Sean Carroll's Mindscape: Science, Society, Philosophy, Culture, Arts, and Ideas - 175 | William Ratcliff on Multicellularity, Physics, and Evolution

Episode Date: November 29, 2021

We've talked about the very origin of life, but certain transitions along its subsequent history were incredibly important. Perhaps none more so than the transition from unicellular to multicellular organisms, which made possible an incredible diversity of organisms and structures. Will Ratcliff studies the physics that constrains multicellular structures, examines the minute changes in certain yeast cells that allows them to become multicellular, and does long-term evolution experiments in which multicellularity spontaneously evolves and grows. We can't yet create life from non-life, but we can reproduce critical evolutionary steps in the lab. Support Mindscape on Patreon. William Ratcliff received his Ph.D. in Ecology, Evolution, and Behavior at the University of Minnesota. He is currently Associate Professor in the School of Biological Sciences at Georgia Tech. Among his awards are a Packard Fellowship and being named in Popular Science's "Brilliant 10" of 2016. Web site Google Scholar publications Packard Foundation bio Twitter "Experimental Evolution of Multicellularity," Ratcliff et al. (2012) "De Novo Evolution of Macroscopic Multicellularity," Bozdag et al. (2021)

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These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease. Hello, everybody. Welcome to the Mindscape podcast. I'm your host, Sean Carroll. Life is complicated.

I don't mean your life, my life. Those are also complicated. But as we all know, biology is complicated. The history of biology is complicated. Here on Mindscape, you know, I'm interested in very big questions like the origin of life. But we also have the evolution of life, right?

That's interesting all by itself. And so there's a bunch of phase transitions that we like to talk about that happened over the course of the evolution of life. One such transition was the origin of eukaryotes, right? Eukaryotes are cells that have nuclei in them, and we think these days that that came about from two different kinds of cells getting together, joining together, sharing their DNA, distributing it in the cell in interesting ways. You have mitochondria, and then you have the nuclear DNA. and it's all a big part of the interesting kinds of life we have on Earth. I shouldn't say interesting. It's all interesting. The complex kinds of life we have on Earth.

Eukaryotes, compared to prokaryotes, which don't have nuclei, have a lot more capacity, a lot more potential for being complex, such as being multicellular. And once you go from a single-cell organism to a multi-cell organism, then the possibilities of complexity grow enormously. So today's guest, Will Ratcliffe, is a biologist who is one of the world's experts on exactly this transition from unicellularity to multicellularity in the evolution of life. Not just studying what happens there in the record of actual life on Earth, but doing artificial evolution, or what some people call directed evolution, doing an experiment in the lab where he takes seeds into group, take yeast cells, and they let them evolve. They give a little bit of selection pressure to grow bigger,

and they say, you know, they don't poke in there the DNA. They just let mutations happen and watch what happens to these initially unicellular yeast organisms. And what happens very quickly is they become multicellular. You know, in some sense, which we'll hopefully try to make clear in the podcast, yeast and many other eukaryotes are ready to be multicellular in some sense. so a little nudge pushes them in the right direction. But there are surprises that come up along the way. You know, there are different ways to be multicellular,

and there's a big difference between being multicellular in the sense of just having a lot of cells sticking together versus being truly differentiated with different organs and things like that. And even though the experiments have not been going on very long, there's still thousands of generations that we have ability to look at, and it's taught us something about this crucially important transition, which might have implications for, you know, life elsewhere, the idea of life, the future of life even,

now that we human beings have the ability to actually go in there and poke at the genome of different kinds of organisms and see what happens. You know, maybe there's whole kinds of genetic information schemes that would lead to different kinds of organisms that evolution was not able to find. I don't know. I'm just a physicist and podcast host, but Will is an expert on this, so let's go. Will Ratcliffe, welcome to the Mindscape podcast. Thank you. I'm delighted to be here.

So, multicellularity, big thing in the history of life. It's no planning on my own part. I don't plan a series of podcasts in any interesting way, but we have been talking a little bit of the origin of life. Recently did Betuel Kachar about what we learned about old life from paleogen. genomics and so forth. But that was all single-celled stuff, basically. So why don't you put us into context? Like, when did multicellularity arise? What do we know about its origins? Yeah. No, I'm happy to be on this sort of path of understanding life's origin and the evolution of complexity. And in some sense, they've already done the hard work, because once you have a cell,

you've already sort of, you know, you've done 95% of the, I actually agree with Nigel Goldenfeld in his recent interview where he said, you know, once you have cell, you have cells. who cares about elephants. To some extent, I agree with them. To the other extent, I've made my entire career on disagreeing with him. So multicellarity is when you have an organism that's composed of multiple cells. And it opens up all kinds of opportunities for complexity because those cells can take on different roles. You can get all sorts of novel ecologies arising and you can have organisms with all these specialized functional parts.

When multicellularity arose is kind of not really a, it's not, there's no one answer. Because multicellularity actually has evolved many times in different lineages on Earth. So we actually have fossils from... Like five times or 500 times? Um, in between the two. Okay. So it's probably in the, in around 100, although we actually don't have a really robust estimate. That's a lot, though.

Okay. So that's... It's a lot. Some lineages, like the green algae, it's evolved more than 25 times independently and just green algae alone. But isn't it true that? life on earth was unicellular for a really, really long time? Yeah. So the majority of life, so life, you know, start, when life started out pre-cellular,

and then once you have sort of it becoming cellular, that's really a sort of almost a sea change in how life, you know, evolves. Information is transmitted vertically, and you have sort of cellular revolution. And that was probably on the order of 3.8 billion years ago or even earlier. Like that's about the origin of Luca, the last universal common ancestor, So the coalescence of all things can be traced back to that point. But there was probably some cellular life before that. But it's very, very hard to study anything that's that old.

And yeah, so the majority of the diversity of life that we have out here today is also single-celled. But different single-cellularity began exploring with multicellularity at different time points. So cyanobacteria began forming chains of cells in which you'd have cells that are specialized for carbon fixation through photosynthesis or nether. nitrogen fixation by basically taking nitrogen from the air and turning it into ammonium, which can be used to building proteins and stuff. And that happened around 2.1 billion years ago. And then you have, that's all, and different types of bacteria played around with multicellularity between 2 billion and 1 billion years ago.

And they've all remained pretty small and simple. Then you have eukaryotes arise. And eukaryotes are a special kind of single cell organism. They're actually the result of a fusion between an archaea and alpha-prose. Protoabacterium. That alpha proteobacterium was at one point a symbiont and became domesticated as our mitochondrian to sort of provide energy for our cells and these eukaryotic cells have really sort of run away with with sort of I don't know

forming different kinds of organisms that bacteria and archaea don't really seem to be able to form. So sorry. What you just said about the origin of the eukaryotes, the the cells with with nuclei in them, you sounded pretty confident. I'm not, for, from my own, you know, naive physicists reading, are we pretty confident that we really know how that started and why it started? Or is this a leading more and more confident? Yeah, okay, good. So there, so as we find these archaea from the Loki Archaeota and other sort of Asgard related to, it's a great acronym, right? I don't actually know what it stands for, but this,

there's this clade of Archaea that have been found in sort of seafloor sediments in the, I guess the near-Arctic. And these things have all sorts of eukaryotes specific cell biology, things that we thought only eukaryotes have. And it turns out that they are archaea. And if you look at them on a phylogenetic tree, which is like a genealogical tree, but applied to vast timescales and not just, you know, your local family, then you can see that they actually nest as a sister group to eukaryotes. So we're pretty sure that archaea And if you sort of begin to disentangle and unpack how eukaryotes arose You really see that we basically have a that eukaryotic cells have a mixture of Archaeal stuff and bacterial stuff like our

The lipid membrane that we have surrounding ourselves is actually bacterial It's not archaeal they use a different kind of chemistry in their lipids which make the membrane of the cell And we think that actually came over from the from the mitochondria. So the nucleus was the archaea, and then the big puffy part was the bacteria or vice versa? So we don't actually, I think those details we don't really know. Those are still the kinds of things we're working out.

But I think a better way of thinking about it is that the cell as a whole, like the macro, like the larger symbiont was the archaea. And that sort of got smaller bacteria living within it. And those bacteria that sort of co-opted being a part of this new eukaryotic cell, they can't divide on their own anymore. They still have a remnant little genome, right? So if you look at it inside a mitochondria, it has its own genome, which is, you know, one of those things that made people realize, you know, 40 years ago, oh my gosh, like these

things were once free living separate cells. Right. So this is, this is, I know probably a lot of people in the audience know this very well, but maybe there are some who don't, and it's so amazing. I really want to just sit and dwell on it that there's two different sets of DNA in every one of ourselves. There's, you know, our DNA and then there's these little free-riding, not free-riding, but symbiotic mitochondrial DNA in every one of ourselves. And so we sort of genetically pass down both from generation to generation. Yeah. And the DNA inside of mitochondria is totally stripped

down. Like if you imagine that it was at one point a car, now it is just the steering wheel, or I don't know what the better analogy is, but, but you know, it's, it's, you know, 20,000 DNA bases when a free-living bacteria is typically on the order of a few million. Cool. So they're just, they're teeny. Yeah. Yeah. And so.

