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Session 3: Evidence of Evolution

Transcript of Part 3: Genetics of Morphology

00:00:00.04	Hi, my name is Hopi Hoekstra and I'm a professor at Harvard
00:00:03.10	University. And in this second segment, what I would like to do is
00:00:06.15	tell you a story about the genetic basis of evolutionary
00:00:09.22	change. And in particular, we're going to focus on the story
00:00:12.06	of a morphological trait. I'm going to tell you this story in the context
00:00:17.15	of making links between environment, phenotype, and
00:00:20.08	genotype. In particular, I'm going to tell you today a three-part story. The first
00:00:24.18	part of the story, I'm going to tell you about a role for natural
00:00:28.09	selection in driving differences and traits in a natural population.
00:00:31.14	And then I'm going to take you from the field into the lab,
00:00:34.11	where we've been doing work to understand the genetic basis
00:00:37.06	or the genes that control this phenotypic difference. And it's
00:00:40.12	not just that we want to know the genes, but I'll tell you
00:00:42.07	about how changes in these genes through development
00:00:44.25	actually produce variations in the phenotype. And then once we
00:00:48.02	made all the links, then we'll have a more complete understanding
00:00:50.14	of the process of adaptation. And here's where I think things can get
00:00:53.24	really fun. Because we can go back out into the wild
00:00:56.22	and ask how these traits evolved in natural populations. So let's
00:01:00.10	start today at the level of phenotype. So the phenotype I'm going to
00:01:03.23	tell you about is one that we've been studying in my group for almost
00:01:06.03	a decade. And that is color variation. So why do we study color?
00:01:10.25	Well, color is one of the primary ways in which organisms
00:01:14.11	interact with their environment. And it varies tremendously
00:01:18.04	between species, and it can also vary between species. And in
00:01:22.04	particular, we know at least in some cases, even small changes
00:01:25.03	in color difference can have a huge impact on fitness.
00:01:27.22	The ability of organisms to reproduce and survive in the wild.
00:01:31.03	So here I've just given you a number of examples of how
00:01:34.21	color is used. It can be used, for example, in terms of reproduction
00:01:38.21	in that it can be used for mate choice. So we have the canonical example of
00:01:41.29	the peacock's tail. But flowers also use color to attract pollinators
00:01:46.26	which is the way that they reproduce. Color can also be
00:01:49.20	used not to attract other species or other individuals,
00:01:52.18	but it can be a way to warn them of let's say a distasteful
00:01:56.29	poison. As is the case in these poison frogs.
00:02:00.29	Now, no good biological story comes without having
00:02:04.16	cheaters. So we also have mimics, those species that
00:02:08.14	are themselves not toxic, but mimic those that are toxic.
00:02:13.09	And thereby are also avoided by predators. But by
00:02:17.00	far, color is used most commonly in terms of camouflage or
00:02:21.23	in terms of crypsis. So that's what I'm going to focus on today.
00:02:25.00	In addition to being ecologically relevant, the other reason we
00:02:29.23	study color is because we can rely on nearly a decade's worth of work
00:02:34.22	by geneticists who have been tracking down genes involved
00:02:37.16	in spontaneous mutations that occur in laboratory populations
00:02:40.28	of mice. So here what I've done is I've made a list, which again
00:02:44.04	I don't expect you to read, but just to appreciate that we know a lot
00:02:47.25	of genes. And if you mutate that gene, it's going to have an effect on
00:02:51.08	color. And here are some of the mutant color differences
00:02:55.00	that have spontaneously arose in laboratory colonies of
00:02:57.26	mice. So in some sense, we have a little bit of a headstart because
00:03:00.26	we know a number of what I'll refer to as candidate genes,
00:03:03.29	genes that contribute to color differences in mammals, but may
00:03:08.05	also contribute to natural variation in color. Now unfortunately,
00:03:12.06	laboratory mice or their wild equivalents, while very common
00:03:15.14	in nature, for example, in our houses, vary a lot in laboratory
00:03:19.23	populations. They don't actually vary that much in the wild.
00:03:22.15	So, instead of studying these mice, we've decided to do
00:03:25.17	a study on a closely related group of mice, mice in the genus
00:03:29.20	peromyscus called deer mice, shown here in their slightly
00:03:32.21	flattened form. So this is a common way that mice are kept
00:03:36.25	in museum collections. And it nicely illustrates the variation in
00:03:40.24	color that I'm going to talk about today. So in this first image,
00:03:43.28	you can see that these deer mice vary tremendously in their
00:03:47.00	dorsal coat. They vary from almost completely white to
00:03:50.23	almost completely black. But they also vary not just in the
00:03:54.28	pigments and individual hairs on their dorsal coat, but they
00:03:57.19	also differ in the distribution of pigments across the body.
00:04:00.10	Or variation in color pattern, as shown here. So these candidate
00:04:05.04	genes that I showed you in the previous slide, many of those
00:04:07.23	are implicated in variation in dorsal color. But in fact,
00:04:11.04	we know very little about how patterns are made. So by studying
00:04:14.10	these mice, not only can we take advantage of this vast
00:04:17.00	range of variation in the color, but we also may learn something
00:04:20.02	new about color patterning. So you may have already noticed
00:04:24.09	that these slides were both taken by someone called Sumner back
00:04:28.07	in the 1920's. Well, this is Francis Sumner, shown here.
