Digging up DNA

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

It wasn’t quite Jurassic Park, but palaeontologists from North Carolina State University announced in Nature on April 30 that they had sequenced collagen from a mummified herbivorous hadrosaur, Brachylophosaurus Canadensis, that died 80 million years ago. It was very similar to collagen from a modern turkey.

Their findings, and their previous analysis of mineralised collagen from a 68 million-year old Tyrannosaurus, have added to evidence that modern birds are actually living dinosaurs, sharing a theropod ancestor with predatory dromaeosaurids like Tyrannosaurus and Velociraptor.

But the genes that coded for the collagen have not survived, and the whole premise of Jurassic Park, that dinosaur DNA preserved in blood-sucking insects in ancient amber can be used to resurrect extinct dinosaurs, is pure science fiction.

How long can sequenceable DNA survive in ancient bones, or in the deep-frozen flesh of an extinct woolly rhino or a mammoth that conveniently interred itself under optimal conditions in Siberian permafrost? Not long at all, on the geological timescale, says Dr Jeremy Austin, of the Australian Centre for Ancient DNA at the University of Adelaide.

And for Australia, which has probably not seen permafrost since the glacial maximum around 20,000 years ago, the answer would seem to be not much more than 40,000 years.

The driest inhabited continent also has few cold, wet, caves harbouring animal bones of the right vintage, so since Austin and Australian Research Council Federation Fellow Professor Alan Cooper established the new centre in 2005, aged nucleic acid has been in short supply.

The University of Adelaide funded the centre, and Austin was recruited from the Museum of Victoria, while Cooper was repatriated from Oxford University to become director.

Its initial focus was on resolving the most controversial issue in Australian paleontology: what wiped out Australia’s Pleistocene megafauna, and when?

In the past decade, evidence has increasingly suggested that species like the cow-sized wombat Diprotodon, the huge browsing kangaroo Procoptodon, and the fearsome, crocodile-sized varanid lizard, Megalania, coincided approximately with the arrival of the first humans in Australia 45-50,000 years ago.

But were such Brobdignagian beasts already headed for extinction when humans arrived, because of epochal climate change that set in around two million years ago, drastically increasing fire frequency and altering landscapes across the continent?

The answer to that question could offer clues to how Australia’s modern flora and fauna will respond to climate changes that are occurring more rapidly than at any time during the Pleistocene, as human activity floods the atmosphere with billions of tonnes of greenhouse gases.

---PB--- Neanderthals and Mungo Man

Professor Svante Paabo’s team at the Max-Planck Institute for Evolutionary Anthropology is preparing to publish the results of its project to sequence the genome of Europe’s extinct Neanderthals, after publishing a complete Neanderthal mitochondrial (mtDNA) DNA genome sequence in 2000.

In 1995, CSIRO and Australian National University molecular geneticists made international headlines when it published a partial mtDNA sequence from an early modern human skeleton found buried in the dunes at Lake Mungo, in south-western NSW.

The age of “Mungo Man” has been variously estimated at between 30,000 and 62,000 years – the upper estimate, which lies outside the range of carbon dating, was obtained with an electron spin resonance.

“Mungo Man” appeared to have an mtDNA signature that pre-dated the archetypal mtDNA signature of “African Eve”, the putative female ancestor of all modern humans.

But Austin says the study is now widely disbelieved, and may have been an artifact of contamination by modern human DNA. “Few people believed it when it came out, and there has been no publication since, suggesting it was a one-off result,” he says.

“The polymerase chain reaction used to amplify DNA is extremely sensitive, and if you’re working with ancient DNA, there are many ways you can get caught out by contamination.”

Austin says the current believable limit for ancient animal DNA sequences under the best conditions – ancient ice cores or permafrost that has not been thawed and refrozen – is around 100,000 years, and no more than twice that figure.

“Some people claim to have extracted ancient DNA from specimens in glaciers and ice cores gong back 300,000 to 400,000 years, but you can choose to believe that or not,” he says. “But in Australia, the maximum age comes down significantly.

“We have some nicely preserved megafauna material from Tasmania, but it’s mostly proved intractable to DNA sequencing. One theory is that Tasmania was the last refuge for the megafauna.

“We’ve had the bones carbon dated, and they come out between 30,000 and 50,000 years, which probably explains why sequencing is not working – it comes out of caves in Tasmania that have variously been wet and dry for long periods of time.”

In 2002, Dr John Long, then with the Museum of WA, discovered an undisturbed, intact skeleton of Australia’s biggest marsupial predator, Thylacoleo carnifex, in a limestone cave beneath the Nullabor Plain. Its remarkable state of preservation gave molecular geneticists hope that it might be relatively recent – mere tens of thousands of years old.

Austin was pessimistic about the prospect of recovering DNA from the fossil. “I’m not a paleontologist, but it looked pretty old to me,” he says.

He was right: it proved to be several hundred thousand years old.

