Lab Activity No.

12
PHYLOGENETIC SYSTEMATICS

Objectives

1. To become familiar with the cladistic approach to reconstruction of phylogenies.

2. To construct a character matrix and phylogeny for a group of very unusual organisms.
3. To interpret the evolutionary history of traits based on a phylogenetic reconstruction.

PART I. INTRODUCTION

The branch of biology known as systematics expressly deals with the evolutionary history or phylogeny of groups of
organisms. Systematists seek to produce a classification or taxonomy, which reflects the evolutionary history of a particular
group of organisms. For instance, two species that are classified in the same genus are considered to be more closely related
(i.e., share a more recent common ancestor) than species in different genera within the same family. Such a system is said to
be hierarchical.
The following hierarchy of classification is used in biology:
Kingdom/Domain
Division or
Phylum Class
Order
Family
Genus
Species
How can systematists be sure their taxonomic classification reflects the true evolutionary pathway? We can’t go back in a
time machine to tell when various groups evolved. Instead, most systematists use general morphology to draw conclusions
about ancestry. For instance, a leopard frog and a mink frog appear to be more similar to each other than either is to a toad.
This observation is reflected in the fact that the two frogs are classified in the genus Rana and the toad in the genus Bufo.

Unfortunately, measures of morphology can be subjective. Two systematists looking at the same organisms may
make different judgements about the relative similarity of those organisms. In the past 25 years, a technique has been
developed which provides a more objective way of assessing evolutionary relationships. The technique, called cladistics, was
developed by the German biologist, Willi Hennig. Cladistics has transformed the way that systematic research is conducted.

In short, cladistics is a method to reconstruct the probable evolutionary pathway of a group of organisms. The
technique is designed to identify clades, groups of organisms that all share a common ancestor. Clades are usually depicted
as branching diagrams called cladograms or evolutionary trees. Let’s consider the simple cladogram below. Time is
measured vertically in a cladogram. This cladogram tells us that the first event was the evolutionary split into two different
lines, one leading to species A and one to the other branch of the tree. The next event was the splitting of B from the line that
leads to C and D. The final and most recent event is the splitting of C and D into two separate species.

You can distinguish three different clades in this cladogram: one clade that includes A,B,C & D because they all share a
common ancestor, a second smaller clade that includes only B, C& D because they are on a separate branch of the cladogram
from A and an even smaller clade including only C & D. A taxonomist might use this information to place C and D in the
same genus, B in a different genus but in the same family as C & D and finally A in a different family but in the same order
as B, C and D.
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How does one develop a cladogram? For a group of organisms, a systematist collects data on as many different
characters or traits as possible. Number of toes, hair color, and the sequence of nucleotides in a particular gene are all
characters that can be measured. The more characters, the better. The particular variations in each character are called
character states. For the character number of wings in insects, there are three possible character states: two pairs of
wings (the majority of insects), one pair of wings (houseflies and other dipterans) and no wings (springtails, silverfish
and other wingless insects).

A character state that is shared by two or more organisms may suggest that those organisms are closely related.
However, not all shared characters are useful in developing a cladogram. We have to distinguish between two types of
characters: primitive and derived. By primitive, we mean that the character in question is present in the most primitive
member of the group. A derived character is one that has changed from the primitive condition. Shared primitive
characteristics are useless in developing a cladogram; shared derived characters are all-important in piecing together
the probable path of evolution.

Let’s consider the evolutionary tree below to understand why primitive characters are uninformative and derived
characters are useful. We will consider flower color for eight different species of poppies.

Blue Red Yellow Yellow Purple Red Orange Orange

Red Yellow Red Orange

Red Red

Red
The actual pathway of evolution is shown in the tree. However, we can only see those organisms that exist at the
present time, namely the eight branch points at the top of our diagram (remember time goes backwards from top to
bottom of a cladogram). If we claim that the two species with red flowers (the primitive condition of the trait) are
closely related we would be making a big mistake. Explain why this error occurs in the space provided below:

There are two cases in this evolutionary tree where flowers share derived traits for flower color. Two plant species have
yellow flowers and two species have orange flowers. It is much more likely that yellow flower colors evolved just once
(remember as modern systematists we do not know what the ancestral flower color was as shown in the diagram) rather
than twice. Cladists call this the principle of parsimony. It’s more likely that a particular character state evolved once
than twice; one should presume only one evolutionary event, not two, when possible. The shared possession of the
same derived character state helps us build a case that the two yellow-flowered species shared a recent ancestor and
thus constitute a clade.
Sometimes, the same character state can evolve separately. In such cases, we say that the character state in
question is an evolutionary convergence. On the other hand, sometimes a derived character state can revert back to the
primitive condition. When this happens it is known as an evolutionary reversal.

