Leveling the Field in Ancient DNA Research

Some stories of history are written in ink, and others are written in nucleotide bases. Ancient DNA (aDNA) analysis – the study of DNA from archaeological or paleontological specimens – is perhaps the closest scientists will ever come to a time machine – a window into the past. In this feature article, we explore how inclusion and representation are critical considerations when we look through that window.

The realization of a “glorious dream”

In 1984, Dr. Russell Higuchi – then a postdoctoral researcher in the laboratory of the late Professor Allan Wilson at the University of California, Berkeley –  led a study often cited as building the foundations of aDNA research. It was, at least, the first academic account of the field’s development. The researchers extracted mitochondrial DNA (mtDNA) from dried muscle samples belonging to a 140-year-old quagga from the Natural History Museum in Mainz, Germany. The evolutionary history of the quagga – extinct as of 1883 – carried physical traits resembling both zebras and horses; to which was it more closely related? Higuchi and his team’s successful sequencing of the two short mtDNA sequences confirmed that, indeed, the quagga was more closely related to zebras than to horses. Specifically, it shared a common ancestor with a mountain zebra (known as the Equus zebra) several million years ago.

In an accompanying commentary to Higuchi’s paper, British geneticist Sir Alec Jeffreys expressed that “any hopes that molecular biology and paleontology can be fused into a grand evolutionary synthesis by studying fossil DNA, still look like nothing more than a glorious dream.” Given the ever-expanding volume of aDNA studies published in the literature today and the field’s recent win of the 2022 Nobel Prize for Medicine or Physiology, you might interpret Jeffrey’s reservations as… well, pessimistic.

However, he spoke to the tremendous challenges associated with extracting and studying aDNA – including DNA degradation and exogenous DNA contamination. It would take the introduction of the polymerase chain reaction (PCR) in the 1980s, and the profound impact of next-generation sequencing technologies (NGS) in the decades that followed to create the flourishing research field we see today.

It seems Jeffreys’ “glorious dream” has been realized. But what has it taught us?

Learnings from the past

Almost four decades have passed since Higuchi and colleagues’ quagga paper. During that time, the breadth and scope of aDNA studies have expanded exponentially. DNA from archaeological samples has helped to reconstruct the evolutionary history of the human species; the first draft Neanderthal genome was sequenced by Professor Svante Pääbo – dubbed the “Dark Lord” of aDNA by some – and colleagues in 2010. Since then, new branches of the human family tree have been identified, such as the Denisovans. Knowledge of our partial ancestor’s DNA code has even helped to understand why some people might be more at risk of suffering certain diseases, like COVID-19, than others.

aDNA, recovered from the teeth of individuals laid to rest in graves inscribed with “pestilence”, shed light on the origins of the Black Death. A bubonic plague outbreak that claimed 50–60% of West Eurasia’s population in just seven years, the Black Death’s “launch point” is now believed to lie somewhere in the wider Tian Shan area, an impressive mountain system that straddles the border between China and Kyrgyzstan. Successful sequencing of the woolly mammoth (Mammuthus primigenius) genome has even made the prospect of “reviving” the extinct species a plausible reality, with a little help from genetic engineering.

Looking to the past might well offer scientists clues on how to handle the inevitable challenges our planet is yet to face, such as the impact of global warming. In late 2022, a study published in Nature outlined the discovery and analysis of aDNA obtained from clay and quartz samples collected from a geologic land point known as the København Formation in northern Greenland. The work uncovered a two-million-year-old ecosystem, one that weathered extreme temperatures. Such a climate would have required adaptation from organisms inhabiting the environment to survive. “It is possible that genetic engineering could [be used to] mimic the strategy developed by plants and trees two million years ago to survive in a climate characterized by rising temperatures and prevent the extinction of some species, plants and trees,” Professor Kurt Kjær, a geology expert based at the University of Copenhagen, and co-author of the study said. “This is one of the reasons this scientific advance is so significant because it could reveal how to attempt to counteract the devastating impact of global warming.”

