Basic Tree Building With PAUP
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
http://paup.csit.fsu.edu/index.html.
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.
Basic Tree Building With PAUP
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 1.
4. 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 1.
5. 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.
Basic Tree Building With PAUP
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
;
end;
D
E
F
G
H
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
0000000000
A 1100110000
B 1100100000
Basic Tree Building With PAUP
C
D
E
F
G
H
;
1101001000
1010000101
1010000010
1010000000
1010000100
1100011000
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 2.
6. 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?
Basic Tree Building With PAUP
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.
2.
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.
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 3.
4. Mark the character state changes in the new tree(s).
What is different /same about the different trees?
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Basic Tree Building With PAUP
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.
Basic Tree Building With PAUP
Figure 1. Diagram of initial grouping of Dendrogrammaceae or Caminalcules species.
Basic Tree Building With PAUP
Table 1. Characters and character states for Dendrogrammaceae or Caminalcules.
Characters
Character State
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Table 2. Character state matrix for Dendrogrammaceae or Caminalcules.
Species
1
2
3
4
5
6
7
8
9
OUTGROUP
A
B
C
D
E
F
G
H
10
Basic Tree Building With PAUP
Figure 2. UPGMA tree
Basic Tree Building With PAUP
Figure 3. Parsimony tree(s).