00:04:32.09	In his field regalia. This is actually the outfit that he would wear
00:04:36.16	when he would go out into the wild and trap mice. He's a
00:04:39.10	classic natural historian, he was associated with initially the
00:04:42.13	University of Michigan natural history museum. And he spent
00:04:46.29	a bulk of his career trying to answer the question of why
00:04:50.10	populations vary so much in nature. And to do this,
00:04:53.25	he would drive around the U.S. and catch mice, and document
00:04:57.20	variation in a number of traits, including color variation.
00:05:02.17	So Francis Sumner's favorite species of deer mouse, and
00:05:05.28	mine as well, is a particular species call peromyscus polionotus.
00:05:09.17	Peromyscus polionotus is often referred to as the old field
00:05:13.19	mouse. And that's because much of its range in the southeastern
00:05:16.27	U.S., as seen here, so Alabama, Georgia, South Carolina,
00:05:20.08	and Northern Florida. It occurs in these old fields, which are
00:05:24.13	really overgrown agricultural fields. Now throughout
00:05:28.13	most of its range, it may look to you like a typical mouse.
00:05:31.13	It's got a dark brown coat, a gray scruffy belly, and a striped
00:05:35.01	tail. But what's particularly interesting to us about these mice
00:05:38.19	is that they've recently invaded these sandbar islands and
00:05:44.15	sandy dune habitats on the Gulf coast of Florida, as well as
00:05:48.03	the Atlantic coast of Florida. So each one of these
00:05:51.16	numbers on this map refers to a different subspecies
00:05:54.13	of what I'll refer to as "beach mice," because the mice actually live
00:05:57.20	on the beach. Now, the first part of the story, I'm going to
00:06:00.29	focus on one of these subspecies. Here number 3.
00:06:04.04	And that is the Santa Rosa Island beach mouse. So let me show you
00:06:07.06	a picture of their habitat. So unlike their mainland counterparts
00:06:12.02	that live in dark loamy soils, these mice live on these beautiful
00:06:15.14	sandy islands off the Gulf coast of Florida. And here's a picture
00:06:20.03	of one of our field sites. So you'll notice that there are two
00:06:24.05	dramatic differences in habitat that these mice occupy.
00:06:26.25	So first, you can tell that the soil, in this case, this granulated
00:06:33.02	sand that's almost like walking on hills of granulated sugar.
00:06:36.07	It's much lighter in color than the dark loamy soils of the mainland
00:06:40.00	subspecies. But in addition, these beach habitats also have
00:06:45.17	much less vegetative cover, so these mice are exposed to
00:06:48.11	really high levels of predation. And I'll tell you more about
00:06:51.05	their predators in a minute. So it may not be surprising then,
00:06:55.01	when we go out and catch mice in these beautiful beaches,
00:06:58.08	the mice look different. So here's a picture of one of those beach
00:07:01.29	mice. And I should mention that this is not to scale.
00:07:04.02	So both of these mice are about the same size and they're about
00:07:06.23	the size of a ping pong ball. But what this slide does serve to illustrate
00:07:10.17	is the dramatic differences in pigmentation. So this particular
00:07:13.25	mouse, you can see is lacking pigment on its nose,on its sides, and if you could see the tail, it's also missing that
00:07:19.17	strong tail stripe. The other thing I want to mention to you
00:07:22.23	about this system is that we know something about the geological
00:07:25.14	age of these islands. They're about 6000 years old, which suggests
00:07:30.12	that the difference in coat color that you see here may have
00:07:33.17	evolved in just a few thousand years. So you may all be
00:07:37.24	thinking that this makes perfect sense, right? That these mice are
00:07:41.00	running around in these beautiful white sand beaches and having
00:07:43.04	a light color coat would make them more camouflaged.
00:07:46.15	And that's a great idea, but we wanted to actually prove that.
00:07:49.23	So as scientists, what we wanted to do is empirically
00:07:53.00	demonstrate that color matters for survival. So actually
00:07:56.07	do an experiment. We wanted to know how much it matters,
00:07:59.03	in other words, we want to estimate the strength of selection.
00:08:01.27	How favorable is it to actually match the background?
00:08:05.17	And then finally, we wanted to know the agent of selection.