“While the majority of ancient DNA research is focused on big-picture, curiosity-driven or ‘blue skies’ questions, there is a growing appreciation that ancient DNA can be used for more applied aspects of science,” – Dr. Nicholas Rawlence, director of the Palaeogenetics Laboratory at the University of Otago, wrote in a 2021 editorial published in Frontiers in Ecology and Evolution.

Year upon year, records of the oldest DNA recovered and sequenced are shattered. Comprehensive reviews of the field, such as Orlando et al’s Ancient DNA Analysis, offer further reading of work that lies beyond the scope of this article.

As the study of aDNA continues to evolve, arguments for greater inclusion, equality and respect for Indigenous communities and their oral histories growing louder.

Professor María del Carmen Ávila Arcos, who leads the International Laboratory for Human Genome Research (LIIGH) in Querétaro, Mexico, is one of the researchers championing sustainable practices in aDNA research.

Challenging underrepresentation in the global catalogs of genetic variation

After her initial interest in aDNA was piqued by the early Neanderthal DNA papers, Ávila Arcos observed how a large proportion of genetics research focused on European or European-descent populations. Sadly, this issue is not confined to the perimeters of the aDNA field. Appeals for increased diversity in genomics research – often used to inform disease risk, progression and treatment in modern medicine – have increased over recent decades. Progress in acting on such calls, however, has been criticized.  

Ávila Arcos chose to direct her time and efforts to counterbalance the unevenness in the field. At LIIGH, her research group explores the genetic history of understudied populations – particularly Indigenous and Afromexican peoples – combining DNA from ancient and modern-day populations. The study of aDNA in this context carries great significance for understanding Mexico’s colonial history, and for providing present-day populations with knowledge of their genetic background, she explains.

During the 16^th century, the Spaniard Hernán Cortés landed on the shore of Veracruz on the Gulf of Mexico, accompanied by “conquistadores” (meaning “conqueror” in Spanish). Cortés journeyed onward to the Aztec capital of Tenochtitlán (now Mexico City), eventually colonizing the region and claiming the Aztec empire for Spain, naming it “Nueva España” – “New Spain”. Hundreds of years of human suffering would follow. The impact on the Indigenous population was devastating. Not only did European colonization inflict extreme massacre through violence and displacement of the Natives, but it also introduced deadly epidemics. It is suggested that the susceptibility of Native populations to “old world” diseases may have even contributed to the European conquest being possible.

Up to 90% of Native populations were lost in some regions, Ávila Arcos explains: “This drastic reduction in the size of the Native populations decreased the amount of genetic diversity in the Indigenous population, and what we observe today in the present-day population is only a fraction of what existed over 500 years ago.”

"To have an accurate notion of the amount of genetic diversity that existed prior to colonization, and how this has changed through time – resulting in the genetics of present-day Indigenous populations (and admixed Mexicans too) – we can leverage the power of aDNA.”

Further negative consequences of colonization – disease

Ávila Arcos is also harnessing aDNA analysis to understand the genetic legacy of the transatlantic slave trade in Mexico. During the Colonial period, millions of individuals from the West Coast of Africa were enslaved and forced to work as laborers in the Americas. “The contribution of enslaved Africans to the construction of our nation has been neglected for centuries, which has resulted in an erasure of past and present Afrodescendants in Mexico,” Ávila Arcos says. “Today, several social movements are demanding a recognition of the contribution of Afrodescendants to our history and to our present.”

The ongoing research – called “The Afro-Mexico Genomics Project” – started nine years ago, and has received support from Stanford University (where Ávila Arcos completed her postdoctoral research) and The National Autonomous University of Mexico (UNAM). “It started from an authentic interest in learning more about the genetic contribution of African genes to our genetic pool, and also from the frustrating realization of the systematic and institutional efforts to erase Afrodescendants from the idealized narrative of Mexico being the product of solely Indigenous and European admixture,” Ávila Arcos explains.