Most of the DNA from the genome of the mitochondria has been exported and is now residing inside our chromosomes. Oh, okay. All right. So we co-opted the useful parts of it and there's some of the useful parts. Other useful parts are still hanging out in the mitochondria. Yeah. Certain elements of it, it seems like it's kind of hard to move them into the mitochondrial through

those membranes into the mitochondria and it's better just to have them made locally. but other stuff, you want top-down host control. And so you just move that stuff into the domain of the host. And it was this, you know, we love phase transitions here on the Minds Day podcast. So this leap from prokaryoticness to eukaryoticness was a big phase transition. And you're saying that that enabled new kinds of multicellularity or made it easier? It certainly seems that way, although we don't have a first principles, you know, universally accepted, explanation for why that's true. So when you look at sort of the tapestry of multicegular organisms,

there are bacterial and archaeal multicegular organisms, but they're very, very simple. They've been around for a really long time, billions of years, and they don't seem to have really done much beyond making small groups of cells. Within eukaryotes, though, you have the entire sort of complex tapestry of life that you see when you look at your window, right? You see trees, you see things growing on those trees, you see insects buzzing around those, you see mushrooms growing off of logs. All of those things are eukaryotic multicellular organisms. And multicellularity, one of the great features of it is it's like the sort of anti-Anakoranina thing, right?

And unlike all multicellular families being alike in the same way, right? They're in fact all very, very different. Every multicellular lineage does multicellularity in its own specific way. You know, free-living algae in ponds that form spherical colonies of multicellular cells that spin around with flagella that keep the modal are very different from plants, which are essentially growing into the air and competing for light, which are very different from animals,

which are very different from fungi. So in some sense, it's a challenge to try to come up with universal rules for understanding how multisageal organisms evolve and become more complex. On the other hand, we actually, we can still do that. But you have to keep in mind that every lineage has its own ways of becoming multisailer

that depend on its specific ecology and, very importantly, the cell biology of the ancestor. Is it just too simplistic to imagine that being eukaryotic provides a bit more flexibility when it comes to being multicellular in the sense that a good, robust multicellular organism will, each cell has the same DNA, but it gets expressed in different ways. And being a eukaryote enables sort of more flavors for that? Yeah, that's actually, you kind of nailed the explanation that people have, which is that eukaryotic cells have a much larger number of sort of types. of gene regulation available to them and it's easier to get cellular differentiation in eukaryotes.

There also tend to be larger cells with more complex forms of cellular morphology. The ancestors of eukaryotes often have like, at least if we look at like the best work in this area has been done by people looking at the unicellular relatives of animals.

Okay. So animals, right, evolved roughly 800 million years ago and, you know, if you go back to the sort of family tree, just once step above animals. There's things which, which, you know, are a common ancestor of animals and things which have stayed single cell. And if you look at those things that have stayed

single cell, they often have these complex life cycles where they can exist as an amoeboid and crawl around, which looks a lot like, you know, one of your blood cells that's going off and trying to kill pathogens, right? It can exist as a flagellated state where it's swimming, which looks a lot like a sperm cell. It can exist in many different

sort of temporal types of cell differentiation, which you could imagine get sort of sort of reworked from being expressed in time to expressed in space. And now you have all these sort of modules to play with for building a multi-segro organism. Yeah. And it's also kind of cool because in some sense, a little bit of complexity at the micro level is enabling much greater complexity at the macro level. Absolutely.

And but, but I just, I want to caution that this is consistent with what we understand, but I don't know if it's, it's not universally accepted yet. Perfectly very good. Because bacteria are, and archaea, are extraordinarily, really complex and diverse. And it's kind of hard to find something that eukaryotes can do that they can't do. That makes sense. Yeah, no, it makes perfect sense.

So this was, so this transition to eukaryotes was about 800 million years ago? That was probably more on the order about 2 billion years ago. Oh, 2 billion years ago. Oh, 2 billion years ago. Yeah, animals were roughly 800 million years ago. I see. Okay. So eukaryotes, so this story that I was, that I have in mind of taking a long time for

multicellularity. to arise sort of gets divided into, it took a medium long time to be eukaryotic and then another medium long time to be multicellular, right? That's right. Yeah. So in the eukaryotic space, you know, eukaryotes arise around two billion years ago. And then roughly a billion years later, you start to see the first lineages of like bona fide eukaryotic multicellular organisms arising and still sort of persisting to the modern day. So you see, and this really occurs in red and green algae. Interestingly enough. So you get these small little algae that form branched, you know, topologically structured like little trees, branched multicellular groups, which if you look at the

fossils and look at modern day ones, oh my gosh, they look basically the same. Yeah. And this whole story is just bizarre to a physicist because we think of systems as being characterized by time scales. And if you don't know any better, there's a certain time scale that governs everything. that happens. And presumably the time scale for a microorganism is, you know, is lifetime or it's time to split or something like that. And those numbers are much, much smaller than a billion years. So for something interesting to happen, but for it to take a billion years to happen is just a reminder that physics is not very good at estimating certain things. Right. And the timescales that things, I think it's something that we kind of keep learning.

in evolutionary biology is that the potential for something to evolve in a certain way is often extreme it's it's often kind of unlimited over sort of geological timescales things can evolve very very very quickly given suitable selection and often when you see stasis it's simply because you don't have any selection in any you know significant selection on the organism's state and so during that you know one two billion years to roughly five hundred and 40 million years before the present, you had an era of very, very low environmental oxygen. And so that actually in some ways changes the expectations of whether you expect to see multicyclarity and what types of multicellularity you expect to see.

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Well, I guess that was going to be my question. You know, I'm a big fan of multicellular organisms myself being one. But from the evolutionary point of view, what was the benefit to becoming multicellular? And I guess you've already started saying something about oxygen. Well, yeah, there's a lot of different benefits to multicellularity. This, again, goes back to the sort of reverse anachron and a problem, right? Where every lineage has its own sort of reasons to become multicellular. So there's no one sort of reason to do it.

in some cases you have organisms that are forming groups in order to avoid being consumed by predators because predators are filter feeding and they have a small mouth and if something bongs against their face and can't get in their mouth then they're not eaten so it's good to form a group in other cases you have organisms that are cooperating metabolically they're excreting stuff into the environment and if you can get a sort of clump of cells that are all cooperating and excreting enzymes into the environment you can degrade environmental stuff much better than if it's a chaotic mix of different species and things that are maybe not producing the enzymes, et cetera. There are, there's actually a whole list.

We're actually working on a review paper on this right now. I could go on and on, but it doesn't really matter. What matters is that there's lots of different reasons to come up to secular. Maybe one that's really relevant to algae or photosynthetic organisms is that, you know, light is this very valuable resource, but it's directional. So if you can overgrow your competitors, you get the food. And that's why we have, you know, 300 feet tall trees. They're not like getting more sun by being 300 feet tall than,

then something's sitting on the ground, they're just getting to it first. Well, they're not being overgrown by somebody else, right, when you're the tallest one in the forest. So it's an arms race, right? Like trees are the result of a 450 million year-long arms race. But then I do want to let you go into more about the oxygen stuff because that clearly was relevant. Yes, yes. Okay, so oxygen is very important in the sense that we really see sort of, if you look back

the history of multisylarity, you see these sort of experiments in, let me just, talk about how oxygen's happened on Earth first. The great oxidation event, which I think is a great term, very much named by geologists, because it took several hundred million years for this event to occur, occurred roughly 2.1 billion years ago in that era, a couple hundred million years, you have cyanobacteria cracking out so much oxygen that we go from a world that basically is anaerobic to one that actually has oxygen in the atmosphere and in the oceans. But it's at very, very low levels. Maybe 1% of modern levels, maybe lower. It's at very low levels.

And it stays low for the next one and a half billion years. And then in the Phanozoic era, which is about 540 million years before now, it starts to really rise. And that's actually concordant with this explosion in multicegular diversity. You have the Cambrian explosion in animals happening right about that time. Plants, land plants kind of invade not that much longer, right? And fungi, they've been around for a while. We actually don't have a great understanding of the origin and diversification of fungi, but fungi seem to really also diversify around this time as well.

And so we've long thought that oxygen was this kind of catalyst for multicellularity, that if you give a system oxygen, you're going to enable the evolution of larger, more complex multicellular organisms. And there's a very good physical reason for this, which is that if cells depend on having oxygen and you form a group, then you become diffusion limited. And those internal cells don't have access to very much oxygen. And therefore, that might set a maximum. size on an organism because, you know, basically if you can't get oxygen to the interior,

maybe those cells can't survive at all. That sets a maximum size to the organism. And so until you can invent a circulatory system, you're stuck with diffusion, and therefore size is just directly a function of oxygen concentration. We thought this way for a long time. This isn't something we have actually tested, though. So a postdoc in my group, Ozahn-Bosdog, did this cool experiment where he took our yeast system. Maybe this is a good time to introduce it. Well, keep talking. We'll go back to the East. Don't worry.