00:08:07.25	Who's doing the selection? And in this case, who are the
00:08:10.17	predators? So first I want to tell you about the experiments we did
00:08:15.10	to try to make this link between color variation and the differences
00:08:18.25	in environment that these mice live in. And in particular, implicate a role
00:08:22.09	for natural selection. Now if I could do any experiment
00:08:25.13	in the world, here's what I'd love to do. I'd love to catch
00:08:28.25	let's say 100 light mice and 100 dark mice, maybe give them all
00:08:32.16	a tag. And let's say release half of them, equal numbers of
00:08:36.11	light and dark, in dark habitat. The other half, equal numbers
00:08:40.06	of light and dark, in light habitat. And then come back, let's say a few
00:08:43.21	months later, and see who survived. And I'd have the expectation
00:08:46.22	that mice that are lighter would survive better in light habitat,
00:08:49.11	and those that are darker in dark habitat. Now for a number of reasons,
00:08:53.03	that experiment is quite hard to do. So instead, what we
00:08:56.16	did is what I'd argue is the next best experiment. And in some ways,
00:08:59.17	it may be even better. So here's the experiment that we actually
00:09:02.22	did. Instead of using live mice, we made mice. So here's
00:09:07.05	a picture of my postdoc, Sacha Vignieri, who along with
00:09:10.05	an undergraduate from Harvard, Joanna Larson, made
00:09:13.09	hundreds of plasticine mice. Half of them were painted
00:09:17.13	dark to mimic the mainland mice, and half of them were painted light
00:09:20.18	to mimic the beach mice. Now this experiment in some
00:09:24.28	ways, as I mentioned may even be better than using live
00:09:27.20	mice, because here the only difference in these mice is
00:09:31.04	their color. So they're made from the same mold, they all look the same,
00:09:34.09	they all smell the same. Whereas with live mice, the beach mice
00:09:37.09	and mainland mice may differ in let's say smell, in escape
00:09:39.28	behavior, in activity patterns. So here, the experiment completely
00:09:44.08	focuses on the difference in color and not correlated traits.
00:09:47.05	But the downside of this experiment is would it actually
00:09:50.18	work? Could we actually fool predators into attacking these
00:09:54.00	plasticine models of mice and not live mice?
00:09:56.29	Well, I wouldn't be telling you about this experiment if it didn't actually
00:09:59.26	work. So what Sacha did was she released equal numbers
00:10:03.22	of these light and dark mice in both light and dark habitats, where
00:10:06.20	live mice of the species actually occurred. And then counted
00:10:10.29	predation events. So here's a predation event. So what you're
00:10:13.29	looking at here is a dark mouse that was put out on light
00:10:16.16	soil. And you may notice that it's missing part of its
00:10:20.03	left ear, and it's got a big chunk taken out of its back.
00:10:23.05	And this is a classic predation event, and in fact, not only
00:10:26.26	can we tell it's been attacked, but we can actually say
00:10:30.08	something about who's doing the attacking. Because these marks
00:10:33.02	are consistent with an avian predator. So here's the results of
00:10:38.10	the larger experiment that Sacha did, where she was counting
00:10:41.14	the number of predation events in different habitat types.
00:10:44.23	So, what you're looking at here are the results of this experiment.
00:10:50.08	So let's first focus on this far panel at the light habitat.
00:10:54.17	What you can see is that there's cryptic and what we'll refer
00:10:57.11	to as non-cryptic mice. And these bars indicate the relative level of
00:11:03.01	predation in both of these two types of mice in this particular habitat.
00:11:06.28	And what you can immediately see is the level of predation here in
00:11:10.19	these cryptic mice is much lower, in fact, it's about half
00:11:14.00	of non-cryptic mice. So what this means is that both mice
00:11:17.22	were attacked by predators, but the mismatched mice were attacked
00:11:21.15	about twice as often. Now when we look at the dark habitat
00:11:24.28	we see a very similar pattern, but in reverse. Here we can
00:11:28.17	see the dark mice survived much better, and while still attacked,
00:11:33.22	they were attacked about half as often as the mismatched mice.
00:11:36.24	So what this first thing tells us is that color seems to matter.
00:11:40.10	And in fact it matters a lot. We can take these numbers and sort
00:11:44.16	of translate that into a selection intensity. I'm not going to go
00:11:48.12	into details about this, but let me just say that color matters
00:11:51.17	a great deal for these mice. And in fact, mice that match their
00:11:55.21	habitat have about a 50% increased chance of survival
00:11:59.21	compared to those that are mismatched. And the final thing
00:12:02.27	I want to say, as I mentioned, we can tell in some cases
00:12:05.08	who's doing the predating. And about half the attacks
00:12:09.16	were due to avian predators, and about half of the attacks
00:12:12.14	were due to mammalian predators like coyotes and foxes.
00:12:16.03	So together, what this experiment is telling us is that
00:12:20.06	the differences between color variation are tightly linked
00:12:22.17	to the environment and that it's natural selection that
00:12:26.01	is playing a role in driving these color differences. So now that
00:12:30.18	we've implicated a role for natural selection, the next thing I
00:12:34.00	want to do is to take you from the field into the lab,
00:12:36.15	where I'll tell you about how we're going about identifying
00:12:39.15	the genes that are responsible for these differences in adaptive color
00:12:42.12	variation. So the first thing I want to do is give you a general sense
00:12:47.23	or an overview of the approach that we're taking. And I don't
00:12:50.22	want you to get caught up in the details, but more appreciate
00:12:53.01	this general approach. So what we're able to do is
00:12:57.05	take these mice from the field and bring them into the lab, so
00:13:00.13	we have both dark mice and light mice. And they have
00:13:03.27	differences in their genomes, in their chromosomes, so
00:13:06.24	I'm going to illustrate this by the dark mice having dark chromosomes
00:13:09.27	and the light mouse having light chromosomes. And what we can
00:13:12.29	do is take a dark mouse and a light mouse, one male and one
00:13:15.05	female. Put them in a cage together and they'll actually
00:13:17.11	reproduce. And they'll produce what we refer to as hybrids.