In collaboration with Afrodescendant communities from the Pacific coast of Mexico (regions called Costa Chica and Veracruz), the project team is sequencing DNA extracted from the saliva of over 300 participants and genotyping them. “In parallel, we started a project to genetically characterize aDNA from samples of individuals very likely of African ancestry, dated to the early colonial period in Mexico,” she explains. Though the work has not yet been published, Ávila Arcos says their current data shows, for the first time, the extent of genetic variation shared between modern African populations present in Afrodescendants from Costa Chica and Veracruz. “We observed higher values in Costa Chica than in Veracruz and have been able to suggest likely places of origin in Africa for this African genetic component,” she explains.

“One important motivation behind our work is the underrepresentation of Mexicans, particularly Indigenous and Afromexicans, in the global catalogs of genetic variation, which dispossesses them from the knowledge of their genetic history and potential beneficial findings,” – Ávila Arcos.

Understanding which viruses may have contributed to the large-scale death of Native populations is another important application of aDNA for Ávila Arcos. Her team extracted and enriched viral DNA from skeletal remains found in mass graves located in present-day Mexico City, dating back to the 16^th century. The application of aDNA analysis in this context is referred to as paleovirology, and it helped the team shed some light on pathogen biology and transmission during this period. “A remarkable finding obtained from aDNA was that some African-born individuals – who were likely taken to New Spain under force as part of the transatlantic slave trade – carried some viruses: hepatitis B and human parvovirus B19, that were also of likely African origin,” Ávila Arcos explains. “This implied that some of the pathogens that were circulating during colonial times had an African origin, highlighting additional negative consequences that the European colonization carried for Indigenous and African populations.”

Paleovirology can also offer evidence on ancient social interactions, as a 2020 study of the “red complex” – a collection of three oral bacteria: Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola – revealed. Known to contribute to periodontal disease, T. forsythia was detected in dentin and dental calculus samples obtained from skeletal remains spanning the Pre-Hispanic and Colonial period in Mexico. Analyzing the T. forsythia genomes, Ávila-Arcos and colleagues noted the presence of specific genes in the Pre-Hispanic individuals that were absent in Colonial individuals – and vice versa. “This study highlights the potential for studying ancient T. forsythia genomes to unveil past social interactions through analysis of disease transmission,” they write.

Towards sustainable aDNA research

Ávila Arcos hopes the impact of her work “brings perspective to our rich and diverse past and present, and the need to level the field for historically oppressed populations.” She emphasizes, “I also hope it reveals the fabulous potential that aDNA carries to study the many aspects of our biological past. Mexico, as a megadiverse county, has a very interesting natural history – and aDNA can help us study it with a temporal lens.”

Leveling the field is not a simple endeavor, however. A consideration for her lab – and the work of any scientist pursuing aDNA analysis – is the cautious handling of ancient material. aDNA analysis requires the destruction of irreplaceable ancient human samples, a responsibility that Ávila Arcos does not carry lightly: “In Mexico, archaeological remains are considered national patrimony. Their destructive analysis should be well justified and carried out responsibly and ethically,” she says. The ability to sequence sedimentary aDNA (sedaDNA) may provide a solution in the future. “We are just starting to realize that soil is a magnificent DNA reservoir of whole ancient ecosystems, and it shows a lot of promise for the study of ancient humans, too. I think in the future there will be a lot more focus on sedaDNA for the study of our population history.” Ávila Arcos adds, “The best part is that we wouldn’t rely solely on destructive analysis of precious human samples to do so!”

Dr. Ávila Arcos explains the genetic ancestry results to a participant from Veracruz. Credit: Pavel Galeana.

Ávila Arcos’ work also carries heavy ethical implications – she deals with incredibly sensitive topics and delving into history can no doubt affect present-day populations. “We need to be extremely careful about how we present and discuss our results. We try to always be very careful not to perpetuate damaging narratives or discrimination,” she says. Alongside colleagues, Ávila Arcos published a perspective article in Frontiers in Genomics, which outlines recommendations for sustainable aDNA research in the Global South. This is a term often used to describe lower-income countries, including those have been historically oppressed by colonialism.

The team state that the appeal of aDNA studies in the media, combined with trends for this work to be published in “high impact” journals, is driving the collection of ancestral human remains with limited or no engagement with local researchers and appropriate communities.

