The genetic basis of evolutionary change in morphology, phenotypic adaptations, and behavior

Transcript of Part 3: Genetics of Behavior

00:00:00;15	Hi, my name is Hopi Hoekstra and I'm a professor at
00:00:03;26	Harvard University. And in the first segment of my talk,
00:00:07;01	I introduced you to the field of evolutionary genetics, and
00:00:09;27	in particular, the study of the genetics of adaptation.
00:00:13;08	In the second segment, I told you a story about understanding
00:00:17;11	the genetic basis of a morphological trait. And in this third
00:00:21;13	segment, what I'd like to do is switch focus and look at the genetics
00:00:26;11	of a behavior. So, we'll be looking at this in the context of
00:00:31;14	making links between environment, behavior, and genotype.
00:00:33;29	And in particular, I'll start by telling you about one behavior
00:00:39;08	and how that may have evolved via natural selection.
00:00:43;24	And then second, and for most of the talk, what I'll focus
00:00:47;01	on is how we're trying to understand the genetic basis
00:00:50;14	of this behavioral variation. Now, one of the big questions
00:00:56;05	in biology is what is the role of genes in producing behavior?
00:01:00;26	And in particular, in recent years, what are those genes that contribute
00:01:04;18	to behavioral variation? I think we have just a few very
00:01:10;00	elegant examples of the connection between genes and behavior,
00:01:12;27	and I'll highlight in this next slide. So, for example, we know
00:01:16;17	that amino acid changes in the Period locus affects courtship
00:01:20;15	song, which in turn affects mating behavior in two different
00:01:26;08	species of drosophila. We also know that changes in the expression
00:01:30;21	of the gene Foraging in these adorable little drosophila larvae
00:01:35;08	affects feeding behavior. There's also a few examples of
00:01:39;23	behavior genes that affect social behavior. Two of my favorites
00:01:42;03	are amino acid changes in the Gp6 locus seems to be associated
00:01:48;12	with the number of queens in a colony. And then most recently,
00:01:52;17	gene expression changes in the face of vasopressin gene seems to
00:01:57;09	affect the affiliative behavior of males in monogamous versus promiscuous
00:02:02;11	voles. Now as I mentioned, making the connection between genes
00:02:06;22	and behavior is a fundamental challenge in biology, yet I've also
00:02:10;09	told you that while we have some examples, we don't have a lot
00:02:14;06	of examples. And the question is, why is it so hard to find genes
00:02:18;14	that affect behavior? And I just wanted to give you a few reasons why
00:02:21;24	this may be. So there are a number of challenges to studying behavior
00:02:27;05	that go even beyond the challenges that I illustrated before
00:02:30;26	in studying morphological traits. For example, we tend to
00:02:34;18	think that behaviors in general have relatively low heritability,
00:02:38;02	that means that genetic contribution to behavioral differences may be
00:02:41;27	quite low. And this may be in part because the environment
00:02:46;02	can have large effects on our behavior. We know that just
00:02:49;00	thinking about human behavior, for example. We also tend to
00:02:52;10	think of behavior variation as having a complex genetic architecture.
00:02:56;25	And what I mean by that is that it could be many genes seem to
00:03:01;11	affect behavior. And so we're not just going after a single gene
00:03:04;27	that affects behavior, but we have many targets to find.
00:03:08;25	Then there's also some practical issues, such as tractability.
00:03:13;00	Especially if we're interested in behavioral evolution in natural
00:03:16;05	populations. We have to study them in a species in which not only
00:03:20;15	do we know there's natural variation in behavior, but where we can
00:03:23;05	get large enough sample sizes. Where we can breed them in
00:03:25;18	controlled laboratory conditions with short generation times.
00:03:28;11	But also that has genomic resources. But I would argue
00:03:31;23	maybe the biggest challenge to studying behavior is simply the
00:03:35;20	challenge of measuring behavior. And so I want to illustrate
00:03:38;08	that by talking about drosophila courtship behavior.
00:03:41;02	So let's say for example, we have two species of drosophila
00:03:43;28	that have different courtship rituals. Now I'm not a drosophila
00:03:48;25	biologist, but let me just say that courtship is actually
00:03:52;00	-- well, at some level very simple, has a number of different
00:03:54;12	components that are illustrated down below. So for example,
00:03:58;05	drosophila courtship involves two flies meeting, approaching
00:04:03;06	each other, wing flapping, there's tapping, there's licking, there's
00:04:06;25	smelling, there's mounting. All of this happens before copulation.
00:04:09;27	So, if there are differences in this sequence between species,
00:04:14;00	how do we measure that? For example, do we measure the
00:04:16;26	number of tapping events? And is that really the most biologically
00:04:19;27	relevant component of the differences in courtship?
00:04:22;28	So, next what I'd like to do is tell you about how we're trying
00:04:25;19	to circumvent this problem of measuring behavior. And that is,
00:04:29;23	not by measuring the behavior itself but by measuring the
00:04:32;29	product of behavior. And this harkens back to what Richard
00:04:38;06	Dawkins referred to as the extended phenotype in a book
00:04:40;20	that he published in 1982. While we tend to think of phenotypes as
00:04:45;28	traits sort of within our own skin or within the skin of an organism,
00:04:49;08	in fact, we can think of phenotypes as extended outwards.
