AP Biology Lab 2: Using Online Molecular Databases to Study Evolutionary Relationships
Background Information:
How does an evolutionary biologist decide how closely related two different species are? The simplest
way is to compare the physical features of the species (their “morphologies”). This method is very similar
to comparing two people to determine how closely related they are. We generally expect that brothers
and sisters will look more similar to each other than two cousins might. If you make a family tree, you find
that brothers and sisters share a common parent, but you must look harder at the tree to find which
ancestor the two cousins share. Cousins do not share the same parents; rather, they share some of the
same grandparents. In other words, the common ancestor of two brothers is more recent (their parents)
than the common ancestor of two cousins (their grandparents), and in an evolutionary sense, this is why
we say that two brothers are more closely related than two cousins.
Similarly, evolutionary biologists might compare salamanders and frogs and salamanders and fish. More
physical features are shared between frogs and salamanders than between frogs and fish, and an
evolutionary biologist might use this information to infer that frogs and salamanders had a more recent
common ancestor than did frogs and fish.
This methodology certainly has problems. Two very similar looking people are not necessarily related,
and two species that have similar features also may not be closely related. Comparing morphology can
also be difficult if it is hard to find sufficient morphological characteristics to compare. Imagine that you
were responsible for determining which two of three salamander species were most closely related. What
physical features would you compare? When you ran out of physical features, is there anything else you
could compare? Many biologists turn next to comparing genes and proteins. Genes and proteins are not
necessarily better than morphological features except in the sense that differences in morphology can be
a result of environmental conditions rather than genetics, and differences in genes are definitely genetic.
Also, there are sometimes more molecules to compare than physical features.
In the following exercise, you will use data in a public protein database of gene products (proteins) to
evaluate evolutionary relationships. In the first part of the exercise, you will be creating a phylogenetic
tree for a group of organisms based on morphological characteristics only. In the second part of the
exercise, you will use an online database to generate a phylogenetic tree for the same groups of
organisms using protein sequences. For the third part of the exercise, you will determine what
evolutionary relationships might exist among a group of organisms you choose based on a protein
sequence of your choice. You will also draw a phylogenetic tree for these organisms.
You will obtain your data from a public online database that contains the amino-acid sequences of
proteins coded for by many genes for many different organisms. Hemoglobin, the molecule that carries
oxygen in our bloodstream, is composed of four subunits. In adult hemoglobin, two of these subunits are
identical and coded for by the alpha hemoglobin gene. The other two are identical and coded for by the
beta-hemoglobin genes. The hemoglobin genes are worthy of study themselves, but today we will just
use the protein sequences as a set of traits to compare among species.
Part One: Determining Relationships Among Groups of
Vertebrates Using Morphologies
In this part of the lab, you will view several examples of vertebrate
organisms and determine what characteristics they have in common
with one another. (Note: vertebrates are organisms with a backbone and spinal cord). You will also make
a note of what characteristics distinguish them from one another. To do this, follow the directions given
below:
1. Your teacher has uploaded a document containing images of the sample organisms to the Wiki
page or projected the images on the Smart Board. You need to view the photographs provided
and fill in the chart below.
Species Name
Organism Type (is it a
fish, bird, mammal,
reptile, etc.?)
Similarities Shared
with at Least One
Organism in the List
(provide 3 and identify
the other organism)
Differences From
MOST Other
Organisms on the List
(provide 3)
Homo sapiens (Human)
Pan troglodytes
(Chimpanzee)
Bos Taurus (Cow/bull)
Anser anser anser
(Greylag goose)
Ovis aries (Sheep)
Canis familiaris (Dog)
Gorilla gorilla gorilla
(Gorilla)
Caretta caretta
(Loggerhead sea turtle)
Trematomus bernacchii
(Emerald rockcod)
2. Using the morphological similarities and differences you recorded above, draw a phylogenetic
tree where each “branch” represents one of your organisms. Organisms that are close together
on the tree should be more closely related than organisms that are far apart on the tree. (Note:
Your answers do not need to be “perfect.” However, they must show an understanding of the
connection between morphological similarity and increased evolutionary relatedness.)
Phylogenetic Tree Based on Morphological Characteristics
Part Two: Determining Relationships Among Groups of Vertebrates Using Molecular Data for a
Known Protein Sequence
3. Now you will use a publicly available database known as Uniprot to find molecular information
about each of these organisms. Go to the following website: http://www.uniprot.org and you will
see the following screen:
4. For the first activity you will do, you will search in the database for information about the
hemoglobin beta protein. Hemoglobin beta (HBB) is a subunit of the hemoglobin protein, an
oxygen carrying pigment with four subunits found on red blood cells. In the box labeled “Query,”
type in the search term: HBB. This will search for all information in the database.
5. Because you are trying to locate specific information about the species listed in your table, you
can also search for HBB species name. You may find this proves to be easier when searching.
Once you have located the species you are searching for, check the box out to the left side of its
name so that its amino acid sequence is chosen for alignment.
6. You will notice that when you check the box next to the species names, a green bar appears at
the bottom of the screen, like this:
This bar will contain the records you have selected as well as a few buttons that you will use
during this activity: retrieve, which retrieves the desired protein sequence; align, which aligns
multiple protein sequences when more than one sequence is chosen; blast, which is used to
align nucleic acid sequences; and clear
7. Once you have selected all 9 sequences, click align. You will get a screen that looks like this:
8. Under the section that says “alignment,” you will see multiple protein sequences. Each letter
represents a different amino acid. Some organisms will have very similar sequences and some
will have very different sequences based on their evolutionary history.
