Biology
Unit 6, Biodiversity/ Evolution, Lab Activity 6-2
In this lab1 you will develop a taxonomic classification and phylogenetic tree for a group of
imaginary organisms called Caminalcules. The purpose of this lab is to illustrate the principles of
classification and some of the processes of evolution. Taxonomists (people classifying organisms) use
various methods to determine which taxa organisms should be placed in. The end product of
classification is a cladogram. The ideal cladogram will reflect the evolutionary relationships between
organisms.
Various characteristics of an organism’s morphology (the structure of an organisms body) are
used to determine relationships between organisms. Animals with four legs are probably more closely
related to each other than to animals with flippers. Morphology can be misleading. Porpoise and tuna
were once grouped together based on their similar morphology. Today we know that they are only
distantly related. To help verify what morphology might suggest as a relationship researchers have
turned more and more to genetic comparisons. By studying the genes and the proteins produced by those
genes we can now get a very clear idea of how closely related a group of organisms are. You will use
both morphology and genetics to determine a cladogram of the fictitious Caminals. We do these
exercises with artificial organisms so that you will approach the task with no preconceived ideas as to
how they should be classified. This means that you will have to deal with problems just as a taxonomist
would. With real organisms you would probably already have a pretty good idea of how they should be
classified and thus miss some of the benefit of the exercise.
Research Question
How does classification show relationships between animals?
Protocol
A
ctivity 1 Classification
Step 1 ) Cladistic Classification of Living Caminalcules based on Morphology
In this step you will produce a cladogram of the living caminals on 11 x 17 inch paper.
1) Cut out and carefully examine the fourteen living species of Caminals and note the many
similarities and differences between them. Use the following characteristics as an initial guide;
Body Shape – Bulb/Long/Oval
Eyes – None/1/2/Stalked
Rear Legs – None/Single/Flipper/Toes
Front Legs – None/Stub/Flipper/Tentacles/Toes
Spotting – Shape/Number/Pattern/Size
1
Caminal figures from ‘Caminalcules’ Exercise by R. Gendron, Indiana University of Pennsylvania
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2) Sort the specimens into like groupings based on body shape. This will give you three different
clades of Caminals. We will call them the sluggers, bulb butts, and ovoids.
3) You will have 19 & 20 (sluggers) in one clade, 16, 1, & 24 (bulb butts) in a separate clade, and
the remaining caminals in the third clade. By definition a clade is a group of organisms that
share a common ancestor. It does not say who the ancestor was, just that they share one. It would
be like two sisters being in a clade. Their mother would be their common ancestor even if they
never knew their mother.
4) Now look at your three clades. Which
two clades are most closely related?
Sluggers
Ovoids
Big Butts
Which two are most distantly related?
Line them up across your table to
show this relationship.
5) Figure 1 shows one way that the three
clades can line up. You may not agree.
That is fine but be prepared to explain
your reasoning. You will notice that
taken together the three clades can be
considered a single clade since all of
Figure 1
the caminals share a common
ancestor.
6) Next sort each of the three clades into
higher level clades based on one of the
other characteristics. An easy clade to set apart is the group that includes 28, 14, & 13. None of
them have eyes and would represent a smaller clade that is part of the ovoid clade. Your
growing cladogram might look like the one shown in Figure 2.
7) Further sort each clade into higher and higher level clades until all of the caminals are separated.
You will have a line running to each caminal when you are done. Ask your teacher to review
your work before you glue the caminals to the 8 ½ x 14 inch piece of paper. Use a pencil to draw
your lines in case you want to change it later.
8) Title your cladogram, "Living Caminal Cladogram." Put your name and class period on it.
Include it with this lab report when you turn in your packet.
Sluggers
Ovoid
eyeless
Ovoid
eyes
Big Butts
Figure 2
Answer Stop question #1-2 before continuing
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EXTRA CREDIT: Make a mobile of your cladogram.
