Phylogenetic Analysis STUDENT 2021

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EXERCISE #7 PHYLOGENETICS – DATA ANALYSIS AND TREE BUILDING WHERE TO MEET Lab (SB 303) ITEMS NEEDED Computer Mesquite software downloaded KEY CONCEPTS Construction and interpretation of phylogenetic trees, phylogenetic trees as hypotheses, character support of a phylogenetic tree TEXTBOOK REFERENCES Chapter 20 – Phylogeny (pages 383-392) LITERATURE REFERENCES Maddison, W. P. and D.R. Maddison. 2014. Mesquite: a modular system for evolutionary analysis. Version 3.0 http://mesquiteproject.org . McCabe, D. 2014. Competitive phylogenetics: a laboratory exercise. The American Biology Teacher: 76:127-131. Meredith, et al. 2011. Impacts of the Cretaceous terrestrial revolution and KPg extinction on mammal diversification. Science 334:521-524 TERMS cladistics, parsimony, homology, homoplasy, morphological characters, outgroup, branch, root, taxon, sister taxa, basal taxon, tree length INTRODUCTION Depiction of the evolutionary process as a tree was a key insight in Darwin’s thinking about descent with modification from common ancestors. In current application, the leading approach to the construction of phylogenetic trees based on common ancestry is cladistics . In cladistics,

the goal is to distinguish shared derived characters to organize taxa. When a character state first appears in the ancestry of a group, it is expected that all species that are derived from the ancestor with the character will share it due to homology . In this way, species are grouped at finer and finer scales based on the origin of derived characters through time. Those species that share the derived character are grouped together. One of the challenges in constructing a phylogenetic tree is determining the pattern of shared derived characters that has led to the relationships among species. Parsimony is a method for making decisions about this pattern of character evolution for a particular tree. Under parsimony, a tree that represents the fewest number of character changes (origins of derived traits) is considered the most likely evolutionary scenario since it is expected that most characters will not change. OBJECTIVES In this exercise you will use the character matrix that was compiled for mammal species in an earlier lab to construct phylogenies that use parsimony and identify an outgroup taxon. The characters that best support your phylogeny will be reviewed and your most parsimonious phylogeny will be compared to the current phylogenetic hypothesis for mammals. METHODS The program that you will use to make your tree is entitled Mesquite (v. 3.7). It is a powerful, open source program that can be run on a range of platforms (Windows PC, Mac, UNIX/LINUX) and can be downloaded from http://mesquiteproject.org/ . Read the information on this website and follow the links relevant to your computer and operating system. You will end up with a folder of files used to run Mesquite, which you may have to unzip before using. NOTE: If you are having trouble getting Mesquite to run, refer to the file “Mesquite_Starter_INSTRUCTIONS.pdf”, which should be in the folder you download. 1. Download the file “Mammal_Morph_Matrix.nex” from Brightspace 2. After launching the Mesquite software, select File  Open File… 3. Navigate to the location where you downloaded the file from Brightspace. The .nex file extension is important for the file to be read and it should appear as shown in Fig. 1.

Figure 1. View of character matrix and key features of the program. 4. As you work through the procedures below, your results will appear as additional tabs that are collectively called a Project. Make sure you save your work as you go along. The filename can be the same as the one you started with. Generate a tree We’ll begin our analysis by making a generic tree and then measuring tree length. Tree length provides a metric of the number of changes in character state that are necessary to explain the relationships in the tree (topology). The generic tree only takes into account the order in which the species appear in the character matrix . Consequently, you might expect the tree length to be high since there is no attempt to organize species relationships using relative differences among character states. 1. Select the menu Taxa & Trees  New tree window  With trees from source. 2. On the window that pops up, select Default Trees and click OK. This will bring up a tree diagram. 3. Measure the number of character changes that occur on this tree to explain the data. a. Select the menu Analysis:Tree  Values for Current Tree… b. On the window that pops up, select Tree value using character matrix  Treelength and click OK. 4. Note the tree length for comparison later on and save your work. 5. Save the tree diagram. Select the menu File  Save Tree as PDF. Name the file and save it in a place you will remember.