00:13:21.04	Then we take those hybrid individuals, and we breed them
00:13:24.20	together. And then what happens is in this next generation
00:13:27.16	is their genomes get shuffled. So some individuals are going to have
00:13:31.16	different parts of their chromosomes that come from the light parent,
00:13:34.08	and some from the dark parent. So we're effectively shuffling
00:13:38.06	up their genomes. Now in this population, this second generation of hybrids,
00:13:42.13	what we do in all those individuals is we measure their coat
00:13:45.16	color pattern, and then using genetics or molecular biology,
00:13:49.16	we're able to sort of characterize their chromosomes and determine what regions
00:13:53.08	come from each of the parents. And then what we do is
00:13:56.16	-- I'll just simplify and say we do a nice statistical analysis
00:13:59.20	and ask, are there regions of the genome that seem to be
00:14:03.05	correlated with different aspects of different color variation?
00:14:06.20	So for example, in these chromosomes here, do all these mice
00:14:10.08	that have the light allele from this parent in this region
00:14:15.04	of the chromosome, if all those mice have, let's say, light
00:14:18.13	tails. That suggests that a gene controlling tail color
00:14:21.20	may reside in this part of the chromosome. And this is what we
00:14:25.11	refer to as a QTL analysis, or quantitative trait locus analysis.
00:14:29.03	But it's really just this simple statistical association.
00:14:32.10	And then what we do with this statistical association is that
00:14:36.06	we take that region of the chromosome, and we lookfor its homologous region, the same region in the mouse
00:14:43.01	genome, which we have the complete genome sequence.
00:14:45.12	We know where all the genes are. And we look for
00:14:48.02	candidate genes, remember that list of candidate genes
00:14:51.07	that I showed you earlier in the talk, and ask do any of those
00:14:54.04	candidate genes fall within this particular chromosome region.
00:14:57.16	With the ultimate goal of finding a mutation in that gene
00:15:01.04	whether it's in the protein structure of that gene or
00:15:04.10	maybe a mutation that controls the regulation of this gene,
00:15:07.21	that we can then link to the color differences between the parents.
00:15:10.24	So now that I've given you this overview, next what I want to do is walk
00:15:14.02	you through each of the pieces. So as I mentioned the first
00:15:17.02	thing we do is this cross. So we brought mice in from the wild,
00:15:21.15	we brought in a mainland species and one of these beach
00:15:24.09	mouse subspecies. And here they are, shown again in their
00:15:26.28	flattened form. You can see a dark parent, and over here
00:15:29.13	the light parent. And here is that F1 hybrid shown here. And you can
00:15:34.16	already see this F1 hybrid has traits from both of the parents.
00:15:38.26	So for example, it doesn't have a tail stripe like the light parent
00:15:41.29	but it has a fully pigmented face like the dark parent.
00:15:45.01	That suggests both dominant and recessive alleles
00:15:49.01	contribute to this light color adaptive beach mouse
00:15:52.23	phenotype. Then as I mentioned, we take these F1 hybrids and we breed
00:15:56.06	them together, and that's when we get these F2s.
00:15:58.23	And this is where we've now with recombination shuffled
00:16:02.00	up their genomes. So, for example, presumably this mouse
00:16:05.22	has more pigment alleles from the light parent, and the mouse over here
00:16:10.08	has more pigment alleles from the dark parent. But what you can
00:16:13.05	see is that there's a continuum of variation. And what this
00:16:16.11	immediately tells us that this color difference we observe
00:16:19.11	between beach mice and mainland mice is not controlled by
00:16:22.06	a single gene, but in fact is controlled by a handful of genes.
00:16:26.10	And the reason I say a handful and not hundreds is because
00:16:29.04	you can see in this population we get mice that look like
00:16:31.15	the dark parent, and we get mice that look like the light parent.
00:16:34.16	And this suggests there are not hundreds of genes, because it would
00:16:37.26	be a very small chance that we would get all the light
00:16:40.11	-- all hundred of those light alleles in one individual.
00:16:43.06	But instead, probably just a handful. And from this variation,
00:16:46.27	we estimate there are about 3-5 genes. But I'll tell you more about
00:16:50.05	those genes in just a minute. So as I mentioned, we then take
00:16:53.19	the color variation in these mice and we measure them in
00:16:57.01	all these individuals. And then we also genotype them to
00:17:00.08	figure out what regions of the genome come from the light and dark parent.
00:17:03.06	We did this using molecular techniques. And here's the results
00:17:08.10	of that. So what this is is each one of those lines shown
00:17:11.29	here represents a chromosome. And each one of these markers is
00:17:15.14	a difference between the light and the dark parent.
00:17:17.12	So in each one of these markers we can tell whether a particular
00:17:20.08	individual has that region of the genome comes from the dark
00:17:23.05	parent or the light parent. Then what we do is the statistical
00:17:27.05	analysis. And what we found was, there are three regions
00:17:30.22	of the genome, which I've highlighted here, that seem to
00:17:33.27	control color. That is there's genes in these regions of
00:17:36.15	the genome that control different aspects of color patterning.