00:04:52;20	For example, an animal artifact that can be treated like any other
00:04:57;01	phenotypic product whose variation is influenced by a gene.
00:04:59;27	So for example, if a behavior results in the production of an
00:05:03;19	artifact, then that behavior is controlled by genes. We can
00:05:06;04	treat that artifact just as we would any other morphological
00:05:08;23	trait. So let me illustrate this by giving you two examples
00:05:11;29	of extended phenotypes. So the first example that I'm showing
00:05:16;16	you here is the Australian bowerbird, in which the males
00:05:19;25	build these very elegant bowers. And the bowers tend to be very
00:05:23;23	similar within a species. So this particular species builds a bower
00:05:26;26	by putting reeds together that make this sort of U-shape.
00:05:29;22	And then he decorates it with these blue artifacts that he finds
00:05:33;06	in the area. And he'll walk back and forth in this ritual
00:05:38;01	to attract a female. But the shape of the bower differs
00:05:42;04	dramatically between species. And what this serves to
00:05:46;05	illustrate is that this bower is produced by a particular
00:05:48;13	species specific behavior. And the output of it is this bower
00:05:52;10	that we can measure just like we would measure let's say
00:05:54;13	the length of a limb or another morphological trait.
00:05:57;09	Another example is the nest that swallows build. Again,
00:06:01;10	they're very stereotyped in the type of material that's used,
00:06:04;12	where they're built, and the shape of the nest that tends to be
00:06:07;28	very similar within a species, but differs between species.
00:06:10;09	And again, we could treat this like any other morphological
00:06:12;24	trait, and try to understand the genes that produce the behavior
00:06:16;13	that in turn produces the artifact. So today I want to talk about one
00:06:20;21	particular extended phenotype, and that is burrows.
00:06:23;17	Now, burrows have evolved in a number of different
00:06:26;28	lineages. Here I'm just showing you a few examples, they've
00:06:29;10	evolved in bivalves, in ant colonies, in prairie dogs, for example.
00:06:34;18	And the fact that it's evolved independently multiple times
00:06:37;16	suggests that it has a function. So what is the function of
00:06:41;02	burrows? Well, burrows of course can be used for a number of different
00:06:43;28	reasons. And I just wanted to provide you with a few examples.
00:06:46;24	And these reasons can affect fitness, the ability of an organism
00:06:51;19	to survive and reproduce. So burrows can be important for
00:06:55;20	predator avoidance, be important for thermoregulation,
00:06:59;03	or if those species let's say meet and mate underground,
00:07:03;02	they can be important for social interactions. And also
00:07:05;21	food storage or growth. So we know that burrows can
00:07:10;10	have fitness effects. But the other reason we study burrows is
00:07:13;24	because we have some hints that genes may be important
00:07:17;09	in the production of burrows, and importantly in the differences
00:07:20;18	between the size and shape of burrows. And this goes back
00:07:23;15	to work done by Carol Lynch who was at the University of
00:07:25;21	Colorado, who was studying burrow building in laboratory
00:07:29;01	mice. So what you're looking at here is an outline of a burrow
00:07:32;09	that was excavated, and here is the entrance tunnel, of a
00:07:35;21	particular species of mouse. And what she showed through a series
00:07:38;24	of crosses is that different components of the burrow tended to
00:07:42;16	be very heritable. That is there seems to be a genetic component.
00:07:46;09	So we know that there's ecological relevance. We know that
00:07:49;16	at least in laboratory mice there seems to be a genetic
00:07:52;02	component, which suggests that this may be a good extended
00:07:54;29	phenotype or a way to try to connect genes and behavior.
00:07:58;02	The other little anecdote I want to tell you about burrows is one of
00:08:01;05	the few behaviors that actually fossilizes. So what I'm showing
00:08:04;14	you here is actually a picture of a fossil burrow that's produced
00:08:08;24	by a now extinct beaver that built these burrows in this very
00:08:12;11	stereotypical corkscrew shape. Well, we're not studying burrows in
00:08:17;21	bivalves or ants or prairie dogs, but instead studying burrows
00:08:20;14	of this particular species here, Peromyscus polionotus.
00:08:23;09	Which tend to live in these very open habitats, like
00:08:27;17	this slide illustrates. And in fact, this is taken from one of our
00:08:31;28	less glamorous field sites. It's a burnt peanut field in Alabama.
00:08:35;13	But the reason we're studying this mouse is because of
00:08:38;22	work that was done by Francis Sumner, who was a natural
00:08:41;04	historian who early in the 1900s documented variation
00:08:45;28	within and among populations of Peromyscus polionotus
00:08:49;24	throughout the range. And one of the traits he documented was their
00:08:53;08	burrowing behavior. And in particular, he noticed they built a very
00:08:58;15	stereotyped burrow. So in a paper that was published
00:09:01;19	in 1929, along with his field assistant, what he showed was
00:09:05;14	that these mice throughout their range produced a very
00:09:09;17	complex burrow that looked like this. So these burrows are
00:09:15;01	characterized by an entrance hole, and soil when it's active
00:09:19;27	there's fresh soil that's excavated out of it. And they have this
00:09:22;25	long entrance tunnel, which leads to a nest chamber.
00:09:26;14	And then a secondary tunnel that radiates up near the
00:09:29;17	surface, but doesn't penetrate the surface. So next what
00:09:33;11	I'd like to do is just tell you how we catch these mice in the wild,
00:09:36;16	which will serve to illustrate two important aspects of this
00:09:40;02	burrow shape. So how do we catch these mice in the wild?