A
ctivity 2 – Phylogeny based on Molecular Evidence
Step 2) Develop a cladogram based on Cytochrome c mutations
1) As a reminder of the process of translation (DNA→mRNA→Protein) use a mRNA code sheet to
determine the amino acid sequence of the following DNA nucleotides. A code sheet may be found at
http://en.wikipedia.org/wiki/Genetic_code
Remember base pairing rules for DNA→mRNA are A to U, T to A, C to G, and G to C
Amino
Glycine
Acid
mRNA 5’G G C
5’
DNA
3’C C G C T A C A G A A G T T T C C T T T C5’
The sequence of nine amino acids you just decoded is the beginning of a much longer polypeptide that
folds into the protein Cytochrome c. Read about Cytochrome c at
http://en.wikipedia.org/wiki/Cytochrome_c
Answer Stop question #3 before continuing
The primary sequences of the 100 amino acids making up the Cytochrome c protein have been
determined for a variety of organisms including all living caminals. The full sequence of each can be
found on your course disk as either a pdf file, Excel file, or as a series of .fasta formatted files (more on
.fasta files in a moment) or on the web on the Falcon Science site under Biology Web Downloads.
Researchers have found that the sequence of amino acids in Cytochrome c varies among organisms due
to periodic mutations in the DNA code. Since mutations accumulate over time it is possible to determine
how closely related different species are by comparing their Cytochrome c sequences. Those with few
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differences in the amino acid sequence had a recent common ancestor while those with a large number of
differences diverged at a more distant time in the past.
Answer Stop questions #4-6 before continuing
2) Compare all of the Caminal Cytochrome c sequences to determine patterns of descent. You can
do this in one of two ways, manually or by using a web based sequence comparison tool.
To compare the sequences manually (this is a lot of work-check out the SHORTCUT) you must;
a) Print several copies of each sequence sheet and compare each Caminal with every other
Caminal.
b) Mark any differences.
c) Count and record the differences in Data Table 1.
d) Once you have completed all comparisons (91 total comparisons) you
6
17
26
can use the information to validate/update the cladogram you
developed based on the morphology of the Caminals. A cladogram
made from this data will use the number of differences in Cytochrome
2
2
c sequences between two Caminals as a measure of the distance
4
between them. (example; The analysis in Figure 3 shows that there
were 4 amino acid differences between species 17 and species 26. This
2
provides a distance measurement for our cladogram . It is 4 mutations
from Caminal 17 to Caminal 26. When we compare Caminal 6 to
Caminal 26 we find 8 mutations have occurred .)
Follow the link;
Figure 3
< http://web.virginia.edu/Heidi/chapter5/Images/8883n05_29.jpg>
To see a cladogram made from a Cytochrome c analysis.
Answer Stop question #7 before continuing
Ninety-one comparisons of 100 amino acid polypeptides is a lot of work. You can; a) do it
yourself, b) split the work up among several of your classmates, or c) do it the easy way by using a
computer based tool developed specifically to compare protein sequences.
SHORTCUT
Using BLAST to compare multiple amino acid sequences
The U.S. government maintains NCBI (The National Center for Biotechnology Information) with
web access open to anyone. The following instructions will walk you through using the NCBI
website to compare your caminal sequences. You may also view the tutorial, ‘Comparing Protein
Sequences using BLAST’, located on your course disk or on the Video Tutorials link of the Falcon
Biology Home page.
1) Go to <http://blast.ncbi.nlm.nih.gov/Blast.cgi> to begin your analysis
2) Scroll down to <Basic BLAST> and select <protein blast>
3) Beneath the entry boxes on the new page click the <Align two or more sequences> box. A
second set of data boxes will appear.
4) Click on the <Choose File> button below the first set of data boxes and navigate to where the
.fasta files that contain the Cytochrome c sequences for the caminals is located. This may be
on your course disk or at some other location if you downloaded the files from the school
website. In both cases they are in a folder called <CaminalculeCytochromeSequences>.