00:17:39.25	And lucky for us, in each one of these regions, there's one
00:17:44.17	of these candidate genes that I mentioned earlier.
00:17:47.00	Oh, I should also mention that the differences in the size
00:17:50.12	of the arrows reflects the amount of variation that a particular
00:17:53.14	locus explains. So the Agouti locus way over here, explains
00:17:58.12	a larger proportion of variation compared to Corin, which explains
00:18:02.05	the smallest amount. So what this is telling us is that there's
00:18:06.06	3 regions of the genome, and each one of those contains
00:18:08.29	what I'll refer to as a candidate gene. So next what I'd like
00:18:12.13	to do is just tell you about what of these genes. And then I'll
00:18:15.22	summarize and tell you about all three of them at the end.
00:18:17.28	So the gene I'm going to focus on is this gene up here,
00:18:21.07	Mc1r, or the melanocortin 1 receptor. And one of the
00:18:24.26	reasons we focused on this gene is because we actually
00:18:27.13	know a lot about the structure and the function of this gene.
00:18:30.03	So, the melanocortin receptor is a classic g-protein coupled
00:18:35.24	receptor. That is, it's found in the membrane, and it's got
00:18:39.18	extracellular and intracellular regions. Each one of these
00:18:43.22	little circles represents a different amino acid, and what
00:18:46.06	I've done is I've color coded those amino acids that we know
00:18:49.23	when you change that amino acid, it has an effect on
00:18:52.29	color, and on particular species. And those that make
00:18:56.20	-- when you make that change, an individual darker, it's shown
00:18:59.24	in black and those lighter is shown in gray. So you can see
00:19:03.12	summarized over a number of different studies, that
00:19:07.13	there are multiple mutations in this receptor that can affect
00:19:11.06	color. And it can either make an individual lighter or darker.
00:19:14.20	So the first thing to note is that there are many different
00:19:17.17	mutations. The second thing to note is that they're found throughout
00:19:20.28	the receptor. And the third thing to note is that even a single
00:19:23.27	amino acid mutation can have an effect on color. So one change
00:19:27.26	can have a big effect on phenotype. Now, the first thing we did is
00:19:32.29	we sequenced this gene in both the mainland and the beach
00:19:37.24	form, and asked are there mutational differences between
00:19:40.15	the two? And in fact, we found one and it's highlighted
00:19:44.03	here in red. And the mutation is a single nucleotide change that
00:19:48.03	caused the change in amino acid, whereas the mainland species had
00:19:51.20	an arginine at position 65, beach mice had a cysteine.
00:19:56.02	And this is a charged changing mutation, so it actually
00:19:58.21	changed the charge of that amino acid so that it's more likely
00:20:01.06	to have an effect on the structure and function of melanocortin
00:20:04.14	1 receptor. Now the unfortunate thing, though, was that it didn't
00:20:09.03	overlap with any of the other mutations that had been previously
00:20:11.07	characterized in other species. But that's okay because we can do an experiment
00:20:14.18	to test whether this mutational change had an effect on the way
00:20:18.03	that this receptor functions. So first thing I want to do is tell you a little bit
00:20:21.25	about what this receptor does. And then I'll tell you about our
00:20:24.25	experiment. So what I'm showing you here is a melanocyte.
00:20:28.19	Now a melanocyte is a pigmentation producing cell.
00:20:31.07	And in mammals, we produce two types of pigment,
00:20:34.03	a dark brown to black eumelanin, and a yellow to blonde
00:20:37.19	pheomelanin. So you can look at your own hair and
00:20:39.12	determine whether you have eumelanin or you have pheomelanin.
00:20:42.25	Now, this melanocyte has the ability to produce both types
00:20:47.21	of pigments. But which pigment it produces at any one time
00:20:50.14	is largely controlled by the melanocortin-1 receptor.
00:20:54.05	Which essentially acts like a switch. When it's turned on,
00:20:57.11	that is when let's say alpha-MSH, which activates Mc1r,
00:21:02.03	is around, Mc1r turns on and it signals by increasing intracellular
00:21:06.11	cyclic AMP levels, and you get the production of dark pigment.
00:21:10.00	By contrast, when Mc1r is turned off, then you get the production
00:21:15.15	of less cyclic AMP intracellularly, and the result is light
00:21:19.21	pigment. So Mc1r is very much a switch that determines which pigment is
00:21:24.27	produced. Now this leads to a nice prediction. So in beach mice we have
00:21:29.03	a mutation in Mc1r that we think leads to light coloration.
00:21:32.14	So our prediction is this, is that mutation of arginine to cysteine
00:21:36.18	change at position 65 will reduce receptor activity, which will result
00:21:41.13	in lighter pigmentation. So we can test this doing an experiment.
00:21:45.02	And so what we did is we took both the light allele and the dark
00:21:49.03	allele, that remember, differs by one nucleotide change,
00:21:51.26	we cloned it into an expression vector, we put it into
00:21:55.03	cells. And then we added alpha-MSH in increasing amounts.
00:22:01.14	To activate Mc1r, and then as a proxy for activity, we measured
00:22:05.13	cyclic AMP. So here are the results of this experiment.
