Smilodon family tree

Mike Thorn (UK)

In his book, “Architects of Eternity: The New Science of Fossils”, Richard Corfield coins the term “reluctant palaeontologists”. He has in mind those chemists, biochemists and biologists who use the techniques and skills from their own disciplines to shed new light on our ideas about evolution. Ross Barnett, of the Department of Zoology at Oxford, might well be considered to be in this category. A biochemist by training, he has recently co-authored a paper on the DNA of three extinct cats which has helped to lay to rest some of the arguments about the feline family tree.

Ross came to Oxford in October 2002, to work on a PhD, after completing his biochemistry degree at Edinburgh. His supervisor, Professor Alan Cooper, was interested in cat genetics and had managed to raise funds to carry out research into the relationships of several extinct cats. In particular, there were questions about where the sabre-toothed cats, such as Smilodon and Homotherium, fitted in.

As Ross explained:

There has been a lot of study done on these animals. For example, there is a huge collection of thousands of individuals of Smilodon from Rancho Le Brea in Los Angeles, so they’ve been really well characterised from their morphology.

What the palaeontologists had concluded from this was that there was a split at the base of the cat family tree between the group that goes on to form the sabre- tooths – the machairodontinae – and the group that forms the modern cats – the felinae.

However, molecular studies that were done back in 1992 by Stephen O’Brien’s group tentatively suggested that Smilodon was part of the modern radiation of cats, the felinae, and that the machairodontinae was not a proper family.”

Another question that Alan hoped would be resolved was the relationships of the American cheetah-like cat, Miracinonyx. Miracinonyx was originally described in the early seventies as being closely related to, or perhaps an ancestor of, the puma. However, in 1979, D B Adams looked at the morphology of the bones and concluded that it was much more cheetah-like, suggesting a relationship with the modern African cheetah instead.

Alan reasoned that both of these questions might be resolved by further DNA studies. The controversial work on Smilodon DNA by Stephen O’Brien and his colleagues had been carried out on a very small sample and was contrary to what the skeletal morphology suggested. The analysis of a larger sample of Smilodon DNA, along with some from a different sabre-toothed cat, would decide the question. If a DNA sample could be found for Miracinonyx, its relationships could also be established beyond doubt. Alan had managed to locate some useful material in the basement of the Zoological Museum in Amsterdam. At the beginning of the twentieth century, bones of the sabre-toothed cat, Smilodon populator, were found in Mylodon Cave, on the southern tip of Patagonia. The remains had been in the cave for around 13,000 years.

However, since the cave was at a constant, cool temperature, they were well preserved. The basement in Amsterdam, where they were subsequently stored, also had a constant, low temperature. An initial analysis of this material suggested that there was a good chance that some DNA might have survived intact and so Alan brought small samples from several bones back to the UK.

To get DNA material for Miracinonyx, the project had to become an international affair. Larry Martin, of the University of Kansas, had recovered a great deal of material from a karst sink-hole in the Bighorn Mountains of Wyoming. The sink-hole, called Natural Trap Cave, has been open for an estimated 50,000 years and is entered through a hole that drops some ten metres to the cave floor below. In ancient times, animals fell through and either died from the fall or were unable to escape and subsequently died of thirst and hunger. Today, it is covered by a grid to prevent palaeontologists suffering a similar fate.

Like the Mylodon Cave in Patagonia, the conditions at the bottom of Natural Trap Cave are constant. It has a cool temperature all year round, which helps to preserve genetic material. The remains of many animals have been found there, including mammoths, American lions, wolves, horses, bison and the well preserved remains of a number of Miracinonyx. It was these bones, radiocarbon dated at around 24,000 years old, that Larry thought could be a source of DNA.

The final set of material came from Dick Harington of the Canadian Museum of Nature, Ottawa. He had collected the remains of the scimitar toothed cat, Homotherium,in the Yukon region. Again, the preservation was good, but the exact age of the material was uncertain. Radiocarbon dating can only date things younger than around 56,000 years and the Homotherium remains were older than this.

With all the samples collected, the Oxford lab began trying to isolate DNA from the material from the three different cats. The analysis of ancient DNA has a rather chequered history. The first scientists to show that DNA could survive after death were Allan Wilson and Russel Higuchi, from the University of California. In 1984, they managed to extract mitochondrial DNA from a 144-year-old specimen of a Quagga, a zebra-like member of the horse family that was hunted to extinction in Africa during the nineteenth century.

