Name: Date: Gash: AP Biology

LAB: Understanding Phylogeny

Between 1990–2003, scientists working on an international research project known as the Human Genome Project
were able to identify and map the 20,000–25,000 genes that define a human being. The project also successfully
mapped the genomes of other species, including the fruit fly, mouse, and Escherichia coli. The location and complete
sequence of the genes in each of these species are available for anyone in the world to access via the Internet.
Why is this information important? Being able to identify the precise location and sequence of human genes will
allow us to better understand genetic diseases. In addition, learning about the sequence of genes in other species
helps us understand relationships among organisms. Many of our genes are identical or similar to those found in
other species.
Suppose you identify a single gene that is responsible for a particular disease in fruit flies. Is that same gene found
in humans? Does it cause a similar disease? It would take you nearly 10 years to read through the entire human
genome to try to locate the same sequence of bases as that in fruit flies. This isn’t practical, so a sophisticated
technological method is needed.
Bioinformatics is a field that combines statistics, mathematical modeling, and computer science to analyze
biological data. Using bioinformatics methods, entire genomes can be quickly compared in order to detect genetic
similarities and differences. Using bioinformatics tools, you can input a gene or protein sequence of interest and
search entire genomic libraries for identical or similar sequences in a matter of seconds.
In this laboratory investigation, you will use bioinformatics to compare several genes, and then use the information
to construct a cladogram. A cladogram (also called a phylogenetic tree) is a visualization of the evolutionary
relatedness of species. Figure 1 is a simple cladogram.
Note that the cladogram is treelike, with the endpoints
of each branch representing a specific species. The closer
two species are located to each other, the more recently
they share a common ancestor. For example, Selaginella
(spikemoss) and Isoetes (quillwort) share a more recent
common ancestor than the common ancestor that is
shared by all three organisms.
Figure 2 includes additional details, such as the
development of particular physical structures called
shared derived characters. Note that the placement of
the derived characters corresponds to when (in a general,
not a specific, sense) that character appeared; every
species above the character label possesses that
structure. For example, tigers and gorillas have hair, but
lampreys, sharks, salamanders, and lizards do not have
hair.
The cladogram above can be used to answer several
questions. Which organisms have lungs? What three
structures do all lizards possess? According to the
cladogram, which structure — dry skin or hair —
appeared first?
Historically, only physical structures were used to create cladograms; however, modern-day cladistics relies heavily
on genetic evidence as well. Chimpanzees and humans share 95%+ of their DNA, which would place them closely
together on a cladogram. Humans and fruit flies share approximately 60% of their DNA, which would place them
farther apart on a cladogram.
Name: Date: Gash: AP Biology

Part 1: Understanding Comparative Anatomy

The phylogenetic tree below shows the proposed evolutionary history of elasmobranch orders (sharks and rays)
based on teeth morphology. Use it to answer the questions that follow.

1. Which order seems to be the most distantly related from the others?
2. Which order has the closest ancestry with mackerel sharks (Order Lamniformes)?
3. What tooth characteristic seems to distinguish the Galean sharks from the Squaleans?
4. Why do you suppose the rays are on a branch that separates it from the other “modern sharks”?
5. Why do you suppose that sharks have such great variations in tooth morphology?
The diagram below is comparing two species of hammerhead sharks. Eusphyra blochii is more primitive than
Sphyrna tiburo, meaning that it appeared on Earth first and is considered a more ancient shark. Use this diagram to
answer the following questions:
6. Shared ancestral characters are those attributes of a plant or
animal which all, or in this case, both members of the group
possess. They can also be called conserved characters because
they are a character seen in the ancestral species that is also
retained by the more modern species. Identify some ancestral
characters seen in these hammerheads.
7. Shared derived characters are advanced traits which only
appear in some members of the group, usually the modern
descendant species. Identify some derived characters seen in
these hammerheads.
Name: Date: Gash: AP Biology

