Kieren Mitchell: Direwolves and their DNA

Kieren Mitchell: Image courtesy of Kieren Mitchell

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Kieren Mitchell: Exploring the evolution and extinction of direwolves

One of the people who’s digging into the past through the use of ancient DNA to understand why a species might have vanished is Dr Kieren Mitchell from the University of Adelaide. His species of choice? The direwolf. Yep, they are real - or rather, they were… 

Kieren: Yeah, that's a surprising thing when you talk to people just to begin with, is that a lot of people don't actually realise that they were a real animal, extinct now, but they were as real as lions and tigers and other things that we see around us today. They're not just mythical animals that you see on TV programmes like Game of Thrones or read about in fantasy novels. So, yeah, that's a surprise to a lot of people straight off the bat is that, yeah, they were roaming around, you know, as recently as ten thousand years ago, maybe when they became extinct, along with a whole bunch of other things like sabretooth cats and giant ground sloths and that kind of thing.

Kat: All the cool stuff. So what were they like? I'm imagining something just like massive teeth slobbering, maybe slightly fluorescent. I don't know. What's a direwolf like?

Kieren: That's maybe where we've been misled a little bit by popular culture as well. So direwolves have this fearsome reputation. They're called direwolves. And they were probably a little bit bigger than living grey wolves. That's the species of wolf that everyone will be familiar with. Maybe they were slightly bigger, maybe ten percent, twenty percent bigger, but more so than dimension wise bigger, they were a bit stockier and heavier than a grey wolf would be. So you certainly wouldn't be riding one into battle or anything like that, but they'd be a pretty formidable sight if you came across one or, more likely, if you came across a pack of them.

Kat: Oh, yeah so like big chunky boys, basically. 

Kieren: Yeah, exactly. Yeah. Big chunky wolves. And they were chunky probably because they'd evolved to hunt all these big chunky animals that were roaming around North America for thousands of years. So things like bison, things like, there were mammoths and mastodons in North America, all of these really big fauna, these megafauna, they would have been specialised to prey on those species. 

Kat: So they are obviously very cool. But what made you decide to try and investigate the natural history and their evolution? Where where did that journey start for you?

Kieren: So for myself, and for a lot of my colleagues , we're interested in finding out what past ecosystems were like. And one that we're particularly interested in is in North America, where direwolves are from, because there are all these different extinct animals that we know from North America. There's direwolves, there's sabretooth cats, extinct species of horses and camels and bison, mastodons and mammoths, all of these things that we're all living together for tens of thousands to hundreds of thousands of years until they abruptly became extinct. 

Kieren: And so they lived in North America through all these long periods of climatic change, all these ice ages. And so there was an ecosystem and a community of animals there that had really evolved altogether into this sort of unique ecosystem that was really resilient to all this climate and environmental change that was happening cyclically over tens of thousands of years. So we really want to know how that ecosystem evolved, how it worked, and then ultimately maybe why it changed and why all those species became extinct as well.

Kieren: And one part of trying to investigate that and to start to answer those questions is to know who all the players are, to really get into the nuts and bolts of how things are related to each other and how extinct species are related to living species, because that can help us to learn about what these extinct species were actually like. If we can find something that's still alive, that's a little bit like that extinct species, we can start to draw inferences then. So we want to know what the direwolf's role exactly was in that sort of ancient ecosystem.

Kieren: And so we've been drawing comparisons for a long time with living grey wolves because we thought that that was probably the nearest living analogue of what a direwolf was. We thought they were probably pretty closely related species or maybe even just subspecies of one single species. So where we were coming from with our study was really just to test that to ground proof and make sure that that was a sensible comparison to be making.

Kat: So here's where we bring the Genetics in. And now, obviously, direwolves are extinct, wolves today are not. How do you go about getting DNA from a direwolf, to try and see if they're actually related to today's wolves? I'm really confused by this, because if they lived so long ago, what sort of DNA can you get? What sort of samples do you get it from? 