00:09:42;20	Well, the first thing we do is go out to a habitat that looks like
00:09:44;25	this. And we look for burrows. So here's a burrow entrance,
00:09:49;20	and this freshly excavated plume of soil suggests that it's an active burrow.
00:09:53;14	The next thing we do is, one of us, and in this case, my graduate
00:09:57;05	student, Jesse Weber, you can see his back right here,
00:10:00;05	lies down on his belly and takes some plastic tubing and
00:10:04;03	weaves it slowly down that entrance tunnel. When hits the
00:10:07;05	nest chamber, he'll either blow into the tube or rattle it
00:10:10;17	around. And the rest of us are not just standing around and
00:10:13;22	watching Jesse, but we're at the ready. Because what's going to
00:10:16;14	happen is that a mouse is going to pop out of that secondary
00:10:18;21	tunnel, which we pounce on it, and then we've caught
00:10:21;25	the mouse. Now I've told you that the secondary -- the
00:10:24;27	first thing I want you to note, is that secondary tunnel..
00:10:27;20	remember I told you doesn't penetrate the surface, yet
00:10:30;04	we're still able to predict with pretty high accuracy where
00:10:33;07	that escape tunnel is going to come out. Because the shape
00:10:36;11	of the burrow is so stereotyped. The second important thing to note,
00:10:40;24	is that secondary tunnel is actually used as an escape hatch.
00:10:44;19	So that when something comes down the entrance tunnel,
00:10:47;06	the mice are still able to escape. The other thing I should mention is
00:10:51;28	in the field, when we find a burrow in which the escape tunnel is visible,
00:10:56;03	and we see the hole, we never found mice in such a burrow.
00:11:00;29	Which suggests that once they've used that escape
00:11:03;02	hatch, then they've abandoned the burrow. So why would
00:11:07;06	these mice build such a complex burrow? Well, the answer may
00:11:10;06	seem obvious. We haven't yet done experiments to
00:11:13;18	demonstrate the fitness effects of different burrow designs.
00:11:17;09	But suffice it to say it's pretty clear that having an escape
00:11:22;19	tunnel, especially when a major predator is a snake, makes
00:11:26;13	a lot of sense. So if a snake comes in the entrance tunnel,
00:11:28;22	these mice can escape out the back door. Okay, so clearly
00:11:34;03	the complexity of this burrow can be important for fitness.
00:11:37;18	The next thing we wanted to do was try to understand the
00:11:41;24	genetic basis. So the first thing we wanted to do was try to
00:11:44;18	study these mice in a controlled laboratory environment where we
00:11:48;09	could minimize environmental effects and focus on the genetic
00:11:51;04	influences. So to do this, what we were able to do was to bring
00:11:55;11	mice from the field into the lab. Now this work that I'm going to
00:11:59;20	describe is really inspired by work by Wally Dawson, who back
00:12:03;24	in the '70s and published in the '80s, showed that you could bring
00:12:06;13	these mice from the field into the lab and they would still
00:12:11;10	recapitulate their burrowing behavior. So we wanted to test
00:12:15;25	if this is still true. So to do this, we built what we refer to as
00:12:19;27	phenodomes. These are in some sense, glorified sandboxes, that are
00:12:23;28	quite large. So they're 5 feet by 4 feet by 3 feet high. And each
00:12:27;29	pheno-dome is filled with a ton and a half of dirt. To start the
00:12:32;03	trial, we put in a single mouse and we let it live in that pheno-dome
00:12:37;12	for about two days, completely undisturbed. So it's allowed to build its
00:12:41;19	burrow over two night time activity periods. So just to give you
00:12:45;06	a sense of what this actually looks like, here are our 10
00:12:48;20	pheno-domes in our controlled laboratory facility.
00:12:52;18	Then once the mouse has gone through its two day time
00:12:56;25	period, we trap the mouse out of the cage and to measure the
00:12:59;25	burrow, what we use is what I'll refer to as pheno-foam.
00:13:02;14	Essentially this is insulating foam that we can squirt into
00:13:06;24	the burrow, let it harden, and then produce this cast of the burrow
00:13:10;02	like the one shown here. And then treat that cast as a
00:13:15;07	morphological trait, an extended phenotype, of a behavior.
00:13:20;03	Thereby circumventing the problems associated with measuring
00:13:23;09	behavior. So let me just show you in this next video of exactly
00:13:26;25	how we do this. So here's Jesse Weber, he's threading that
00:13:30;24	tubing down the entrance tunnel. And what you see is out pops
00:13:35;11	the mouse. And you'll notice that you had no idea or no indication
00:13:39;24	before where that escape tunnel was until the mouse popped
00:13:42;10	out. And after the mouse is taken out of the cage, we fill
00:13:46;09	this burrow with this insulating foam, or pheno-foam.
00:13:50;21	Which you'll see here as it's squirting out of that escape tunnel.
00:13:53;19	We thread it through, filling it with insulating foam along the way.
00:13:57;28	Then what we do is we wait a few hours, the foam will be allowed
00:14:02;13	to harden, and then we dig that out of the burrowing box.