All right. Good, good. In any case, although, okay. You can't explain it without. It's hard for me to explain our change in thinking about oxygen without referencing the experiment because that was what helped us how to say. Okay. Should we go back and just talk oxygen in general?

Yeah. No, let's just like note that the point is that, you know, when I talked to Betuel, we talked about the great oxygenation event two billion years ago. And what you're emphasizing is that's nothing. There was another growth of oxygen by a factor of 100 later on. And there's this, you know, it's not completely clear why that happens. And we're going to illuminate maybe why it happens by talking about yeast. So let's pause there and talk about yeast for a while.

You know, what's a yeast and why do you care about it? So yeast are a single-sled fungus. And I care about them because they're a really wonderful model organism for laboratory experiments. And we all, we're in the same. the middle of pandemic and everyone is baking bread. So we're all familiar with yeast. But so this is a, so it's a fungus, which is, what kingdom is that in? That's a plant. No, sorry, not a plant. It's a fungus, obviously, but it's a eukaryote. It's a eukaryotic fungus, single-celled. It's a single-sode eukaryote. And they actually, the funny thing about the yeast, yeast is a generic term for any single-celled fungus.

The yeast that we use is Saccharomycese Saravisier. And so there's many different yeast, and they can actually be very, very diverse. There's yeast from, you know, very different branches of the fungal tree of life. It actually seems to be kind of a common strategy, and this is the only multicellular group that does this, where you'll have things which formed filamentous multicellular fungi. They'll actually evolve to form single-celled yeast. And in fact, the yeast that we work on, Saccharomycese Sarvasiae, which is the baker's yeast, it's the most common. yeast used by humanity. It's sort of domesticated by humans for brewing beer and baking bread. It's also in many ways, like one of the most well-studied and extensively used workhorses of

molecular biology in the research community. And it's very, very easy to make it do what you want. You can basically do crazy synthetic biology experiments very easily in yeast because you can just kind of stick genes in them and they'll express them and it works. I mean, that's clearly very useful. And also, I get the impression that, you know, even if yeast are unicellular, they're kind of living on the edge of wanting to be multicellular. I think that's probably true. Certainly in our case, our yeast derived, descended from a multicellular ancestor, but that was several hundred million years ago. So what does that even mean?

And during that time, they've really lost much of their genome that we know multicellular fungi have. They seem to have turned jettisoned at all. Okay. So they devolved inside. in some sense. Yes. Yes. Yep. And this is very rare in the, in this pallet of multicellularity. You don't see unicular descendants of animals or plants running around nature. But fungi seem to do this all the time. I see. So the yeast you're dealing with were formerly a multicellular organism and now they like to be unicellular. Yeah. They've been unicellular for a long time.

And then you, so it's not completely surprising that you can sort of prod them in different ways and mutate them and get them back to being multicellular. Yeah, it may not be that surprising, though I don't think they're actually reactivating like latent ancestral pathways of multicellularity. I think it's actually just pretty easy for them to, in our case, we understand the cell biology of how they become multicellular in our lab very well. And you can just break the machinery that normally allows daughter cells to separate from a mother cell after they're done dividing.

And then you just get these tree-like, you know, multisulmonary. secular, you know, groups forming. So you're not actually bringing back to life the multicellular organism that it used to be. You're sort of creating a different kind of multicellular yeast. Exactly. And these are the snowflakes. Is that right? Yes.

So tell us about the snowflakes. Okay. So about 10 years ago when I was actually a fifth year grad student, sort of, I guess, avoiding writing up my thesis. I started a clover. I think you've known them. I know them in my lab now. Sorry, if they're listening to this.

I started a collaboration with Mike Travisano at the University of Minnesota to basically, we had a coffee chat where we're like, what's the coolest thing we could do in the lab? After we discussing origin of life as being too difficult for us, biologists and we're not chemists, so we don't know how to do that. Maybe we should play around with evolving multicellarity. And so over December, I started doing these experiments selecting on single cell yeast to form groups.

and we did a really simple selection experiment in the lab. And these selection experiments are kind of like selective breeding in the way that we have, you know, we've domesticated dogs and we've made wheat and corn from wild relatives. It's basically a selective breeding experiment. The fancy term now is either experimental evolution or directed evolution. And so we did this really simple selection experiment where we took our yeast and every day, you know,

we dilute them 100-fold into fresh media. Those cells grow up and then we put them through sort of erase, to get to the bottom of the test tube. Okay. So we give them, you know, a few minutes sitting on the bench to get to the bottom of the test tube, and then any of the ones that are at the bottom of the test tube get transferred to fresh media. Everyone else is killed. And so this really incentivizes them to form groups because groups settle through liquid media faster than single cells.

So, sorry, just to be clear, the mutations in the DNA are completely natural. You're not inducing them. But this selection pressure is what you are. That's the directed and directed evolution, right? Exactly. Exactly. And we don't really think necessarily that settling faster is a reason that things will become multicellular in nature, although there's recent work out of Omaya Dodin's lab showing this for a unicellular relative of animals. But the way that we think of it is it's really just a very expedient way to select on size.

And that's what all these sort of lineages that are experimenting with multicellularity are getting advantages from. There has to be an advantage to size. Otherwise, things stay single-celled. Like most images stay single-celled. Some of them have an advantage of forming groups, and those ones are selected to form groups. And so we're just doing that selection in a really efficient way. You know, we can select on a million clusters in a few minutes of sitting on the bench and a 10-second pipette transfer. So you're not, I mean, alternatively, in a synthetic biology sort of context, we might go in there and remove a bit of DNA, a gene, and replace it with something else.

but you're, in this particular case, you're actually just letting a mutation happen. So in the very first experiments, we didn't know what happened, and we just, you know, we just let these mutations occur. And within a few weeks, these populations had all formed these beautiful little groups that we call snowflakes. We came up with that term, actually, because they settle, much like sort of snow settling through the liquid media, and they actually have this cool fractal-like growth form, where if you break off a small branch, it's self-similar to the morphology of the larger group. You can imagine that they're growing as a tree.

And if you break off a sort of small branch of the tree, it looks a lot like the bigger, the bigger overall tree growth form. And also, we were in Minnesota in December, so there was a lot of snow around. But, you know, actually all of our experiments that we do now, we start them out with using the synthetic approach to basically delete a gene that was the one that was most often mutated in the natural experiments at the beginning of our, to start our experiments off, we just remove that bit of DNA to make them all snowflakes in the same way. And then we can basically replay multiple parallel runs of our experiments, but they're all starting

out in exactly the same multicellular state, these simple ancestral snowflakes. But is it safe to say that it's just one gene you have to remove to make a unicellular yeast into a snowflake? I mean, that gives evidence that it's right there on the edge of being ready to be multicellular. It's super easy, but that's probably true for most unicyclature organisms. Okay. Oh, okay. So, yeah, we know, we've played around with algae.

and doing similar experiments in Clamidomonas, which is a model green alga. It's also very easy to form groups. And you can do similar things with bacteria and get them to form chains. And basically, cell division has to be done correctly in order to get one cell to pop off a daughter cell and have them both separate. And there's a lot of ways to kind of break that division process in a way that gives you a simple group of cells. So, yeah, so basically it's very similar to being unicellular, but you're sticking together, literally sticking together. Exactly. Like, I think of them as naive. Like, they are not a multicegular organism. They're a dumb clump of cells. Right, okay.

And in fact, to become a multicegular organism is an evolutionary transformation. Okay. And do these at all, they must occasionally appear in nature if that's the case. Like, you must get a mutation that creates a yeast snowflake. I mean, certainly they do. This mutation rates fairly high. But we don't typically see them floating around, you know, on, on, on, on, rotting fruit or in the sap of oak trees, which is where you typically find Saccharomyces in its natural habitat, you don't really find snowflakes. And I think the reason for that is that it's ecologically costly for them in this environment.

If you're, you know, if you're making a living as a yeast and basically being moved from one sugar source to another, dispersal is a premium virtue. Right. And if you and, you know, your next eight generations of offspring are all stuck together in one big group and a fly doesn't come down and peek you up on its foot and transfer you, you're dead. So it's every cell for itself. reverse bed hedging. Okay.

And, okay, good. Are we then ready to talk about the oxygen, the role of oxygen? Sure. Why it was helpful? Yeah. Yep. So our lab specializes in using experimental evolution to study key steps in the evolution of multicellularity and multicellular complexity.

And our main model system are these snowflake yeast. So getting back to oxygen, like looking at the tapestry of oxygen on the history of Earth, we know that it sort of went from zero to maybe one percent. 2 billion years ago, stayed very low for the next billion and a half years, and then it smashed up to modern levels around 500 million years ago. And we've long thought that sort of oxygen was this fuel for the multicellular fire, that because it diffuses more deeply into tissues, the size of a simple multicegular organism without a circulatory system is just dependent on the concentration of oxygen in the environment, and therefore you have this sort of monotonic dependence.