Kieren: That's a great question. And so my colleagues and I, at the Australian Centre for Ancient DNA and other ancient DNA labs around the world, we specialise in really getting this genetic information from some pretty old samples. Because what happens basically is when any animal dies, the DNA in your body, in your cells, doesn't immediately degrade or evaporate. It sticks around for a while. They're big molecules. They're actually pretty resilient molecules as well, that the double helix of the DNA lends it some stability. It doesn't disappear overnight. What happens is that it gradually decays away over years, tens of years, centuries. And that all depends on the environment that the bone is actually in. So, you know, if this animal dies in a really cold part of the world and its bones fall into a cave or a crevasse in the ice or something like that, those are perfect conditions for DNA preservation. Just like you would put a piece of meat in your freezer and, you know, hopefully it would be just as good when you take it out maybe twelve months later.

Kat: Not ten thousand years later though.

Kieren: Perhaps not ten thousand years later. Although if you really do have great conditions for DNA preservation, you can push up to about a million years and still get DNA from those bones. Unfortunately, in the case of the direwolf, they actually don't live in very cold parts of the world. Despite what you might see on Game of Thrones, they were much more tropical or temperate animals. And so what that means is that most of the bones that we have from direwolves are from much warmer parts of the US in particular. And that means that we can only really easily get DNA from them. So it sort of decays and erodes away much faster than it would in cold climates. 

Kieren: And so we're happy or we're lucky to be able to get DNA from things that are sort of fifty or sixty thousand years old. And so what we do is we have to go out and collaborate quite often with museum curators to find bones that we think are going to preserve this DNA. And then we take little pieces of that bone back to our ancient DNA laboratories. So we might only need a tiny, tiny little piece of bone. We dissolve it down and that releases the DNA out of these little pockets in the bone. And then we can centrifuge it really, really, really quickly. And that lets us then pellet down and spin down all of the other stuff that was on the bone, like little bits of dirt and little bits of leftover.

Kat: The crap! 

Kieren: Yeah, exactly. Yeah. 

Kat: That's a scientific term. 

Kieren: That is. Yeah. And then we can get rid of that stuff, the crap that we've centrifuged down and then we just have the DNA from that extinct animal in a tube of clear liquid. 

Kat: Pop it through a sequencer. Bob's your uncle. You've got the direwolf DNA sequence.

Kieren: Exactly, Whiz Bang.

Kat: So when you started looking at the sequence, the DNA that you're getting from the direwolves, my first question is, was it any good? Because obviously DNA does degrade and how did you start piecing it together? And then what did it actually look like when you did think that you had a sequence from direwolves here?

Kieren: So the DNA, it wasn't great. We tried and tried a lot of samples. I think the final figure, we were looking at fifty or sixty bones of direwolves from all across the United States. And so any of your North American listeners are probably familiar with the fact that the La Brea Tar Pits is probably the most famous place for getting direwolf fossils. There's hundreds and hundreds of direwolf fossils that have been recovered from the La Brea Tar Pits. Unfortunately, none of the samples that we tried from the La Brea Tar Pits gave us any DNA at all, probably because California's probably a little bit too warm to be able to get DNA, especially if the bones have been sitting in like a nice bath of tar for thousands of years as well.

Kieren: So we had to go a little bit further afield to be able to find some samples we could actually get the DNA from. And we ended up with five samples out of our sample of fifty or sixty that we could get some DNA from. And so that included mainly places a little bit further north. So one of the samples came from Wyoming, two came from Idaho, another one from Ohio. So all states that are a little bit more northerly in the U.S. where the DNA preservation was just a little bit better, and that allowed us to piece together just enough of the genomes of these animals to be able to make the comparisons that we needed. We weren't looking at the mammoth quality DNA here that they get from mummies dug out of permafrost. We were really having to put together this jigsaw of really old and fragmented molecules to build a picture of how direwolves are related to other species.

Kat: So then how did you make that comparison? So what did you start comparing them with and what did you find?

Kieren: So what we need to do then is once we have our DNA from the direwolf, that we've sort of reconstructed on a computer, we need to compare that to genomes from the other species that we think they might be related to. And so what we did is to get a whole lot of genomes from living grey wolves that we think were probably their closest relative. But then we need other comparisons as well to kind of establish just how close a relationship that might be. 

Kieren: And so we also had DNA data from different species of Jackal, from the African wild dog, from coyotes and a number of other species as well of just these wolf-like animals. And so all we're doing then is looking at the number of A's, C's, G's and T's that are the same between different species and that differ between different species. Because the more differences you have, the more distantly related you are. And it tends to be that the more similar your genomes are, the more similar your relationship is. 