00:14:07;14	And then again, we treat this cast as a morphological representation
00:14:12;02	of burrowing behavior. So today what I want to do is focus on
00:14:16;15	just a few aspects of the burrow size and shape when we're
00:14:20;18	talking about its underlying genetics. So the burrow shape,
00:14:25;11	all of which we've measured off of these casts, consists of
00:14:29;16	a number of different aspects. So today I'm going to talk about
00:14:31;29	the total length of the burrow, shown here, and in particular,
00:14:37;01	the entrance tunnel length. And then refer to this as the escape
00:14:41;15	tunnel, or the presence or absence of that escape tunnel.
00:14:43;19	So when we started this experiment the first thing we wanted
00:14:47;02	to make sure of was that these mice now 30 years or 60
00:14:52;00	generations after Wally Dawson's original study, still built
00:14:55;04	these complex burrows in the lab. So what we did was we simply
00:15:00;03	put a mouse in the dirt and asked, will it burrow?
00:15:02;11	And as this video illustrates, these mice hit the dirt and
00:15:06;09	immediately start digging. What this means to us is that this
00:15:10;14	behavior is not strictly learned. In other words, these mice have never
00:15:14;23	seen dirt. Their parents have never seen dirt. And upwards of
00:15:17;25	let's say 60 generations, they've never seen dirt.
00:15:20;17	So, the building of this complex behavior is not one that's
00:15:24;02	strictly learned. So what this means to us is that there's an
00:15:27;05	innate component. And in fact, there may be a strong genetic
00:15:30;09	component to this burrow building behavior. Okay, there are several
00:15:35;27	other very basic questions we had that we tested in the lab.
00:15:39;17	The first one was what happens if you take a mouse and put it
00:15:42;12	in for one trial, and then put it in for another trial, and another trial?
00:15:45;23	How consistent is the burrow size and shape? Well, to do this,
00:15:49;28	we simply ran that experiment three times in a row, giving them
00:15:53;13	two day trials. And what we saw was that they always built
00:15:56;25	a complex burrow, and that in general, the length was quite
00:16:01;21	consistent over trial. So what you see here is that there is a slight
00:16:05;05	trend over multiple trials, that the burrows get longer, but they're not
00:16:09;25	statistically different. And this trend could be associated with
00:16:12;28	either their learning to get better over time, or the first couple
00:16:17;16	of trials, they're still acclimating to this new environment.
00:16:20;09	In either case, the results I'm going to tell you about are all
00:16:25;02	focused on the best burrow, which tended to be the third trial.
00:16:28;06	Okay, so despite the fact that there are some differences,
00:16:31;15	it is still remarkable how consistent in both size and length
00:16:34;24	the burrows were within individuals across trials.
00:16:38;09	The next question we had was, what about differences
00:16:41;16	between the two sexes? Did the males or the females dig
00:16:44;10	different size burrows? Well it turns out when we did these
00:16:47;29	trials, we found absolutely no difference between the burrows
00:16:50;29	that were built by males and females. So again, all the data
00:16:53;14	I'm going to show you is going to include both males and
00:16:56;16	females together. So these are some of the basics that we learned by
00:17:00;11	studying these mice in the lab. Next I want to tell you is
00:17:03;07	what we learned about studying these mice in the field.
00:17:06;25	So we sent Jesse out to the field, and he would go out
00:17:10;26	into this burnt peanut field in Alabama. He'd find burrows
00:17:14;09	like this one with a fresh plume of excavated soil, suggesting it's
00:17:17;24	an active burrow. He trapped out the mice and then made
00:17:22;03	a cast of the burrows in the wild. So here's after a lot of
00:17:27;06	excavation, you can see a rather large burrow. For those of you
00:17:30;02	who are molecularly oriented, right here you'll see a 15ml
00:17:33;19	conical, which gives you some sense of scale. So he did was
00:17:37;29	excavated burrows like this throughout the range of these mice.
00:17:41;28	And in different habitat types or in different soil types, some that had
00:17:46;10	low silt content or more sandy soil, compared to those that had
00:17:50;09	high silt or more hard packed, for example more clay. And
00:17:55;12	by doing this and digging out different burrows, we learned
00:17:59;12	a number of things about variation in the wild. The first thing we
00:18:03;18	learned was that burrow shape is conserved, that is in all of these
00:18:06;26	different habitat types, we always saw burrows with an entrance
00:18:09;29	tunnel and an escape tunnel. So that shape is highly conserved.
00:18:13;13	But we did see some differences. That is even though the length
00:18:17;13	of the burrows and the shapes of the burrows were conserved,
00:18:19;20	the depth differed. That is when the soil got harder packed,
00:18:23;12	the mice stopped digging down, but interestingly, the total
00:18:28;05	length didn't change. So what that means is the angle
00:18:31;05	of the burrow would change depending on the soil, but the
00:18:33;29	length stayed constant. In other words, we think the mice
00:18:37;06	somehow, and we don't know how yet, are measuring length
00:18:40;26	of the burrows, and that's consistent. The other things we learned
00:18:44;25	and you may have noticed this, is that wild burrows are
00:18:47;21	longer or bigger than those built in the lab. And there's a number
00:18:51;05	of reasons this could be. For example, maybe we didn't give them
00:18:54;16	enough space in the lab or we only gave them two days in the lab.