As you add more oxygen, you get bigger organisms. But no one's really ever tested this, and there's a couple reasons, but one of them is that we haven't had a model system that we could evolve of multicellularity, something which we could put through 1,000 generations or more in the lab and actually explore its evolution. We haven't had a model system where we can toggle the way that they use oxygen, because this actually turns out to be very important. If you assume that the cells in your organism all have to use oxygen to grow, well, then you sort of really limit that you you've put an assumption to the system that's very much going to

dictate the outcome, which is if you say, if you don't have any oxygen, that cell dies, well, then you really restrict them to being to being small when there's not much oxygen. But it turns out that the ancestral state of eukaryotes were called mixotrophs. That is, they could ferment without oxygen when they had fermentable carbon, or they could switch over to using mitochondria to respire when there was oxygen available. And that's a pretty sensical strategy in a world where oxygen is very, very low. You don't want to be an obligate arrow if there's not much oxygen in the world. So just to, again, to sort of linger on this because it's kind of cool.

So since there is oxygen, if there is oxygen in the world, respiration is a useful way to bring energy to your internal cellular workings. And the alternative you're saying is fermentation, which, again, a lot of alcohol-related terminology here. But what exactly is fermentation in this context? Fermentation is just a metabolic strategy in which you break apart carbon, you know, polymers, and you get some energy out of that. Okay. But you don't use oxygen to sort of burn them. So before there was a surfeit of oxygen, you could get energy from fermentation. If there was enough oxygen, it's more efficient to just do respiration.

Yeah, so oxygen provides two huge benefits. One, you can get a lot more energy out of the resource, up to 18 times more energy. You know, so it's, you can get a lot more out of it. Yeah. And two, some kinds of carbon compounds cannot be fermented, but they can be respired. So in that sense, it's kind of a co-factor in letting you eat something that if you don't have the oxygen, you can't eat it. Okay. Which is actually true for getting back to, you know, alcohol. You know, yeast can eat alcohol, but they need oxygen to do it.

So when you're, when you're making your beer, people have the airlock on there and they're trying not to let oxygen get into their, into their, you know, fermenting. And that's because they don't want the yeast to eat their alcohol. You know what's wild? We can video chat from space. Order groceries just by talking out loud. And men have like 17 options for hair loss alone. But a bra that actually fits and feels good, apparently too much to ask. Until now, meet the uplift bra from Nix.

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Okay, yeah. So what we have is a question. How does the evolution of multicellular size depend on the presence of oxygen? We've had this long assumption that it's monotonic. The more oxygen you give them the bigger they can get. And we have now this yeast model system where we can evolve them in the lab, and we can go through about five generations of evolution per day. So, you know, in a year, you're, you know, over a thousand generations.

You can really push their evolution fast. And we can select on size very easily. And then we can actually mess with yeast. Yeast are a great model organism. And we can basically make them fermentation only. So we can basically make them anaerobic. And we do that by basically breaking their mitochondria. They have mitochondria.

We can break them. So, okay, their mitochondria don't work anymore. They can only ferment. then we can actually, we can make them respire only so they can only grow using oxygen. Or we can let them do both. We can let them be the native state of yeast, actually, is to be a mix of drift,

just like the ancestral state of eukaryotes. They can either ferment or respire depending on what that cell wants to do. So we took all three of these different kinds of snowflake yeast, which were otherwise identical at the start of the experiment, and we evolved them with selection for larger size. And we didn't get what we expected.

We expected that the more oxygen they have, had oh and and I should say we also did this um with with different environmental oxygen okay so we gave them low environmental oxygen or high environmental oxygen and we gave them high by basically just bubbling that stuff through through their tubes so so there's a lot going on here but what happens is when we evolve them what we find is that unlike the expectation that the more oxygen they have the bigger they get we find that when they have no oxygen that is they're an anaerobobe or they have a low amount of oxygen. It doesn't matter if they're a mixotrofe or an obligate aerob or they have high amounts of oxygen, right? We end up getting this Nike swoosh. It's not a monotonic increase. They get really big

when they have no oxygen. They evolve to be really big when they have lots of oxygen. And when oxygen is limiting, they actually stayed small. It didn't really get very much bigger at all, despite a thousand generations of us every day selecting for bigger and bigger size. But is it only the fermenters that get big when there's no oxygen? So actually, that's how we gave them no oxygen. So actually, that's how we gave them no oxygen. Rather than removing oxygen from the test tubes, we just remove their ability to use it. So the fermenters are essentially in the no oxygen world in our experiment. And those guys got big. The ones which could use oxygen, if they had a little bit of oxygen, they were actually much smaller than the fermenters. But if we pumped in tons of oxygen to their test tubes, then they

became very large. So what does that imply for us about evolutionary history? You're saying that, you know, As oxygen increases, first it gets worse for the yeast. If we sort of anthropomorphies a little bit and think that getting bigger is good, right? It indicates a healthy kind of unicellular yeast. As oxygen comes into the atmosphere, it's initially bad? Is that a safe conclusion? Yeah, first let me say, rather than saying bigger is healthier, I would say that bigger is necessary for evolving complexity.

Okay. it provides opportunities for cells to evolve different roles and functions. And in fact, Andy Knoll has written very persuasively that size itself is a driver of increased complexity. Like once you have a big organism, now you really have an incentive to evolve a circulatory system and mechanisms of cellular communication and resource transport within the organism. So size probably came first and complexity probably followed, which is one reason that we're so interested in size. But there is, this is a provocative statement. So there is a chicken and egg problem.

Is it that once the cells get big, they want or need to be complex so they can differentiate? Or is it that once the cells get big, they have the capacity to become complex, and that's intrinsically useful? I would say neither. I would say once the groups get large, there is an evolutionary incentive to evolve the kinds of behaviors that drive increased complexity. Okay. So they don't necessarily want to. There's no internal, you know, desire in a yeast cluster or, you know, a clump of cells in nature. But there is an advantage associated with that kind of differentiation once you have size.

Because once you have size, you have all of these diffusion limitations that make growth very difficult. And also that provides advantages, right? You have the opportunity for an inside, outside environment. You know, you can localize food to being in the center of your organism and therefore doesn't leak out. I mean, there's all sorts of reasons why once you have a big group, there are advantages to evolving essentially the kinds of interactions and integration amongst the cells and the organism that typically drive increased complexity.

Okay. And so then getting back to the evolutionary history, it sounds like we're on the verge of saying that the first great oxygenation event didn't really help because a little bit of oxygen slows you down, but once it went way up, then it became beneficial. This is what your yeast experiments are teaching us, then it became beneficial to become more multicellular.

exactly right. That at first, we probably would have been better off without the great oxidation event. And in fact, geologists call that period of time between two billion years and one billion years ago, the quote-unquote boring billion. Because, you know, there's not that much happening in the biosphere that's at least apparent in the geological record. I'm sure there's all sorts of of really interesting, you know, single-cell microbial biochemistry and evolution happening. But you don't see that really in the fossil record. And so during that sort of, you know, low-oxygen-boring billion, oxygen may in fact have acted as a constraint. And I'll just throw one more thing in there. The reason why we think that we got this Nike swoosh effect is not just artifacts of metabolism

or some sort of, you know, short-term proximate explanation. But we actually built an evolutionary model to understand this effect better. And it turns out that if you build a very simple first principles model, then you can recapitulate this behavior and it's extraordinarily robust. And the intuition actually is that oxygen is a valuable resource. And so if you have no oxygen, well, there's nothing to compete for. If you give a system a little bit of oxygen, well, now all of a sudden, those groups that can better utilize that oxygen have an advantage. And in fact, that provides an incentive to get smaller and have a larger proportion of the

cells in that group be capable of utilizing that oxygen to grow faster, to divide more. They tend to win in these evolutionary simulations. And then as you, despite their lower survival, because we're always saying bigger provides better survival, right? Despite their lower survival, they still win by getting smaller. But then you pump in enough oxygen to the system that they can both get big and have high survival and use the oxygen well. And there's no longer a conflict. They don't have to get small to use the oxygen. And that's really what we think is driving this suppression of multicellular size from oxygen.

That small amounts effectively is saying like, here's a valuable resource. but if you're big, you don't get to use it very well. And so that's a very strong selective incentive to get smaller or stay small. And you can remove that by removing oxygen entirely or pumping tons of it into the system. So one of the lessons that I got from talking about too was these transitions that have a big effect on life like oxygen, there's a give and take, right? There's sort of geological or even astrophysical things that happen that change conditions on Earth that make the environment. for the living organisms different, but then living organisms feed back

in quite an impressive way to what is going on on Earth. So can we say something about which is more important for this second big boost in oxygen? Was it that life made more oxygen, photosynthesis, or was it something more geochemical going on?

