Last week, Joel talked a lot about evolutionary trees and showed you a lot and showed you some tips on, how evolutionary trees are made. What I'd like to do right now, is demonstrate two free software packages that you can use, for different purposes in looking at and teaching evolutionary trees. is going to be is in morphological characters so, so physical characters of organisms and we'll, I'll show you how to make a matrix of those characters and code, for the presence or absence of those features. And then use a software called Mesquite to allow you to manipulate the tree and come up with best tree, we'll talk a little about parsimony and what that means. And then after we do that, we're going to look at, how to make trees from DNA sequences. So a DNA, each base in the DNA chain is basically one character, but you have to do you can't build this tree the same way, as looking at a matrix of characters because you may have a thousand or more characters. And so, we're going to use a software called SeaView, and I'll show you how to download some genetic sequences align them, and then build the tree, using SeaView. So, I've already opened up Mesquite and built a character matrix, there's a protocol for you in the classroom resources section, and if you're following along, I'm already on page two, part b, where it says your tree. So I've put together a character matrix, using only 5 taxa, five organisms, and four characters and you know, these characters may seem a little obscure to you. Mammals are often classified based on their teeth, and I got this, from one of our scientists here for another project that we worked on. So, I want to set up a possible tree that I can use to examine, how these characters might help us look at the evolutionary relationships of these organisms. So I'm going to go to the Taxa and Trees, this is all if it goes too fast, don't worry, its in the protocol. Make new trees and use the default trees, to set up some trees that I can play with. So I'll click yes here and, and so here's a tree. The organisms right now, the way they're listed are in the same order, that they were in my matrix. So this doesn't really tell me anything right now, about the evolutionary relationships of these organisms, based on those characters. I have some different trees I can choose from, you know I'll just stick with the first one, just for demonstration purposes here. So I need a way to evaluate the relationship based on the characters in my matrix, so I'm going to go to analysis and look at values for the current tree. And then tree length is basically, the fancy way of, of looking at the changes, and what I get is a little window that tells me my tree length. So basically, this tree, this five tells me, that somewhere in my tree, there's five changes in characters that, that resulted in this tree. And what, the nice thing about this program is, I can take the mule deer and move them to a different part of the tree and see how that changes the tree length. So, by moving the mule deer over, you know, next to the mountain lion, I got a tree length of four. So this tells me that this is a better tree, so there's less evolutionary changes that had to occur, you know, in order for the computer program to draw this tree. So what we want to do, we want to find the tree, we want to hunt around and try to find a tree that gives us the best representation, of what may have been the evolutionary relationships. So I chose mule there for a reason. I, I know that the black bear, the mountain lion, the weasel and the wolf, are all carnivores. So I'm going to take the, the deer and I'm going to move it all the way to the root of my tree, and I'm going to call that my outgroup. Alright, so I've now got the, the mule there on the outside. So I need a little bit more information. So I'm going to go back up to the analysis window, and open up the character history bar and I'm going to use the stored characters and, there's different ways to look at it, we're just going to stay simple here, and use parsimony, which is looking for the simplest tree with the fewest number of evolutionary changes. and the nice thing about this is, now I get a character chart down here, that allows me to see where different characters changes. So, for character one I just have a change here and a change here. And so what I want to try to do, is figure out a place where that only changes once. So I'm going to move the weasel over here, with the black bear, and for character one now, I only have one place on the tree, where this evolutionary change had to occur. And my tree length is, is still at four though, so I wonder if there's a simpler way. So I'm going to look at the other characters. So for number two, I have some kind of crazy stuff going on here too, so I'm going to there's obviously there's, there's different changes that had to happen here, so I'm going to try to simplify it, so I can get a place where and move that down there so that now, you know, this still looks like there's two characters going on here. Maybe if I put it over here, I can get a better tree. So now, I actually get one place on this tree, where that character two changed. And now I've got my tree down to three evolutionary changes. For Character three, have