LABORATORY ACTIVITY 3 | PHYLOGENY

INTRODUCTION

Scientists make hypotheses about evolutionary relationships based on observations and

gather evidence to test those hypotheses. We refer to this field of making testable
hypotheses of evolutionary relationships as phylogenetic systematics.

Hypotheses about the evolutionary relationships among groups of organisms are based
on the idea that they are arranged together by shared characters that have been
inherited from a common ancestor.

These hypotheses are often represented by branching diagrams called phylogenetic

trees.

There are many types of data that can hypothesize evolutionary relationships and build
phylogenetic trees. Morphological characteristics, DNA/molecular data, ecological
information, behavioral characteristics, and development can all be useful. The more
information that can be collected about the group or taxon, (plural taxa) that we are
interested in, the more likely we are to build an accurate representation of how these
organisms are related.

In this laboratory activity, we will be building a phylogenetic tree using morphological

characters and molecular data. You will also learn the characteristics of a phylogenetic
tree.

MATERIALS / SPECIMENS

PROCEDURE

The Anatomy of a phylogenetic tree

(a) The figure above shows an example of a phylogenetic tree depicting the relationship
between Species A, B, C, D and E, which are positioned at the ends of the branches.
The pattern in which the branches connect represents our understanding of how the
species in the tree evolved from a series of common ancestors. Each branch point
(also called an internal node) represents a divergence event, or splitting apart of a
single group into two descendant groups.
(b) At each branch point lies the most recent common ancestor of all the groups
descended from that branch point. For instance, at the branch point giving rise to
species A and B, we would find the most recent common ancestor of those two
species. At the branch point right above the root of the tree, we would find the most
recent common ancestor of all the species in the tree (A, B, C, D, E).

(c) In a phylogenetic tree, the relatedness of two species has a very specific meaning.
Two species are more related if they have a more recent common ancestor, and less
related if they have a less recent common ancestor.

(d) For instance, suppose that we wanted to say whether A & B or B & C are more
closely related. To do so, we would follow the lines of both pairs of species backward
in the tree. Since A and B converge at a common ancestor first as we move
backwards, and B only converges with C after its junction point with A, we can say
that A and B are more related than B and C.

(e) There are some species whose relatedness we can't compare using this method. For
instance, we can't say whether A & B are more closely related than C & D. That’s
because, by default, the horizontal axis of the tree doesn't represent time in a direct
way. So, we can only compare the timing of branching events that occur on the same
lineage (same direct line from the root of the tree), and not those that occur on
different lineages.
(f) You may see phylogenetic trees drawn in many different formats. Some are blocky,
like the tree on the left. Others use diagonal lines, like the tree on the right. You may
also see trees of either kind oriented vertically or flipped on their sides.

(g) The three trees above represent identical relationships among species A, B, C, D,
and E. You may want to take a moment to convince yourself that this is really the
case – that is, no branching patterns or recentness of common ancestors are
different between the two trees. The identical information in these different-looking
trees reminds us that it's the branching pattern (and not the lengths of branches)
that's meaningful in a typical tree.

(h) All the trees we've looked at have had nice, clean branching patterns, with just two
lineages (lines of descent) emerging from each branch point. However, you may see
trees with a polytomy, meaning a branch point that has three or more different
species coming off it. In general, a polytomy (shown above) shows where we don't
have enough information to determine branching order.

Constructing a Cladogram using Morphological Characters

(a) The underlying principle is Darwin’s idea of “descent with modification” in building a
phylogenetic tree. Basically, by looking at the pattern of modifications (novel traits) in
present-day organisms, we can figure out—or at least, make hypotheses about—
their path of descent from a common ancestor.

(b) Let us create a cladogram using the Table below

Species C01 C02 C03 C04 C05

Outgroup 0 0 0 0 0
A 1 0 0 0 1
B 1 1 0 1 0
C 1 0 1 1 0
(a) Initially, the cladogram of all species would be found below. Let us use the different
characters to split them into groups

(b) Let us go with Character 1 wherein it shows that Species A, B and C possess
character 1 but not the outgroup. The figure below demonstrates the grouping of
species A, B and C except for the outgroup. What do you call character 1?