00:18:58;01	But I didn't tell you one important fact in these mice, and that
00:19:02;18	is that Peromyscus polionotus is one of the few monogamous
00:19:05;18	species. And when we go out in the wild and we catch these
00:19:10;04	mice, and we put tubing down the burrows, usually it's not
00:19:12;29	just one mouse that pops out, but usually two mice. And usually
00:19:16;11	a male and a female. This isn't surprising if these species are
00:19:19;29	monogamous. But it does raise the question of, in the wild
00:19:23;01	maybe it isn't a single mouse that builds a burrow, like our
00:19:26;02	tests in the lab, but instead two mice, maybe a male and a female.
00:19:30;08	So, I just want to illustrate what we learned about studying
00:19:34;27	this in the lab. When we put two mice in a box, we ask the question,
00:19:41;03	do they build two independent burrows or do they cooperate
00:19:43;09	and build a single burrow? And this little clip illustrates the
00:19:47;24	main result, and that is, the two mice build only a single burrow.
00:19:51;25	And that burrow is about twice as long than what each individual
00:19:56;05	burrower built. So there is some aspect of cooperation,
00:20:01;04	a term I'm using loosely here, in terms of building burrows.
00:20:05;00	Okay, so let me just summarize what I've told you so far.
00:20:08;15	So, what I've shown you so far is that burrowing behavior appears
00:20:15;06	to have a genetic component. And this is because we've tested
00:20:18;12	them in a common environment, and these are mice that again
00:20:20;29	have never seen dirt before. So this is not learned behavior.
00:20:24;16	I've shown you hints that it's similar across trials, so the same
00:20:30;11	individual produces similar burrows. There's no difference between
00:20:32;28	the sexes, and among individuals within the species, there
00:20:36;16	are actually very little differences. What we learned from
00:20:40;12	studies in the wild is that burrow shape and length tend to be
00:20:43;06	very consistent, but the depth will vary depending on the type of
00:20:45;18	soil used. And then finally, I showed you just some hints that
00:20:49;05	in fact, in the wild, males and females may cooperate to build these
00:20:52;03	burrows. Okay, the next question is -- so everything I've told you
00:20:56;13	about really focuses on this one species, Peromyscus polionotus,
00:21:00;03	but what we really want to know is how this behavior evolved.
00:21:03;13	So what happens if we look at other species of Peromyscus?
00:21:06;20	So, what we did was, we did the same types of trials across
00:21:12;01	a number of different species of Peromyscus, which are illustrated
00:21:15;07	here. Peromyscus polionotus, which I've been talking about is shown
00:21:19;02	at the bottom. So I'm just going to highlight the types of burrows
00:21:23;15	that each of these species built, with these cartoons.
00:21:26;08	So some species built no burrows, others very small burrows.
00:21:30;10	But only Peromyscus polionotus built these large burrows
00:21:33;28	with this escape tunnel. Let me point out that these are not
00:21:36;25	to scale. So this is just to serve to illustrate an example of the type
00:21:40;23	of burrow these species built. So this is interesting for a
00:21:43;29	number of reasons. First, it suggests that the Peromyscus
00:21:47;07	polionotus burrow, and in particular, the escape tunnel is really
00:21:50;11	unique to this species. So we're looking at the evolution of the
00:21:53;11	gain in complexity, compared to other species in this genus.
00:21:56;24	The second thing to note is that here, as showing you the
00:22:02;00	relationships among these species, and you can see that
00:22:04;17	even among closely related species, you can have very big
00:22:07;14	differences in burrowing behavior. And in particular, we
00:22:11;22	were focusing on these two species down here. So even
00:22:14;25	though polionotus is closely related to maniculatus, as indicated
00:22:18;11	by this phylogeny. You can see they build very different burrows.
00:22:22;07	But what's particularly exciting to us is that these two closely
00:22:26;11	related species, while good species in the wild, if we bring
00:22:29;12	them into the lab and give them no choice, they'll interbreed and
00:22:33;29	produce offspring. This allows us to now take a genetic approach
00:22:38;02	in the lab to try to dissect and identify the genes that contribute
00:22:42;13	to the differences between these two species. So next, what
00:22:46;00	I'm going to do is tell you a little bit more about the burrows that
00:22:48;16	maniculatus builds in comparison to polionotus.
00:22:50;13	So, I've told you already that polionotus build long burrows
00:22:54;24	with an entrance tunnel and an escape tunnel, this is just showing you
00:22:58;16	some data on their length. By contrast, maniculatus build these
00:23:02;16	little burrows. They're like the size of a little baby sock.
00:23:05;09	They're not very long, they have an entrance tunnel,
00:23:08;27	again which is shorter than the entrance tunnel you see in
00:23:11;23	polionotus, and they never build an escape tunnel. Okay, so
00:23:14;26	these are very clearly distinct types of burrows. But as I mentioned,
00:23:18;03	these two species, we can make hybrids in the lab. The first
00:23:22;01	question is, well what type of burrows do the hybrids make?
00:23:24;06	Well, quite surprisingly I think, hybrids make burrows
00:23:29;08	that are absolutely indistinguishable from the complex
00:23:32;07	burrows of polionotus. That is they're long, they have a long
00:23:35;21	entrance tunnel, and they always have an escape tunnel.
00:23:37;19	So what this suggests is that the gene or genes that control the
00:23:41;13	differences between the simple burrows and the more
00:23:44;25	complex burrows, are dominant. Okay. So now to get the
00:23:52;03	genes, what we can do is take those F1 hybrids, now cross them
00:23:55;25	back to the simple burrowers, and look at the next generation.
