Constructing Phylogenies
Introduction
This case study is
designed to give students some experience with protein data bases and the
analyses of such data and is based upon and largely excerpted
from an article by Bilardello and Valdes (1998).
Phylogenies or evolutionary histories of organisms help in classification,
show possible evolutionary relationships, and are based on a variety of evidence,
including fossils, morphology of living organisms, embryology, and molecular
traits. Among the latter are the base sequences in DNA and RNA and amino acid
sequences for homologous proteins in different organisms. The purpose of this
case study is to constructing a phylogeny using molecular traits, and then
compare this to a phylogeny based on morphology..
When using molecular trait data to develop phylogenies, differences in the sequence of DNA/RNA bases or of amino acids of a specific protein are determined. This information is available on the internet at www.expasy.ch. Swiss-Prot is a protein sequence database. The number of differences indicates the evolutionary distance between the organisms being investigated. For example, the greater the number of differences, presumably the more distantly related, are the organisms, i.e. their common ancestor existed longer ago than did the common ancestor of two organisms with fewer differences in their amino acid sequences.
For example, the amino acid sequence in cytochrome c for five different organisms is presented in the following table:
|
Human |
GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP
NLHGLFGRKT GQAPGYSTYTA ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE
RADLIAYLKK ATNE |
|
Horse |
GDVEKGKKIF VQKCQCHTV EKGGKHKTGP
NLHGLFGRKT GQAPGFTYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE
REDLIAYLKK ATNE |
|
Equas |
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP
NLHGLFGRKT GQAPGFSYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE
REDLIAYLKK ATNE |
|
Mouse |
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP
NLHGLFGRKT GQAAGFSYTD ANKNKGITWG EDTLMEYLEN PKKYIPGTKM IFAGIKKKGE
RADLIAYLKK ATNE |
|
Dog |
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP
NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKTGE
RADLIAYLKK ATKE |
|
|
D:
aspartic W: tryptophan H: histidine K: lysine
R: arginine F:
phenylalanine I:
isoleucine L: leucine V: valine Y: tyrosine M: methionine A: alamine C:
cytosine Q: glutamine
N: asparagines E: glutamic
acid P: proline S: serine T: threonine
G: glucine |
To develop a phylogeny of the five species, you must first develop a similarity matrix illustrating the similarities in amino acid sequences, as shown below.
|
Similarity matrix |
|||||
|
|
Hu |
Ho |
E |
M |
D |
|
Hu |
100 |
88 |
89 |
91 |
89 |
|
Ho |
|
100 |
99 |
94 |
94 |
|
E |
|
|
100 |
95 |
95 |
|
M |
|
|
|
100 |
96 |
|
D |
|
|
|
|
100 |
There is a website (/http://clustalw.genome.jp/) which helps you to do this. It is an alignment tool. Next a phylogenetic tree is developed from this information.
Figure 1. Dendrogram based on similarities in amino acid sequences.
Project
1. Use netscape to find the amino acid sequences of the protein hemoglobin a for the following species:
HBA_ALLMI american alligator
HBA_AMMLE Barbary sheep
HBA_ATEGE Black-handed spider monkey
HBA_DANRE zebrafish
HBA_HETPO Port Jackson shark
HBA_Human human
HBA_LATCH coelacanth
HBA_TARGR roughskin newt
Choose two more species from different groups (e.g. a bird)
2. Create a similarity matrix for hemoglobin a for the ten species.
To do this, open the web site for ExPASy, type in the name of the protein you wish to compare at the top of the page in the cell following Search Swiss-Prot/TrEMBL for ___________. In our case this will be hemoglobin a. Then select Go. The list of sequences produced is long and may seem confusing. For our species, go down to, for example, HBA_HUMAN. Click on the sequence. This brings up the sequence entry. Scroll down to the sequence information. Click on FASTA format. You need to copy the sequence (the entire sequence) in this FASTA format and then go to CLUSTALW
(http://clustalw.genome.jp/. Use another browser for this) and paste the sequence into the window
provided. You need to do this for the other nine species. Then execute the multiple alignment by hitting the execute multiple alignment button at the bottom of the page. A CLUSTALW results form will be presented.
| |
Hu |
Ho |
E |
M |
D |
| Hu |
0 |
17 |
20 |
20 |
23 |
| Ho |
|
0 |
3 |
22 |
26 |
| E |
|
|
0 |
24 |
28 |
| M |
|
|
|
0 |
26 |
| D |
|
|
|
|
0 |
You can then develop a tree using these results by going to the bottom if the CLUSTALW page to the select tree menu, select dendrogram, and execute. The branching tree developed will be based upon the differences in the amino acid sequences. Compare this to the method used above. Are your results the same?
3. Show the results of your evolutionary phylogeny based upon these differences or similarities in heloglobin a amino acid sequences..
4. Compare this to the development of cladograms in chapter 23 of your text by developing your own cladogram based upon morphological features.
5. Does your molecular cladogram compare to a cladogram you developed based upon morphological features? Explain.
6. Submit this as a report using WORD.
7. You must work with a partner for this project and each of you independently fill out and hand
in a Peer Evaluation Form
8. Grading:
Table 1 = 3 points
Table 2 = 3 points
Table 3 = 3 points
Table 4 = 3 points
Figure 1 = 3 points
Discussion = 3 points
Peer evaluation forms (2) = 7 points
Project due March 19at 11:00 AM for section 2 and 2:00 PM for section 1. Minus 25% each day late.
References
Bilardello, N. and Valdes, L. 1998. Constructing phylogenies. The American Biology Teacher 60 (5):
369-373.