Section 14
Evolution of Populations
Molecular Lab
BIOLOGY 6.0
1) What morphological features do bats share with fish, with birds, with mammals? Fill in Table 1.
2) Generate a DISTANCE MATRIX for the β-globin polypeptide for two bird species, two bat species,
two non-bat mammal species and one fish species. The fish species and one species from each of the
other groups are already assigned. The second one from each group is your choice. Finish filling in
Table 2 and 3, following the steps listed below.
STEP 1 – Open a new Word document. DO NOT CLOSE THE DOCUMENT. Later you will be copying
your protein sequence data into this document.
STEP 2 – Now go to the web site for the Swiss-Prot data base (http://www.uniprot.org/)
Swiss Prot is a Swiss database of amino acid sequences of many proteins from many species.
STEP 3 – In the “Query” box, type “Hemoglobin HBB” This will give you a list of over 650 organisms
along with their amino acid sequence for the beta hemoglobin gene.
STEP 4 –If you are given a specific organism to search for you, such as in table 1, “haddock” you may
go up to the search query and type in “hemoglobin hbb haddock” and your organism should show up. In
the table where you can choose the organism, refer to your excel spreadsheet to see the options. Once you
find an organism, then you can go and type keywords in from the name into the query box as well. (Make
sure to still keep the “hemoglobin hbb” in the query box.
STEP 5 – Once you locate the organism, click on the blue hyperlink associated with the specific organism.
STEP 6 - This screen contains a lot of information. Scroll down until you reach the section labeled:
“Sequences.” This is the order of amino acids found in the beta hemoglobin protein for that
organism. Above the sequence there is a blue link “FASTA.”
STEP 7 - Use your mouse to copy the information, and paste this into your word processing document.
The copied information will look something like:
>sp|P02087|HBB_DASNO HEMOGLOBIN BETA CHAIN - Dasyous novemclinctus (Three
banded armadillo).
VNLTSDEKTAVLALWNKVBVBHGGZALGRLLVVYPWTQRFFESFGDLSTPAAVFANSKVKAHGKKVLTSFGEGMNHLDN
LKGTFAKLSELHCDKLHVDPENFRLLGNMLVVVMARHFGKEFDHWMHACFQRVVAGVANALAHKYH
STEP 9 - Repeat the above steps until your word processing document contains the FASTA formatted
sequenced for all of the organisms listed in Tables 2.
STEP 10 - Aligning the sequences and determining how similar they are can be done manually but this
is very tedious. Instead, go to the web site listed below that will do this for you in seconds.
http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=lalign
STEP 11 - Copy and paste one sequence from your word-processing sheet into the first sequence box
and another sequence into the second sequence box. Copy ONLY the protein sequence and
not any of the identification information. Make sure you keep track of which two species you
are comparing.
STEP 12 – Diagonal right of the first box, you will see three check boxes. By default the box two check
boxes are checked. Uncheck them, and make sure to then check the “show identity” checkbox.
Click on “Align Sequences.” The computer will return a set of information including “the
percent identity in the 146 aa overlap.” Record that information in Table 3. This value
represents the percent of amino acids that are similar. If all of the amino acids are the
same, the percent similarity is 100%. Not only does Lalign give you the percent similarity, it
also shows you the actual alignment of the two sequences. Identical amino acids are marked
with two dots (:). If there is one dot (.), then the change has been conservative (both amino
acids have similar properties and charge). If there are no dots. then the change has been
non-conservative (the two amino acids have different biochemical properties).
STEP 13 - Continue to make all pair wise comparisons between species and enter these date in Table 3.
For each comparison use the percent identity for the overlap of all the 146 amino acids. You
have now constructed a Distance Matrix Table. This information will be used to answer
questions 1, 2 and 3.
PART II
A friend has read that the ancestors of whales were terrestrial, four-legged, hoofed mammals
(ungulates). This friend looked up some information in an encyclopedia and found that there are two types:
perissodactyl (odd-toed) and artiodactyl (even-toed) ungulates. Your friend is interested in determining
which group might have been the ancestor. Unfortunately, whales do not have feet so one cannot examine
their morphology to figure out which is the more likely scenario. You and your partner are to use
hemoglobin beta chain sequence data to determine which is the more likely.
Repeat the process used in PART 1, but this time construct a distance matrix for one whale, one
fish, two perissodactyl mammals and two artiodcatyl mammals. Fill in Tables 5 and 6. This information will
be used to answer question 4.
PART III
Some phylogenetic systematists contend that the class Reptilia is improper because it does not include
birds. In technical terms they consider the class Reptilia to be PARAPHYLETIC because it contains some,
but not all, of the species that arose from the most recent common ancestor. Many phylogenetic
systematists believe that a group should contain ALL the relatives of the most recent common ancestor (in
technical terms, they believe the group should be MONOPHYLETIC). If the class Reptilia is monophyletic,
then all the reptiles should be more closely related to the crocodile than to any non-reptilian group. If any
non-reptile is more closely related to the crocodile than another reptile, then the group is papaphyletic.