00:23:58;25	So the first thing I want to do is show you some results from
00:24:04;20	this next generation of mice and their burrowing size and shape.
00:24:09;13	So first, let's just focus on one trait, total burrow length.
00:24:12;06	So here, this is the parental generation, the F0 generation.
00:24:16;18	And on this lower axis, what we have is length in centimeters.
00:24:20;27	And then frequency on the vertical axis. So what you see is
00:24:25;22	maniculatus build very small burrows compared to polionotus,
00:24:29;20	and their distributions are non-overlapping. Now as I mentioned
00:24:33;25	in the hybrids, the first generation hybrids, built polionotus-like
00:24:38;01	burrows in length, although there's a few stragglers over here.
00:24:40;15	So then we took these guys and bred them to these guys.
00:24:43;29	And asked that next generation, what types of the burrows do they have?
00:24:48;01	Now remember, they're going to have more maniculatus DNA
00:24:50;19	in them. Because these are half maniculatus, half polionotus.
00:24:53;23	And we crossed them back to maniculatus. And what we see is
00:24:57;07	that there's a range of -- or a distribution of lengths of the burrows.
00:25:02;04	So they tend to be more maniculatus-like, not surprising.
00:25:06;05	They have more maniculatus DNA. What's surprising is we still get
00:25:09;15	mice down here that are building polionotus-like burrows.
00:25:12;17	So this distribution tells us two things, one it's not a single
00:25:15;29	gene. We don't see a 3:1 segregation of long and short burrows.
00:25:19;16	But they're multiple genes. But because we can recapture the
00:25:23;18	polionotus-like burrow, this suggests there's a handful of
00:25:27;07	genes, and not hundreds of genes controlling it, which is quite
00:25:31;13	exciting. So the first result is that there are multiple genes,
00:25:33;26	not hundreds, maybe a handful that contribute to differences
00:25:37;12	in burrow-length between the two species. Now this is just
00:25:40;18	length, what about shape? So now we're focusing on that
00:25:45;15	second generation again, and asking what shape burrows
00:25:48;09	do they make. Now they make a range of shapes, some
00:25:51;25	build these very small burrows like the maniculatus parent.
00:25:55;23	Some build burrows like this one shown here, which is like the
00:25:58;29	polionotus parent. But what you can see is sometimes we have
00:26:02;03	burrows that are small with an escape tunnel, and other
00:26:04;25	ones long without an escape tunnel. And if we simply count
00:26:08;06	up the number of individuals who build an escape tunnel, compared to
00:26:12;27	those without an escape tunnel, and ask how does that segregate in that
00:26:16;10	second generation. What we see is that they're almost equal
00:26:19;04	numbers. This is not distinguishable from a 1:1 ratio.
00:26:23;10	What this suggests is, or at least consistent with the role
00:26:27;25	of just a single gene controlling these differences. Controlling the
00:26:32;28	presence or absence of the escape tunnel. Now let me say
00:26:35;06	this is data early on, and we've done more experiments that
00:26:38;23	suggest that maybe it's a little more complicated, but again,
00:26:42;05	this suggests that maybe the presence or absence of the escape
00:26:44;20	tunnel has a relatively simple genetic basis. Okay, so what I've
00:26:49;17	shown you is that in terms of size and shape of the burrow,
00:26:52;01	we're looking maybe for a handful of genes. But of course the
00:26:55;25	big question is, what are these genes? Now to do this, we took all
00:26:59;25	of these mice which we've characterized their burrow-building
00:27:02;09	behavior. Now we've genotyped them with a number of markers
00:27:05;02	throughout the genome, and asked if there a correlation
00:27:07;20	between what regions of the genome they get from the
00:27:11;04	complex parent and the simple parent, in terms of burrowing,
00:27:13;22	and the burrowing that that hybrid exhibits. So here what you're
00:27:17;25	looking at is a linkage map. I showed you one of these in segment
00:27:20;29	two, as well. So each one of these lines represents a different
00:27:24;01	chromosome. Each one of the dashes represents a different
00:27:26;21	marker that tells us whether it comes from the maniculatus
00:27:29;10	parent or the polionotus parent. And the main thing I want you
00:27:32;12	to take away is it seems like there are four regions of the
00:27:36;17	genome, indicated by these black arrows, that seem to control
00:27:39;26	burrowing behavior. So again, this is consistent with what
00:27:44;05	I've told you before. There's probably a handful of genes involved.
00:27:47;16	What's interesting again, is that it seems like some of these
00:27:52;03	genes, like this one shown here, seem to be largely associated
00:27:55;13	with escape behavior, which these other three genes seem to control
00:27:59;09	burrow length. And again, consistent with the results I told you
00:28:01;13	before. Okay, so what are these genes? So I'm just going to
00:28:05;09	focus on some very preliminary data, and tell you about
00:28:08;17	how we can go from this resolution down to the resolution
00:28:11;07	of genes. So now what I'm going to do is I'm going to focus
00:28:14;00	on this one right here. And now I'm taking this chromosome,
00:28:18;04	I'm going to turn it on its side. So here's the chromosome, here's
00:28:22;05	position along this horizontal axis. And on the vertical axis,
00:28:25;23	we have LOD score. You can think of this as the statistical
00:28:28;18	association between genotype and phenotype. You can see as we
00:28:31;07	move down the chromosome, we get to this peak of statistically
00:28:35;02	significant peak of high LOD score, which suggests that this
00:28:38;15	region of the chromosome has a gene that seems to influence
00:28:42;06	burrowing behavior. And in particular, length of the burrow.