Kieren: And so we performed all of these comparisons between the direwolf and all these living species as well. And what we found was not at all what we expected. Because based on about a hundred years worth of work, of palaeontological research, the consensus had kind of been that the direwolf must be a very close relative of the grey wolf. And that's sort of the basis for our whole comparison and how we think about direwolves as members of that ancient ecosystem. 

Kieren: So what we found, though, was that direwolves are not at all close relatives of grey wolves. In fact, they aren't particularly close relatives of any living species that we compared them to.

Kat: That is so bizarre. So they look like wolves as far as we know from the bones. This should be a wolf. But when you actually look at the DNA, it's just not.

Kieren: Exactly. So that came as a big surprise. And so we spent a lot of time really making sure that that result was correct. And really that's really helped having those five different samples that we had the DNA from.

Kat: So now you've had this big family revelation that wolves are not the same as today's wolves and that they've gone on their own evolutionary journey. What does then this tell us, coming back to your original question about what is their place in the world of the time? And the other question as well, why don't we have them here anymore? Why did they go extinct? 

Kieren: So based on our results, we came up with a couple of interesting observations or hypotheses about exactly that question. So one thing that our results tell us is that the direwolf, has a very, very long evolutionary history in North America. Much longer than we'd previously maybe appreciated because, based on the fossil record, we'd thought that maybe the direwolf had evolved and had been living in North America for fifty thousand to one hundred thousand, maybe two hundred thousand years. 

Kieren: What our results really show us is that it's probably more like five or six million years that the direwolf has been there in North America, co-evolving with all of these other species around it. So that lets us imagine it as maybe a much more specialised animal than we'd previously thought, because the thinking up until our study had been that direwolves, if they are different to grey wolves, just kind of a really recent offshoot of that lineage of grey wolves. And so grey wolves, they have a bit of a reputation for being very generalist and very flexible and adaptable animals. So you find them in all sorts of habitats, eating all sorts of things around the world today. And so that was a bit confusing when we were thinking about a close similarity between the direwolf from the grey wolf.

Kieren: So if a grey wolf is like a direwolf and the grey wolf is really adaptable, then shouldn't the direwolf be really adaptable as well? And if that's the case, then why did it become extinct? So in a lot of ways, it actually makes sense that they're these distantly related, very specialised lineage that didn't have that same level of adaptability as grey wolves. And maybe that really is what did them in, in the end, is that they couldn't adapt to changes in their ecosystem when all these other species around them that they relied on to eat were becoming extinct. They maybe couldn't adapt to other sources of food. And so they became extinct as well.

Kat: So they became extinct because basically, they're picky eaters.

Kieren: I think so, I think that that's kind of what we have to conclude.

Kat: Wow. And it's really fascinating to hear the stories of what we can find out about the past, about this completely different time from these ancient bones. And I find it fascinating that you can start to understand the context of what was going on so long ago by comparing species that are alive today and what we know about their evolutionary journeys. So are there any other animals that you are applying this kind of idea to? Tell me about some of the weirdoes that you're investigating from the past. 

Kieren: Well, being from Australia, I have a Southern Hemisphere bias, I guess. Because a lot of these charismatic, enigmatic Northern Hemisphere species get a lot of the attention. You know, mammoths, the direwolf now, sabretooth cats, all of these things that you see very prominently in popular culture. 

Kat: They've got good PR.

Kieren: Yeah, they've got good PR people. I'm interested in some better representation of some of our cool Southern Hemisphere extinct species. And so one of the projects that I'm working on at the moment is on sequencing DNA from some of Australia's giant short faced kangaroos. Which a lot of people probably have no idea even existed. 

Kat: Wow. 

Kieren: So these things, some of them were so big that they wouldn't have actually been able to hop anymore because the size of them would have meant that hopping would have placed too much pressure on the tendons in their legs and basically would have snapped their legs. So you have to imagine these giant two metre tall kangaroos just striding around the landscape. So that's one project that I'm really interested in, is how can we learn more about these weird extinct kangaroos using ancient DNA?