00:22:09.17	So here what we did is you can see we added increasing
00:22:14.16	amounts of alpha-MSH, which turned on Mc1r, and you can
00:22:18.21	see that its signaling higher and higher cyclic AMP
00:22:21.22	until it sort of plateaus out. And this is a normal sigmoidal response curve
00:22:26.09	that we see in the mainland mouse. Now when we did the same
00:22:30.02	experiment with the beach mouse allele that differs by again
00:22:32.22	that one nucleotide change, what you can see is there's a dramatically different
00:22:36.14	pattern of activity. And that is no matter how much alpha-MSH
00:22:40.28	we added, there's still a relatively low level of receptor activity.
00:22:44.21	So what this suggests is that one nucleotide change changes the
00:22:48.26	function of the receptor, and it's in the direction we expect.
00:22:53.04	That it has lower activity, which is consistent with producing
00:22:55.21	lighter pigmentation. So at this point, it's worthwhile stepping
00:22:59.07	back and sort of thinking about what I've just showed you. So here I've
00:23:02.22	showed you a single nucleotide change affects the activity of the receptor,
00:23:07.04	we know this receptor affects color variation, and we know
00:23:11.08	that color variation affects survival in the wild. So what we
00:23:14.13	have done here is made a link between a single nucleotide
00:23:17.14	change and fitness or survival in the wild. But I don't want
00:23:22.24	to leave you with the impression that this is the whole story, because
00:23:26.28	as I mentioned, color difference is controlled not by a single
00:23:29.03	gene, but by multiple genes. And so the two other genes,
00:23:33.23	if you are paying attention you might have noticed, actually
00:23:37.02	interact with Mc1r. So Agouti, for example, represses Mc1r
00:23:42.02	activity. And what we see in beach mice, is that higher
00:23:45.26	levels of Agouti expression are associated with lighter pigmentation.
00:23:49.07	And so the allele in beach mice has increased expression
00:23:52.13	of Agouti, which leads to lower activity of Mc1r and light pigment.
00:23:57.02	Corin is the third gene, and while this gene is just newly
00:24:02.04	discovered in the last few years. We don't know its exact function
00:24:05.14	but we know it interacts with Agouti, and again, increased
00:24:09.05	expression of Corin is associated with this light pigmentation
00:24:11.26	in beach mice. So these genes are interacting together
00:24:15.11	to produce light pigmentation. So what we've done in this second part
00:24:20.28	is to tell you about the genes that affect coloration and a little
00:24:25.08	bit about how changes in those genes actually cause
00:24:28.09	these differences in color patterning. And now that we have a much
00:24:32.20	more complete picture of adaptation, here's where I think things can get
00:24:36.08	really fun. So I'm going to take you now back from the lab into the
00:24:39.16	wild. And we're going to talk about how these traits may have
00:24:42.05	evolved in natural populations of beach mice. So to do this, this
00:24:46.14	involves us actually going back to the field. So here's a picture
00:24:49.15	of me and my postdoc, Vera Domingues, when we're out
00:24:53.04	on the Atlantic coast catching mice. Here's a mouse
00:24:57.07	currently being weighed. So we hang it by its tail on a little
00:25:00.09	pesola. We take measurements including their weight,
00:25:03.15	the size of their ears and feet, et cetera. And what Vera's doing here
00:25:07.20	is measuring their coat color. And what's nice about this
00:25:11.23	work in the wild is we take these measurements, we measure their
00:25:14.21	coat color, we also give them a little ear tag, and we take a little
00:25:18.05	snippet of DNA from their tail, and we release them back
00:25:21.19	in the wild. So here we have a DNA sample from each of
00:25:24.12	these mice and we have a record of their coloration.
00:25:26.24	So next what I'd like to do is tell you about what we've learned
00:25:30.02	about natural populations of these mice and how these color
00:25:33.15	differences may have evolved. So just by way of reminder,
00:25:36.29	so far what I've done is I've focused on only one of these
00:25:40.22	populations, that is population number 3, the Santa Rosa
00:25:42.29	Island beach mouse. But next what I'd like to do is tell you about
00:25:46.02	variation among these subspecies. So the five subspecies
00:25:49.26	on the Gulf coast and the three subspecies on the Atlantic coast.
00:25:53.13	So, when we go out and catch these mice and record their
00:25:58.12	color differences. We find some very striking patterns, which I'm going to
00:26:02.19	show you in the next slide. So here what I'm showing you
00:26:05.18	are cartoons that represent the different subspecies of
00:26:10.12	beach mice. So each one of these cartoons shows you the typical
00:26:14.09	color of a beach mouse from each of these populations, compared
00:26:17.07	here to a mainland mouse. So the first thing you may notice
00:26:21.19	is that all the beach mice are much lighter in color
00:26:26.00	compared to the mainland mouse. But that each of these
00:26:28.07	subspecies differs in their color pattern, and in fact,
00:26:31.23	they're so distinct that if you went for let's say spring break
00:26:35.06	down to the Gulf coast of Florida and you brought me back a
00:26:38.13	beach mouse, I would say with about 95% certainty
00:26:42.03	just by looking at the color of the beach mouse, I could tell you
00:26:44.16	what subspecies it is. But, instead if you went to Florida and you
00:26:49.09	didn't tell me if you went to the Atlantic coast or the Gulf
00:26:51.28	coast, and you just brought me back a beach mouse, I'd probably
00:26:55.08	have a 50/50 chance of knowing what subspecies it was
00:26:58.17	and that's because the mice on the Atlantic coast are very similar
00:27:02.09	to mice on the Gulf coast. So let me highlight that here.
