) and other biotechnologies. The film captured the imagination of the public as well as the scientific community, sparking renewed interest in ancient DNA. In a communication in 2009, evolutionary molecular biologist Alan Cooper of the University of Adelaide noted that while it is not possible to retrieve DNA from dinosaurs that became extinct more than sixty-five million years ago, as depicted in Jurassic Park—even under the most amenable, deep-frozen conditions, the upper limit of ancient DNA survival is approximately five hundred thousand years—genetic data gleaned from ancient DNA taken together with fossil records make it possible to study much older events by extrapolating backward.

As such, ancient DNA may be employed to study how species and populations evolved when impacted by climate change and mass extinctions. For example, while ice cores provide a great deal of data about past environmental conditions, paleontologists are now more likely to use ancient DNA to assess the effects of climate change on various species. Research on permafrost-preserved megafauna in Alaska and Canada showed that the last glacial ice age, which occurred more than twenty-two thousand years ago, ended with the extinction of the giant bison and other species.

Isolation and Analysis of Ancient DNA

The degree of DNA degradation of an ancient specimen is a function of age and the environmental conditions under which it was preserved. Samples a few thousand years old will typically yield very viable DNA. Of greater concern than viability is the possibility that the ancient DNA may become contaminated with modern DNA. Fossils and ancient remains, such as teeth and bones, are potentially at risk of contamination from pollen, bacteria, fungi, or the skin cells of the person extracting the DNA. Even minute quantities of modern DNA may contaminate the sample, resulting in faulty data. If the sample has been encased in ice or rock, contamination is unlikely; if it was exposed to air, contamination is highly possible.

Since ancient DNA is usually found in fossils in very small amounts, amplification with PCR is a necessity. This presents yet another possible source of contamination. Erroneous nucleotides may be introduced during the PCR process and mistaken for mutations in the ancient DNA when compared to a sequence of modern DNA. Thus, the researcher must take special precautions to protect the specimen from contamination from the time it is extracted and isolated through analysis and sequencing. Upon retrieval, specimens should be placed in airtight, sterile containers by researchers wearing sterile gloves and face masks and using sterilized laboratory equipment and reagents. This should be performed in a “clean room,” which is a room with a low level of environmental contaminants. Detailed guidelines exist that scientists may follow to prevent contamination of ancient DNA during PCR and sequencing.

In 2008, Mélanie Pruvost and colleagues outlined protocols to be used with PCR to minimize or prevent mutations, contamination, and post-excavation degradation. In 2009, Svante Pääbo and colleagues at the Max Planck Institute published several articles on the recovery of Neanderthal DNA in which they examined recent technological advances in preserving ancient specimens and protecting them from contamination during sequencing (Green et al.; Maricic and Pääbo). They discussed the creation of genomic libraries (sets of DNA fragments containing the whole genome of an organism) under clean-room conditions, DNA sequence tags with unique markers attached to molecules of ancient DNA in clean rooms, and radioactively labeled DNA to identify and alter steps in the sequencing procedure where losses of ancient DNA have been shown to occur. With proper execution and precautions, these technologies can dramatically reduce contamination as well as the amount of fossil material required for sequencing, so that less than half a gram of bone was required to produce the draft sequence of three billion bases in the Neanderthal genome.

Ancient Microbial DNA

One of the most fertile areas of ancient DNA analysis has been in the study of the origins of human diseases. In the past, archaeologists relied on physical evidence such as bone scars, deformities, and dental remains to determine whether an ancient human had suffered from a particular disease. More recently, the ability to recover ancient bacterial DNA from Egyptian mummies helped establish the presence of skeletal tuberculosis (TB) in finds dated as early as 3000 BCE. The bubonic plague, caused by the bacillus Yersinia pestis, was thought to be responsible for a series of epidemics that occurred during the sixteenth century but could not be confirmed without adequate medical records. In 1998, however, French researchers unearthed the skeletal remains of persons who presumably died from the plague in the sixteenth century; using PCR to amplify a gene from Y. pestis extracted from dental pulp, along with sequencing, the scientists obtained proof that the plague did indeed exist in France at the end of the sixteenth century.

