RAISING THE
DEAD

Tasmania’s marsupial wolf was declared extinct 66 years ago. Now scientists are trying to create a live animal from a pickled one, a project that has garnered a surprising amount of success—and criticism.

By Scott Weidensaul

The small animal is wrinkled and gray, its forelegs curled against its chest in an unintentionally protective position and a long, open incision running the length of its stomach. Stored in a clear jar of alcohol, it hardly looks like the stuff of high-tech science and acrimonious debate. This young thylacine, the marsupial "tiger" of Tasmania, was taken from its mother’s pouch in 1866—at first a curiosity from a weird and newly settled land, and later a pitiable relic of a species recklessly driven to extinction.

But now, 136 years after its death and 66 years after its species was declared extinct, the preserved baby sits at the junction of molecular biology, conservation ethics, and endangered-species politics—and also at the locus of humanity’s guilt and hopes in dealing with the natural world. That’s a lot to pin to a dead creature you could easily cup in two hands, but ever since 1999, when the Australian Museum in Sydney announced its intention to clone a living thylacine from this pickled specimen, the reaction, both pro and con, has been surprisingly fierce. Critics have lambasted it as science fiction that will drain money from more important work; proponents see it as a way to mitigate a grievous wrong committed against the planet, while burnishing Australia’s languishing scientific reputation.

The man at the center of the storm is Don Colgan, a soft-spoken evolutionary biologist who heads the museum’s cloning team. He admits the odds of ever producing a live thylacine are long, but his group has already managed to extract unusually good-quality DNA from the preserved baby. Several other cloning projects around the world have focused on long-extinct species, but only the thylacine team has posted any significant success to date (see "Born Again?," page 61). If Colgan succeeds where so many have predicted failure, he will not only have beaten his own odds but restored—at least in facsimile—one of the natural world’s most unusual masterpieces.

To understand why the museum’s project has attracted such attention, it’s important to understand the significance of the thylacine from both a biological and a cultural perspective. Australia’s unique, marsupial-dominated fauna has long been a textbook example of convergent evolution, and in this the thylacine is Exhibit A. Though more closely related to kangaroos and opossums, it was molded by the demands of its predatory lifestyle into a close analog of the wolf—a lean hunter with a short brownish coat, a stiff tail, and more than a dozen dark stripes along its back and hindquarters. At roughly 65 pounds, the thylacine was the largest of the carnivorous marsupials and an accomplished hunter of wallabies and other grazing species in the coastal scrublands, eucalyptus forests, and alpine meadows of Tasmania—"Tassie" to most Australians.

English settlers in the early 19th century—the first Europeans to have any close dealings with the thylacine—variously dubbed it the marsupial wolf, a native cat, or even a hyena. But the name that stuck was Tasmanian tiger, even though there was nothing remotely feline about the animal. (The name thylacine, coined by scientists, comes from the Greek words for "pouched dog.") Regardless of what they called it, sheep farmers accused the thylacine of attacking their flocks, and after a century of trapping, shooting, and poisoning, the last known thylacine died in a zoo in 1936—ironically, less than two months after the Tasmanian government extended legal protection to the species.

But Tassie has been reluctant to let go of its tiger. The beast has become an icon—stylized on automobile license plates, gracing the label of the state’s best-selling beer, adopted as the symbol of everything from a regional television network to local sports teams. Of the animal itself, though, there is nothing but specimens locked up in museum cases or gathering dust on shelves. Most consist of stuffed skins or skeletal material, but a fair number are pouch babies, the nearly hairless neonates too young to be out on their own. The usual method for preserving such soft-tissue specimens is to submerge them in formaldehyde, but the Australian Museum’s now-famous baby is embalmed in alcohol. That makes all the difference, Don Colgan explained, as he guided me through the warren of narrow corridors and cluttered labs in the museum’s research wing.

Formaldehyde is hard on DNA’s double helix of amino acids, Colgan said, but alcohol is much gentler—meaning that it has been possible to extract relatively high-quality DNA from several tiny samples of the thylacine’s heart, liver, and muscle. Colgan pulled a large X ray from a file and held it up to the light, showing me five lines a couple of inches long and heavily crossbarred with light and dark bands—the DNA, treated with radioactive nucleotides and photographed. With his index finger, he indicated one of the blurry streaks.