Part 2: Building Cladograms with Morphological Characters

In cladistics, similar characteristics that come from a
common ancestor are used to divide organisms into
groups. A cladogram will begin by grouping organisms
based on a characteristic displayed by all the members
of the group. Subsequently, the larger group, or clade,
will contain increasingly smaller groups (clades) that
share the traits of the clades before them, but also
exhibit distinct changes as the organism evolves. The
example to the right represents a cladogram.
To make a cladogram, scientists first collect data on
the features of all the organisms they hope to classify.
This data is then analyzed to determine which
characteristics were present in what could have been
a common ancestor, and which might have been
developed in later times. Observe how the data in the
table below the cladogram were used to eventually
make the cladogram pictured:
• Note that the characteristics studied (#1-7) are
listed as columns in the table.
• The organisms being compared are listed in the
rows.
• If an organism has the characteristics, it gets a
score of “1” in that column. If it does not have that
characteristic, it gets a “0”. For example, amoebas
are eukaryotic but lack all of the other
characteristics.
• Notice how the sum of the scores for each row
determines how the organisms are arranged in the
cladogram. For instance, amoeba has a total score
of 1, which is the lowest for all the organisms.
Therefore, it goes on the first branch of the
cladogram. The sponge has a total score of 2 (1+1)
and goes on the next branch. The organism with
the highest score (in this case the cat; score=7)
would be added to the last branch of the
cladogram.
1. Use the following data to construct a cladogram of the major plant groups:
Name: Date: Gash: AP Biology

2. Now it is time to apply this method to hardware. Students will use common nails, screws, and bolts to simulate
the process of applying cladistics to living organisms or fossil life forms. Use the following instructions to make
a cladogram for your set of hardware “organisms”.
a. Use the key provided to identify the different pieces of hardware at your table.
b. List your pieces of hardware under the “Organism” column in a table like the one below.

c. Add up the scores for each piece of hardware (“organism”) and then use these scores to build a cladogram.
d. What is the most shared ancestral character in your cladogram?
e. What is the most shared derived character in your cladogram?
f. Where did you include your assigned “mystery specimen” in your cladogram? Explain your reasoning.
g. Why did hardware “evolve” over time?
h. Explain why hardware can be an example of adaptive radiation?
i. Identify an example of “coevolution” in your hardware.

Part 3: Building Cladograms with Molecular Data

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is an enzyme that catalyzes the sixth step in glycolysis, an
important reaction that produces molecules used in cellular respiration. The following data table shows the
percentage similarity of this gene and the protein it expresses in humans versus other species. For example,
according to the table, the GAPDH gene in chimpanzees is 99.6% identical to the gene found in humans, while the
protein is identical.

1. Why is the percentage similarity in the gene always lower than the percentage similarity in the protein for each
of the species? (Hint: Recall how a gene is expressed to produce a protein.)
2. Draw a cladogram depicting the evolutionary relationships among all five species (including humans) according
to their percentage similarity in the GAPDH gene.
Name: Date: Gash: AP Biology

Part 4: Determining the Origin of Whales Using the Cytochrome B Molecular Clock
A molecular clock is a technique which uses rates of molecular change to determine when two species diverged.
The molecular data used for such calculations is usually nucleotide sequences for DNA or amino acid sequences for
proteins. One protein that is commonly used in this technique is cytochrome b. This membrane protein is a good
model to use because of its prevalence in nearly all living organisms. In this model, species with more similar
cytochrome amino acid sequences would be more closely related than if their cytochrome sequences were
different.
There is a large database of sequences available known as SWISSPROT. We will access the SWISSPROT database,
download the sequences of interest, and then use another tool available from SWISSPROT to align the sequences
to derive a measure of their similarity.
In this part, we will attempt to determine the phylogeny of whales. All cetaceans, including whales, dolphins and
porpoises, are considered descendants of land-living mammals of the Artiodactyl order (even-toed ungulates). Both
descended from a common ancestor, the Indohyus (an extinct semi-aquatic deer-like ungulate) from which they
split around 54 million years ago. Primitive whales probably first took to the sea about 50 million years ago and
became fully aquatic about 5-10 million years later.
1. Identify the common names for the families listed in the table below.

2. Visit: https://web.expasy.org/sim/ . Enter the Humpback code under Sequence 1. Enter the Bovidae code
Under Sequence 2. Scroll down and click submit.
3. On the results page, scroll down to the Alignments.
a. How identical are the two cytochrome proteins that were compared?
b. How many amino acids in those proteins overlap with each other when compared?
c. What do all of the letters represent?
d. What does the asterisk show?
e. Record the “score” in your data table.
4. Repeat this same procedure for the other whales and land mammals in the data table
3. Which family of land mammals had the highest score with all of the whales and what does this mean about
their cytochrome protein structure?
4. Based on this, which ungulate family shares the most recent common ancestor with the whales?
5. What does this suggest about the origin of whales?
6. Notice that two of the whales have higher scores than the other two. What does this suggest about these
whales? (Hint: look up whale suborders)
7. Use this information to make a cladogram that includes the animals in the table and the two suborders of
whales.