They used a technique called bacterial cloning in which bacteria are encouraged to take up genetic material from a sample and then, as they multiply, sufficient copies of the cloned DNA are made to allow analysis. This technique was very time consuming and difficult, so further advances in this field were slow in coming.

Polymerase Chain Reaction

At around the same time that Wilson and Higuchi were publishing their results on the Quagga, Karry Mullis, then working at the Cetus Corporation in California, came up with a new technique called Polymerase Chain Reaction (PCR). Polymerases are enzymes present in all living things and are responsible for copying genetic materials as well as “proofreading” and correcting any errors in these copies. PCR exploits this natural function to rapidly and accurately amplify small amounts of DNA up to quantities that can be analysed. PCR revolutionised DNA studies, inspired the book Jurassic Park and brought forth a flurry of scientific papers. Claims were made of DNA extracted from Egyptian mummies, insects preserved in Miocene amber and even from dinosaur bones. Unfortunately, most of these turned out to be flawed: PCR was so good at amplifying tiny amounts of DNA that even the slightest contaminants from modern DNA sources were picked up.

When Ross and his colleagues were extracting and analysing their DNA, they took precautions to prevent such contamination spoiling their results.

Ross explained that:

We have a separate building, which Alan got money to build from the Welcome Trust, called the Ancient Biomolecules Centre. It has nightly UV lights that come on to help destroy stray DNA. It’s got positive air pressure to stop pollen grains and so on being sucked in when people open doors and it gets wiped with bleach daily. We wear sterile gloves, full body paper suits and masks. When we extract the DNA, we have two tubes, one with the bone sample and one without. The tube without a sample goes through all the same processes that the tube with the sample does and if, at the end, we find DNA in the control tube, we know we have contamination.

As a final check, we sent out bone samples to Ian Barnes, at University College London, to see if he could replicate the results. Ian was the best person for this, as he had been involved with the project from the beginning. During his post-doctoral stint with Alan at Oxford, he had been the first to try amplifying sequences from our Smilodon samples.” A reasonable quantity of DNA was extracted from the Smilodon and Miracinonyx material. As it was a lot older, the Homotherium remains yielded less genetic material but a short fragment of DNA was obtained.”

Another member of the team, Matthew J Phillips, carried out phylogenetic analysis of the genetic material that had been recovered. This analysis strongly supported the placing of the sabre-toothed cats in the machairodontinae and not the felinae, something the team had been expected all along.

As for Miracinonyx, the genetic studies provided strong evidence for it being a close relation of the puma. So, why did the morphological studies place it close to the cheetah? The answer lies in parallel evolution. Based on their findings, the Oxford team believes that the ancestors of the puma migrated from the Old World to the Americas at around six million years ago. Around three million years ago, this ancestor gave rise to two lineages, one which led to the modern puma and one which led to Miracinonyx. At about this time, the prairie was beginning to spread across North America and the team believes that Miracinonyx adapted to fill the niche of high-speed hunter that the cheetah occupies in Africa and Asia today.

This is not the first time that DNA studies have helped to resolve issues that could not be decided by morphological studies alone, as Ross explains:

“Alan [Cooper] has done a lot of work on the Moa in New Zealand. There are thousands of bones there and, until they got a look at the nuclear DNA …, everyone had assumed that there were three species of Moa based on their size. It turns out that there was just one species, which showed extreme sexual dimorphism, with big females and smaller males.”

Smilodon images provided by the Department of Earth Sciences, University of Bristol (with whom copyright remains).

Further reading

Evolution of the extinct Sabretooths and the American Cheetah-like cat”, by Ross Barnett, Ian Barnes, Matthew J Phillips, Larry D Martin, C. Richard Harington, Jennifer A. Leonard and Alan Cooper is published in Current Biology 15(15), R589-R590.

For further reading about the evolution of the big cats, see The Big Cats and Their Fossil Relatives, by Alan Turner, with illustrations by Mauricio Anton, Columbia University Press, New York (1997), ISBN 0-0231-10229-1.

For a popular account of the discovery of PCR, see Architects of Eternity: The New Science of Fossils, by Richard Corfield, Headline Book Publishing (2001), ISBN 0-7472-7179-8.