5) Select the amino acid sequence file for the Caminal you want to analyze. After clicking on the
file you will be returned to the BLAST search page.
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6) Scroll down to the <Choose File> button in below the second set of data boxes and
Navigate to, then select, the .fasta file, “Caminalcule AADatabase.” After clicking on the
database file you will again be returned to the BLAST search page.
7) Scroll down to the
button and select it. The selected sequence will be
automatically compared to all of the sequences of the other living Caminals. Scroll down to
the box titled, “Sequences producing significant alignments:”.In the, “Ident” is the number
of matching amino acids. Subtract that number from 100 to obtain the number of differences.
Record the differences in Data Table 1.
Once you have compared all of the different Caminals you can have the BLAST sequencer produce a
phylogenetic analysis of the data as follows;
9) At the top of any of your amino acid comparisons select [Distance tree of results]. A
phylogenetic tree will be produced. Select <slanted> above the tree display to change the style of
the tree. Use a screen capture tool to paste the cladogram into a word processor for printing.
Turn your BLAST cladogram in along with the cladogram you produced by comparing caminal
morphology.
10) Compare this BLAST cladogram with the Morphology cladogram you produced in Activity 1.
11) Produce a third cladogram that is a combination of the cladogram you produced based on
morphology (Activity 1) and the one produced by comparing the Cytochrome c sequences
(Activity 2). Include all three cladograms in your lab report packet.
Data
Table 1 Cytochrome c Analysis
1
2
3
4
9
12
1
2
3
4
9
12
13
14
16
19
20
22
24
28
13
14
16
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20
22
24
28
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Stop Questions
1) What defines a clade? _____________________________________________________________
2) What do the nodes of the cladogram represent? _________________________________________
3) What function that is carried out by all cells is Cytochrome c involved in? (hint: we studied this
process in unit 2) ___________________________________________________________________
4) Changes in the order of amino acids are due to periodic mutations in the coding sequences of the
protein. The first amino acid in the sequence you just decoded is glycine. What would happen if the
RNA sequence that coded for glycine was mutated from GGC to GUC?
____________________________________________________________________________________
5) What would happen if the mutation was from GGC to GGU? ________________________________
6) Do all mutations cause a change in polypeptide sequences? ___________________________
7) Using the example in figure 3, page 4; how many mutations are there between Caminal #6 and #17?
______ ? Is Caminal #6 more closely related to #17 or to #26? __________. Explain your reasoning (it
is this explanation that will earn you credit).
___________________________________________________________________________________
Analysis Questions
The analysis for this lab includes;
1) Your cladogram (11 x 17 sheet) based on Caminal morphology.
2) Your Cytochrome c Analysis Data Table 1
3) Your printed Blast Cytochrome c cladogram.
4) Your combined Morphological / Cytochrome c cladogram.
Conclusion
1)
2)
3)
4)
You have just used two methods to determine the phylogeny of a group of living
organisms. They almost never agree. Explain why the two methods do not always
agree. _______________________________________________________________
Which method do you have the most confidence in? __________________________
Explain your reasoning. _________________________________________________
____________________________________________________________________
Why do you have more confidence in the method you chose (#2 above)?