In 1999, Charles L. Greenblatt of Hebrew University reported the isolation of DNA in several types of bacteria from 120-million-year-old amber. Comparisons of the DNA sequences of ribosomal RNA (rRNA) lent credence to the claim that he and his colleagues had actually isolated ancient DNA and not contaminants. While studies from 1994 to 2006 had shown that amber is a useful medium for conservation of soft-bodied microorganisms, viable specimens older than 135 million years were very rare and had not included microbes. In 2006, however, a group of German and Italian researchers, led by Alexander R. Schmidt, discovered a “microworld” of 220-million-year-old microbes preserved in Triassic amber as old as the first dinosaurs. Droplets of amber were found to contain protozoa, fungi, bacteria, and algae comparable to extant genera, thus providing insight into the evolution and paleoecology of the Lower Mesozoic (the Triassic, Jurassic, and Cretaceous periods, from 251 to 200 million years ago) microorganisms, which have apparently undergone little or no change from the Triassic to the modern era. The largest known deposit of Triassic amber was discovered near the Italian Dolomites, with bacteria the most prevalent microorganism. Examples of all trophic (nutritional) levels were found: bacteria and algae as producers and food sources, protozoa as consumers, and fungi as decomposers. The researchers’ discovery of association among various protozoa or with other unicellular organisms with shells indicates that they had settled outside huge bodies of water. Unchanged since the Lower Mesozoic era, these protozoa had obviously survived the age of the dinosaurs as well as the diversification of mammals and birds.

Evolution and Ancient DNA

Neanderthals, the closest relatives of modern humans, dwelled in Europe and Asia until they became extinct at least thirty thousand years ago. For more than a hundred years, anthropologists and paleontologists have attempted to demonstrate their evolutionary relationship to modern humans, who emerged about four hundred thousand years ago. Pääbo made the first major contributions to the understanding the genetic relationship of modern humans to Neanderthals when he sequenced Neanderthal mtDNA in 1997. In 2009, Pääbo, who heads an international consortium of researchers called the Neanderthal Genome Project, announced that the group had completed a first draft of the complete Neanderthal genome, which can now be compared to earlier sequences of human and chimpanzee genomes in order to obtain initial insights as to how the Neanderthal genome differs from that of modern humans. In 2012, Pääbo's research group used ancient DNA evidence from a finger bone and two teeth to introduce a new race of humans known as the Denisovans. The scientists were able to map this race's entire genome and by doing so were able to add complexity to what was known about ancient humans and Neanderthals and their connection to each other. Scientists hope that new technology will shed new light on these connections and human evolution in general. In the 2020s, researchers began moving beyond analyzing mtDNA to using new techniques in whole genome sequencing. This enables researchers to compare the DNA of people today with the DNA extracted from very old skeletons.

Also in 2009, evolutionary anthropologists lent credence to the theory that a Neanderthal known as Shanidar 3, whose skeletal remains were unearthed in the late 1950s and early 1960s, was killed by a human being who was capable of using a projectile weapon, rather than another Neanderthal, whose weapon of choice would have been a thrusting spear. This finding contributed to a body of evidence that contact between the two species was most often violent, eventually resulting in the extinction of the Neanderthal. Fernando Rozzi of the Centre Nationale de Recherche in Paris found evidence that humans were both violent and cannibalistic with their Neanderthal neighbors. Many conflicting theories explaining Neanderthal extinction exist, however, such as that climate change adversely affected their hunting grounds, causing the species to starve. Another theory posits that Neanderthals became extinct because they bred with human beings, a premise based on a 2006 discovery of thirty-thousand-year-old skeletal remains in Romania that had both Neanderthal and human characteristics as determined by genetic analysis. However, it may be that the extinction of the Neanderthals was a foregone conclusion and the species was apparently doomed by its genome. In July 2009, scientists from the Max Planck Institute for Evolutionary Anthropology found scant genetic diversity among DNA samples gleaned from six Neanderthal fossils and concluded that the species “teetered on the brink of extinction” with a population that never exceeded ten thousand.

Researchers uncovered the skeleton of a teenage girl in a cave on Sulawesi in 2015. Her remains had been buried in the cave for 7,200 years. They later concluded that she was part of the Toalean culture, prehistoric hunter-gatherers who lived in the area about 8,000 years ago until about the fifth century CE. The teenager, a female, is the first largely complete and well-preserved skeleton associated with the Toalean culture. The teenager's DNA showed that she had descended from the first wave of modern humans to enter Wallacea, an island region between Asia and Australia, 50,000 years ago. Her bones are the first to be discovered in the region.

