Ancient DNA throws a ‘scientific hand grenade’ into the discussion about the arrival of humans in North America

Lundbeck Foundation researchers have analysed 30,000-year-old material excavated from a Mexican mountain cave – where the discovery of stone tools suggests that the first humans arrived in North America at least 15,000 years earlier than thought. The findings have been published in an article in Nature. These DNA analyses will also be used in a major Danish study on brain disorders.

Donkeys are essential when it comes to carrying technical equipment, food and drinking water up to the Chiquihuite Cave in northern Mexico. Neither humans nor machines are up to the job because the cave lies in extremely rough terrain, halfway up a mountain slope. From the dry valley, where the journey begins, there is a steep 1,000-metre climb to the cave. And when – after winding through the piles of boulders that litter the slope – you finally arrive at your destination, 2,750 metres above sea level, an astounding sight awaits.

Because how would you actually define a mountain cave? Well, it could be a small hole in the mountain, perhaps two or three metres deep. You could call that a cave. But it could also, as is the case with the Chiquihuite Cave, be two connected, almost ballroom-sized caverns, each measuring around 50 x 15 metres with a ceiling height of 20 to 30 metres, resulting from aeons of erosion and deposits from the soft limestone wall.

The Chiquihuite Cave made a serious appearance on the scientific radar around ten years ago – the first test excavations were conducted in 2012 – and things have moved on rapidly since then.
The hypothesis is that this cave, which has gradually filled up with detritus from its ceiling and dust from outside, may be an archive; an archive of evidence that could add new facets to the evolutionary history of humans.

And, as indicated in a major research article recently published in Nature, one of the world’s most prestigious scientific journals, this hypothesis stands up to scrutiny. The article is written by 28 researchers – a team of archaeologists and DNA experts – from Mexico, the UK, Denmark, the USA, Brazil and Australia. To give a very condensed version of their discoveries in the Chiquihuite Cave:

The first humans arrived in North America at least 30,000 years ago – around 15,000 years earlier than science previously thought probable. And, referring to the 1,900 stone tools found in the North Mexican cave, the researchers explain that these people had already developed techniques for making tools as far back as then.

‘The Nature article is a scientific hand grenade. The fact that it pushes the date of the first migration to the American continent back significantly is sure to ignite heated debate,’ says Professor Willerslev, head of the Lundbeck Foundation GeoGenetics Centre at the University of Copenhagen (UCPH).

Together with two of his colleagues, Associate Professor Mikkel Winther and Associate Professor Martin Sikora, Eske Willerslev made up the Danish contingent of the international team. The three researchers from UCPH were responsible for the DNA analysis of the ancient remains of animal and plant material that were found during excavation of the Chiquihuite Cave.

A three-metre thick layer
To date, science has assumed that the initial migration to North America began around 15,000 years ago, at the earliest. At this time, a narrow, ice-free corridor appeared along the northern part of the Pacific coast, making it possible to walk from Siberia down through the American continent. There were no other access routes to the continent since North America was covered by a thick ice cap that did not melt sufficiently for passage until much later – around 13,000 years ago.

The unique feature of the Chiquihuite Cave is its ‘floor’, which is made up of six layers of detritus and dust – all in all, a three-metre column of ancient remains. The layers are so compressed and stable that they can be dated one by one, from top to bottom, using a variety of sophisticated measuring techniques. In each layer, the researchers have found deposits of stone tools such as knives, scrapers and arrowheads, which they were also able to date.

‘The cave finds are extremely interesting. These archaeological finds are so far the oldest in America. And the excavated stone tools are of a type unique to America,’ says Professor Ciprian F. Ardelean, of the Universidad Autónoma de Zacatecas, Mexico.

30,000 years ago, when the first stone tools were left in the Chiquihuite Cave, the massive ice cap had not yet covered all of North America. This means that, at this time, it would have been possible to walk from Siberia down through the American continent, Eske Willerslev explains:

‘And that’s how we must understand the presence of these humans in Mexico at this particular time – unfortunately, though, we have no idea who they were. Because although we searched very thoroughly for human DNA in the samples we gathered during the ten days we spent at the Chiquihuite Cave, there were no human traces to be found. However, they could still turn up – we gathered hundreds of earth samples, and we’ve not yet had time to analyse them all.’

Mikkel Winther Pedersen was in charge of the DNA analyses conducted in the laboratory at the Lundbeck Foundation GeoGenetics Centre. Among the finds that have been DNA-profiled, there are numerous plants, grasses and animals. He elaborates:

‘For instance, we found DNA from an American black bear, many different rodents, several types of bat, sparrows and falcons. These are all animals we would expect to find in Mexico at the time. And we were also able to ascertain how the plant composition changed over time, in line with the climate changes that occurred during this period.’
Mikkel Winther Pedersen and Eske Willerslev scraped the samples from the walls of the excavations in the cave. They used sterile scalpels and wore space suits while they worked to prevent contamination of the samples with present-day DNA.

The earth samples were collected in test tubes which were then sealed. In the laboratory in Copenhagen, each sample was examined individually using a special DNA extraction method. Mikkel Winther Pedersen explains:

‘This particular method applies so-called shotgun sequencing. So, we collect all of the DNA from the sample – from animals, plants and microorganisms – and determine where it comes from by comparing it against specific reference libraries. Therefore, if there’d been human DNA in the samples we’ve examined so far, we would have found it.’
As Mikkel Winther Pedersen says, the ancient DNA they are using shotgun sequencing to find in a earth sample is usually extremely fragmented and damaged: ‘It comes from cells, hair, urine and faeces, and it is excreted via plants, animals or humans who were in the locality at the time – in this case in the Chiquihuite Cave. Of course, if we find a bone or a tooth, we perform DNA analyses on these, too.’

Brain disorders
In 2018, the Lundbeck Foundation granted DKK 60 million to a project to investigate the interaction between human DNA and neurological and psychiatric disorders through the ages.
Two of the questions the project seeks to answer are: Where do disorders such as Alzheimer’s disease and schizophrenia come from? And why do these brain disorders get ‘stuck’ in our DNA when they, quite clearly, make life difficult and cause great suffering?

This research is coordinated by the Lundbeck Foundation Centre for Disease Evolution, and the work includes sequencing of 5,000 samples of ancient DNA. The oldest material in the samples will be over 10,000 years old. DNA information from this ancient material needs to be compared with present-day samples, and it is hoped that important evolutionary patterns will appear over time.
The project is headed by Professor Eske Willerslev and Professor Thomas Werge, who is head of the Institute for Biological Psychiatry, Capital Region of Denmark. The analyses that will be conducted to shed light on the origin of neurological and psychiatric disorders must also take into account the environmental factors present at the point in history from which the samples originate.

Environment is potentially always an accomplice if you investigate disease issues from a DNA perspective, says Eske Willerslev:

‘It’s therefore vitally important to be able to conduct precise environmental analyses of localities where ancient DNA has been found. We must be able to see and understand the DNA based on the environment from which it comes. The Chiquihuite Cave will play an important role here, as a model and quality parameter, because the analyses that have been performed are so thorough – in terms of both dating and DNA profiling,’ says Professor Willerslev, and he adds:

‘If we find human DNA in one or more of the cave samples we have yet to analyse, that will just make the story even better.’


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