00:28:45;20	So then here are our markers, shown below. Okay, so
00:28:50;26	now let's narrow into this region and ask, what genes occur
00:28:53;24	in this region? Well there are a number of genes, about -- let's say
00:28:58;04	about 50 or so. But there's one in particular, the one I've
00:29:01;05	highlighted in red, that we're excited about. So here are
00:29:05;20	a number of genes that occur in this region. These are just the
00:29:08;20	ones that are brain-expressed genes, which we think are
00:29:11;07	especially good candidates. But next what I want to tell you
00:29:14;14	is just a hint of the type of gene that may be involved in
00:29:17;18	this behavior, and that is what we refer to as Chrm5.
00:29:21;08	So why are we so excited about this particular gene, Chrm5?
00:29:25;19	Well, it's based in part on what we know about this gene from studies
00:29:28;26	in both mice and humans. So this muscarinic acetylcholine
00:29:34;11	receptor 5 has been studied in laboratory mice. We know it's
00:29:39;08	expressed in the brain, and in particular, in dopamine containing
00:29:42;13	neurons in the basal ganglia. It's expressed in particular in
00:29:46;06	two regions of the brain that are associated with reward circuitry
00:29:49;23	and another region associated with motor controls, spatial
00:29:52;26	learning, and addiction. We know that in human studies,
00:29:57;16	in genome wide association studies, this same gene has
00:30:01;04	been associated with nicotine addiction in humans. And if we
00:30:04;18	knock it out in mice, it's associated with morphine addiction.
00:30:07;09	So our current hypothesis is that these mice that build longer
00:30:11;19	burrows, may in fact be addicted to burrowing. Now we haven't proved
00:30:16;14	this yet, but we do have some tantalizing preliminary data.
00:30:20;11	So we've looked at the expression of this gene in our Peromyscus
00:30:23;09	mice, and have shown it's expressed in the same regions
00:30:25;26	of the brain that have been shown in laboratory mice.
00:30:28;23	But particularly is exciting is that there's a difference in
00:30:31;05	the expression level between the polionotus and the
00:30:34;29	maniculatus. In particular, that it's expressed higher in the
00:30:37;12	complex burrowing species. So while this remains to be shown
00:30:43;15	whether this gene is truly involved in these differences in
00:30:46;08	burrowing behavior, this does give you some sense about
00:30:48;17	how we're going after the genes that are involved. And ultimately,
00:30:52;29	to functionally prove that they are causally controlling behavior
00:30:57;14	differences. So in this second part, I've shown you that
00:31:00;03	burrowing differs among closely related species, which is
00:31:03;13	quite exciting. Because we can take advantage of this
00:31:06;11	to try to get to the underlying genetics. I showed you that
00:31:09;25	the polionotus complex burrows are unique, so we're looking at the
00:31:13;01	gain in complexity in this behavior. And because we can cross these
00:31:16;19	closely related species, we've shown that F1 hybrids produce these
00:31:20;11	complex burrows that suggest these gene or genes are largely
00:31:23;28	dominant. And then I've showed you that with continued
00:31:26;27	crosses in the second generation, complex burrows may be
00:31:30;00	controlled by just a few loci, compared to the simple burrowers.
00:31:33;16	And that different aspects of the burrow seem to be controlled
00:31:36;22	by different genes, that is that there are different genes that
00:31:39;14	control length of the burrow, compared to presence or absence
00:31:42;13	of the escape tunnel. And then I've hinted at, at least a type of
00:31:46;19	gene that we think may be contributing to these differences
00:31:49;20	in this naturally occurring behavior. Which may be one of the
00:31:52;22	-- we may be onto one of the first examples of a gene
00:31:56;23	controlling a complex mammalian behavior. But I want to end
00:32:00;18	today by telling you about the work we're doing not just to
00:32:04;03	understand the genetics of the burrow architecture, but
00:32:07;04	actually what the mouse is doing. So the last question
00:32:10;10	I want to ask is, how did the mouse's behavior evolve to
00:32:13;14	result in these different extended phenotypes?
00:32:16;02	In other words, do the complex burrowers, Peromyscus polionotus,
00:32:20;27	do they dig at a faster rate? Do they dig more efficiently?
00:32:23;12	Do they dig for longer periods of time? Now answering these
00:32:27;04	questions is actually quite complicated because these mice
00:32:29;21	are nocturnal. They're doing their behavior underground.
00:32:32;12	So how do we study this? Well, my postdoc, Brant Peterson,
00:32:36;01	has come up with a nice ingenious way of studying the mouse
00:32:38;25	behavior more directly. And that is he built what we refer to
00:32:42;12	as an ant farm, or what we refer to as the Brant farm.