Should birds be considered a type of reptile? You will evaluate this question using the β-globin
chain. You will use a BLAST (Best Local Alignment Search Tool) search. The information obtained from this
analysis is to be entered into Table 7. The steps for obtaining these data are listed below.
STEP 1 - Access the SWISS-Prot database as you did in Parts I and II.
STEP 2 - Find a hemoglobin beta chain for any species of crocodile.
STEP 3 - A BLAST search takes a particular sequence and then locates the most similar sequences in the
entire database. A BLAST search will result in a list of sequences with the first sequence being
closest to the one entered and the last sequences being least similar. The easiest way to do a
BLAST tab found next to the SEARCH tab. Once you click on the blast tab, the amino acid
sequence for that organism will be placed into the box. Simply click the Blast button.
STEP 4 - The next screen will have a list of sequences in order of similarity. List those species in Table 6
beginning with the first most similar species that is not a crocodile until the table is completed.
These data will assist in answering question 5.
Section 14
Evolution
Mole Evolution Computer Lab AS
BIOLOGY 6.0
TABLE 1. ANIMAL COMPARISON (answer + for yes or – for no)
FEATURE
Insects
BIRDS
BATS
.
.
.
Feathers
.
OTHER
MAMMALS
Scales
Hair
Wings
Homeothermy
4-chambered
heart
Mammary glands
TABLE 2. PART I – Species List
SPECIES
COMMON NAME
Fish species
Haddock
SCIENTIFIC NAME
Bat Species # 1
Tomb Bat
Taphozous georgianus
Emperor penguin
Apenodytes forsteri
Mealanogrammus aeglefinus
Bat Species # 2
Bird Species # 1
Bird Species # 2
Other Mammal Species # 1 Atlantic Bottle-nose Dolphin Tursiops truncates
Other Mammal Species #2
TABLE 3. PART I – Distance Matrix (% similarity)
FISH
BAT #1
BAT #2
BIRD #1
BIRD #2
MAMMAL
#1
FISH
100
BAT #1
XXXXXXXXX 100
BAT #2
XXXXXXXXX XXXXXXXXX 100
BIRD #1
XXXXXXXXX XXXXXXXXX XXXXXXXXX 100
BIRD #2
XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX 100
MAMMAL
#1
XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX 100
MAMMAL
#2
TABLE 5. PART II Species List
SPECIES
Whale Species
COMMON NAME
SCIENTIFIC NAME
Fish Species
Odd-toed #1
Equus Caballus
Horse
Odd-toed #2
Ceratotherium simum
White Rhinoceros
Even-toed #1
Ovic aries
Sheep
Even-toed #2
Bos Taurus
Bovine (cattle)
Table 6
WHALE
SPECIES
FISH
SPECIES
ODD-TOE
#1
ODD-TOE
#2
EVENTOE #1
EVENTOE #2
WHALE
SPECIES
100
FISH
SPECIES
ODD-TOED
ODD-TOED
EVEN-TOE
EVEN-TOE
MAMMAL#1 MAMMAL#2 MAMMAL#1 MAMMAL#2
XXXXXXXXX 100
XXXXXXXXX XXXXXXXXX 100
XXXXXXXXX XXXXXXXXX XXXXXXXXX
100
XXXXXXXXX XXXXXXXXX XXXXXXXXX
XXXXXXXXX
100
XXXXXXXXX XXXXXXXXX XXXXXXXXX
XXXXXXXXX
XXXXXXXXX
TABLE 7. PART III BLAST Order
SIMILARITY
First most similar (do not use crocodile)
Second most similar
Third most similar
Fourth most similar
Fifth most similar
Sixth most similar
Seventh most similar
Eighth most similar
100
SPECIES NAME
Ninth most similar
Tenth most similar
Eleventh most similar
QUESTIONS
1. Which two animals in Tables 2 and 3 have the most similar β-globin chains and what is their %
similarity?_______________________________________________________________________________
_____________________________________________________________________________________
2. Which two animals in Tables 2 and 3 have the least similar β-globin chains and what is their %
similarity? ______________________________________________________________________________
_ _____________________________________________________________________________________
3. Do you believe that bats should be classified as mammals? Explain what data in tables 1 and 3 helped in
making that determination
_______________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
4. Is the whale hemoglobin beta chain more similar to that of odd-toed or even-toed ungulates? _________
Was the degree of difference enough that you believe it to be significant? _________________________
Explain. ______________________________________________________________________________
____________________________________________________________________________________
5. Do the data obtained from your BLAST search suggest that Reptilia is paraphyletic or monophyletic?
______________________ Explain the data that helped you most make that decision. ______________
_____________________________________________________________________________________
_____________________________________________________________________________________
6. What is the correlation between amino acid sequences and the evolution of a species?
_______________________________________________________________________________________
_______________________________________________________________________________________
_______________________________________________________________________________________
Reflection: