Investigating Polar Bear and Giant Panda Ancestry
(Adapted from Maier, C.A. (2001) “Building Phylogenetic Trees from DNA Sequence Data:
Investigating Polar Bear & Giant Panda Ancestry.” The American Biology Teacher. 63:9, Pages642646.) Adapted for NGWB by Mark Alan Miller January 2009.
Introduction:
This activity will allow you to use the tools used by geneticists and evolutionists located and
maintained by the National Center for Biotechnology Information. These tools will allow you to
compare the isolated and sequenced genes from species that are stored in GenBank. Each unique
sequence is identified by an “accession number” as unique as your social security number. You will
also be able to compare sequences and generate phylogenetic trees using Biology Workbench that
contains specific application such as CLUSTALW (sequence similarity tool), CLUSTALDIST
(generates a genetic distance matrix) and DRAWGRAM (creates phylogenetic trees). The gene
sequence that is being used in this activity is the 12s ribosomal RNA gene sequence from several
bear species and the giant panda. The 12s gene is a good candidate for study because it doesn’t
undergo recombination (not subject to meiosis) because the gene is found on the mitochondrial
chromosome* which is inherited only from the mother via the egg.
* Check out this link for a discussion of the use of mitochondrial DNA and if it really is only from
the mother. Athena Review †Vol.2, no.2: †Recent Finds in Paleoanthropology Molecular clockwork
and related theories http://www.athenapub.com/molclock.htm
Instructions for Accessing 12s rRNA Gene Sequences
PART 1:
a. Open a new web page or new tab and Enter: http://www.ncbi.nlm.nih.gov
b. In the Search bar; select "nucleotide" from the drop down menu
c. Enter the accession number, to obtain each of the following 12s rRNA gene sequences one at a time,
and select “GO” to the right of the bar. The information for the bear will appear in the window, click on the
accession number.
Species
Accession Number
American Black Bear
Y08520
American Brown Bear
L21889
Spectacled Bear
L21883
Asiatic Black Bear
L21890
Polar Bear
L22164
Giant Panda
Y08521
e. Find the bar with “GenBank” to the right of the “Display” use the drop down menu and select "FASTA"
to bring up the sequence in FASTA format.
f. Open a new web page or new tab so you can toggle between the two pages and go to: SWAMI, The
Next Generation Biology Workbench: (http://www.ngbw.org/), set up a free account (if you need help;
http://www.ngbw.org/help/register.htm). Once your registration is complete, you will be logged in to your
personal area.
g. The NGBW allows you to store data and tasks in folders just like MS Outlook, or other a mail clients.
So before working here, you must create at least one folder. Click on the button that says “Create New
Folder”. Name your folder, and save the name. If you get stuck, here is the help link
(http://www.ngbw.org/help/create_folder.htm). The folder will appear on the left side of the screen.
h. Each NGBW folder has a data area and a tool area. For now, we are going to upload some sequences,
so click on the data area icon. It will open the Data management page; now click on the Upload/Enter
Data button.
j. Go back to the NCBI page; highlight the entire FASTA sequence (including the “>”). Copy the
highlighted sequence.
i. Go back the NGBW Data Upload page.
j. In the “label” box, (at the top of the upload form), type in the type of bear.
k. Position the cursor in the top left of the data entry box and “Paste” the sequence. You will need to
replace the FASTA description with the name of the bear, making sure the “>” symbol is still the first
symbol. Use the dropdown boxes at the bottom of the page to tell us what kind of data you are
uploading. Entity Type is “Nucleic Acid”, the Data Type is “Sequence”, and the format is “FASTA”. Select
“SAVE”.
l. Return to GenBank (on the other web page). Copy and Paste the remaining 12s rRNA gene sequences
into the NGBW in exactly the same way.
Instructions Part II: Analyze the 12s rRNA Gene Sequences
1. Align the five different Bear 12s rRNA Gene Sequences (not the panda)
a. In the NGBW, click on the “Tasks” icon in your working folder. When the Task management pane
appear, click on the “Create New Task” Button. Enter a description for the task and click the “Set
Description” button.
b. Now click on the “Select Input Data” button. Check the click boxes to the left of all the bears (not the
panda yet). Click the “Select Data” button.
c. When the Task Creation Pane re-appears, click the “Select Tool” button. From the Nucleic Acids
Sequence Tools tab, choose “CLUSTALW_N” (the tools are alphabetical). Now click the "Save and Run
Task" button.
d. A new page will load that lets you follow the progress of your jobs. Click the “Refresh Tasks” tab near
the top of the page, until the “View Status” button on the right turns into “View Results.” Click on the “View
Results”: tab, and a page showing your results will appear. Click on the link “outfile.aln”, and the results of
your alignment will be exposed. Click the “Save to Current Folder” button.
e. A window will appear that lets you name and specify the kind of data you are saving. Enter a data
Label, then select Entity Type: “Nucleic Acid” Data Type: “Sequence Alignment” and Format: “Clustal”.
Then click the “Save” button.
2. Determine the Genetic Distance Between Sequence Pairs
a. Now go back to the Tasks area of your folder. Click the “Create New Task” button. Give the task a
description and “Set the Description”, just like before. Click the “Select Data” button, and find your
alignment data, check the box to the left of the alignment, and click “Select Data.” When the task creation
page reloads, choose Select Tool, and find “CLUSTALW_DIST” under the Phylogeny/Alignment Tools
tab. When the task creation page appears, click the “Save and Run” button.
b. When the Task management page reloads, use the “Refresh Tasks” button to monitor when the job
completes. When the “View Output” button appears, click on it, and expose the results. Click on the
infile.dst link to expose the Distance Matrix results. Record the distance matrix in your journal to examine
later.
3. Build a Phylogenetic Tree of the Bear Species
a. CLUSTALW_DIST also outputs a phylogenetic tree. Under the “View Output” for CLUSTALW_DIST
click on the infile.ph link. Save this data to your current folder, just like in 1.d and 1.e. But this time chose
Entity Type: “Taxon” Data Type: “Phylogenetic Tree” and Format: “Newick”
b. Now go find this data item in the Data area of your folder, under the tab “Phylogenetic Trees.” Click the
data item, and it will open up, revealing these two links “Show/Hide Data Contents | Draw Tree.” Cllck on
the Draw Tree link, and you will see an interactive view of the Tree. Record the diagram in your journal.
4. Determine the relationship of Giant Panda to the Bear Phylogenetic Tree
a. In GenBank (if you haven’t already) access the giant panda 12s rRNA gene sequence and import it into
the your NGBW Data area.
b. Run a new alignment with all five bears and the panda sequence. Import the alignment into your data
area, use “CLUSTALW_DIST” to create a distance matrix and a phylogenetic tree. Import the
phylogenetic tree into your data area do see the tree. Record all information.
Analysis: (due typed)
1. Create a table showing the distance matrix of the five bears and the panda (Please note that a
distance of 0.00 indicates identical sequences and as the difference in gene sequences increases so
does the number).
2. Provide a phylogenetic tree of the bears and the panda. Label the lines on the tree with the
corresponding distances.
3. Explain and provide support for the conclusions made by the program (Biology Workbench).
4. Red Panda are reported to be more closely related to raccoon. Find the relationship (provide both
the distance matrix and tree) between the Red Panda and raccoons to the others you’ve already
examined. Is the first statement supported?
5. Pick a few animals and run their 12s rRNA patterns to see how they are related. Record this
information.