___________________________________________________________________
Which method would you use to determine the relationships between living and
fossil Caminals? __________________ Explain your reasoning ________________
____________________________________________________________________
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Caminal Cytochrome c sequence Sheet #1
1
2
3
4
5
0
0
0
0
0
1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0
2 Z Z Z Z Z Z Z Z G D V F K G K K I S I M K C S H C K T E E K G G K Q V T G P N L H Y L F G R K T G Q
0
1 Z Z Z Z Z Z Z Z G D V E K G K P I S I M K G S Q C K T E E K G G K Q V T G P N L H G L F H R K K G Q
9
9 Z Z Z Z Z Z Z Z G D V C K G G K I S I M K C S P C H N V E K K F K H K C G P N L H G L F V WG T G Q
4 Z Z Z Z Z Z Z Z G D V E C N G K I S D M K C S P C H N E D K G C K H A C P P N L H G L R G L K V G Q
3 Z Z Z Z Z Z Z Z G D V E C N G K I S D M K C S P C H N E D K G C K H A C P P N L H G L R G L K V G Q
2 Z Z Z Z Z Z Z Z G D V E C N G K I S D M K C S P C H N E V K G G K H A C G P N L H G L F G L K V G Q
2
1 Z Z Z Z Z Z Z Z G D V E C G G K I S I M K C S P C H N E V K G H K H K C G P N L H G L F G L K V G Q
2
2 Z Z Z Z Z K Z Z G D V E K G G K I S I M L C S P C H N E E K G G K H K C G P N L H G L F G L K V G Q
1 Z T Z D M Z A Z G D V E K G G K I S I M K C S P A H N K E K G G K H K C G P N L H G L F G L K V G Q
6
2 Z Z Z D M Z A Z G D V E K G G K I S I M K C S P A H N K E K G G K H K C G P N L H G L F G L K V G Q
4
1 Z T Z D M Z Z Z G D V E K G G K I S I M K C S P C H N K E K Q G K H K C K P N L H G L F G L K V G Q
1 Z Z Z Z Z Z Z L G H V P L G N K I F I M K C S Q K I T V A K G G K H K T T P N L H G L F G S K T G Q
4
1 Z Z Z Z Z I Z Z G D V K L G K T I F I M K C M Q C H T V M P G G K H L Q G I N L H H L F G S K T G Q
3
2 Z Z Z P Z Z Z Z G D P Q L G K T I F I M K C M A S H T V E K G G H H I T G T N L H G P A V S K T G Q
8
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Caminal Cytochrome c sequence Sheet #2
6
7
8
9
0
0
0
0
0
0
1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0
2 S Q G Y S Y T A A N T V K G L I S G E D T L M E Y L E N P K K Y I WA T K M I F P M I K K N E R A D
0
1 A P G Y MY T A A N T V K G I K C G E D T L M E Y L E N P K K Y I P A C K M I F P M I K K N E R A D
9
9 A P G Y S Y T A A N MN K G Q Y WG E D T L M E Y L E N P K K Y N A D T K M I F V G I K K I E R A D
4 A P G Y R C P A A N K N H G I I WS E D T L M E Y L E N P K Q Y I P G K Q M I F A G N K K E E R A D
3 A P G Y R C P S A N K N H G I I WS E D T L M E Y L E N P K K Y I P G K Q M I F A G N K K E E R A D
2 A P G Y R C P Y A N K N H G I I WS E D T L M E Y L E N P K K Y I P G K Q M I F A G N K K E E R A D
2
1 A P G Y R Y P Y A N K N H G I I WS E D T L M E Y L E N P K K Y I P G K Q M I F A G N K K E E R A D
2
2 A P G Y R Y P Y A N K N H G I I WS E D T L M E Y L E N P K K Y I P G K Q M I F A G N K K E E R A D
1 A P WY S Y T A A N K N H G I I WS I D T L M E Y L E N P K K Y I P G T K Q I F A G N K K E E R A D
6
2 A P G Y S Y T A A N K N H G I I WS I D T L M E Y L E N P K K Y I P G T K Q I F A G N T K H E R A D
4
1 A P WY S Y T A A N K N H G I I WS E D T L M E Y L E N P K K Y I P G T K Q I F A G N K K E E R A D
1 A P G Y S Y T A A N K N K G I I WA E D T L M E Y L E N P K K A C E G T K M I F D WI K K E E R A D
4
1 A P G Y S Y T A A N K N K G I E WA E D T L M E Y L E N P K K Y S R L T K M I F V G M WK E E R A D
3
2 A P G Y S Y T A A N K N K G I I WA E D T L M E Y L E N P K K Y I P K T K M I F I G I K K L E R A D
8
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