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Section 14
Evolution of Populations
Molecular Lab
BIOLOGY 6.0
EVOLUTION: A MOLECULAR APPROACH
Evolution is the central unifying phenomenon of all biology. Such an important concept deserves
considerable attention. In this exercise you will use a molecular database to investigate important
concepts/phenomena as you attempt to answer evolutionary questions using molecular data.
How does an evolutionary biologist decide how closely related two species are? The simplest way is
to compare physical features of the species under consideration. This is the COMPARATIVE METHOD. This
method involves comparisons rather then experiments and the researcher neither manipulates nor controls
the conditions. Generally speaking, any study that asks how traits arose among species or how closely
related species are must use the comparative method. Why? Generally speciation (there are exceptions)
cannot be observed in a human lifetime and manipulating conditions so as to affect speciation are difficult
or impossible.
This method is much like comparing two people to determine how closely related they are. One
generally expects that siblings will be more similar to each other than two cousins. If you construct a
pedigree chart, you find that siblings share common parents, but you must look farther back on the tree to
find shared ancestors for cousins. Cousins do not share the same parents; rather, they share some of the
same grandparents. Another way of looking at it is that the common ancestor of two siblings (parents) is
more recent than the common ancestor of two cousins (grandparents). In an evolutionary sense this is why
we say two siblings are more closely related than two cousins.
In a similar way, evolutionary biologists might compare fish, frogs and salamanders and infer that
frogs and salamanders had a more recent common ancestor than frogs and fish. While this methodology
has more merit, it also has its problems. Two very similar looking people are not necessarily related.
Certainly many people have made use of the fact that they look like famous people. Also, you have
probably seen siblings who are so different it makes you wonder about the vagaries of genetics. Likewise, it
is possible that two species with very similar features, attributes, etc. are not closely related.
Imagine that you are responsible for determining which of two of the many Drosophila species are
most closely related. What physical features would you compare? When you run out of physical traits, is
there anything else you could compare? In the past, some might have turned to the fossil record, (if
possible),
behaviors, geographic distribution or ecological factors such as food sources and habitat preference. More
recently, many biologists have turned to genes or gene products (proteins). These more molecular
approaches are not necessarily better than using morphological features except in the sense than using
morphological differences are sometimes due to environmental conditions rather than genetics, while genes
and proteins are definitely genetic. Also, there often are more molecules to compare than physical features.
Usually the best approach is to use data from as many areas as possible.
In this exercise, you will use molecular data to evaluate evolutionary relationships. These data are
available in a public protein database.
Which molecule should you choose? This is not always an easy task. Comparisons can be rendered
meaningless by lateral gene transfer or by convergent evolutionary processes. Comparisons can be
hampered if the same gene or protein is of different lengths in different species or if the molecules are too
variable or not variable enough. In the following exercises you will work with the hemoglobin beta chain (βglobin). As you are aware, normal adult hemoglobin is composed of four subunits. Two of these are
identical and coded for by hemoglobin alpha gene (α-globin). The other two are identical and coded for by
the beta globin gene.
PART I
Some years ago, in the comic strip CALVIN and HOBBS, Calvin was trying to convince his classmates that because bats have wings, they are really birds. In this section you will attempt to refute Calvin.
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://expasy.proteome.org.au/sprot/).
Swiss Prot is a Swiss database of amino acid sequences of many proteins from many species.
STEP 3 – Find the link “Uniprot Web Site”
STEP 4 – In the “Query” box, type “Beta Hemoglobin” This will give you a list of over 1500 organisms
along with their amino acid sequence for the beta hemoglobin gene.
STEP 5 – To quickly search for specific organisms, you may click on the orange download button. I
would place the organisms into an excel spreadsheet by RIGHT CLICKING the “OPEN” hyperlink. Open this
link into either a new tab or a new window, therefore you can refer back to it each time instead of clicking
the back button on the web browser. Expand column “F” so you can see the scientific name and the
common name for all organisms. Find the first organism from table 2 – “Mealanogrammus aeglefinus” on
the spreadsheet. That organism is on row 585 on the excel spread sheet, so if organisms are grouped into
25’s, you can divide 585 by 25 and that should give you an approximate page number of where that
organism is found, which is actually 23.4. Go to page 23 and that organism should be found there. Once
you find the organism, click on the blue link to the left. (If you find an easier way to quickly locate each of
the organisms, by all means do it.)
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 and 3.
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/lalign.htm
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 - 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 pairwise 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
FISH
.
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
MAMMAL
#2
XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX 100
MAMMAL
#2
XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX XXXXXXXXX 100
Sect 14 – Molecular Evolution Computer AS – Page 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
100
SPECIES NAME
Eighth most similar
Ninth most similar
Tenth most similar
Eleventh most similar
Twelfth most similar
QUESTIONS
1. Which two animals in Tables 2 and 3 have the most similar β-globin chains? _______________________
____________________________________________________________________________________
2. Which two animals in Tables 2 and 3 have the least similar β-globin chains? _______________________
_____________________________________________________________________________________
3. Do you believe that bats should be classified as mammals? Explain how 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: