Basic Tree Building With PAUP

Transcription

1 Phylogenetic Tree Building Objectives 1. Understand the principles of phylogenetic thinking. 2. Be able to develop and test a phylogenetic hypothesis. 3. Be able to interpret a phylogenetic tree. Overview One of the oldest biological activities of humans involves identifying and grouping other living things. The earliest efforts to categorize living things were very simple and tended to identify which plants and animals could be used for food or natural products, as well as which ones were toxic or dangerous and should be avoided. Over time, early human cultures developed folk taxonomies which are simple ways to organize life into different groupings (i.e. fish, dogs, shrubs, fruit trees) based upon general features. While both have high utilitarian value, they do not contain specific information about relationships of species within or among groups. Likewise, these types of artificial classifications have low predictive power regarding many of the traits a species in a group may have. In contrast, scientists now use natural classifications which group organisms into a hierarchical system based upon their characteristics using specific criteria. Rather than focusing on utility, these classifications group organisms in a manner that reflects their overall similarities and differences in many traits. Because of this, natural classifications have higher predictive powers both in terms of what features members of a group will have, as well as into which groups a newly identified species should be placed. Furthermore, natural classifications strive to organize groups in a manner that shows evolutionary relationships among groups and among individual species within groups. This exercise focuses on how biologists collect and analyze data to organize biological diversity and investigate relationships among species. You will work with species from a group of simulated plants called the Dendrogrammaceae (Duncan, Phillips, and Wagner, 1980) or a group of simulated animals called the Caminalcules (Sokal, 1983). Working in pairs, you will look at your group of organisms, choose the characters you think will be informative, collect data (called scoring characters), and, finally analyze those data to produce diagrams that propose relationships among different species. For the analysis you will use a program called PAUP (Phylogenetic Analysis Using Parsimony) and compare two different tree building approaches (UPGMA and parsimony). Further information about PAUP can be found at Note: Two sets of sample taxa are given in the files Dendrogrammace & Caminalcules 1 & 2. Comple collections of hypothetical organisms can be found in the files Dendrogrammaceae set and Caminalcules set.

2 I. GROUPING, CHARACTERS, & SCORING 1. Your T.A. will assign each pair of students to either the Dendrogrammaceae (plants) or Caminalcules (animals) group. Look through the drawings you have been given of plants or animals labeled A - H and the one labeled OUTGROUP. These drawings represent different species in your study. Species A-H will be assumed to be in the same genus, and they will collectively be referred to as the INGROUP. The OUTGROUP species is a member of a closely related genus. It represents the characteristics most likely found in the common ancestor of the ingroup and outgroup lineages. 2. You next need to choose the traits you will use in your analysis. Looking at the pictures of your study group, identify some traits you think will be useful for grouping the species in your analysis. In this type of research, traits you describe are called characters, and the condition of that character in a particular species is called the character state. For example, stem number could be a character you identify for a plant, and one stem or more than one stem would be the character states. For animals, a character could be hands and the character state could be present or absent. The character and its particular character state are essentially the phenotype of a species. 3. Looking at species A-H, how would you organize them into different groups? Which species do you think are most similar or more closely related to one another? Sketch out your preliminary groupings and relationships in the area labeled Figure Discuss the characters you have identified with your partner. Decide on a list of 10 characters that you will use for these species and list them in the column titled characters in Table Assign character states for the 10 characters you identified to the ingroup and outgroup. To conduct the analysis, you need to determine which are the ancestral (primitive) character states, and which are the derived (advanced) character states. Extensive research and familiarity with the group would normally be necessary to determine ancestral and derived character states. For our purposes, we are assuming that this species was chosen as the outgroup because it has character states most similar to those of the common ancestor of the ingroup and outgroup. 6. Next, you need to score the character states in a binary system (0 or 1). Start by describing all of the character states for the outgroup species. Code all of the outgroup traits as 0 to indicate the ancestral character state. Record these codes in Table 2. Next, score and code the character states for each species of the ingroup. Using the character states you recorded in Table 1, score the character state as 0 if it is similar to the outgroup and 1 if it is different.

3 II. Creating a PAUP Data File 1. Now that you have created a data matrix, you need to enter it into PAUP. To do this, Open the folder named PAUP on the laptop computer desktop. 2. Double click the PAUP application icon. PAUP will automatically launch a dialog box. 3. Click on File and select the file named BIOL1134.nex from the folder named sample NEXUS data. To open the file in PAUP s data editor, first change the mode from Execute to Edit in the lower left corner of the dialogue box. Then click Open/Edit. The PAUP nexus file has two blocks of information, which are delimited by the words begin and end. The word following begin defines the block-type. *Note: File names showing text within brackets [ ] should be replaced with your own file name or data. You will need to save these files on a flash drive or similar device. The Input Block Begin by specifying the number of taxa or species types in your data set (ntax= ). It should be 9 in this instance. The next information in the file is the list of taxon names (letters will be fine). Be careful to not erase any of the syntax, such as the semi-colons (;). Also be sure to watch for spelling errors. #NEXUS begin taxa; dimensions ntax=9; taxlabels OG A B C D E F G H ; end; The Data Block Enter your data matrix into this block. For simplicity, edit the sample data file we have provided. Check to confirm that the number of characters in the matrix are the same as what you have scored (dimensions nchar= ). In this case it should be 10. The format symbols should not be changed. The outgroup is indicated by OG and should read 0 across the matrix. You will not need to edit this line. Complete the matrix for the remaining taxa A-H so that it matches your information in Table 2. sample data file begin characters; dimensions nchar=10; format symbols = "01"; matrix OG A B