the same thing. Character four is something that they all had, so really doesn't tell me much about it. So it looks like the simplest tree that we can make using this small matrix, is a chart that has three evolutionary changes. Shows that the black bear and the weasel have a common ancestor, right about here. The wolf has a common ancestor back here, and then the mountain lion has a common ancestor here. And then the mule deer has a common ancestor to all of these back here, and this is actually what a lot of scientific evidence shows us. As I said before, building phyogenetic trees with DNA sequences, is a little bit different because you have a longer sequence. the last tree we looked at, we only had a few characters and a few taxa or organisms to look at. in, in using DNA sequences we may, we need to be able to compare thousands of base pairs at a time. Sometimes the mathematical algorithms are very complicated. There's, you know, quite a bit of science that just goes into the computational problems that are involved in building these trees. We don't have to worry about that. all we need to do, is figure out how to download some sequences, format them and, stick em into a program that will, will spit out a tree for us. And so, here's how that's going to work. So, now we're going to build a tree, using genetic sequences. There's a protocol, in the classroom resources section, for, for using the SeaView, which is a free program, it's one of many different programs that you can use, to build phylogenetic trees. One of the things that's really important in building trees using genetic sequences, is to make sure that you're choosing the same gene for all your organisms, and the choice of your organisms, is really up to you. It's best to have a hypothesis. You may want to look at the relationship between carnivores. You may the tree that we're going to look at is, is a tree that, is going to investigate the relationship between primates. so, I need to, put, first put together a, a set, file with, my sequences in it, so I can input it into Seaview. the place you want to go to, is GenBank, which is a, a fantastic database of genetic sequences. There's literally thousands and thousands of, of genetic sequences stored in many different forms. we're going to use the gene search here, and I'm going to search for one type of primate which is a human, and I'm going to use for all my sequences, I'm going to use the cytochrome oxidase, one gene, so the, the abbreviation for that is Cox1. It's being used quite commonly in what's called DNA barcoding. So trying to, to find the, DNA signature that helps identify all different organisms. So there's a lot of cytochrome oxidase sequences in GenBank. So I'm just going to click search and I come with a couple different humans here. This one actually isn't the one I was thinking I'd find, but we have homosapiens neanderthalensis, so it's an extinct form of human. I'm going to click on this and open up the sequence information for homo sapiens, which is the species I'm interested in, and you get a lot of information here. so, based on the protocol, you're going to go to the screen button. So, I'm going to right click on the green sequence and go to views and tools. I want to make a fasta file, so I'm going to look for the fasta format down here and I'll click that. And I should get a bunch of As, Cs, Ts, and Gs, which I do. So what you'll need to do in order to make a file, you need one document that has all the files that you want to look at, in it. So you'll just cut and paste this, the information here, you're going to strip most of that out. The carrot has to stay, all this information is in the protocol as well, and we're going to name this set of As, Cs Ts and G's, according to what you want to appear on your tree. So, you'll cut and paste and, and you, you'll use a text editor, not dot word, like Microsoft Word because that adds a lot of characters into the formatting that, that will interfere with the phylogenetic program. so, when you're finished, you'll get something like this. So this is my, my sequence of primate genes. So I have my human cockswine. You'll see how I've changed the title right here, I've kept the carrot. There's a return right after this. If I put a space here, then the cockswine's not going to show up on my tree. If I wanted that to show up, I'd have to put a little underscore there, right. So you have your carrot, your name, one return right here and then another return down here. And then we'll, we'll keep going and so I've got, Bonobo chimpanzees the, the other chimpanzee, I've got gorilla on my tree, a green monkey, a capuchin monkey, a lemur, so I've got a more primitive primate, a baboon. And then I want some out groups, I want some things that aren't, primates to help me organize my tree. So, I've got a horse here, which is another mammal that's not in the primate group, and then just to make sure, I've included a crocodile, which is, you know outside the mammal group. So now that I have my file set, I'm going fast to file set, I'm going to open SeaView. And, works very simply. Where it says alignment file drag zone, you're just going to drag your file in, and let it go, and, and I see all my sequences and they're color coded