The rates of evolution in Adélie penguins have been studied using ancient DNA. Adélie penguins, a species common to the Antarctic coast and neighboring islands, have dwelled in the same areas of Antarctica for many thousands of years. Through excavation of various colony sites, researchers retrieved partially fossilized bones covering a range of up to almost seven thousand years. By comparing the sequences of a portion of mtDNA among samples of various ages with modern samples, researchers were able to estimate rates of evolutionary change in Adélie penguins. Because the samples were only thousands of years old, the results were deemed more reliable than those from older fossils. Where did these penguins come from, and why were these mobile birds bound to the Southern Hemisphere? Competing hypotheses posit that the penguins either originated in tropical, warm temperate waters or species-diverse cool temperate regions, or even in Gondwana about one hundred million years ago, when it was located farther north. (Gondwana, or Gondwanaland, is one of two composite continents, along with Laurasia, that are believed to have formed from the breakup of the supercontinent Pangaea. It comprised Antarctica, South America, Madagascar, Africa, India, parts of south Asia, and Australia.)

To test their hypotheses, researchers in 2005 constructed a phylogeny of extant penguins from 5,851 base pairs of both mitochondrial and nuclear DNA. They concluded that an Antarctic origin of extant taxa was highly likely; using molecular dating techniques, it was estimated that penguins originated about seventy-one million years ago in Gondwana, when it was farther south and cooler. The researchers hypothesized that as Antarctica became covered with ice, penguins migrated via the circumpolar current to oceanic islands that are close by Antarctica and later to the southern continents. Thus, global cooling played a major role in penguin evolution, as it has for vertebrates in general. Penguins reached cooler tropical waters in the Galápagos only about four million years ago and have not crossed the equatorial thermal barrier since.

Future Research: New and Endangered Species, Climate Change

In 2011, a study published by Camilo Mora and colleagues, funded by the Census of Marine Life project, estimated there are 8.74 million eukaryotes (plants, animals, and fungi) in the world, of which approximately 1.2 million had been documented at the time of the article. Every year, scientists across the globe discover new animal and plant species. Recently documented were an Ecuadorian salamander that resembles the film character E. T., a jumping spider, and the fossil of the oldest gecko species trapped in amber, dating from one hundred million years ago. A flying frog, the world’s smallest deer, and an emerald green viper are among the more than 350 new species found in a decade of research in the eastern Himalayas; the discoveries from 1998 to 2008 put the region on par with the island of Borneo in Indonesia as a “biological hotspot.” The findings point to the importance of protecting the area, which covers northern Burma and India, Bhutan, Nepal, and Tibet.

One may ask how species are defined and why is it important to identify them. In the nineteenth century, Charles Darwin studied species by observation using taxonomic systems: Did the animal have fur, fins, or feet? It was not until the early twentieth century that scientists began to compare the genetic differences among species. This led to the notion that a species could be defined by the barrier to reproducing with other species. However, since some species do not reproduce, one of the strongest notions that rivaled that of biological “species” was the phylogenetic concept, which replaced sexual reproduction with origin from a common ancestor. In the twenty-first century, biological diversity can be ascertained by obtaining both modern and ancient DNA samples and tracking how a species descended from such an ancestor.

While the debate is ongoing, the question “What is a species?” must be answered in the future in order to determine which species are considered endangered. In the face of the current era of global climate change, which has already led to the extinction of many plant and animal species, the ultimate goal is to preserve the biological diversity of Earth and prevent existing species from being lost forever.

Key Terms

• DNAa long linear polymer found in the nucleus of a cell, formed from nucleotides and shaped like a double helix
• DNA polymerasethe enzyme that produces a complementary strand of DNA using a DNA template
• mitochondrial DNA (mtDNA)believed to be of bacterial origin before it was transferred to the eukaryotic nucleus; in the most multicellular organisms, maternally inherited, most likely present in ancient specimens, and useful in tracing human origin; used to compare closely related species such as Neanderthals and human beings
• primeran oligonucleotide (short strand of nucleotides typically eighteen to thirty bases long) used as a starting point for Taq polymerase to produce a complementary copy of a DNA template strand; two primers are needed that flank the DNA sequence being amplified
• ribosomal RNA (rRNA)the central component of the ribosome, the protein manufacturing machinery of all living cells, thereby providing a mechanism for decoding messenger RNA (mRNA) into amino acids; in contrast to mtDNA, rRNA genes are used to compare distantly related species
• Taq polymerasethe enzyme that was first isolated from the hot spring bacterium Thermus aquaticus, which is stable at temperatures close to the boiling point of water (100 degrees Celsius, or 212 degrees Fahrenheit); it is used as the DNA polymerase in PCR cycling, which must reach high temperatures to separate the strands of DNA
• thermal cyclera machine that raises and lowers the temperature of the DNA sample in preprogrammed steps; high temperatures separate the double strands of double-stranded DNA (dsDNA), and low temperatures are used to anneal the strands