Kat: That just absolutely blows my mind. This giant shuffling kangaroo shuffling around. Anything else on your shopping list of weird extinct species?

Kieren: So another fun project was one recently in the last few years where we were looking at an extinct species of rhino from Eurasia. So through across Siberia. And it has a complicated scientific name. But the most popular common name that it's known by is the Siberian unicorn. And so it's clearly a rhino. We know that from the fossils, but it would have had an absolutely enormous single horn protruding basically out of the centre of its head. So in contrast to some of our living rhinos that have two horns a lot of the time and the horns are kind of further down the snout, this thing would have looked a bit more unicorn-y than perhaps some of our living species with this enormous horn. 

Kieren: And so one part of the study that we did a couple of years ago was not only to sequence the DNA from this Siberian unicorn and establish that, yes, actually, it definitely is a rhino. But what we did was to radiocarbon date a whole lot of the bones of this animal to try and figure out exactly when it became extinct, because that was a big question as well, is did this species of rhino, this Siberian unicorn, become extinct a hundred thousand years, maybe even three hundred thousand years ago? Or was it more recent than that? And so we found that it actually became extinct maybe thirty five, thirty six thousand years ago. And the interesting part of that is that it means that early, anatomically modern humans leaving Africa might have actually encountered some of these animals.

Kat: Unicorns are real 

Kieren: Yeah. So it's fun to think about maybe this Siberian unicorn had some lasting impact on the zeitgeist and somehow got passed down through oral histories to become the mythical unicorn. Of course, there's probably much more likely candidates for that. So things like our living species of rhino, narwhals and mutated deer with only single horns and something like that, it's still fun to think that, well, maybe there's a little bit of this extinct rhino in the mix as well.

Kat: All these species that lived in the past, we know that evolution is a tangled web. And when we think about human origins and human evolution, there's been some funny business. I think it's the best way of putting it in our evolutionary history. Do you see any evidence of hybridisation, odd liaisons in the kinds of species that you've been looking at from the past?

Kieren: That's a great question. And what we do find is that, yes, that story about the tangled web that we see emerging as part of our human history seems to be replicated across all different animal species as well. We look at some of these extinct species and we find that, oh, actually, some of our living spaces still have ancestry, so things like living grizzly bears, brown bears still have a little bit of ancestry, some of those populations are from extinct cave bears.

Kieren: So one thing that we were really interested in looking at is whether there was any trace of ancestry from direwolves in any of our living species of canids? So any of these other wolf-like species. Particularly, could we find any direwolf ancestry in living grey wolves or coyotes from North America where you'd think that they were living there in the same place at the same time? If something's going to happen, it's going to happen. So that was another part of the comparisons that we did between the genomes of all these animals.

Kieren: And we're a little bit surprised to find that there was no signal, no evidence for any direwolf. ancestry in any living population of wolf like species, whether it's the grey wolf, the coyote, jackals or anything like that. No evidence at all. Which is surprising because on the spectrum of types of animals that will hybridised with each other, wolves and their relatives are really far up. They're just not fussed, let's say.

Kat: Dogs, generally not fussy.

Kieren: Exactly. Exactly. So that was a real surprise to us. And that we sort of started to think might play into the extinction of the direwolf, as well. Because one thing that can happen, so hybridisation a lot of the time is maybe just a happy accident kind of thing. It's a by-product it's not necessarily evolutionarily meaningful when it happens, but occasionally adaptive genes can transfer between species. And so a species can acquire new variation or new genes from a species that they've hybridised with and that can help them to adapt to their environment.

Kieren: So maybe that was just another nail in the direwolf's coffin, that they couldn't interbreed with grey wolves and they couldn't interbreed with coyotes or they couldn't or they didn't. And so another path towards them being able to adapt to changes in their environment was maybe closed off to them so they'd maybe spent so long being specialised, lost interest in all of these other species, just focussed in on eating or their big prey animal and maybe that's what did them in in the end.

Kat: So picky eaters and picky shaggers and then you’re gone. 

Kieren: Exactly. Exactly right. Yeah, that's it.

Kat: Not exactly scientific, but certainly memorable… Thanks to Kieren Mitchell from the Australian Centre for Ancient DNA at the University of Adelaide in Australia.