I am not the expert on this. I would defer to somebody like Chris Reinhardt who really studies this problem. But my understanding of it that's kind of a layman's understanding is that it's a combination of the two that you had big changes in Earth's temperature. You had snowball Earth occurring at about that time. You have big

changes in the composition of the oceans being bacterial dominated to algal dominated. You have sort of these combination of biotic and global geochemical cycles occurring, which combined drove this transition to far higher atmospheric oxygen concentrations. Okay, good, good. All right, so here we are. How long ago again? We started these experiments. in the very beginning, about 10 years ago. I mean, how many millions of years ago was the... Oh, sorry, sorry. Was which one?

Was the more oxygenation creating multicellular life? Yeah, about 500. 500 million years ago. Okay. So more oxygen and at least, if not multicellularity, then at least growth in the size of the unicellular organisms would be expected by then. And as you've made the case already,

this is a good... allower, or it opens the floodgates to becoming multicellular in interesting ways. So did the act, do we know? Did the actual yeast like the fungi you're looking at become multicellular? Or is it other things that did? You mean around 500 million years ago? Yeah, 500 million years ago.

Yeah, so that's the time period in which I think there's sort of a flowering, a renaissance of multicellularity around the world. Now, to be clear, I think a lot of things had already been multicellular, but they were kind of largely small, sort of unimpressive, things you wouldn't really notice. You know, animals probably evolved to form small multiserable groups 800 million years ago, but it wasn't until about 550 million years ago that there's just this huge explosion in animal diversity. You know, land plants didn't really begin to colonize, colonize land and, you know, begin this arms race of getting larger and more complex until around 450 million years ago. And in terms of like the seaweeds, for example, the seaweed's like red algae and green algae,

they had been around since over a billion years ago, but those are very small. And you really see this explosion in seaweed diversity and size around the same time. And then the brown algae, like the dominant kelps that you see if you go to the ocean and look at the, look at the sand that's covered with these big brown kelp, those actually are kind of the most recent kid on, they're a new kid on the multi-secondary block. They evolved roughly 200 million years ago, and we don't really have that much evidence of what their actual ancestor looked like, despite the fact that that's one of the most recent multicellular transitions. I'm so glad I'm not a biologist. It's just too complicated. I don't know how you keep the different colors of algae in mind.

But good. This might be a slight digression, but maybe it's exactly the right time to talk about the fact that not only do the sizes and stickiness of your cells and your yeast change. But the shapes change as well. And you've done interesting work on sort of the physics behind packing cells together and statistical mechanics and things like that. So it's always important to remember that as organisms evolve, it's not just changing the genome and giving yourselves new capacities. There's real physics constraints here that shape what is possible and what is not. That's totally true. And I think that's true for everything and it's very, it's maybe more true than on almost any other biological system that I know of for the early evolution of multisilularity in the sense that, you know, forming a group of cells requires these simple multisylular organisms to confront to confront, to confront stresses that act over length scales that they have no history of dealing with.

They're used to dealing with stresses that act over a few microns. They're not used to stresses that act over 50 microns, 100 microns. They're not used to dealing with avoiding how cell packing causes strain to accumulating groups and causes them to fracture. So understanding how multicellular organisms evolve is not just a question of biology and understanding cellular differentiation and gene regulation and all these things that biologists tend to think about. But it's also a question of how multicellular bodies become these sort of biophysically tough, robust structures. So in a sense, you can think of this through the lens of sort of Darwinian. materials. Okay.

That a body is a Darwinian material. And it's something which evolves and started out pretty bad and gets good. How do they do that, right? We kind of understand how modern organisms make, you know, strong, tough bodies. But they probably do it very differently from the way that nascent multicegular organisms would have done it. Because modern organisms are essentially built through a pattern, a process of development, which can take physical strain to account, which can minimize, which can minimize, which can, optimize cell packing in a way that reduces strain accumulation.

It's all coordinated. Early multicegular organisms, they don't have this coordination. So how do they do it? And so this is a long-time collaboration with Peter Yunker, who's a soft matter biophysicist in Georgia Tech. And we recently have a, we put a paper on the archive with Ray Goldstein, who's at Cambridge, looking at the statistical mechanics of cell packing in multicellular organisms. Now, we focused really on just two model organisms to do our experiments in. Number one, snowflake yeast, which is a dumb clump of cells at this point.

We're talking, you know, essentially the original snowflake east. It hasn't evolved yet for any appreciable amount of time in the lab. And also a green algae, volvox, which you may have seen pictures of this in various textbooks or just online. They form this cool sphere of cells where the entire center, it's like a marble of everything. extracellular matrix, and the cells are actually located around on the surface. Okay. So we thought about sort of what are the fundamental properties of a simple group of cells, right? Multicircular organisms really differ.

If noise arises during growing growth and development, that can be catastrophic to an organism. We sort of know how existing organisms do it, but we don't know how nascent ones do. So we looked at the distribution of cell-free volumes within these clusters. And the reasoning for that, the logic was that despite the fact that multicellular organisms are all very different, you know, they can have cells growing like a tree, or they can have cells on the surface of a sphere of extracellular jelly. They all share one thing in common, which is that cells take up space. And if you sort of go back to what we understand about the packing of materials like sand grains or something, it turns out that you get universal packing statistics for things where their particles take up space. and in fact this arises from maximum entropy considerations, and the distribution of cell-free volumes

or the amount of free space around each particle is distributed with a K-gamma distribution. And that's fact indicative of this maximum entropy-driven cell package. You'll have to explain what a K-gamma distribution is. I think you're probably, I'm not a physicist, so you're probably the better place to explain that than I am. I actually don't know that specific one, but basically what you're saying is that there's

some probability distribution over different ways you can arrange yourself, and it's like some power law or something like that. And it's predictable based on saying, given the macroscopic constraints, let's maximize our entropy. Exactly. If every possible configuration of cell packing were equally probable, you would get a distribution of cell packings that is very, in fact, quite likely and not a very unlikely distribution based on just this very simple assumption where there's no free parameter fitting. You just need to know a few things about your system, and you can get a predicted distribution of cell packings based on this assumption. And what does that have to do with the... Well, sorry, I guess what I should say is, are the differences between different kinds of macroscopic morphologies,

mostly down to the shape of the individual cells, or is it the stickiness of the cells with each other, or something more complex? I think the biggest difference has to do with how these cells arrange themselves in the group. So there's a sort of, there's a sort of palette of different ways that multifaciered groups can form. You can have trees arising where essentially daughter cells bud from a mother's cell and remain attached. So you can imagine that, you know, you're adding ping pong balls onto existing ping pong balls and you just continue percolating the growth of this group, right? Or you can have something where you have a sphere of cells and, you know, a spherical group in the cells are arranged on the surface.

Or you can have cells that are sticky, actually sticky, and come aggregate together into a big blob because they're, you know, they're attractive to each other. Or you can have something where you have one cell that gets really big and then undergoes a series of repeated smaller and smaller divisions until it forms a group of nested cells within a sort of exterior cell wall. All of these things actually occur in nature as routes to multicellularity. One of the cool things about multisylarity is that it's very, very diverse. And when we looked at the Snowflake East,

which is basically a brand new model organism, that's had no time to adapt to being multicellular. It's not even really a multicellular organism. It's a dumb clump of cells. And we look at Volvox, which had been around for 200 million years and have development on a very bona fide multicegular organism. They both fit this maximum entropy distribution really, really well.

And then if we simulate the growth of different groups, according to those four different things that I just described, we can grow them through different rules and look at them and then partition space, they all fit this maximum entropy distribution very, very well. well. And this is cool. It's cool for a couple of reasons. Number one, it tells us that there's sort of a multicegular ground state, that if you're going to form a group of cells, the distribution of cellular packing is probably going to be predicted by this maximum entropy prediction. And

you can probably get away from that to some degree with coordinated multicellular development, but it's a starting point. And number two, it helps us understand, I think, one of the larger conceptual challenges in understanding how multicellularity arises, which is it helps us understand how groups of cells get heritable traits, even without multicellular developmental programs. Maybe I should unpack that a little bit. Yes. I mean, this is exactly where I wanted to go, because, I mean, let's phrase it in the following way. As I understand the snowflake yeast, they're multicellular in the sense that you got a bunch of cells

and they're sticking together to make a big snowflake kind of shape. But it's not the kind of multicellularity that we're familiar with in the macroscopic world where there's huge cell differentiation and there's organs and things like that, even though the underlying DNA is the same. And so even if you say, okay, I've made a transition to multicellularity, there's this other big transition looming in front of us about how you have cell differentiation within what you would call an organism. them. And that raises the question of what even counts as a true multicellular organism.

Right. And I'm going to sidestep the philosophical discussion here unless you want to have it because it can take about 10 minutes. We'll come back to it. But yeah, do the biology discussion first. Yeah. So I think the way to think about multicellularity is that forming a group of cells gives you a multicellular group. But it's not a multicellular organism. Right. And in order for something to be an organism, you need to sort of have, there's actually a philosophical literature on what counts as an organism. And it kind of boils down to, you know, the organism contains interacting components in which, you know, there's essentially mutual adaptation and, you know, and essentially functionality.