(c) We still need to split further Species A, B and C by what character? It would be
Character 4 wherein Species B and C possess that character

(d) What happens with Characters 2,3 and 5? They are only found in specific species.
What do you call these characters?

(e) In the laboratory exercise, please use the previous table of characters of plants &
fungi as well as invertebrates including chordates to create a cladogram along with
the characters found.
Using Molecular Evolutionary Genetic Analysis (Molecular Data)

(a) Download the MEGA Software version 11 using this address:

https://www.megasoftware.net/ Follow the steps such as identifying your computer
requirements, MEGA user agreement, and personal information.

(b) Once installed on your computer, click the MEGA desktop icon to start the program.
In MEGA’s main window, choose to Open a File/Session from the File menu. Choose
the sequence file (practice.fasta)

(c) In the resulting dialog choose Align. The Alignment Explorer shows a name for each
sequence at the left, followed by the sequence, with colored residues. Now is the
time to align the sequences.

(d) Two alignment methods are provided: ClustalW and MUSCLE. Either can be used,
but in general, MUSCLE is preferable. In the toolbar, near the top of the window,
ClustalW alignment is symbolized by the W button, and MUSCLE by an arm with a
clenched fist to “show a muscle.” Click Muscle from the Alignment menu and choose
Align DNA.

(e) Choosing an alignment method opens a settings window for that method. For
MUSCLE, I recommend that you accept the default settings. Click the OK button to
start the alignment process.

(f) When the alignment is complete Save the session as practice_alignment.mas

(xxx.mas file).

(g) MEGA cannot use the .mas file directly to estimate a phylogenetic tree, so you must
also choose Export Alignment from the Data menu and export the file in MEGA5
format where it will get a .meg extension. You can leave the title blank and you will
also be asked whether they are coding sequences. Save the file as
practice_alignment.meg (xxx.meg file).

(h) In MEGA main window, choose to Open a File/Session from the File menu and open
the .meg file that you previously saved. There are several widely used methods for
estimating phylogenetic trees (Neighbor Joining, UPGMA, Maximum Parsimony,
Bayesian Inference, and Maximum Likelihood). We will be concentrating on UPGMA.

(i) From the Phylogeny menu choose Construct/Test UPGMA…. . First, Set the Test of
Phylogeny to None. Substitution Type is Nucleotide. Model/Method should be
Maximum Composite Likelihood. Rates among Sites should be Uniform Rates.
Patterns among Lineages should be Same (Homogenous). Lastly, Gaps/Missing
Data Treatment should be Pairwise Deletion. Save the tree from the File menu as
practice_alignment1.mts.

(j) It is desirable to know the reliability of the phylogenetic tree. The most common way
to estimate the reliability of a phylogenetic tree is by the bootstrap method. To
 perform the bootstrap test return to the Analysis Preferences dialog. Under
Phylogeny Test, set Test of Phylogeny to “Bootstrap Method,” then set No. of
Bootstrap Replicates to an integer between 100 and 2,000, please set yours at 1000.
Click compute. A window with a progress bar shows how the analysis is proceeding.

(k) When the analysis is complete, a tree will appear with numbers on every node.
Those numbers, bootstrap percentages, indicate the reliability of the cluster
descending from that node; the higher the number, the more reliable the estimate of
the taxa that descend from that node. The bootstrap test does not estimate the
overall reliability of the tree; instead, it estimates the reliability of each node. That is
actually advantageous because it tells you which parts of the tree you should trust
and which parts you should not take seriously.

(l) Save the tree from the File menu as practice_alignment2.mts

(m) Do these steps for selected invertebrates and plant & fungi sequences. The two files
are invertebrate_exercise.fasta and plant_exercise.fasta.

(n) How do your molecular trees compare to your morphological trees?