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Phylogenetic Analysis

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PART II: Conducting a Phylogenetic Analysis


In this part of the lab, teams of 4 students will conduct a phylogenetic analysis on a group of organisms using characteristics of their skeletal remains.  Using a software package named “Mesquite” you will first generate a character matrix.  This matrix is a simplified representation of the morphological differences you see in the various organisms.  Your team will have to define characters and character states and enter these into a Mesquite Matrix file. Next, Mesquite will generate a small subset of all possible trees that represent the most plausible phylogeny (based on a defined selection criterion, like Parsimony).  Then you will manipulate the trees to further investigate unresolved characters – those characters that have no clear best solution.  Finally, you will save the trees that you believe are the best models for evolutionary relationships within your group of organisms.  These trees will serve as the results that you will present in a PowerPoint presentation to the class.  (See Lab Manual – Part IV for details on the description of the PowerPoint presentation).



Building and Analyzing Phylogenetic Trees Using Mesquite

(Due to Bug in Java periodically use FileàReset Menus)


Setting Up a New File

1.      Select File from the top menu bar and choose “New”

a.       Name and Save file to desktop

2.      In the next window that will ask if you want to include new taxa:

a.       Check the “Make Taxa Block”

b.      Name the taxa block

c.       Designate the number of taxa

d.      Deselect/uncheck “Show Tree Window”

e.       Check “Make Character Matrix”

f.       Press OK

3.      Name New Character Matrix

a.       Designate the number of characters (type 5 or so, you can add more later)

b.      Select “Standard Categorical Data”

c.       Press OK


Building a Character Matrix

1.      Double click on taxon in rows to rename your taxa.

2.      Double click in the empty cell below column numbers to name your characters. (Remember characters are general descriptions like eye color and character states are blue or brown eyes).

3.      Enter your character states into the matrix.

a.       For example, Taxa 1 has blue, round eyes and Taxa 2 has brown, narrow eyes and you coded your characters as follows: 0= blue, 1= green, and 2= brown for eye color; and  0=round, 1= narrow, 2= oval and 3=square for shape.  You would enter (0, 0) for Taxa 1 and (2, 1) for Taxa 2.

b.      Be sure to type up a key to your Characters and Character states for use in your presentation…in a Word document.

4.      You may add characters or taxa to your matrix by choosing Matrix on the menu bar, then selecting “Add taxa” or select “Add characters”.



Displaying and Analyzing Trees

1.      Once your matrix is finished, select Taxa & Trees from the menu bar and choose “New Tree Window”. Use “Simulated Trees” – OK. Then select “Uniform speciation (Yule)” and OK for Tree Depth.

2.      From Tree Window, select Analysis and choose “Tree Legend”, then scroll down to “tree length” and select.

3.      Press OK for “Current Parsimony Model” in next window.

4.      Select Analysis from menu bar and choose “Trace Character History”.

a.       Select “Stored characters” in window prompt. Then, select “Parsimony Ancestral States” in next window.

5.      Click on the Forward and Back arrows in the Trace Character view box to see how your characters map on to the tree.  As you move through the different characters traces take note of the characters that had to arise more then once, i.e., the color of the trace is on two or more separate branches.  These are signs of multiple evolutionary events for the same character, which is not likely (not parsimonious). It is evidence of convergent evolution, which is much less likely (in most cases) then a single evolutionary event within a related group/clade.  When you see this, try moving the branches together to make one clade of that particular character.  Just click on the branch and while holding the mouse button, drag to the branch you wish to attach it to.  (See demonstration in class).

6.      When you swap branches notice the “Tree Length” value.  To obtain the most parsimonious and thus most likely tree topography you want to minimize the “Tree Length” value.  The tree length reflects the number of evolutionary changes that need to occur in order to map all the character states onto any given tree (any given topography).  The more convergences the longer the tree length because one character state will have had to arise more than once.  Keep swapping branches around until you find the lowest value for tree length and the tree pattern (topography) that reflects the evolutionary relationships that make the most “sense” in your group of organisms.


Altering the Tree Color and Style

1.      Go to Drawing on the menu bar and play with any of the color or style option you feel enhance your tree or use colors to highlight a particular character of taxa you may wish to talk about in your presentation.


Saving Trees for your Presentation

1.      Go to File on the menu bar, select “Save Tree as PDF”, then name and locate the file where you can find it.

2.      Save as many trees as you want to include in your PowerPoint presentation.

3.      Current trees you are working on may be lost when you close Mesquite.  Be sure to capture PDF versions of all the Trees you may want.  You can always choose not to use them, but better to save many trees for your presentation.


Inserting your Trees into PowerPoint

1.      The PDF file of your saved trees will have a very large blank border.  Consequently, if you try to insert the entire PDF file into PowerPoint it will look awkward.

2.      Therefore, open the PDF file, use the “image copy” tool to select only the tree parts of the file and then paste into a PowerPoint slide.



PART III: PowerPoint Presentation Assignment

DUE during the labs of March 31st – April 2nd .


In groups of 4 students, prepare a 5-10 minute PowerPoint presentation summarizing the phylogenetic analysis that you ran in lab.  See Part III of the lab for procedures on conducting your analysis.  Be sure to save trees that will help you report your findings.  Your PowerPoint presentation should focus on two aspects; 1.) the characters and character coding you came up with for your group of organisms and 2.) a comparison of your trees to current published relationships.  The presentations should include a minimum of the following slides, 1.) a title slide with student names; 2.) an intro/background slide (what organisms did you analyze); 3.) a slide(s) of the characters and character states that you used; 4.) a results slide or several, showing tree(s) you came up with, including the character states mapped onto the tree 5.) a comparison to published results (textbooks or primary literature)  and 6.) a conclusion/discussion slide(s).


Group Presentations will be evaluated as follows:


% Total





completeness of presentation; covered all relevant results



organization; tables, graphs/charts, etc. clear, easy to read; legends complete, self-explanatory



conclusions; consistent with results in scope and meaning



overall communication; effective and clear oral presentation



participation; active in presenting Powerpoint talk




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