"The DNA represented on this line would be about 40 copies of every gene in the thylacine genome," Colgan said. "That doesn’t sound a lot, but it was extracted from probably a match-head-size piece of tissue." Scientists measure DNA fragments by the number of base pairs they contain—the rungs that form the twisted ladder of a DNA molecule. This thylacine’s DNA contains 1,200 to 2,000 base pairs per section—badly fragmented when compared with samples from living organisms but 10 times better than is normal with ancient DNA.

Given the surprisingly good results, the museum’s team is preparing to create a bacterial genomic library by inserting the thylacine genes into bacteria, which will allow the scientists to grow as much of the genetic material as they need. Beyond that, they hope to have the Tasmanian tiger genes sequenced—their genomic code "read" in its proper order. "It’s like putting a jigsaw puzzle together; if you have large pieces covering the same area as a number of very small pieces, obviously it’s a lot easier with the large pieces," Colgan said.

In the near term, genomic sequencing promises several scientific payoffs, he said, including genetic comparisons with other marsupials. But it is the prospect of cloning—creating a living, breathing thylacine—that raises the greatest expectations and presents the most serious challenges. In normal cloning, the nucleus of a host egg cell is removed and another living cell, containing the DNA blueprint for the organism being cloned, is inserted. (Because each clone is an exact genetic copy of the parent specimen, one must have multiple specimens of both sexes and a variety of family lineages to create a reproducing population. Colgan noted that there are hundreds of thylacine specimens in the world’s museums, many of which may provide equally usable DNA.)

The trouble is that with ancient DNA, there is no living cell nucleus to serve as a starting point, so unless someone invents a way to tease a dead cell back to life, Colgan and his team must use what he calls "the brute force approach." This entails reading what they can of the thylacine genome and filling in any gaps with DNA from other marsupials, then creating artificial chromosomes, packaging them in artificial membranes, and inserting them into an egg from a closely related species. The Tasmanian devil and the numbat, the latter a small termite-hunter, are prime candidates for supplying both missing DNA and a host egg.

If that sounds a bit like science fiction, you’re close. Creating an artificial chromosome would require assembling roughly 50,000 pieces of DNA, each containing about 2,000 base pairs, in exactly the correct order for each of the thylacine’s chromosomes, which may number between 30 and 80—a task currently beyond anyone’s capability, and likely to remain so for the foreseeable future. What’s more, scientists are learning that even with conventional cloning, random errors seem to creep into the genetic code and create all manner of flaws in the cloned animal, from obesity and heart defects to immune-system malfunctions.

The quality of the DNA extracted from the preserved pup makes at least the sequencing part of the job easier, Colgan said, and he is confident that decoding the tiger’s genome is simply a matter of time. He’s exploring partnerships with large sequencing companies, which could run through the thylacine’s 3.5 billion base pairs in a few years. But the price tag would be high; while the initial work has been relatively inexpensive, just sequencing the genomes of the thylacine and potential host species may run to $15 million each. (Funding so far has come from the Australian Museum, the government of New South Wales, and a private trust set up by two brothers specifically to support the cloning effort.)

Even assuming his team can sequence the tiger’s complete genetic library, Colgan pegs the chances of cloning a live thylacine at only 30 percent over 200 years. "If the project ever does come to fruition and we have a bounding baby thylacine, it’ll be because of advances we simply can’t conceive of at the moment," he said.

Another scientist who is using cloning technology, however, says the roadblocks facing the Australian Museum are almost insurmountable. Alan Trounson heads a small team at the Monash Institute for Reproduction and Development, in Melbourne, that’s working to clone the critically endangered northern hairy-nosed wombat. Trounson told me the thylacine project faces two barriers, one technical and the other biological. First, he said, barring advances in the synthesis of artificial chromosomes, the alcohol that preserved the DNA so well for sequencing also negates the chance of ever using those cells directly for cloning, since there is no way to rehydrate them once the alcohol is removed and the cellular structure collapses.