4 C D E F G H ; end; 4. Save the nexus data file on a flash drive with a new name. This will be one of the two data files you need. Files saved on the computer may be erased so do not use this as your only data storage area. 5. PROOFREAD YOUR DATA MATRIX WHEN FINISHED!!! 6. Repeat steps 1-3 above. This time delete the OG taxon and all scoring for it. Likewise change ntax=8. Save this file with a new name indicating no outgroup in the data file. 7. Close the data file after saving. III. PHENETIC ANALYSIS You will start your analysis using a method called UPGMA (Unweighted Pair Group Method with Arithmetic Mean). The tree generated by this analysis, called a phenogram, indicates overall similarity among the study taxa. Before you start, look at your data and make a prediction about which taxa will be grouped together. Executing your data file for phenetic (UPGMA) analysis 1. From the File menu, choose Open and double click on your file that does not have the outgroup data (the file/open mode should be on Execute this time). 2. After executing the file, PAUP displays comments and some general information about the data. In our case, PAUP provides the source, dimensions of the data matrix (# of taxa, # of characters), and character coding (binary: 1 s and 0 s). 3. To generate a UPGMA tree, type the following commands into the box with the blinking cursor at the bottom of the screen. Type: UPGMA Click on Execute 4. This will generate a UPGMA tee showing overall similarity among the different taxa. 5. Draw this tree in the area labeled Figure Mark the character state changes in the UPGMA tree. IV. CLADISTIC ANALYSIS Next, you will conduct a cladistic analysis of the species. This analysis uses the outgroup to evolutionarily root the ingroup species in the tree, thereby making it possible to compare primitive and advanced traits and how they have changed. The diagrams produced through cladistics are called cladograms. Based on cladistic theory, what do you predict will be the groups that will form?

5 Executing your data file for cladistic analysis 1. From the File menu, choose Open and double click on your file that does contain the outgroup data (the file/open mode should be on Execute this time). 2. After executing the file, PAUP again displays comments and some general information about the data matrix and character coding (binary: 1 s and 0 s). 3. To generate a cladogram(s), you will define the optimality criterion (parsimony) used to generate the tree: Type: set criterion=parsimony Click on Execute 7. Define a search strategy Type: hsearch Click on Execute 1. Once the search is started, PAUP displays information about the options and assumptions being used during the search. When the search completes, PAUP displays general information about the search results. 2. Note the number of rearrangements, the number of different trees found, and which tree was the best tree. A value called the consistency index can also be found The consistency index (CI) is a measure of homoplasy and will equal 1.00 if there is no homoplasy in the tree, and you have obtained the shortest, most parsimonious tree. CI is calculated by dividing the minimum number of possible steps (character changes) to allow for evolution of each character state once by the actual number on the tree generated. 3. Click close to dismiss the search status dialog box. Viewing trees and related information 1. Having completed the analysis there should now be a single best tree currently in memory. To display the tree, do the following: Type: showtrees 2. (If there is more than one tree, you can display all trees (type: showtrees all) or a specific tree, e.g. tree number 4 (type: showtree 4). 3. Draw the best one or two trees in the area labeled Figure Mark the character state changes in the new tree(s). What is different /same about the different trees?

6 Describing Trees The showtrees command draws a simple picture of the branching order of taxa. 1. To get a more detailed picture of the tree in phylogram format with branch lengths, do the following: Type: describetrees 1/plot=phylogram brlens=yes 2. Record the tree length and consistency index values. 3. Using the best tree you have drawn, use your data set (Table 2) to indicate the character state changes in both the UPGMA tree and your cladogram. NOTE: PAUP can save trees in several formats. We will save the tree in NEXUS format with branch lengths: Type: savetrees file=[yourchosenname.tre] Print Results Print your results to a.pdf file. Save this file for preparing your report. You can use the snapshot tool in Adobe acrobat to copy and paste trees into your final report document. Questions 1. Having completed the UPGMA analysis, what do you think it indicates about the relationship among the species? 2. How do the UPGMA results compare to your initial prediction of the relationship among the species? Explain. 4. What do your trees show about character state changes in these taxa? 5. How do character state changes compare between the UPGMA and parsimony trees? V. REPORT YOUR FINDINGS Prepare a brief (1 page) description of how the phenetic and cladistic methods produce different trees describing these species. In your explanation, address the following issues: 1. How do the trees compare to your predictions? To one another? 2. Why do you think the trees are different? 3. Do the two analyses suggest different relationships among the study species? 4. What is the difference in information content of a phenogram versus a cladogram? 5. What do your analyses indicate about character change in these different taxa? 6. What do your analyses indicate about phylogenetic relationships among these taxa? Explain.

7 Figure 1. Diagram of initial grouping of Dendrogrammaceae or Caminalcules species.

8 Table 1. Characters and character states for Dendrogrammaceae or Caminalcules. Characters Character State Table 2. Character state matrix for Dendrogrammaceae or Caminalcules. Species OUTGROUP A B C D E F G H

9 Figure 2. UPGMA tree

10 Figure 3. Parsimony tree(s).