9. Scroll down until you see the section that says “Guide tree.” The database has created a
phylogenetic tree based on percentage similarity between the protein sequences for the nine
organisms. You should see something that looks a little like this:
Copy the tree you see into the space below by hand, taking care to replace the accession code
(the numbers at the end of the branches) with the actual species name of the organism.
Phylogenetic Tree Based on Amino Acid Sequences:
Part Three: You Choose!
Now you will choose a protein sequence to align for at least 6 different organisms using the UniProt
database. When choosing organisms, you should choose at least one bacterial species, one plant
species and if available, one fungal species in addition to the animal species you choose. Formulate a
hypothesis about which organisms you think will be most closely related, and which organisms you think
will be most distantly related to one another.
Protein Choices:
Protein Name
Cytochrome C
Gene names
CYTC, Cytc1
Superoxide dismutase
SOD1
Catalase
CAT, kata
Aquaporin
AQP-1, AQP1, AYP1
Cyclin T1
CCNT1
Function of this protein
A cytochrome protein involved in
cellular respiration
An antioxidant enzyme important
in preventing cellular damage by
reactive oxygen species
An enzyme that catalyzes the
breakdown of hydrogen peroxide
to water and oxygen gas
A protein channel that regulates
water flow into and out of cells
A protein that regulates cell
division
Designing Your Investigation:
Protein You Have Chosen
Organisms You Have
Chosen (write the scientific
name)
1.
2.
3.
4.
5.
6.
Hypothesis (does not need
to be in “If, then” format)
Results
10. Use the same method you used in Part 2 to align the sequences for the protein you have chosen
for your six different organisms. Use the “Guide Tree” function to generate the correct
phylogenetic tree based on this protein. Copy the tree you see into the space below, taking care
to replace the accession code (the numbers at the end of the branches) with the actual species
name of the organism.
Phylogenetic Tree Based on Amino Acid Sequences:
Analysis Questions, Part 1: The answers to the following questions should be integrated into the
discussion/conclusion section in your lab report.
1. Based on your morphological analysis, which species are most similar and why?
2. Which species are most dissimilar and why?
Analysis Questions, Part 2: The answers to the following questions should be integrated into the
discussion/conclusion section in your lab report.
1. Which two species above seem to be most closely related based on the phylogenetic tree created
using amino acid sequences? How can you tell?
2. Which two species above seem to be least closely related based on the phylogenetic tree created
using amino acid sequences? How can you tell?
3. How does the predicted phylogenetic tree you filled in based on morphological data differ from the tree
generated from the molecular data (the amino acid sequences)?
4. Do you think the phylogenetic tree generated from the molecular data more accurately represents the
evolutionary relationships between these nine organisms than the phylogenetic tree generated from the
morphological data? Why or why not?
Analysis Questions, Part 3: The answers to the following questions should be integrated into the
discussion/conclusion section in your lab report.
1. Was your hypothesis supported by your data? Why or why not?
***Modified from a lab created by Ms. Lee Ferguson***
Writing Your Mini Lab Report:
Directions: You will be including an introduction section and a discussion/conclusion section in this mini
lab report. You will also turn in your lab packet so that your phylogenetic trees can be evaluated
according to the criteria in the results section of the rubric below. You mini lab report should be titled
“Mini Lab Report: Using Online Molecular Databases to Study Evolutionary Relationships.” This report
should be typed, 12 point font, and double spaced. Make sure to include your name, date, and class
period at the top right corner of the page. Please include “introduction” and “discussion / conclusion”
subtitles so that I know which sections are which. You may find that you need more than one paragraph
to fully meet the requirements for each section. You will have two grades in the grade book. There will
be one grade for the introduction section (weighted as 33% of the assignment) and one grade for the
results and discussion/conclusion sections (weighted as 67% of the assignment).
Rubric:
Section
Introduction
(Science
Practice 3)
Results
(Science
Practices 1
and 2)
Discussion
and
Conclusion
(Science
Practice 5)
Criteria
A. The introduction thoroughly discusses the purpose of the three
parts of the lab (2 points for each part)
B. The introduction clearly outlines a logical hypothesis for Part 3.
C. The introduction connects the material of the course to the lab
activity by providing the reader with key terms (2 points) and
background information (2 points) about classification and
phylogeny.
A. The phylogenetic tree you created in Part 1 demonstrates a clear
understanding of the connection between morphological similarity
and evolutionary relatedness.
B. The phylogenetic trees you copied in Part 2 and Part 3 are
correctly recorded in your lab packet with appropriate labels for
species names.
A. Your discussion section includes information about specific
morphological similarities and differences for your nine organisms
that indicate a close or distant evolutionary relationship. (See
Analysis Questions, Part 1, #1-2)
B. Your discussion section describes how your Part 2 phylogenetic
tree is organized to show close and distant evolutionary
relationships between your nine organisms. (See Analysis
Questions, Part 2, #1-2)
C. Your discussion section identifies differences between your Part 1
and Part 2 phylogenetic trees. (See Analysis Questions, Part 2, #3)
D. Your discussion section identifies the type of phylogenetic tree
(one based on morphological data vs. one based on molecular
sequence data) that most accurately depicts evolutionary
relationships between organisms. Justification is provided for your
choice. (See Analysis Questions, Part 2, #4)
E. Your discussion section clearly states whether your phylogenetic
tree from Part 3 supports or refutes your hypothesis. Specific
evidence from the phylogenetic tree is referenced. (See Analysis
Questions, Part 3, #1)
Teacher Comments:
Point
Breakdown
/6
Total Grade
/2
/4
____/12 = ____%
/4
/2
/4
/4
/2
/4
/4
____/24 = ____%