"I am very supportive of their looking at the DNA sequence and perhaps comparing it to the DNA of existing Australian mammals—I think that could be very valuable," Trounson said. "But if they’re talking about cloning a living animal, there’s no way they can use cells that have been fixed in alcohol. I know what alcohol does to cells, and I can’t for the life of me see how they think they’re going to do it."

But even if they progress beyond that, there’s a bigger hurdle remaining.

"If it were technically feasible, and we had cells that were theoretically alive—that is, the nucleus was whole and functional—then what do you put it in?" asked Trounson. He dismisses the idea of using Tasmanian devils or numbats as surrogates, noting that the thylacine split off from other marsupials an estimated 30 million years ago, leaving it with no near cousins—unlike the wombat, which has a very close relative to serve as a surrogate mother.

Others in the Australian conservation community look askance at the thylacine project, not on scientific grounds but for reasons of priority and ethics. I left Sydney and flew the 600 miles southwest to Hobart, the capital of Tasmania, to meet with Michael Lynch, the executive director of the Tasmanian Conservation Trust. He didn’t mince words when asked for his opinion.

"It just seems so loopy," said Lynch, a bluff, open-faced guy in his late 50s with a full head of curly, gray-white hair. "It’s a bit like boys with their toys, like men with their big guns and their bulldozers. It’s about science for the sake of science.

"Like the United States, we’ve got thousands of species on the endangered list," Lynch continued, "and we are just so poor in terms of the ability of the commonwealth and the states to fund threatened-species-recovery programs. If somebody gave the Tasmanian Conservation Trust that amount of money, I could run 50 recovery programs, and with the people I could have at my disposal, I could guarantee a bloody good success rate. And these are species that are here now, that are being threatened."

A third batch of critics contend that trying to clone extinct species is tantamount to playing God, an act of almost Frankenstein-esque hubris. Others, however, see the issue in a very different light. A conference of bioethicists, scientists, scholars, and native tribes convened in New Zealand in 1999 to consider the plan to clone an extinct crow-size bird called the huia eventually concluded that the project was a moral imperative, an act of "restorative justice."

Perhaps the ultimate irony about the strange and continuing tale of the thylacine is that while one group argues about the propriety of bringing it back from extinction, other experts contend that reports of its demise are premature. More than a few people think the animal survives—not in a metaphysical sense in test tubes in Sydney but in the forests, button-grass plains, and fern gullies of northern Tasmania, where a steady trickle of sightings keeps alive the hope that tigers still linger in their mountain fastness. And as I discovered, after leaving Sydney for two weeks of bush walking through Tasmania, just the idea of surviving thylacines adds to the luster of an already spectacular landscape.

It was a cool, breezy day, with random shafts of sunlight illuminating the thick carpets of green moss that cloaked the ground and tree trunks of the rainforest. I was hiking high in the Great Western Tiers, a range of rugged mountains that frame Tasmania’s Central Plateau—a steep escarpment covered with old-growth eucalyptus, dense stands of deciduous beeches, and tree ferns with fronds like ostrich feathers.

I was here in part because of a conversation I’d had some days earlier with Eric Guiler, a retired zoology professor from the University of Tasmania who has been trailing the thylacine’s fading track for nearly 50 years. Now in his 80s but still an active field researcher, Guiler is the undisputed expert on the Tassie tiger, having mounted several major expeditions in the 1950s and 1960s to search for the animal, and he has written extensively on the subject. He has been publicly critical of the museum’s cloning project in the past—at one point, he called its proponents "bloody idiots"—but when I asked him about it, he simply shrugged it off, saying he doesn’t know enough about molecular biology to evaluate its merits.

To Guiler, the bigger question is whether the tiger still haunts wild Tasmania. He is confident the thylacine survived long past its official extinction date of 1936—he interviewed old opossum trappers who admitted snaring tigers through at least the 1950s, though they disposed of the carcasses for fear of prosecution. Reports of live thylacines come in to the state wildlife department on a monthly basis—many of them obvious cases of mistaken identity or wishful thinking but others hard to dismiss, like that from a Tasmanian Parks and Wildlife ranger who said he saw a tiger in the beam of his spotlight in 1982.