“In the Australian context, we’re pretty much limited to material from species that have gone extinct or died since the last glacial maximum 20,000 years ago. We’re looking at thylacines and Tasmanian devils from Tasmania and the mainland, where both species went extinct around 4-5000 years ago. We’ve got material from Western Australia and south-western Victoria, on the border with South Australia.

“We’re trying to recreate the population history of both species, to determine whether their extinction on the Australian mainland was a fast or drawn-out affair, and whether it was due to the arrival of dingoes in the continent from south-east Asia.

“It’s not easy material to work with, but the Tasmanian devil material from the mainland is showing less genetic diversity than we had expected, which suggests the species underwent some population bottleneck a long time ago, before something similar happened in the surviving population in Tasmania.

---PB--- Mass extinctions

DNA studies of devils in Tasmania had shown that, in the northern and eastern areas of the island, Sarcophilus harrissi is almost as deficient in genetic diversity as Africa’s cheetah, explaining its susceptibility to the lethal orofacial cancer that has decimated the species in its last stronghold.

During the Pleistocene, Australia had three devil species: S. harrissii, S. moornaensis and the largest of the three, S. laniarius, which weighed 10kg or more.

“The lack of genetic diversity in the Tasmanian devil could have been the result of a founder effect, from a small starting population, but we know that Tasmania has been repeatedly connected and disconnected from the mainland during the past few hundred thousand years, so there were opportunities for infusion of new genetic diversity from the mainland populations.”

Another species of interest for the centre was the giant flightless bird Genyornis. Large deposits of fragmented Genyornis eggshells have been found in settings that suggest human activity, and the species’ rapid decline 50,000 years ago is the best evidence for human involvement in the Pleistocene mass extinction. But Genyornis fossils are again too old to yield sequenceable DNA.

Given the difficulty of finding ancient DNA from Australia, the centre has turned its attention overseas for projects – and over the Tasman Sea, in New Zealand.

“We’ve got a PhD student looking at the population history and demographics of moas,” Austin says. “There seems to be a large amount of genetic diversity in moas, and Nic Rawlence is looking at two or three species.

“It’s not really clear what role climate change played a role in the extinction of the moas over the past 40,000 years, but the big advantage of New Zealand is that humans didn’t get there until very late, so the impact of climate change after the last glacial maximum is much easier to distinguish.

Austin says that when Alan Cooper was at Oxford, he sequenced DNA recovered from the bones of New Zealand’s extinct Haast’s eagle, Harpagornis moorei, the largest eagle that ever lived.

The huge eagle, which is thought to have gone extinct around 1400AD, several centuries after the ancestors of the Maoris colonised New Zealand, is believed to have preyed on moa.

Although Haast’s eagle had a wingspan of up to three metres, comparable to Australia’s huge wedge-tailed eagle, females weighed up to twice as much – around 12kg.

Cooper’s DNA analysis produced a surprise: rather than being related to wedge-tailed eagles, Aquila audax, Haast’s eagle appears to be a descendant of Australia’s smallest eagle, the little eagle, Hieraaetus morphnoides.

Despite the size disparity, Haast’s eagle shared its Australian ancestor’s stocky, muscular build, and probably originated as a trans-Tasman blow-in. “It shows how rapidly species can evolve in new environments,” Austin says.

---PB--- Pre-European diversity

One of the things the centre is most interested in is Australia’s pre-European biodiversity. It’s not as flashy as working on mammoths and other extinct megafauna, Austin says, but for better or worse, Australia has suffered one of the largest fauna extinctions in the world since European colonisation – second only to New Zealand.

“The early naturalists collected a lot of specimens of species that are now extinct, or locally extinct, and it’s a very valuable resource for people trying to preserve what remains of our biodiversity,” he says. “It’s also helping to identify pre-European hotspots for biodiversity, and where species contracted to. “So much has gone missing that we have to draw conclusions based on the small fragments of what is left – but it’s doable, because we have the museum collections.

“I’ve been working on eastern bettongs, which went extinct on the mainland in the 1920s, but are doing very well in Tasmania. The problem is that since foxes were introduced into Tasmania, eastern bettongs are now at risk.

“The Department of Primary Industry and Water instigated the project, and it will answer important questions, including how closely the Tasmanian and mainland bettongs were related.

“That will tell us whether re-introducing the Tasmanian bettongs to the mainland might be successful – there’s a big project out in east Gippsland, the Southern Ark Project, which aims to drastically reduce fox populations in the longer term, which would make it possible to establish bettongs there.

“Within Tasmania, we’re looking at whether there are distinct sub-populations and the extent of gene flow between them, which will help people in Tasmania to manage the local bettongs.”

A project to determine the genetic relationships between the Tasmanian and mainland thylacine populations is making slow progress. “We now have a few small thylacine nuclear DNA sequences, and two complete mitochondrial genomes in GENBANK, but without a living specimen to design PCR primers from, it’s slow work.”

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.