The story of dire wolves goes beyond de-extinction

Their names are Romulus, Remus and Khaleesi, and they’re the first dire wolves to walk the Earth in over 10,000 years — or so one biotech company and a flurry of recent headlines say.

On April 7, Colossal Biosciences announced what they called the “world’s first de-extinction,” the births of three dire wolves, extinct animals that lived during the ice ages of the Pleistocene. The pups were instant icons. With snowy-white coats and muscular bodies, they looked like they could have walked straight out of the fantasy TV series Game of Thrones.

News reports heralded the animals as “scientifically seismic” and said “the dire wolf is back.” Still, some scientists aren’t buying it. “That is no more a dire wolf than I am Wonder Woman,” says Jacquelyn Gill, a paleoecologist at the University of Maine in Orono.

The issue largely boils down to genetics. Colossal didn’t create the animals from a fully reconstructed dire wolf genome. Instead, the company relied on the genetic instruction book of a gray wolf, making changes to it based on ancient DNA recovered from two dire wolf specimens. But, Gill says, “for something to be a dire wolf, it should have the full genetic blueprint of a dire wolf.” What Colossal has done, she says, is simply create a genetically modified gray wolf.

But that type of thinking “kind of misses the point,” says Beth Shapiro, chief science officer at Colossal Biosciences in Dallas. Colossal’s goal wasn’t to create something genetically identical to a dire wolf, she says. Instead, researchers wanted to resurrect the animal’s core attributes, like size, body type and face shape. By making a handful of genetic changes, “we’ve brought these extinct genes back to life in a living animal,” she says. Ultimately, Shapiro says, “I’m happy to call that a dire wolf.”

And a new preprint to be posted soon counters one common critique of the work, showing that dire wolves may be more closely related to modern wolves than previously thought.

Colossal is no stranger to controversy. Last month, the company garnered acclaim with its announcement of “woolly mice,” luxuriously tressed mice with genetic modifications inspired by woolly mammoths. The rodents’ golden-brown fluff made them internet stars, but some scientists were skeptical that such a creation brought the field any closer to bringing back woolly mammoths.

“At the moment, I think they’re creating interesting zoo-like novelties,” says Paul Wilson, a wildlife geneticist at Trent University Ontario in Peterborough. Even so, he finds the company’s technology impressive. It goes beyond crafting a dire wolf, he says. Other Colossal research on the red wolf shows how new tools might aid last-ditch efforts to save species that are critically endangered. “I think there’s a huge range of applications, both for medicine and conservation,” he says.

Science News spoke with half a dozen scientists to learn more about Colossal’s recent work, the implications for conservation and what’s next for the new pups. Here’s what we found.

Colossal put together genetic puzzle pieces to create their pups

Long before Romulus, Remus and Khaleesi made their debut, dire wolves (Aenocyon dirus) roamed the Americas during the Pleistocene Epoch, which stretched from roughly 2.5 million years ago to about 12,000 years ago.

Ancient DNA from two of these animals ended up informing the new pups’ biology. Colossal scientists extracted DNA from a 13,000-year-old fossilized tooth from Ohio and a 72,000-year-old inner ear bone fossil from Idaho.

But it can be tough to get a clear read of an animal’s genome from ancient DNA. As soon as an animal dies, its DNA “starts to get chopped up into really tiny pieces,” Shapiro says. It’s not at all like the DNA you might get after doing a cheek swab on yourself, she says.

If scientists analyzed that cheek sample, they’d see hundreds of millions of DNA letters, like long paper streamers. “Ancient DNA is more like confetti,” she says. Most of the dire wolf DNA snippets the team recovered were about 35 letters long. But by lining up billions of these snippets on a computer and comparing them with the genome of a gray wolf (Canis lupus), scientists can get an idea of what makes the animals different.

The team used this information to identify genetic tweaks that might make the gray wolf more dire wolf–like. The researchers modified a coat color gene called CORIN, for example, which gave the pups their light fur. And they tweaked genes linked to body size and ear and skull shape.

Altogether, the team made 20 edits to the genomes of gray wolf cells in the lab. Then, it removed the DNA-packed nuclei of those cells and transferred them into denucleated dog egg cells, which developed into embryos. The researchers implanted the embryos into surrogate hounds, which gave birth via C-section: twin males, Romulus and Remus, born in October, and a female pup, Khaleesi, born four months later.

The gene editing technology “is fascinating and cool and exciting,” Gill says, “but the problem now is that the public is going to believe that an animal that lived 10,000 years ago is back. And it is definitely not.”

Dire wolves may be more closely related to gray wolves than scientists once thought

Across the internet and social media, critics of Colossal’s work have been vocal about dire wolves not actually being wolves at all. That argument seems to be based largely on a 2021 Nature paper that attempted to reconstruct the animals’ evolutionary history. After analyzing the ancient genomes of five dire wolves, researchers concluded that though the animals looked similar to gray wolves, they were highly different, forging separate evolutionary paths nearly 6 million years ago.