Like, so those elements are baked into what an organism is. And in order to, and essentially, the way I like to think about multicellularity is that in order to get all this. these complex features, which is frankly why we care about multicellularity. If multicellularity was just little clumps of cells, like we wouldn't really, we'd be talking about biofilms. We wouldn't be talking about multicellular organisms. So in order to get those features that make multicellularity so rich as a system to have

the sort of power to change the planet's biosphere, which it did, you need, you know, these dumb clumps of cells to evolve to become more complex. and therefore this actually becomes a evolutionary description where these clumps of cells actually gain these increased features across generations via Darwinian evolution. And so that means that groups of cells actually need to essentially have the properties of a Darwinian entity. And the cool thing about a Darwinian entity is that it's an algorithm.

This applies to anything, living or non-living. You only need a few things to be true in order to have a population of things. evolve via Darwinian evolution. So you need to have, you know, whatever entity it is, replicate. It needs to have variation amongst different entities. That variation needs to be at least somewhat heritable. And their success, either their ability to reproduce or survive, their persistence in the population, their frequency in the population, needs to depend on those traits that are varying

and heritable. And if you have all of those things met, then your population will evolve, right? Essentially, if selection is acting on a trait and it's heritable, then your population will change over time to have more of that trait. That's true for single cells. It's true for ants. It's true for dogs. It's true for chemistry. It's true for computer programs.

This is just a feature of the world, right? It's a logical argument. And so in order for a multicellular group to have those properties, right, you need to have some way that that group reproduces and makes more groups. It needs to have variation in multicellular traits. That variation needs to be heritable. and selection needs to act on those multicellular traits. And if those properties are true, then over generations, you can see those multicellular groups evolving.

And in fact, over long periods of time, I think that's how complexity arises. Okay, but... Hey, everyone. It's Cal Penn. I'm the host of Earsay, the Audible and I-Heart Audio Book Club. This week on the podcast, I am sitting down with Ray Porter, the narrator of Andy Weir's audiobook Project, Hale. Mary, massive sci-fi adventure about survival and science. And what happens when you wake up

alone very far from Earth? I really had to make a decision because I caught myself getting that frog in my throat and starting to get teary as I'm narrating some of these sections. And it's like, okay, yo, yeah, yo, is this indulgent? And I really thought about it. I was like, no, at this point, it would kind of be betraying the trust the author and the listener have in telling this story if I don't go through it. But there's places in this book that deeply emotionally affected me, and I left it on the mic. That's great.

Because it served the story. People will say like, oh my God, I cried at the end. It's like, yeah, dude, me too. Listen to Earsay, the Audible and IHeart Audio Book Club on the IHart Radio app or wherever you get your podcasts. You know what's wild? We can video chat from space. Order groceries just by talking out loud. and men have like 17 options for hair loss alone,

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So you get a subtle boost without wires, digging, or that, why am I still wearing this moment at 3 p.m. You asked for it. We delivered in a push-up bra that doesn't dig, poke, or pinch. And with an extensive size range from 28A through 42E, you'll be sure to find your perfect fit with the uplift. Try the uplift bra at nix.com and use code perfect fit 15 for 15% off your first order. That's k-n-i-x.com code perfect fit 15, nix.com. I think I'm on board with everything you just said. but at the sort of I guess that's a high level description of what we want to happen.

Is there a sort of nitty gritty description of at what point you go from a snowflake where basically if I understand the snowflakes, every cell is identical. You know, it's just except for their location in the snowflake. So at some point there has to be a way of saying, no, you become this kind of cell and you over there become that. kind of cell. Is that very far away from where we are now? No, I think that's already happening, actually. Oh, okay. Yeah. But it took, you know, thousands of generations of laboratory evolution to get to that point. Well, I mean, maybe this is a good point to be a little bit more explicit about the long-term evolution experiment. Sure. Yeah. So about five years ago in my lab, Ozahn, Bostock, a staff scientist in my group, set up this cool experiment where we

we'd actually been trying for years to figure out how to do a long-term yeast evolution experiment for, you know, selecting on a multicellular trait. And we had kept, we kept running into these problems where they basically would, they would evolve to get larger, and then they plateau, and then they just sit there for, you know, half a year. And nothing was changing. And we were getting ready to throw up our hands and think, maybe, maybe this is not a Darwinian system. Maybe it doesn't have the properties that I just described. And then he actually started playing around with these oxygen experiments, which was actually,

led to a key breakthrough, which is that it turns out that we had been doing all of our experiments in a low oxygen environment. And that oxygen was constraining our system. So they just could, there was no advantage to getting larger. And so it was pegging it at a small size. So we redid the system with anaerobic yeast. So yeast with no functional mitochondria, with aerobic yeast and with mixedotrophic yeast. And we did this thing where we grew them every day. And we, you know, we basically let them sit on the bench and we took the ones that got to the bottom and transferred them to fresh media.

And we did that, you know, every day we've been doing it now for over 1,000 transfers, which is around 5,000 generations. And we kind of escalated the rate, the length of, the strength of settling selection. So now we're only letting them settle for 10 seconds. So they just fall like rocks to the bottom of the tube. Otherwise, they're killed. And over this time, we saw the evolution of snowflake yeast that are visible to the naked eye. So they're macroscopic.

They're bigger than a fruit fly. And this is this was pretty surprising because it required So number one this this already tells us just connecting to what I was talking about This already tells us that they are that that they are a Darwinian entity right that they have these properties in order to allow them to evolve Because they're getting tens of thousands of times larger than their ancestors and we can actually freeze we freeze our populations in a minus 80 deep freezer About every 50 generations and so we can we can actually go back and pull out our living fossil record. So we know exactly how they're changing. We can compete existing organisms with any of their ancestral time points at any time that we want. We can sequence them. We know what all the mutations

are. We know what many of them are doing, not all of them, but a large number of them. We can really understand the system. And we can understand the biophysics of how these guys evolve to get big. So there's a lot of different routes that we could take in the discussion at this point. We could talk about, we just sort of talked a little bit about how physics informs the origin of heredity. So maybe it's a good time to sort of touch back to that. And then we can talk about the physics of how they evolve these bigger, much tougher bodies. Well, first I just want to mention, you know, it's, we're all familiar with selective breeding, right? We make different breeds of dogs and things like that. And am I right to say that the difference here is

you're not being that selective? Like you have a single thing you're doing. You're letting things fall to the ground. But you're not sort of fiddling in a kind of interventionist way with like this yeast versus that yeast. You know, it's basically, you've put them in a harsh environment and watch them evolve according to their own lights, not because you're pre-selecting some kind of traits. That's a really, really good point. In fact, people in the field often make the distinction between artificial selection where you would be looking for a specific trait and picking those individuals and they'd

be constituting the next generation or sort of experimental evolution in the lab, where you have a selective scheme that you're imposing, but you are letting them figure out the traits. and phenotypes and things that sort of, you know, best optimize fitness in that environment. And you're just letting them run. And it's definitely the latter. Right. We are not picking winners. And we should give some credit to the inspiration from Richard Lensky's long-term evolution experiment.

That's entirely why we're doing this. I don't think we would have even had the thought to do this without his word. You know, tell the audience what that experiment was. Yeah. So Rich Lensky is in many ways the sort of founder of the field of experimental evolution. and he has this incredible long-term experiment in E. coli that's been going on now for maybe almost 80,000 generations. I know it's between 75,000 and 80,000. And so it's by far the longest-running continuous evolution experiment.

And it's been going for 30 years. And so, you know, throughout this really unique data set, we've learned a lot of fundamental things about the way evolution works. Because you can't look at any other system in nature and look at so many generations. in this exact same environment. Yeah. You know, we've learned that basically adaptation never stops. It's a power law. It does an asymptote.

You know, we've learned cool things about how novel gene function arises. We've learned cool things about just basic microbial evolution. We've learned cool things about the origin of and maintenance of diversified microbial strategies. And because he's done this in 12 replicas populations, we've learned a lot about chance and, you know, sort of necessity in the process of evolution. And so you, you, of course, in your version of the experiment, I mean, number one, I should say, kudos because this is clearly a very long-term commitment for a lab or for a person or whatever. And you have to keep it up, right? I mean, you can't just leave it. Seven days a week. Seven days a week. You still take off holidays. Someone's got to be in a lab. And the difference being that you have a window onto this multicellular transition, presumably, whereas Lensky is a lot. looking at just, he's stuck with a unicellular ecoli that are evolving in their own ways. Yeah, that's right. I think that's one of the really exciting things about our experiment is that,

you know, we are doing, we are in year three of 30 or so. And, and I do, I really, and I agree, it's hard work really hats off to ozone for being, you know, this completely consistent, utterly committed scientist who has come in, you know, a thousand days in a road to do these experiments. We really don't like to pause the experiment if we can help it because it throws off the dynamics of the way that the populations are evolving. Well, I was just going to ask, is it even possible in principle to pause it? Can you just like put everything in the freezer and come back? Okay. Exactly. And we've had to do it several times, you know, the incubator overheats and everything dies or you get contamination and you have to go back to a time point.

