MacClade and Maximum Parsimony Trees
Introduction:
Evolutionary relationships between species are often diagrammed as trees. The
trees consist of a root that represents a single common ancestor for the whole tree,
internodes that represent either real or hypothetical common ancestors for specific
lineages within the tree, branches that describe relationships between ancestral species
and their descendents, and terminal nodes that represent the taxa being studied.
taxa
Terminal nodes
Internodes
Common ancestor
of Aliopo and
Tilonotila
Branch
Common ancestor of all
four taxa
DNA, RNA and protein data can all be used to generate such trees, (as can more
traditional characters, such as the presence or absence of a placenta). The assumption
that is made in the molecular analyses is that closely related species will have more
similar gene or protein sequences than distantly related species. Furthermore, since
mutations are both rare and random, it is assumed that the same mutation is unlikely to
arise in two different lineages. Thus, if you have four species with the following DNA
sequences: GAATTC, GATTTC, GATCTC, and GAGTTC, it would be more
parsimonious to arrange the tree as shown in “A”, where the AT mutation at the third
position only happens in one lineage, and the tree itself only requires three mutations,
than in “B”, where it there are four total mutations required to generate the tree, and the
AT mutation happens in two separate lineages.
GATTTC GATCTC
GAGTTC
TC
AG
GATCTC
AT
TC
GAGTTC
GATTTC
AG
AT
AT
GAATTC
Tree “A”
GAATTC
Tree “B”
In this exercise you will use MacClade to generate cladograms or phylograms
(you can choose) that have the smallest tree length (analogous to the total number of
mutations needed to account for the tree) while minimizing the number of times that a
single mutation arises in multiple lineages. You will then compare your MacClade tree
to the one automatically generated for you by the Phylogenetic analysis in the Rarely
Reclusive exercise.
Procedure:
Save the National Biomedical Research Foundation (NBRF) Format File from the
syllabus (filename mitochondrial.NBRF) to your desktop by typing control+click on the
file name.
MacClade Analysis of Mitochondrial Sequences
Open MacClade
1. Double Click on the MacClade Icon.
2. Choose ‘Open File’ (Your computer may open a Finder Window
instead of allowing you to choose Open File..this simply takes you
directly to step 3, so proceed!).
3. Open ‘mitochondrial.NBRF’ from your desktop.
4. Click ‘OK’ to verify that your file is an NBRF DNA file.
5. You should now see that most of your sequence files (minus a few that
had poor sequence quality) have been uploaded into the MacClade
program as seen in the picture below:
6. While this window contains all of your sequences (the taxa in this case
are the individual students, and the characters are the individual DNA
bases at each position of the sequence), the sequences are not yet
aligned, so we need to do that right after you save your data.
Save Data
1. Under FILE choose SAVE FILE AS, and give your file a name and
save it to the desktop. This will make sure you do not lose your data if
something goes wrong during later manipulations. Don’t forget to throw
away all of your files and to empty the trash before you put your computer
away for the semester.
Aligning DNA Sequences
1. In order to analyze how closely related these gene sequences are using
MacClade, you need to make sure that each sequence is properly lined
up.
2. MacClade’s Alignment Tool can be seen in the toolbox in the bottom
left of your MacClade window, as indicated by the arrow below:
3. Click and hold on the Alignment Tool. In the popup box, select ‘slow
method using less memory’.
4. To align mito124 with mito126, make sure that the alignment tool is
selected. Now, click on mito126, and drag up to mito124. Release.
The computer will now line up the two sequences so that they match
as closely as possible. Save your data. I recommend you save after
every alignment step below (sometimes this program crashes)
5. Next, align mitoTP10 with mito126 by dragging the alignment tool
from mitoTP10 up to mito126 and releasing. Note that the
mitochondrial sequences have to shift and insert gaps to find their best
matches with the other sequences.
6. Repeat, until you have finished aligning all of the sequences to the
sequence above them in a pairwise fashion (yes it’s a pain to only
align them pairwise…but that is all this program can do).
7. When you finish, you should be able to see (the bases are each colorcoded) that the sequences line up really nicely now. You are ready to
generate a phylogenetic tree.
Manipulate Data
1.
Generate tree
a) Under WINDOWS choose TREE WINDOW
b) Choose DEFAULT LADDER - a tree should now appear! This tree makes no
assumptions at all about how your microbes are related. In fact, if you look at the
tree, it simply places the organisms in the same order in which you entered the
sequences.
c) Under TRACE choose TRACE ALL CHANGES. This will provide a color
visualization of the number of nucleotide changes between a common ancestor
and the next ancestor or terminal taxon. If you place the cursor arrow on a tree
branch, the number of unambiguous nucleotide changes in that branch will be
written in the small box on the bottom right.
d) Under Display choose ‘Tree Shape and Size”. The choices, from left to right
below are either an angled or a square branch cladogram, or a phylogram.
2.
Minimize Tree
a) Find the most parsimonious tree by clicking on branches, and then dragging
them to new tree locations, remembering as you do so that you are changing the
assumptions about how the species are related. Your goal is to search for the tree
that gives the lowest number of “steps” (analogous to the total number of
nucleotide changes necessary to account for the proposed evolutionary
relationships - so lower is better). If you want to know what the theoretical
smallest tree is, you can choose    minimum possible from the upper
program bar. Both the current tree length and the minimum possible tree length
will be displayed in a small box in the lower right hand corner of your tree. Try
to get as close to the minimum as possible.
b) Try manipulating the tree so it looks like the ‘ideal’ tree from your Dolan
DNA Center analysis. Have you found the minimum yet?
c) Now that you’ve played around with the tree a bit, it is time to tell you a little
secret – the computer will actually help find the smallest tree for you. Simply
click on the ‘search above’ icon in the toolbox, and then move the cursor to the
root of the tree. When you click on the root, the program will search for, and
display, the shortest tree above that spot.