00:32:46;00	The Brant farm is essentially a two dimensional burrowing
00:32:49;29	structure. So we have plexiglass on either side, it's filled with
00:32:52;29	dirt, and it's about the width of a mouse. We can illuminate
00:32:56;22	this burrowing structure with infrared lights, and then we
00:33:01;08	can record the mouse behavior. So I just want to give you an example
00:33:05;10	of what a mouse burrowing looks like. So you can see here
00:33:09;04	the mouse is digging its burrow, it's got to the point where it's
00:33:12;09	starting to dig out its nest chamber. It turns on its back,
00:33:14;29	it starts digging upward, and so by videotaping them
00:33:20;00	in this new setup, we can actually watch exactly what
00:33:24;28	the mouse is doing. Now we got really excited about this
00:33:28;08	particular approach, because it was another way to study
00:33:31;22	the genetics of not just extended phenotypes, but the mouse behavior
00:33:35;02	directly. But one of the things that we learned very quickly
00:33:38;26	that while it's very easy to capture these videos, actualy
00:33:42;02	scoring these videos became very hard. So this goes back to
00:33:45;03	this problem of how do we measure behavior. So it takes a lot
00:33:49;03	of graduate student, undergraduate hours, to actually watch
00:33:52;28	these videos. But luckily, Brant, being ingenious, came up with
00:33:57;13	another idea, and that was to automate this behavior.
00:34:00;22	So here what you're looking at is another mouse digging
00:34:05;08	in the ant farm again, but this time, it's all completely automated.
00:34:10;09	So there goes the mouse down the hill, we can watch this
00:34:13;15	and record this over time. We can look at the burrow, shown here
00:34:17;25	in red, we can see where he's about to dig, shown in blue.
00:34:20;17	We can also measure the change in the slope of the hill, so
00:34:23;29	the amount of digging and excavating is correlated with the amount
00:34:28;04	of sand that comes out, etc ... So, by automating this, not only
00:34:32;07	is it a lot simpler, it doesn't require as many man hours,
00:34:35;12	but we also do this in an unbiased way. So I just want to show you
00:34:39;10	some results from this output, and hint at some early results
00:34:44;02	from this experiment. Okay, so what we're looking at here
00:34:47;20	now is the result of that automated behavioral analysis.
00:34:50;27	So here what we're looking at on the bottom is time,
00:34:53;17	and time is color-coded, so you can see throughout the course
00:34:57;12	of the night we get to more blue. So what you're looking at here is
00:35:00;24	this slope, and here's the tunnel or burrow that polionotus
00:35:04;14	dug. And here, you can see over time, the soil that's excavated
00:35:08;14	goes up. And this cloud-like images tells you about where
00:35:13;22	the mouse is spending its time. So the denser the cloud,
00:35:16;18	the more time a mouse has spent there. So you can see the mouse
00:35:19;01	has been running up and down, and it's also spent time digging
00:35:21;17	out its burrow. But what's really neat about this is we can
00:35:25;23	compare what a polionotus is doing, compared to a simple
00:35:28;28	maniculatus. And there are some very simple differences.
00:35:32;05	The first thing is, as you can see on this axis, which is time,
00:35:35;21	above the line is showing you when they're digging, below the
00:35:39;15	line is activity that's not digging. You can see the mouse is active
00:35:42;29	throughout the whole night, but this mouse starts digging
00:35:45;24	immediately during the night, when it's put in the dirt box.
00:35:49;27	And then finishes its burrow early on and maybe does a little
00:35:53;06	bit of fine tuning later in the night. But this is very different
00:35:56;13	from what a maniculatus does. Whereas a maniculatus that builds a
00:35:59;09	teeny little burrow, as shown up here, what it does is it doesn't do any
00:36:03;02	digging to about two hours before lights come on, and then it digs
00:36:07;23	its burrow very quickly. And then it almost doesn't do anything
00:36:10;13	else, and just sits in its burrow for the last hour before the lights
00:36:13;13	come on. So, this is just to illustrate that these are new features
00:36:17;26	of the mouse's behavior that we're starting to uncover
00:36:21;16	that we never would've uncovered just by looking at
00:36:23;23	the extended phenotype along. And so these are other traits
00:36:27;29	that we're now trying to dissect genetically, just in the
00:36:31;01	same way that we've been looking at burrow architecture.
00:36:33;15	So what I hope I've done today is to convince you that this is
00:36:37;21	a very exciting time in the field of biology, in the sense that we're
00:36:42;01	now at a point where we can make these links between environment,
00:36:46;07	behavior, and genotype. Understanding how those behaviors
00:36:49;29	evolved in the wild, and also the genes, and how those genes
00:36:54;03	work through neurobiology to produce variation in the behavior
00:36:58;09	that's important for fitness. Before I end, I want to acknowledge
00:37:02;22	the people that did the work. As I've hinted at, Jesse Weber, a
00:37:06;21	former graduate student in the lab, worked on the genetic
00:37:09;13	basis and also did some studies on cooperation. Wenfei Tong
00:37:13;24	was a collaborator on the cooperation study. Brant Peterson
00:37:17;11	has been working on the genetics and the mouse behavior.
00:37:19;28	And a graduate student, HIllery Metz, has been working on the
00:37:23;03	genetics and starting to work on the neurobiology of this
00:37:25;21	behavior. But I would say everybody in our lab group
00:37:28;16	shown here, at one point or another, has gotten their
00:37:31;14	hands dirty, quite literally, by helping us dig out burrow casts.
00:37:36;02	We've also been lucky to have an amazing team of undergraduates
00:37:40;00	that have helped with this project, as well as various funding
00:37:42;29	sources. So thanks again for your attention, and thanks for
00:37:48;04	joining me today.