00:27:05.26	So for example, these two subspecies, even though they're
00:27:08.29	separated by over 300 kilometers, are very similar
00:27:12.18	in their overall color pattern. And in fact, I can't tell them
00:27:16.10	apart. Likewise, these mice are very similar. And these mice
00:27:20.26	are very similar. So what this suggests is that on the Atlantic
00:27:26.02	coast and Gulf coast, the mice have convergent color patterns.
00:27:31.08	And so what we wanted to do first was ask the question,
00:27:34.16	did these mice evolve these similar color differences independently?
00:27:39.14	And if so, did they use the same genes? So the first
00:27:44.02	thing I want to show you is a tree, or a topology that shows
00:27:48.24	you the relationships among these different subspecies.
00:27:52.00	So this is a simplified version of a tree that we generated
00:27:54.28	using molecular data, but it highlights the relationships
00:27:58.12	among these subspecies within peromyscus polionotus.
00:28:02.07	And what you can see is the Gulf coast beach mice, shown here,
00:28:06.08	all five of those subspecies cluster together. They're very
00:28:09.09	closely related. But they're actually not that closely related
00:28:12.16	to Atlantic coast beach mice, shown here. In other words,
00:28:17.11	it looks like light coloration has evolved independently
00:28:20.02	on two coasts. So the Gulf coast beach mice probably
00:28:23.07	arose from a dark colored ancestor sometime in the past,
00:28:26.22	that was probably from the Panhandle of Florida. Whereas,
00:28:29.23	the Atlantic coast beach mice independently evolved light
00:28:32.20	coloration, probably from an ancestor in Central Florida
00:28:35.26	that was dark in color. Okay, but here's the cool part,
00:28:40.04	now that we now at least in one of these Gulf coast subspecies
00:28:43.07	that the melanocortin-1 receptor is involved, we can
00:28:45.22	ask in these independently evolved light colored beach mice
00:28:48.22	on the Atlantic coast, is that same gene and same mutation
00:28:51.15	involved? So to do this, we returned back to this melanocortin-1 gene.
00:28:56.21	And we sequenced the DNA in the mice that we collected
00:28:59.19	on the Atlantic coast and asked, do we find that same
00:29:02.24	arginine to cysteine change at position 65? So we simply
00:29:07.28	genotyped that one particular site and asked, is it present in the
00:29:12.00	Atlantic coast? Now, despite the fact that mice from the Gulf
00:29:15.09	coast and the Atlantic coast are so similar in coat color,
00:29:18.06	let me just say that we never found that cysteine change in any of the
00:29:22.09	mice from the Atlantic coast. So what that tells us is that
00:29:26.01	the same mutation isn't involved in that convergently evolved
00:29:29.20	light coloration on the Atlantic coast. So you may be thinking,
00:29:32.13	well, it's not the same mutation, but maybe it's a new mutation.
00:29:36.09	Remember there's lots of mutations in Mc1r that can cause
00:29:39.08	color differences, I told you this earlier. So maybe a new mutation
00:29:42.12	in Mc1r is causing light coloration on the Atlantic coast. So
00:29:45.23	it may not be the same mutation, but it could be the same gene.
00:29:48.18	So we went back to the Atlantic coast mice and sequenced
00:29:50.20	the entire melanocortin-1 receptor, and asked are there any
00:29:54.08	new mutations that are correlated with color. Well, in fact,
00:29:57.08	we found four new mutations in the melanocortin-1 receptor.
00:30:01.02	But none of them were perfectly correlated with color, and
00:30:04.11	when we did those pharmacological assays like the ones
00:30:07.04	I showed you earlier, none of them had an effect on the
00:30:11.17	activity of Mc1r. So what this tells us is that it's not just
00:30:16.12	the same mutation, it's also not the same gene that's
00:30:19.11	responsible for the convergent evolution of light coloration in the
00:30:23.10	Atlantic coast mice. So this is the case for melanocortin-1 receptor,
00:30:26.25	but we're still checking these populations for changes in Agouti and Corin.
00:30:31.27	So what I've done today is I've told you a story about
00:30:37.00	how identifying not only the ultimate causes of phenotypic
00:30:41.12	variation, but also the genetic causes can tell us something about how
00:30:44.13	traits evolved in the wild. And this story revolved around a single
00:30:47.24	species, in which different mutations, at least in regards to
00:30:52.04	the melanocortin-1 receptor, are involved in generating
00:30:55.04	similar coat color patterns on two coasts of Florida.
