Stone-age bears used to urinate in a Mexican cave: Danish researchers only needed a drop to reconstruct their DNA

The Danish-led study was recently published in the scientific journal Current Biology – and it indicates that soil samples greatly increase our potential to study the evolution of animals, plants and microorganisms.

For years, the USSR and the USA battled to become the first nation to put a man on the moon.

The competition was ruthless, and there was much sweating in the laboratories of the two superpowers from the late fifties until the USA and Neil Armstrong finally declared themselves the winners in July 1969!

This intense determination to be the first across the line was no less in the competition between several scientific teams specialising in analysis of ancient DNA. They have all been tackling this tough challenge for almost a decade, using increasingly sophisticated methods of analysis and tools for DNA sequencing to recreate the genomes – basically, the entire genetic code – of animals, plants and bacteria, based on microscopic fragments of DNA.

These DNA fragments could have been buried in the earth since the Stone Age and may even have been there for longer.

Two teams came in first – they did it! And they recently shared the gold medal – that is, the honour – when the results of their projects were published in the scientific journal Current Biology.

One of these projects was led by researchers from the University of Vienna, the other by Professor Eske Willerslev and Associate Professor Mikkel Winther Pedersen, both from the Lundbeck Foundation GeoGenetics Centre at the University of Copenhagen.

Researchers from the USA, the UK, Canada, Mexico, Norway and China also worked on the Danish-led project.

And it really was a race, explains Professor Willerslev:

‘All over the world, everyone scientifically involved in the study of ancient DNA recognised the impending need to reconstruct genomes from fragments of ancient genetic material. If we could do that – and this is what we demonstrate here – it would suddenly give us access to a wealth of opportunities.’

This is because working with highly fragmented DNA from soil samples taken from caves, or perhaps Stone-Age settlements, means that we are no longer dependent on samples taken from bone or teeth to give us sufficient genetic material to recreate a profile of ancient DNA.

‘What’s more,’ says Eske Willerslev, ‘in the future, sophisticated analyses of fragmented DNA found in soil could have the potential to expand the narrative about everything from the evolution of species to developments in climate change.’

Associate Professor Mikkel Winther Pedersen, first author of the team’s article in Current Biology, sees the new method as the dawn of an “entirely new era” of population genetics:

‘So far, studies of ancient DNA have been decidedly limited. Fragmented DNA from a soil sample could only tell us whether a specific species lived in a certain locality at a certain time, but it gave us no concrete details about the individual in question. So, we couldn’t compare this individual with present-day individuals of the same species. But we can now. For example, in our article, we publish for the first time a DNA profile of an American black bear that lived in a mountain cave in northern Mexico in the Stone Age. I don’t believe I’m exaggerating when I say that the potential to extract this type of information from a soil sample of a mere few grams will most likely revolutionise our field,’ says Mikkel Winther Pedersen.

In the cave

Last year, the Mexican cave played a starring role in a scientific article in Nature, also written by Eske Willerslev and Mikkel Winther Pedersen. And Chiquihuite, as the cave is called, is fascinating in more ways than one:

It is located in northern Mexico, 2,750 metres above sea level, high up a mountain slope with a spectacular view over a valley.

 

It takes some climbing to get to, but the effort is well worth it. Three connected caverns await, each measuring around 50 x 15 metres with a ceiling height of 20 to 30 metres.

The cave is the result of aeons of erosion and deposits from the soft limestone wall. Over time, all this material – plus the dust that has blown in from the valley – has created a three-metre thick ‘floor’, a column made up of six layers of soil and detritus, all of which can be dated.

In the 2020 Nature article, the researchers were able to demonstrate, among other things, that the first humans found their way to the Chiquihuite Cave around 30,000 years ago. The discovery of flint tools in an untouched layer dated to this time was proof of this – and the find had a huge impact on the dating of human migration to North America. Previously, scientific documentation had indicated that the earliest migration to the continent only took place around 15,000 years ago.

No human DNA was found in the cave excavations on which last year’s Nature article was based. However, the DNA of Stone-Age humans could easily come to light once the researchers at the Lundbeck Foundation GeoGenetics Centre analyse the many hundreds of soil samples taken from the cave, which have been lying in the University of Copenhagen’s freezers.

On the other hand, the 2020 Nature article revealed that, in addition to the DNA of mice and other rodents, the researchers found Stone-Age DNA of the American black bear, a species living today, and of the extinct short-faced bear (Arctodus simus).

The short-faced bear, which lived in North America and died out around 12,000 years ago, was a gigantic predator. It was almost two metres tall on all fours and could weigh up to 1,000 kilos.

The genomes we managed to create were able to tell an extremely detailed story

‘The bear DNA from the soil samples taken from the cave played a key role when we attempted to reconstruct the genomes of the two species for the article we have just published in Current Biology,’ Eske Willerslev explains.

‘And the genomes we managed to create were able to tell an extremely detailed story. With regard to the American black bear, they proved that the black bears living in the Chiquihuite Cave at that time are ancestors of the black bear population currently living in eastern North America.’

When it comes to the short-faced bear, the researchers made a remarkable discovery when they compared the reconstructed Stone-Age DNA from the cave with a DNA profile extracted from a 20,000-year-old bone from the species found in north-western Canada. Mikkel Winther Pedersen explains:

‘The short-faced bears that lived in northern Mexico were distinctly different from the population of black bears living in north-western Canada. And this is actually an excellent example of the new knowledge that suddenly becomes available when you reconstruct genomes based on DNA fragments extracted from soil.’

Hair, Urine and faeces

But where do these DNA fragments found in the soil come from? And how are they formed?

As Eske Willerslev explains, there could be a number of sources, but hair, urine and faeces, in particular, play a key role. They all provide genetic material which, in the right conditions, can survive for much longer than 10,000 years:

‘When an animal or a human urinates or defecates, cells from the organism are also excreted. And the DNA fragments from these cells are what we can detect in the soil samples. Using extremely powerful sequencing techniques, we can now reconstruct genomes – genetic profiles – based on these fragments.’

Fragments of this kind can be found all over, particularly in dry, cold climates. Damp and heat, on the other hand, tend to break down DNA rather quickly.

Eske Willerslev believes that, in a Danish context, it is realistic to expect to find fragmented DNA from animals, humans, plants and microorganisms in many former Stone-Age settlements, and he adds:

‘Imagine the stories those traces could tell. It’s a little insane – but also fascinating – to think that, back in the Stone Age, these bears urinated and defecated in the Chiquihuite Cave and left us the traces we’re able to analyse today.’

_{DOI: 10.1016/j.cub.2021.04.027}