But those things happen. But we try to minimize it because actually the population dynamics are are really interesting and take days to reestablish and are really non-trivial, especially when you get large groups that have different probabilities for survival, stuff like that. And are you also doing sort of separate parallel versions of it to see differences in ways things can evolve? So we have basically 15 evolving populations. Five of which can't use oxygen. So five are replicates of a genotype that started out with broken mitochondria, anaerobics. Five of which are mixatrose and five of which are obligate aerobes.

And are they all in the same environment or are they different environments? They're all in the same environment. Okay. Yep. And are you hopeful that someday the ones that can't use oxygen will discover how to? Is that something to imagine? I don't think so. In fact, if we sequence them, you look at their mitochondrial genome and it's all gone,

except for a few origins of replication. So they're like, there are, like, there are, you know, an order of magnitude, more mitochondrial genomes, but they are several orders of magnitude smaller. Right. I mean, this is... So they pretty much... They're mitochondria are gone. It's one of the issues with evolution, right? Is that it generally proceeds by small steps.

So if there's a place you could imagine getting to that would be better for you, but the only route to get there is through a valley that is worse for you, evolution will, roughly speaking, never get you there. That's right. That's right. That's why a horizontal gene transfer can be, you know, very powerful and that you can bring in things from totally different lineages. And actually, that's the reason that synthetic biology, I think, has potential to do really cool things that you wouldn't expect to evolve. Yeah.

Because you would have never been able to put these things together, either valley crossing or just moving machinery from one disparate lineage into another. Finally, some intelligent design after, you know, it's been thought about for hundreds of years. Okay, so, good. So what you wanted to say with that on the ground on the table, something about the physics of, all with the name for this experiment? Okay, so in order, so we're selecting for larger size. And again, over, I guess I should say our listeners can take a look at our preprint to read about this work.

It's on the bioarchive. Or if you just find my Twitter thread, Twitter feed, just Google my name, Will Ratcliffe. I sort of walked through the paper in that Twitter thread, so you don't have to read a scientific paper to sort of see what we're doing. But it actually has a bunch more cool visuals. I can link to both in the show notes, so people should check them out on preposterousuniverse.com slash podcast, yeah. Excellent.

Show notes. Perfect. So over the course of 5,000, you know, generations of experimental evolution, the anaerobic yeast evolved to get, you know, 20,000 times bigger than their ancestor. The aerobic and mixotrophic yeast, which can use oxygen,

are actually pegged at just a few times bigger than their ancestor. So, you know, we've selected on them for 5,000 generations. They don't do anything in terms of size, which is kind of neat. I think it's a nice follow-up to the oxygen work that I mentioned before, a nice long-term evolution experiment. So the anorobes, they've gotten huge. And we actually know a lot about the physics of how multicellular groups grow to a certain size,

how snowflakey's grow to a certain size before they break. And it turns out that fracture, in this case, is driven by cells dividing into the interior of the group, causing packing, strain accumulating from cell cell packing. and when that strain accumulating from packing exceeds the strength of a cell-cell connection, you break a branch. And much like cutting a branch off a tree, when you break a branch of a snowflake, it floats away. And that's actually cool because connecting to the Darwinian algorithm argument I talked about before,

this is a way that one group can make multiple groups. As it grows, it breaks off branches, those branches, then grow back to approximately the parent size. They begin to break and you get this sort of life cycle. And there's actually been a fair amount of work devoted to understanding how multi-segulical life cycles arise. And I think this physical packing causing strain mechanism is extremely simple one. It's robust. It gives a very nice life cycle.

And it does something which is actually very important in the context of biology here. It introduces genetic bottlenecks. If you imagine that, like, you know, you have a branch of cells where imagine sort of like you have a ping pong ball and you pot a ping pong ball on the top of it. And each of those cells has a new ping pong ball on top of it. And then all of them get another ping pong ball popped off. That bottom ping pong. ball is the sort of parent of everything downstream, right?

It's the progenitor cell. And it's, you know, three generations back, but it's the progenitor cell. So if you take, if you imagine that that's actually just a branch in a bigger snowflake and you were to break that branch off, then that cell at the base of that branch is actually the genetic parent of everything downstream. So as mutations arise within these branches and you break the branches, you're actually segregating genetic variation between groups. That actually turns out to be profoundly important.

for allowing mutations to arise, which change the properties of cells, which have emergent properties that change the properties of groups. And selection can act on the groups themselves, but because all the cells in the group have the same genotype, they have the same mutation. Now there's a statistical correlation between the group level trait and a genetic trait in the cells. And so if you select on that emergent group trait, well, there's an underlying genetic basis, and now you can actually drive continued Darwinian evolution. So this is, though, not what we see in cell differentiation in big organisms, where the underlying DNA is all the same, but they're just expressed differently.

This is true genome differentiation between different parts of the snowflake, and then maybe a part can break off and really be a different kind of organism. I think the way that I would sort of compare this to extant organics. organisms is that this is similar to having mutations in a fertilized egg. And so, or like in a lineage of cells that leads up to a fertilized egg. And so the egg is now the bottleneck. That entire organism develops with the same genetic under basis. And so, you know, in any organism that has single cell propagules, you have a bottleneck proper.

In our organism, you don't have single cell propagules, but you have these branches that actually act kind of in the same way. That it basically, in fact, you can calculate like the maximum waiting time for genetic variation to be part of between groups, and it's not very long. It sort of sets up an expectation that sooner or later, any mutation that arises, will be partitioned off into its own group, and then you'll never have admixture between that group and another group again. So it's a one-way direction towards breaking up, you know, mutations that arise within an organism and making it between organisms.

And then is there a competition in the test tube between the different? Oh, yes. It's an arms race. Just like the trees getting taller is an arms race. In our case, we don't transfer all of the clusters that survive in a given set of time, like 30 seconds of sinking. We only transfer the bottom of the group of cells that are racing to the bottom. And so there's a continual arms race dynamic here where it's not good enough just to get to the bottom in 10 seconds or 30 seconds. You've got to beat everyone else trying to get to the bottom in 30 seconds.

Have you ever read the short story by Theodore Sturgeon called Microcosmic God? No. Oh, you should. It's from many decades ago, but it's basically a genius inventor buys his own island and then creates some microorganisms and subjects them to really, really harsh conditions that they evolve really quickly and develop intelligence, and then he makes them invent things for them and he sells them to the rest of the world and becomes rich and complications ensue. But I'm just saying that, you know, if this academic thing doesn't work out for you, there might be

another way to, you know, earn a living. Might take a few more transfers. Well, yeah, because it does, I mean, it's pretty rapid your evolution, but it's not that rapid. I mean, but it's still, you know, generations, several generations per day, not a million generations per day. Yeah, we're getting five generations a day and, you know, it's frustrating because it's a log of the, of the, you know, numerical number increase.

So you have these hundredfold expansions of population size a day. That's six and three quarters generations. And it's not even really a hundredfold. It's like 60-fold. Well, but it goes... It goes back to my point that, um, physicists would be puzzled by how something that has a time scale measured in minutes or hours, uh, can change over a time scale of a billion years in a profound way.

Um, but of course, the reason why, if you forced a physicist at gunpoint to say, how could that happen? Well, there must be, you know, the, the, the billion year, time scale must be the small time scale times a big number, a big dimensionalist number, right? And that big dimensionalist number must have to do with the space of possibilities for these genomes to evolve, right, which is just huge. There's a whole bunch of different mutations that could go on. So, I mean, how do you, I guess in part you're dealing with that just by making the environment really harsh and therefore, you know, nudging them to evolve quickly. But is there

some, how do you deal with the fact that even though you're doing a lot of generations, it's nothing compared to a billion years worth of evolution? So, yeah, so we can do some cool tricks to get around some of these things, which is we can use synthetic biology tools to bring in features that evolved over millions of years and see what they do in our system, which is really fun. So, for example, Tony Burnetti, a postdoc in our group, who is a synthetic biology whiz, has been basically engineering our yeast to express myoglobin, which is an oxygen-binding molecule.

And we actually took the sequence from a sperm whale. We optimized it to be expressed in yeast, stuck it into yeast, and it works. They're making sperm whale myoglobin. They're pink. They're pink yeast. And so, you know, we basically think that these oxygen-binding molecules, their whole thing is not like holding on oxygen as a battery. it's to increase the diffusion rate of oxygen through a tissue.

And so we can test the hypothesis that increasing diffusion rates of oxygen through a tissue would interact synergistically over evolutionary time to drive something to become bigger by removing constraints of size that are associated with oxygen. And sure enough, that's what it appears to be doing. I mean, these papers are not out yet. This is work that's ongoing. But, you know, we took myoglobin from a, from a, from a, sperm whale, myohemorrhithrin from a peanut worm, which lives in deep sea sediments and has a very

low oxygen environment. And then we actually took a, there's a published paper from Joe Thornton's group where they, they use ancestral sequence reconstruction to identify the, the ancestor of myoglobin and hemoglobin. So, you know, we made that gene too. Stuck it into our use. So, you know, so it's not just sitting around twiddling your thumbs waiting for evolution to happen in the test tubes. You are, you're sometimes taking an active role and poking at it and seeing what can happen. Absolutely. Yeah.