There are other derived characteristics in the example above: blue flowers, purple flowers. Are these traits useful in
developing a cladogram? Explain your answer below:

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You can see that examining flower color gives us some information about the evolutionary relationships of these plants
but does not permit us to reconstruct the entire cladogram. We need to examine many characters to develop a
parsimonious cladogram (one that requires the minimum number of evolutionary changes). We achieve that end by
finding shared derived features.

How does one determine what the primitive state of a character is? One has to identify and justify an outgroup, a
species or group of species that are closely related to the organisms you are classifying but not directly related. For
instance, if you were doing a cladistic analysis of birds, you might choose a lizard as your outgroup. Therefore, for the
character of skin covering, scales would be the primitive character state because they are present in your outgroup and
feathers would be a derived character state.

To show how one finds the best cladogram, we will do a simple example with only three species (plus an outgroup).
For three species, there are only three possible pathways, shown in the three diagrams below.

We have gathered some morphological data on three species of mythical mammals and the outgroup; these data are
presented in the table below.

Species Number of toes Eye color Tail Number of molars

Outgroup 4 Brown Present 12
A 5 Blue Absent 10
B 4 Blue Present 12
C 5 Blue Absent 10
Now our task is to discover which of the three trees above is the most parsimonious, representing the most likely
pathway of evolution. We determine the best tree by fitting the data in the table to each tree and calculating the number
of evolutionary changes that are required. To facilitate our calculations, we will use the table below. The numbers in
the table indicate the number of evolutionary changes that are required for each character in each tree.

Tree 1 Tree 2 Tree 3

Number of toes 2 1 2
Eye color 1 1 1
Tail 2 1 2
Number of molars 2 1 2
Total changes 7 4 7

It is clear from the table that Tree 2 is the most parsimonious, requiring only four evolutionary changes for the four
characters chosen. We reject Trees 1 and 3. In the future, other systematists could gather other data to see if Tree 2
remains the most parsimonious tree in an independent test.

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 PART II. PHYLOGENY OF HYPOTHETICAL FROGS
(Parts II and III based on a laboratory exercise developed by Stacey Lance)

The best way to begin understanding how cladistics works is to go through an exercise in which you build a phylogeny
using a cladogram. To do this we will use the group of hypothetical frogs shown below. This group consists of a
defined outgroup (O) and an ingroup of six species (frogs A-F) for which we want to build a cladogram. We will build
the phylogeny using the list of characters and character states on the following page. Based on the characters and frog
pictures, fill out the character matrix on the following page for all seven species.

Above images come from Gergus EWA and Schuett GW, 2000. Labs for Vertebrate Zoology an Evolutionary Approach, 2nd edition. Biological
Sciences Press, Michigan.

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Character Matrix
Character Species Species Species Species Species Species
Character States Outgroup A B C D E F
Claws +/-

Chin hair +/-

Horn +/-

Tail +/-

Spikes +/-

Digits 4/5

Spots +/-

Tympanum +/-

Lateral fold +/-

Nostril +/-

Total number of derived traits ---------- >

Now, determine which states are ancestral and derived based on the outgroup. Circle the derived traits and write the
total number of derived traits for each species in the boxes at the bottom of the table. Note that the species that is
most closely related to the outgroup will have the fewest number of derived traits, while the species most distantly
related to the outgroup will have the largest number of derived traits.

Next fill out the following table noting how many derived traits are shared for each pair of ingroup species.

Species A Species B Species C Species D Species E Species F

Species A X

Species B X X

Species C X X X

Species D X X X X

Species E X X X X X

Species F X X X X X X

Use the above matrix to draw a cladogram depicting the phylogenetic relationships among all seven species. Start by
grouping the pairs that have the most shared derived traits and then linking groups together. This is difficult to describe
so look at the example matrix and cladogram below and then just give it a try, and ask for help if you get stuck. We
will go through this as a group before lab is over to make sure everyone understands. Look at the hypothetical example
first.

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 Example Matrix Of Shared Derived Traits:

Species Species Species Species

A B C D
Species A X 1 1 1
Species B X X 2 2
Species C X X X 3
Species D X X X X

Building a cladogram from above matrix:

Step 1. Species C and D share the most derived traits so link them together

Step 2. Species B shares 2 derived traits with C and D, but only 1 with A. A shares 1 with C and D, so the
next step is to group B with C and D.

Step 3. Add the remaining species (A) to the group

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 Step 4. Examine your cladogram. Each node shows a presumed common ancestor. Find the monophyletic
groups. A monophyletic group is a group of species that includes an ancestral species and all of its
descendants. Thus in the following cladogram, C, D and their common ancestor form a monophyletic group.
B, C, D and their common ancestor are another monophyletic group. If you tried to group B, C and their
common ancestor together they would be considered paraphyletic. A paraphyletic group is a group of
species that includes an ancestral species but not all of its descendants. In this case you are excluding D,
which shares a common ancestor with B and C.