Dr. Gabriel Renaud, associate professor at the Technical University of Denmark (DTU) – whose research interests center around aDNA sequencing and population genomics – offers his thoughts on the challenges in this space: “The discussion about having 10 or so large ancient DNA labs, mostly found in wealthy countries, that use material from less wealthy countries and publish articles in prestigious journals to tell the history of cultures – that they are often only vaguely familiar with – is a movie that we have seen over and over. It reeks of 19th-century ‘archeology’,” he says.

“We need to strike the right balance between giving back to less wealthy countries – especially to the local scientists that provide the expertise – the need to be careful about telling the story of marginalized populations that have suffered from either genocide or colonization, and first-world scientists who want to do the right thing,” Renaud adds. 

Much of the discussion and debate surrounding ethical practices in aDNA stem from Indigenous populations claiming respectful treatment of their ancestors’ remains and respect of their oral histories, Ávila Arcos says: “Many studies (some stemming from the big labs Renaud mentions) publish papers without proper consultation with Native communities, and do not include them in the discussion, or even check if their findings are somehow in conflict with their oral narratives.” Given the violent colonial history of abuse, exploitation and marginalization, minimizing Indigenous voices is a further perpetuation of historical damage, she adds.

How do we strike the balance proposed by Renaud? A “global” approach to aDNA research in the Global South is put forward by Ávila Arcos and colleagues in their Frontiers perspective: “This would entail applying global premises of sustainability and justice and maintaining awareness of the historical harms caused by scientific colonialism, extractivism and other forms of exploitation of Global South nations by Global North researchers,” they write. “I think my Indigenous colleagues have done a great job at highlighting these issues,” Ávila Arcos states. However, their fight for sustainable practices is far from over.

The challenge is part of the research, not a distraction

Earlier this month, a commentary was published in Human Genetics and Genomics (HGG) Advances by Kowal et al. The article is a response to guidelines shared in Nature on “the ethics of DNA research on human remains” by Alpaslan-Roodenberg et al. in 2021. According to the Nature paper, the guidelines are the culmination of a widespread agreement that globally applicable ethical guidelines are needed for aDNA research, but that recent recommendations are not generalizable worldwide.

However, Kowal et al argue that “these guidelines do not sufficiently consider the interests of community stakeholders, including descendant communities and communities with potential – but yet unestablished – ties to ancestors.” The HGG commentary highlights three concerns: the separation of “scientific” and “community” concerns, the commitment to open data which “ignores the principles of Indigenous Data Sovereignty” and the potential risks of not consulting communities that do have established – or potential – ties to ancestors.

“Indigenous consultation for aDNA research is still not a standard in many Global South countries. For this reason, we highlight the need to open spaces for Indigenous scientists and stakeholders to debate these pressing issues,” Ávila Arcos says.

Such open spaces include the SING (Summer Internship for INdigenous peoples in Genomics) consortium workshops that have been conducted in the U.S., Canada, Australia and Aotearoa (New Zealand). SING is “working with leaders to change the narrative of Indigenous genomics”, and Ávila Arcos is proud that the most recent consortium took place in Oaxaca, Mexico: “One of the debates was ethical aDNA research in the context of Mexico. I was pleased to hear from the Indigenous and Afro-descendant students who took part in the workshop. They are the people that really need to be leading these discussions. I hope to incorporate some of the learnings that stemmed from these discussions in my future studies,” she says.

Kowal et al emphasize that aDNA researchers must not focus on the bare minimum research practice that is legally necessary. Rather, aDNA scientists must lead efforts to ensure communities from across the globe are identified and “engaged in research that affects them”. They acknowledge this task will not be without its challenges – nevertheless, “the key difference between our approach and that of Alpaslan-Roodenberg et al is that we see these challenges as part of the research, rather than a distraction from the scientific endeavor,” they write.

“I envision a future in which collaborations between resourceful aDNA labs and local research groups are more horizontal and mutually beneficial,” Ávila Arcos concludes.