At the time, “we thought that there was enough genetic divergence that they should not be in the genus Canis,” says Julie Meachen, a vertebrate paleontologist at Des Moines University in Iowa. Canis is the group that includes wolves, dogs and coyotes. 

But those analyses were based on relatively low-quality DNA sequences. Colossal scientists revisited two of the original samples, re-extracted the ancient DNA and then digitally stitched the pieces together on a computer to create a more complete map of the dire wolf genome.

That new map suggests that dire wolves are more wolf-like than once thought, though still distantly related, Shapiro’s team reports in an upcoming pre-print that has yet to be peer-reviewed. “I am not sure if they’ll be put back into the Canis group,” Meachen says. The author list includes Meachen and other scientists on the 2021 paper, as well as George R. R. Martin, who wrote the fantasy series that vaulted dire wolves into pop culture fame.

The new finding lends some more credence to the idea that the new pups are dire wolves, says Heather Jay Huson, an animal geneticist at Cornell University. Either way, Colossal’s news has kept scientists buzzing at the Joint Symposium for Working K9s, a meeting Huson is currently attending in Atlanta. “The big question I’ve been getting is ‘Are they really dire wolves?’” she says.

For Meachen, Colossal’s dire wolf project falls into the “gee whiz” category. “It’s sensational, and I think it was meant to be.” But it doesn’t mean the company isn’t doing important science, she says — especially in conservation efforts for endangered species. The team can also use their technology to boost the genetic diversity of modern critters. That, Meachen says, could one day make endangered animals “better capable of withstanding the environmental pressures humans are creating.”

Colossal’s technology might help save endangered animals

One thing that’s been overshadowed by the dire wolf hubbub is Colossal’s work with endangered red wolves (Canis rufus).

In the 1970s, the U.S. Fish and Wildlife Service recognized that these animals were on the brink of extinction. It began capturing the remaining wolves for a captive breeding program, later releasing red wolves into the wild. But the animals have struggled to maintain a wild population. And the red wolves living today — fewer than 20 in North America — are all descended from only about a dozen individuals. That means they lack genetic diversity, which is a problem for wild populations, says Matt James, Colossal’s chief animal officer.

Colossal identified a group of wolves on the Gulf Coast of Texas and Louisiana that genetically match red wolves, though they’re not officially recognized as such. These “red ghost wolves,” as Colossal calls them, carry in their genomes some ancestral red wolf DNA that’s been lost in wild populations.

The team cloned four of these animals from red ghost wolf cells in the lab. The pups could one day be introduced to the wild population to increase its genetic diversity, James says. Or scientists could edit ancestral red wolf gene variants into today’s red wolves. That could help “make them more adaptable to climate change and disease and all the challenges that wild species face,” James says.

Huson is less hesitant about this kind of work compared with Colossal’s de-extinction efforts. “I think the concept of conserving species that are critically endangered is fantastic,” she says. “These animals are already in the ecosystem, and we know their role.”

That’s not true for animals that have been extinct for thousands of years, which raises some ethical questions, she says. When animals go extinct, other species fill in the gaps in the ecosystem, taking advantage of their newly changed world. Scientists don’t know how bringing back an extinct species may affect the ones living today, she says. “That’s the part that makes me very cautious” about bringing back dire wolves, woolly mammoths or other long-extinct animals, she says. “There could be unintended consequences.”

Romulus, Remus and Khaleesi won’t be on view to the public

It’s unclear where exactly the pups are living, but according to a Colossal news release, they’re housed on an 800-hectare preserve and have 10 full-time caretakers. “We’re going to keep their location secret, at least for the time being,” James says. The team’s concerned about their welfare. “Even with the woolly mice, we had a few fans that wanted to get too close,” he says.

As of yet, there’s no plan to breed the pups, though that might happen in the future. And James doesn’t foresee any plans for a Pleistocene animal attraction open to visitors, a la the fictional Jurassic Park. “I don’t think the animals will be readily available to the public.”

“We’ve brought these extinct genes back to life in a living animal…. I’m happy to call that a dire wolf.”

Beth Shapiro
Colossal Biosciences chief science officer

Scientists outside of Colossal, though, are eager to examine the pups. “I’d really like to see these animals,” says Adam Hartstone-Rose, a comparative anatomist at North Carolina State University in Raleigh. He studies dire wolves and wants to compare the new pups’ teeth and bones to those of ancient animals.

Hartstone-Rose is also concerned about Romulus, Remus and Khaleesi’s behavioral and ecological well-being. Dire wolves long ago “lived in a completely different world,” he says. Parts of the planet were much colder then and the animals that lived alongside them, like giant species of horse and bison, have also gone extinct. What’s more, Colossal doesn’t currently have a breeding population of the animals, so their pups don’t really have a pack to live and hunt and play with.

“Imagine,” he says, “being the only members of your species to exist on the face of the Earth.”