00:30:57.25	But I want to end by telling you a story about convergent
00:31:01.14	evolution. Because sometimes the same genes and same
00:31:04.13	mutations are involved in similar phenotypes in very different
00:31:08.01	organisms. So, if you think about mammals, think about what
00:31:13.02	is the most different mammal from a mouse. Often the answer
00:31:17.03	I get is an elephant, and it's close. I'm going to tell you a story about
00:31:21.11	mammoths. So, mammoths were the subject of intense
00:31:26.17	study, especially about five years ago when there was a big
00:31:30.06	interest in sequencing ancient DNA. And mammoths were
00:31:33.28	a great target because they occurred in Siberia, and
00:31:38.05	their DNA was essentially frozen in permafrost about 14,000
00:31:43.03	years ago. So this is almost like keeping DNA in a giant freezer,
00:31:46.15	which is the best conditions possible. So about five years ago,
00:31:50.07	the goal was to sequence an entire gene from an extinct
00:31:53.25	organism. Now today of course, we're sequencing entire
00:31:57.03	genomes of extinct organisms, but just five years ago, we
00:32:01.00	wanted to sequence an entire gene from the nuclear genome.
00:32:04.29	So this is what a group in Germany, in Leipzig, headed by
00:32:10.12	Michael Hofreiter did. And the gene they chose to study
00:32:14.00	was the melanocortin-1 receptor, because it's a very
00:32:17.13	simple gene. Remember I told you, we know a lot about its
00:32:20.27	structure function, it's only about 1,000 base pairs in length,
00:32:23.05	and there's no introns. So it's very simple and it's a great starting
00:32:26.05	point. So they sequenced the melanocortin-1 receptor,
00:32:29.10	in DNA extracted from mammoths. And here's what they found.
00:32:35.14	They found a mutation, and what mutation was it?
00:32:39.01	Well, it was an arginine to cysteine change at the exact
00:32:41.19	same position that we found in beach mice. Now of course
00:32:45.15	the DNA it was extracted from was bone, so they didn't know
00:32:48.18	the phenotype or the coat color of mammoths, but based on our
00:32:52.06	work in beach mice, what this suggests is that mammoths
00:32:55.01	like beach mice, may have been polymorphic in color. Now the
00:32:59.07	question of why they were polymorphic in color, we don't know the
00:33:01.26	answer to. This could be due to survival differences, other
00:33:06.25	suggested that it's due to sexual selection. So when this
00:33:10.07	work was published, the press line was that blonde mammoths have
00:33:14.15	more fun. But I'm not going to give you any explanation, I'll leave that
00:33:19.08	up to your imagination. So in this case, we have very
00:33:22.21	divergent organisms, mice and mammoths that use
00:33:26.03	not only the same gene, but the same mutation.
00:33:29.12	And in fact, as I alluded to earlier, melanocortin-1 receptor
00:33:33.06	is involved in a number of different color variants.
00:33:36.05	And a number of organisms that you may be familiar
00:33:38.24	with. So in this case, it was the same mutation, but we know
00:33:42.25	that other changes in the melanocortin receptor can also
00:33:46.21	cause differences in color through different mutations. So I just wanted to
00:33:51.02	give you some examples. So some work that I've been doing with a colleague
00:33:54.12	at UC Berkeley, Erica Rosenblum, shows that changes in the
00:33:57.26	melanocortin-1 receptor is responsible for the production of these
00:34:00.22	very adorable lizards that differ in color, when they occur
00:34:05.14	in White Sands, New Mexico. So you can guess which
00:34:09.02	lizard occurs on white sands and which one occurs off white sands.
00:34:13.06	Color differences are also involved in animals you may see
00:34:17.09	every day, including cows. Also, many of you may know
00:34:22.12	of or even have a labrador dog. Well, they come in
00:34:26.21	generally two colors, there's the black labs and blonde labs,
00:34:30.12	again this is caused by a change in the melanocortin-1 receptor. And much more recently, again work out of Germany,
00:34:36.23	this time Svante Paabo's group, has shown that color
00:34:41.06	changes in the melanocortin receptor, in addition to being
00:34:45.20	responsible for human color differences, may also be
00:34:48.29	responsible for color differences or hair differences in neanderthals.
00:34:53.03	So they showed that changes in the melanocortin-1 receptor
00:34:56.02	or variants in the melanocortin-1 receptor were found in
00:34:59.24	neanderthals, suggesting even back in those days,
00:35:05.03	there could have been redheads as well. So what I hope to have done
00:35:09.07	today is to suggest to you that by making the link between
00:35:13.23	environment, phenotype -- in this case a morphological trait,
00:35:17.22	and genotype, we're able to say something about
00:35:21.05	how organisms evolve in the wild. The work that I presented
00:35:25.12	today was done by a large number of people. These are some
00:35:30.22	of the people in my lab group that contributed to the work,
00:35:34.05	shown here looking their mousiest. And in particular,
00:35:38.00	the work I talked about today was done by folks like
00:35:41.21	Cynthia Steiner, Marie Manceau, Vera Domingues, Sacha
00:35:44.23	Vignieri, Holger Rompler, and Lynne Mullen, as well as
00:35:47.29	an undergraduate, Joanna Larson. And we were funded by
00:35:52.03	a number of sources shown here, as well. And with that,
00:35:55.10	thank you for your attention. I hope you enjoyed this segment,
00:35:58.03	and you'll stick around for segment three. Thank you very much.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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