Bibliography

Curry, Andrew. "'Phenomenal' Ancient DNA Data Set Provides Clues to Origin of Farming and Early Languages." Science, 25 Aug. 2022, www.science.org/content/article/phenomenal-ancient-dna-data-set-provides-clues-origin-farming-early-languages. Accessed 4 Sept. 2024.

D'Alessio, Vittoria. "Ancient DNA Brings Us Closer to Unlocking Secrets of How Modern Humans Evolved." Frontier Research, 5 Sept. 2022, ec.europa.eu/research-and-innovation/en/horizon-magazine/ancient-dna-brings-us-closer-unlocking-secrets-how-modern-humans-evolved. Accessed 4 Sept. 2024.

DeSalle, Rob, and David Lindley. The Science of Jurassic Park and The Lost World; or, How to Build a Dinosaur. New York: Basic, 1997. Print.

Green, Richard E., et al. "The Neanderthal Genome and Ancient DNA Authenticity." EMBO Journal 28.17 (2009): 2494–502. Web. 21 Jan. 2016.

Jones, Martin. The Molecule Hunt: Archaeology and the Search for Ancient DNA. 2001. New York: Arcade, 2002. Print.

Kenneally, Christine. The Invisible History of the Human Race: How DNA and History Shape Our Identities and Our Futures. New York: Viking, 2014. Print.

Kerner, Gaspard, Jeremy Choin, and Lluis Quintana-Murci. "Ancient DNA as a Tool for Medical Research." Nature Medicine, vol. 29, 15 Mar. 2023, doi.org/10.1038/s41591-023-02244-4. Accessed 4 Sept. 2024.

Lane, Nick Power, Sex, Suicide: Mitochondria and the Meaning of Life. New York: Oxford UP, 2005. Print.

Marchant, Jo. "Ancient DNA: Curse of the Pharaoh’s DNA." Nature 472.7344 (2011): 404–6. Academic Search Complete. Web. 15 Jan. 2016.

Maricic, Tomislav, and Svante Pääbo. "Optimization of 454 Sequencing Library Preparation from Small Amounts of DNA Permits Sequence Determination of Both DNA Strands." BioTechniques 46.1 (2009): 51–57. Web. 21 Jan. 2016.

Mithen, Steven. Singing Neanderthals: The Origins of Music, Language, Mind, and Body. 2005. Cambridge: Harvard UP, 2006. Print.

Mora, Camilo, et al. "How Many Species Are There on Earth and in the Ocean?" PLOS Biology 9.8 (2011): n. pag. Web. 21 Jan. 2016.

Pennisi, Elizabeth. “A Shaggy Dog History.” Science 298.5598 (2002): 1540–42. Academic Search Complete. Web. 15 Jan. 2016.

Pruvost, Mélanie, et al. "DNA Diagenesis and Palaeogenetic Analysis: Critical Assessment and Methodological Progress." Palaeogeography, Palaeoclimatology, Palaeoecology 266.3–4 (2008): 211–19. Print.

Schmidt, Alexander R., et al. "A Microworld in Triassic Amber." Nature 444.7121: 835. Academic Search Complete. Web. 21 Jan. 2016.

Strickland, Ashley. "Ancient DNA from a Teen Girl Reveals Previously Unknown Group of Humans." CNN, 15 Aug. 2021, www.cnn.com/2021/08/25/world/wallacea-skeleton-dna-discovery-scn/index.html. Accessed 4 Sept. 2024.

Stringer, Chris. Lone Survivors: How We Came to Be the Only Humans on Earth. 2011. New York: Holt, 2012. Print.

Wayne, Robert K., Jennifer A. Leonard, and Alan Cooper. “Full of Sound and Fury: The Recent History of Ancient DNA.” Annual Review of Ecology and Systematics 30 (1999): 457–77. Academic Search Complete. Web. 15 Jan. 2016.

Zimmer, Carl. Evolution: The Triumph of an Idea. New York: Harper, 2001. Print.