according to which base they are. so there's one more set, before I can make a tree here, because I want to make sure that I'm comparing the right parts of the DNA sequence, to each other, so, apples to apples. So, I'm going to go to alignment and I'm going to make sure it's on Clustal for now. MUSCLE's a different alignment program, Clustal seems to work better for my computer, here. and I'm just going to click align all and take a few seconds, Great. So now that its aligned, I'm going to build a tree. So I'm going to use parsimony, distance method is another thing you want, you might want to play around with. Basically the length of the tree is going to show you the difference between the As and Cs and Ts and Gs, but since we've been talking about parsimony, I'm just going to go with that. Program's going to do, exactly what you did with Mesquite, only each one of these is a character, and so we have, if we look all the way to the end here, we have over 1500 characters, we're now comparing. So that would be pretty hard to do in Mesquite, so now I'm going to go to tree, use parsimony, and I'm going to build a tree. And here's what I come up with. So, this may or may not look like something, that makes any sense to you. We have our human, you know, with relationship right here, to the chimpanzee group, which seems to make sense, the gorillas here, the crocodiles down here with the horse, so this is a little hard to read. The relationship seems to be right, but we want, we're used to seeing things with a specific root, and we included the crocodile for a reason, so that we could root our tree. So what I'm going to is I'm going to go to the re-root button up here and I'm going to make the crocodile my out group, so the crocodile's going to be at the base of my tree. And when I do this, it turns into something that makes sense. If you can imagine the trees that you saw before, that there's a root coming out, going to the left over in this part, so it makes more sense. So we have our crocodile as our out group, the horse, being a mammal, shares a common ancestor with everything here, which makes sense. The lemur, as a primative primate, makes sense over here. If we see who's related the humans, it's the chmipanzee and the bonobo, which makes sense. We know that they're our closest living relative and the gorilla is next, so that makes sense. And we know that bonobo and the chimpanzee are each others closest living relative, and so they're on the same branch of the tree, in the same plate if you will. So this tree seems to make sense and gives a good depiction of, you know, what we know about evolutionary relationships. So there's a couple of other things you can do, and it's why I like this set of software for teaching. If we want to flip nodes around, so as Joel said, you can rearrange the tree, but it still shows the same evolutionary relationship. So I can, you know, if I want to flip the horse down to the bottom, I can do that, I want to put the lemur above that. So, I can keep changing the nodes, until I get a tree that looks something like this. It's actually why I like this tree because you know, if you looked at just this, you'd say, oh, humans are, are you know, this elite branch on the top of the tree. But by moving just a couple branches around, as Joel mentioned, we can see that the humans are just another species on a tip, somewhere on the tree of life. The issues that go into these two pieces of software, are really key to getting your kids to understand, how trees are built, and what trees show you. So a tree basically is a hypothesis for an evolutionary past, and you know, the evidences, the characters, whether it's DNA or whether it's morphological characters, and so Having a good understanding of what a tree actually shows, is really important. So as you're, you're doing this with your students you can allow them to do a little bit of trial and error, but it's really key that the characters that are selected, eventually, actually show a true evolutionary relationship. Things like color often don't do that. As you saw in Mesquite, we're able to move the branches of the tree around, so this really gets to the idea of parsimony. So, where did each of those changes happen along the tree, and how can we arrange the tree, so that we minimize the number of changes? that may or may not be the true evolutionary path, but you know, it's the, the simplest explanation for what we see in nature. And then for DNA sequences, there's a you know, a lot of information in GenBank out there. make sure you and your students are choosing the same gene to do the comparisons. And finally, it's very important that your students explain their results. So, you know, when they get a tree, does it look like a, a, real depiction of what's going on? Do they accept the evidence that, that's presenting? or, you know, do they think that there may be another, you know, way that they need to, attempt to try things, so that they get a better result. And so I urge you, to have your students come up with hypotheses of how organisms are related, use GenBank, use SeaView, or any of the other you know, software that's out, that's out there that, that will help you build trees and have them investigate and do their own science.
