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.