Most of the reports, Guiler told me, center on two areas—the rugged northwestern corner of the island, including the Western Tiers, and northeastern Tasmania, where he thinks it is significant that the number of alleged sightings has dwindled to a handful in recent years—a sign, perhaps, that the last tigers are winking out there. Yet when I asked what should be done to nurture any survivors, his frustration flashed to the surface.

"There’s nothing you can do," Guiler said flatly. "You’ve got an animal about whose behavior we know virtually nothing, about whose movements we know nothing, about whose general physiology we know nothing. We know so little about it, it’s impossible to devise a management plan. We don’t even know where the damned things are living. How do you devise a management plan for something like that?"

In the end, although he remains defiantly hopeful, even Guiler can’t be sure the Tasmanian tiger still moves, wraithlike, in the shade of the swamp gums and stringybarks, sending the wallabies scattering in fear, as it did for millions of years.

After several hours of climbing through dark forest, rising higher and higher toward the edge of the escarpment, the trail I was on broke into the open. The small river I’d been following went crashing down a sheer, 400-foot cliff broken in the middle of its drop by a single ledge; with a little careful climbing I was able to scramble out that shelf to sit beside the rushing waterfall. The view down the valley was breathtaking: Bare talus fields of orangish dolerite rock littered the sides of the high peaks, below which grew a solid blanket of eucalyptus and beech forest. The long valley, gouged into a wide U by ice age glaciers, faded to blue in the distance, with rank after rank of mountains, hazy and indistinct, on either hand.

Once this was tiger country—and perhaps it still is, if Guiler and the other die-hard optimists are right, though I find it hard to believe that in all these years, no thylacine has stepped in front of a car, like the hundreds of wallabies and opossums that die each night on Tassie’s winding roads. And so let us say for the moment that the thylacine exists only as museum specimens. If, by a miracle of molecular science, Don Colgan and his colleagues succeed in creating a living, breathing animal, what will they have? One individual does not a species make, and even with many old specimens to mine for DNA, building a population of a few dozen unique tigers would be the labor of decades and many more millions of dollars.

Today the thylacine is both a wraith and a warning, clad in a striped coat. Clone one, the skeptics say, and it becomes a novelty; clone a couple of dozen, and they become a conservation headache—a population that needs a home, and management, and oversight, just another endangered species in a world already parceling out limited resources to so many others. It might also deaden the public’s worry over critically rare organisms, by planting the comfortable but incorrect notion that there’s a scientific quick fix even for something as permanent as extinction.

There is also a less tangible question, one that strikes me hard as I sit in the bright sun and look out over Tassie’s beautiful mountains. How much of what made a thylacine a thylacine—the way it moved, the way it hunted, the way it integrated itself with its prey and its environment—was a matter of hard-wiring in the genes, and how much was a matter of cultural transmission from parent to offspring, in links that spanned 30 million years but are now irrevocably broken? How much came from within—and might be reconstituted from strings of synthesized amino acids wrapped in an artificial membrane—and how much came from without, from the tiger’s connection with the land itself? What relation would a cloned tiger have to the animal that met the first Aborigines 40,000 years ago, or watched with hungry eyes as the tall sailing ships unloaded their bleating cargo?

Yet despite the doubts, the roadblocks, the uncertainties, and the astronomical price tag, it’s hard to argue with another feeling I had that day, and on many days in the forests of Tasmania—the almost visceral absence of a capstone predator, which even the proj- ect’s critics acknowledge may provide the noblest motive for the drive to resurrect the thylacine. It made me think of a comment Michael Lynch had made, his eyes staring off somewhere over my shoulder as he spoke.

"It seems to me that one of the things in play here is a recognition that we, the human race, caused these animals to go to extinction," he said. "And at some level we’re saying, if we can find one out there in the wild, or if we can clone one, we’re assuaging that guilt somehow." Perhaps we need the thylacine more than the thylacine, sleeping in its jar of alcohol, needs us.

Scott Weidensaul’s travels to Australia for Audubon provided a springboard for his newest book, The Ghost With Trembling Wings: Science, Wishful Thinking, and the Search for Lost Species, due out in June.