There's actually a whole lot more crazy synthetic biology that Tony is up to. I could tell you if you're interested. Yeah. But I kind of also want to get back to the physics of this. I'm sorry. Yeah. I never really went down that path. Got off that trail.

But yes, let's complete the physics discussion. Okay. So initially, what all of our yeast that are evolving to become macroscopic do is they make more and more elongate cells. And we really understand how this. affects size and by affecting the, essentially the amount of free space around individual cells and the amount of jamming that occurs inside a cluster. If you imagine before that you were building a snowflake use by adding ping pong balls

onto the tree of ping pong balls, you can see that would get very dense very quickly. It would kind of jam and break apart. But now imagine you're doing the same thing with hot dogs. You have a, you know, the same group has a lot more free space. It's a lot more fluffy. And as a result, you don't have as much jamming. And when they do finally jam, they're at a much, much larger size. So all of our replicate populations are basically getting mutations,

which increase the length of cells, which increases group size. And this slowly, you know, this increase in group size is fairly slow. Over several hundred generations, we're getting, you know, tenfold increase in size. And then we see this surprising break from the sort of linear march towards slightly increased size. We see things that are orders of magnitude bigger with just a little bit more cell length. And if we, in fact, look at how these things are packing. Normally, we expect that more elongate cells are fluffier and there's less

densely packed. But if you make these things have slightly longer cells and all of a sudden, they're packing more and more densely within these clusters, which is not what we expect based on sort of physical first principles. And so it turns out what's happening is we're having this shift, which, in fact, if you're into phase transitions, I think this is something which probably is one. Sounds like it. Yeah.

working on this to know if it really is. We see this shift from cells being basically branches on a tree that if you break a single cell, the entire branch falls away to cells being entangled like vines, such that the cells wrap around adjacent cells. And now, if you want to move that one cell, then you're moving its entangled components and its entangled components, entangled components. And it turns out that that entanglement percolates throughout the entire cluster. And these groups become orders of magnitude.

more tough. In fact, if you look at the materials property of them, they go from being a hundred times weaker than gelatin as the ancestral snowflake use. If you break a single cell-cell bond, the material breaks apart, which is great for getting a life cycle, but very bad for making a tough organism. You go from a hundred times weaker than gelatin to snowflake yeast that are as tough and strong as wood. And it's like a cord that is made from, you know, wrapping thread, you know, various strands of thread. Yeah. Exactly. And, Is that primarily, or is this an answerable question, is that primarily because of the configuration space and some physics, or is it because you're selecting for bigness and toughness and the yeast discovered how to do that in a new way? I think it's the latter, but I'd like to talk to Peter Yonker or my physics collaborator more to be sure he sees it the same way I do.

But we are providing a massive, you know, fitness incentive to get big and out-compete your competitors if you get big. And these things that are entangled are, you know, thousand times bigger than anything that came before them. Like, they're humongous. Are you going to have to increase the size of your test tubes at some point? Are they going to become too big? Your yeast? I doubt it.

And there's a reason baked into the way that we transfer them. We don't actually let the entire test tube compete to settle at the end of every day. We subsample 10% of it and those yeast compete to be transferred. So they actually have a 90% chance of, dying without ever even having a chance to compete for faster settling. And that, I think, actually sets a sort of maximum size to which they can grow. Okay. Before dilution just kind of gets the better of them.

Which, and actually that's very important because something which was able to grow forever would actually be a very boring organism. It would probably kind of break our system. We want things that actually reproduce and die. Yeah. Yeah. And we kind of enforce that through this mechanism. Okay.

So, but getting back to phase transitions, I think that this is something. we're working on. But I think that this entanglement idea is probably an example of a phase transition that as soon as something has the potential to entangle its neighbors, that percolates throughout the entire cluster. And you go from something where you're one fracture away from reproduction to having to break hundreds of bonds. And there's not very much middle ground. And it sounds like something that is actually taking advantage of the feature of multicellularity. It's not just getting bigger by adding more cells. It's getting a good survival characteristic by doing something that a multicellular organism can do that a unicellular organism can't. That's right. And in fact, if you look,

we spend a lot of time thinking about entanglement in biology and it turns out it's a relatively understudied research area. But if you look at the largest fungi that have ever existed, there's something called prototaxides, a fungus from the Devonian 400-ish million years ago that formed these three foot tall, 20, sorry, three foot wide, 25 foot tall columns of fungal mycelium. And if you look at, and they're fossilized

so well, you can take thin sections of them and look at them. And you see, I mean, they're beautifully entangled. You're basically going in two different directions. They're wrapping around each other. It's gorgeous. And actually, as Peter and myself and perhaps David who as well, who's at Georgia Tech and works on the mechanics of biology, we're beginning to think more about, like,

how prevalent is entanglement actually in biological systems? You know, for example, is rhino horn an example of a good example of an entangled material? It's made from hair that's kind of all meshed up and matted down in this horn, and those hairs are not just, like, perfectly parallel. So, you know, how much is entanglement actually underlying a lot of the materials properties of multidis organisms? But we don't typically think of it that way because, you know, they have glue or, you know, they have bonds between cells or something like that.

But it's clearly, you know, a nice way that the possibility of cooperative behavior is changing the qualitative features of the organism, right? That's right. So getting back to something you said earlier, the way we introduced talking about the multicellularity long-term evolution experiment was, are we going to see cellular differentiation, right? When are we going to see cells performing different things? And we're actually investigating this in the context of intelligence. entanglement right now in the sense that our yeast cells are all genetically the same,

but that does not mean that they're physiologically the same. In fact, we see big differences in evolved, differences that have evolved over our experiment in the behavior of young cells that are sort of weighted towards the surface and older cells that are in on the interior of the cluster. Turns out that older cells are much more likely to divide from the middle of the cell, making a right angled branch, which is something which drives entanglement. new cells, young cells, first generation cells, are much more likely to be popped off the tip of the cells.

And in fact, it looks like this is, to some extent, being coordinated by changes in the expression of chaperone proteins, which have a very broad role in the cell. But if we play with the expression of these chaperone proteins, we can actually change these phenotypes. So basically what we see is at over-cellular age, these things are growing outwards. So the internal cells are old, the external cells are young.

we're seeing the evolution of new age-dependent cellular behaviors, which may be, in fact, important for driving the entanglement phenotype and giving these groups really robust materials properties. So this should be an example. Yeah. Is it too much to say, like, this could be the hint of the beginning of true cellular differentiation and expression? That's exactly how we are thinking of it.

Is that it sort of like, if they don't have, this is one way to get. get this, right? Like, there is a reliable distribution of, like, age provides a reliable signal for where the cell is. If it's young, it's probably on the outside. If it's old, it's probably on the inside. And we know that East actually have all these things that are expressed differentially in age already. So that's actually a lever that's easy for them to pull. And what we seem to be seeing is then pulling on this lever of changing the expression of this chaperone protein, which changes with it the distribution of budding, which then changes how entanglement proceeds. It's pretty amazing. It's pretty cool.

Yeah, and the most amazing thing is that you're not taking billions of years to see it happen. By just nudging it a little bit, you're able to see it in the lifetime of a postdoc. Yeah, that's right. And the other thing that's really cool from my perspective is that a lot of this stuff is stuff that I would never have been able to predict before doing the experiment. A lot of experiments in biology are hypothesis driven where you already have a pretty good idea of the way the system works before you even propose the experiment. You set it all up to usually test some very low-dimensional linear effect. and ignoring all the complications of the real world. And is this additive effect significant?

Yes, it is. And those experiments are great. Don't get me wrong. But they're also limiting that you have to already kind of understand your system well enough to be able to propose that kind of experiment before you can do it. And in our case, one of the beautiful and fun things about experimental evolution is that you don't actually necessarily have to know what you're going to get. And you can be really surprised by the twists and turns that your system goes on.

Well, I think that is the perfect place to draw this to a close because you've given us an enormous amount to think about. And that's what science is all about, being a little bit surprised about what you learn. And, you know, I'm hoping that your experiment goes on for at least 30 years. I think you're being modest and it could be at least 300 years. You never know. That would be awesome. I will happily hand the reins over to someone else. Well, unless they solve aging by then, you can still be the boss, you know, for the next 300 years.

I don't know. Oh, yeah. Well, that sounds horrible. No, no. In fact, maybe even a better thing would be to encourage other people to start their own, you know, forks. We can also do that. Oh, that's true. I was going to say their own experiments, but of course, we can share, right? You can pass these around and have it evolved differently in different labs. That sounds good to me. All right, good. A project out there for some of the folks listening to the Mindscape podcast. So, Real Radcliffe, thanks so much for being on Mindscape. Thank you so much. I had a great time. You're confused about your credit score. One site has one.

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