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Generate a tree using parsimony The next step is to apply parsimony during tree construction for comparison to the generic tree. With even a moderate number of taxa to consider, the number of possible trees is immense. For example, for 10 species there are over 34 million possible trees! To make this manageable, Mesquite has a tree searching algorithm that we will make use of to limit the computational time. An additional interesting feature of parsimony and other criteria for tree construction is that there can be multiple trees that are equal in the number of character changes. To produce a single tree, one way to condense many equally parsimonious trees is to select the branches that are found in a majority of the trees. Trees produced in this way are by majority rule consensus . 1. Run the parsimony analysis a. From the top menu select the menu Analysis  Tree Inference  Tree Search  Mesquite Heuristic (Add & rearrange). b. On the window that pops up, select Treelength (about half way down the list) and click OK.  NOTE: The option “Treelength” is different from the option “Tree value using character matrix  Treelength” c. On the next window that pops up, select SPR Rearranger and click OK. d. Select MAXTREES to 100 and click OK. e. Next, choose the option “NOT separate thread” and click OK. 2. You now have up to 100 trees that are equally parsimonious. On the tab entitled ‘Trees from Mesquite’s heuristic search’, you can click through them using the arrow in the upper left. 3. Create a consensus tree a. Return to the first tree on the ‘Trees from Mesquite’s heuristic search’ tab. b. Select the menu Tree  Store Tree. This will save the tree so it is not erased. c. Next select the menu Taxa&Trees/  Make New Trees Block from  Consensus Tree. d. On the window that pops up select Stored Trees and click OK e. On the next window select Majority-Rule Consensus and click OK f. On the next window, select the following settings, then click OK:  Check yes to consider tree weights  Required frequency of clades = 0.5  Check yes to write group frequency list  Treat trees as rooted or unrooted as specified in the first tree g. Next, choose the option “NOT separate thread” and click OK h. On window ‘Use which tree block’ select Trees from Mesquite’s heuristic search and click OK.  NOTE: This step may not appear – this is alright. i. On window ‘Trees ready’ select Yes 4. Measure the number of character changes that occur on this tree to explain the data. a. Select the menu Analysis:Tree  Values for Current Tree…

b. On the window that pops up, select Tree value using character matrix  Treelength and click OK. 5. Note the tree length and save your work. 6. Save the tree diagram. Select the menu File  Save Tree as PDF. Name the file and save it in a place you will remember. Figure 2. Example of a consensus tree with treelength reported. Designate the outgroup An important goal in constructing a tree under cladistics is to assure that the scoring of the characters stems from an ancestral standard or baseline. In other words, it is helpful to include a species that is known to be related to the other species but outside of the group containing them. We call this species (taxon) the outgroup and it ensures that the rest of the tree will be monophyletic . Here we will use the opossum as the outgroup to see if it impacts the relationships within the tree. The opossum is a good choice because it is a mammal, but it is also a marsupial. Marsupials have very different reproductive biology, skull characteristics, and pelvic anatomy when compared to eutherian (placental) mammals. These data, fossil evidence, and molecular data suggests that the two groups split from a common ancestor over 65 million years ago.

1. Make sure that your Consensus Tree tab is active. 2. On the toolbar to the left of the screen, select the Reroot tool. 3. Find the opossum your consensus tree. When you mouse over the branch it will be highlighted yellow. Click on the branch. 4. Note any changes in tree topology. 5. Save the tree diagram. Select the menu File  Save Tree as PDF. Name the file and save it in a place you will remember. Map characters onto the rooted tree Now that you have a rooted tree, it is possible to see how characters are hypothesized to have evolved by mapping them on the tree. Such mapping can also help investigators understand which characters support the tree. Recall that a fundamental assumption of phylogenetic reconstruction using cladistics is that similarity in character states among species is due to shared ancestry or homology. If this is the case, characters should change rarely and when they do change all species above the change are expected to share the derived character. Another way to think of it is that a change in character state defines an ingroup within the tree. Conversely, characters may be similar due to convergence, reversal to an ancestral state, or loss of a derived character. Such characters are referred to as homoplasies . They have similarity that is not due to common ancestry and therefore do not support the tree. 1. Select the menu Analysis:Tree  Trace Character History. 2. On the window that pops up, select Parsimony Ancestral States and click OK. 3. A box will appear entitled ‘Trace Character’. Grab a corner of it and move it to an empty area below the tree (Fig. 3). 4. Within the box click the arrows show the hypothesized evolution of each character. The colors indicate the character value for each branch on the tree. 5. Save tree diagrams for the mapped characters that your instructor identifies. Select the menu File  Save Tree as PDF. Name the file and save it in a place you will remember. Reroot Tool

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Figure 3. Window showing character states mapped on the rooted tree. In this case a post-orbital bar pointing in the forward direction is considered derived. Does this character support the tree? Is it homologous or homoplasious?

Figures and Data 1. Present a tree based on the order that species are listed in the character matrix. Tree length should be reported. 2. Present a majority consensus tree based on parsimony. Report tree length. 3. Present the majority consensus tree when designating the opossum as the outgroup taxon. 4. Present two majority consensus trees with characters mapped. Your instructor will inform you of which characters to present. Discussion Questions 1. Describe the difference between a tree using parsimony and the default tree that is simply based on species order. Which tree is better? Why? 2. Describe how the inclusion of an outgroup changes the relationships in the tree. 3. Of the two characters that you mapped, which supports the tree and which does not? For the character that does not support the tree, explain how it appears to have evolved. 4. Compare your majority consensus tree with the outgroup to the currently accepted phylogeny that was provided. Are there differences? What can explain those differences?