An important thing to realize is that although the nodes represent an ancestral population it does not mean that there
was a single species that directly gave rise to species C and D. Rather, tracing back from D toward the common
ancestor of C and D, there could be several other species along the lineage (sequence of ancestor-descendant
populations). For example, imagine that species D is a chimpanzee and C is a gorilla. The cladogram does not suggest
that there was a single species that evolved into chimps and gorillas. What it does mean is that there was an ancestral
population that gave rise to two distinct lineages. One lineage eventually gave rise to chimps and the other to gorillas.
The lines of a cladogram represent the lineages and time. The deeper into the cladogram you go, the further back in
time you go. Thus the common ancestor of B, C and D occurred before the common ancestor of C and D. Remember
that cladistics is concerned with the timing of divergence. In this cladogram, C and D are the two most closely related
species because they shared a common ancestor more recently than any other clade.

Use the space below to draw a phylogenetic reconstruction for the hypothetical frog species. Once you have a
cladogram you feel confident about, use lines and labels on cladogram to indicate where character states changed.
How many evolutionary changes occurred in your phylogeny? Is there evidence of an evolutionary convergence
having occurred in your phylogeny? How about evolutionary reversals?

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 PART III. PHYLOGENY OF BIRDS
(due in lab next week)

General Instructions

For this project you should be working in groups. Try to do as much work as you can in class, then finish the
rest on your own time. Everyone should hand in the assignment independently (please write the names of all of your
group members somewhere on the assignment).

Assignment

For the purpose of this exercise, you are going to be exploring the phylogeny of 6 bird species (below),
using a California Gull as an outgroup for your comparisons.

Species: A) Cassin’s Vireo

B) Red Fox Sparrow
C) Cape May Warbler
D) Cedar Waxwing
E) Golden-Crowned Kinglet
F) Pyrrhuloxia
Outgroup: G) California Gull

Cassin’s Vireo Red Fox Sparrow Cape May Warbler

Cedar Waxwing Golden-Crowned Kinglet Pyrrhuloxia

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 California Gull

Complete each of the parts of the assignment below.

1) Using the following characters and character states, as well as the pictures of the birds available above, create
a character matrix for your 6 species and the outgroup. This should have the characters listed down the left
side and the birds across the top.

CHARACTER CHARACTER STATES

1) Bill color Yellow (Y) Black (B)
2) Bill bent at end Bent (B) Sharp pointy tip (S)
3) Crest on head of male Yes (Y) No (N)
4) Legs Pink (P) Not pink (N)
5) Black stripe THROUGH eye Yes (Y) No (N)
6) White bar or patch across wing coverts* White patch (W) None (N)
7) Distinct ring at least 3/4 way around eye Ring (R) None (N)
* coverts are small feathers that cover the bases of the wing feathers.

2) For each of the 3 phylogenies on the next page, map the positions of the changes between character states for
each of the characters in your matrix. Label each change with the number of the character. The first character
has been done for you on the first tree. Make sure you use the most parsimonious mapping for each character
(each character should change the minimum number of times). Sometimes a character will have two mappings
that are equally parsimonious – in this case just choose one.

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A) Pyrrhuloxia Sparrow Vireo Waxwing Warbler Kinglet Gull

B) Vireo Sparrow Pyrrhuloxia Waxwing Warbler Kinglet Gull

C) Pyrrhuloxia Sparrow Vireo Waxwing Warbler Kinglet Gull

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 3) For each tree, count up the number of character changes and write it next to the tree. Circle these
numbers. Which tree(s) are the most parsimonious?

4) Pretend it’s a year later, and you have collected more data to make a more detailed phylogeny. You notice that
some of these species of birds are sexually dimorphic (males have significantly different plumage than
females, generally much brighter!). You also notice that in some of these species both parents feed the
offspring, while in others only the female feeds the offspring. You imagine that having bright males near the
nest may tend to attract predators to the young.

You want to distinguish between two hypotheses to explain the patterns you see:
1) Males not feeding the young is an adaptation to dimorphism
2) If males don’t feed the young in a species, it will allow them to become brightly colored

To begin to address this question, map the characters changes in dimorphism and males feeding the
young onto each of the trees on the previous page.

CHARACTER SEXUAL DIMORPHISM MALES FEED YOUNG

Cassin’s Vireo No No
Red Fox Sparrow Yes No
Cape May Warbler No No
Cedar Waxwing No Yes
Golden-Crowned Kinglet No Yes
Pyrrhuloxia Yes No
California Gull No Yes
Note: This data is fictional

5) Which of the hypotheses in part 4 is consistent with your analysis? Explain why in a brief paragraph.

6) Did the addition of the new characters change the overall parsimoniousness of the three trees on the
previous page? Explain your answer.

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