Investigating Bear and 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, Pages 642-646.
We have already used some molecular databases to examine evolutionary relationships among reptiles,
bats, and birds, as well as conducting our own studies of evolutionary relationships. We have used
amino acid sequences for proteins to examine these questions. We will now go “one level deeper” as we
explore DNA sequences and how these can also be used to create evolutionary relationships.
You might wonder, “how do I decide if to use protein or DNA sequence data, and for which sequences?”
The answer is to use as many resources as you have. You may not obtain the SAME result when using
different resources. That is, a phylogenetic tree based on hemoglobin amino acids may not look the
same as a tree based on DNA encoding rRNA. Likewise, trees based on morphology may not agree with
molecular based trees. But scientists look at all these data, and draw conclusions based on overlaps
and consistent patterns between the various data sources.
For this exercise you will investigate these questions: 1) “What is the relationship between the polar bear
and the American Brown bear?” The geographic ranges of these two species are in close proximity. Did
the polar bear evolve from a population of American brown bear that were isolated far north (allopatric
speciation)? 2) How about the giant panda? This species superficially resembles the bear family and
yet its diet, behavior, and genital structure are quite different (Morris and Morris, 1981). 3) Finally, red
pandas may not be named appropriately. Although facial features look “panda-ish,” the life history is not
that much like a panda and the tail certainly isn’t bear-ish. Is the giant panda a bear or did it branch from
some other lineage? We will address these questions by building genetic distance matrices and
phylogenetic trees based on molecular sequence data.
It is possible to create a molecular phylogenetic tree of the panda and bear families because the 12s
ribosomal RNA gene sequences from several bear species and the giant panda are available from
GenBank (a molecular database). Since the 12s ribosome gene is carried on the mitochondrial
chromosome, it does not undergo recombination and is inherited directly from mother to offspring
through the egg. These characteristics make mitochondrial genes excellent tools for studying the family
trees of closely related species (Cann et al, 1987).
You will be performing two sets of analyses. First, you will look at several bear species and examine their
DNA (and therefore evolutionary relationships). Next, you will include the giant panda and determine its
relationship to other bear species. From your findings, we will discuss whether the data support the giant
panda and red panda being classified among the bears, or whether they belong in separate groups.
Procedure:
These are the steps to access bear and giant panda 12s rRNA sequence from GenBank, import them
into Biology workbench, then use Biology Workbench nucleic acid tools to align chosen sequences,
create genetic distance matrices, and develop phylogenetic trees. Because you have experience
searching for sequences by name, we have provided you with accession numbers to make things go
faster (see table on the top of page 2).
1. Access the 12s rRNA sequences and import them into sequence analysis software. Go to
www.ncbi.nlm.nih.gov. Select “Nucleotide”, enter the accession number for the following 12s
rRNA gene sequences one at a time, and select “GO.”
1
Animal
12s rRNA Accession Number
American Black bear
Y08520
American Brown bear
L21889
Spectacled bear
L21883
Asiatic black bear
L21890.1
Polar bear
L22164
Giant panda
Y08521
Red Panda
S80939.1
Dog
Y08507.1
Raccoon
Y08510.1
Crocodile skink
AY169595
Cow
AY676873
NOTE: you can access other 12s rRNA sequences by searching using
the terms “12s rRNA AND Critter-of-choice NOT complete” (the NOT is
used because for some organisms the ENTIRE mitochondrial DNA is on
record and you would have to search for the 12s rRNA gene)
2. Click on the accession number.
3. Next to the “Display” window, select “FASTA” to bring up the gene sequence in FASTA format.
4. Highlight and copy the entire FASTA sequence, up to and including the initial “>” symbol.
Paste into a WORD document.
5. Repeat steps 2-4, finding, displaying, highlighting, copying and pasting until all 12s rRNA sequences
have been found.
6. SAVE your WORD document!!!
7. From the WORD menu, under FILE, select SAVE AS. In the resulting window, look for the format
option, then select TEXT ONLY (the default is a formatted WORD document with hidden codes),
then click on SAVE. This step saves your sequences as a simple text file, which is preferred by most
bioinformatics application. The hidden formatting commands in WORD often cause errors in
sequence analysis. You should now have two copies if your 12s rRNA sequences; one as a word
document (file name ends in .doc, such as bears.doc) and one as a text document (file name ends in
.txt, such as bears.txt).
7. Go to Biology Workbench. http://workbench.sdsc.edu (Notice: NO www)
8. Select “Session” then “Start New Session,”
9. Name the session then select Start
10. Select “Nucleic Tools”
11. Select “Add New Nucleic Sequence” then click on “RUN”
12. In the Label box, name the session then click on RUN
13. Now you could toggle back and forth from NCBI to WORKBENCH and copy and paste FASTA
sequences but you saved them all in a WORD document, so let’s explore another way to add
sequences to WORKBENCH. Click on “Browse” and find your TEXT document (bears.txt), select it,
then select “Upload File.” Just like magic, your sequences will all end up in separate windows. IF
they don’t, you have probably selected the bears.doc instead of bears.txt.
14. Edit the FASTA label in the “Sequence” box (the box containing all the A, T, C, and G). Leave the >
but delete all but the name of the critter – in other words the names you want to appear on the tree.
15. Select “SAVE.” Now you have a session with a bunch of sequences, all individually listed. You can
select two or more as you need them.
Align the Bear 12s rRNA Gene Sequences.
1.
2.
3.
4.
For now, select all the bears but omit the raccoon, dog, and skink.
Choose CLUSTALW then “RUN” then “SUBMIT”.
Copy and Paste the Phylogram into a WORD Document. This is data you will be turning in.
Click on “IMPORT ALIGNMENT” to enter the Alignment Tools section of the Biology Workbench.
2
Determine the Genetic Distance between sequence pairs.
1. Click on the box to the left of the imported and aligned sequences, then choose “CLUSTALDIST,”
“Run”, and “SUBMIT.” Print the distance matrix so that you can examine it closely later.
2. Copy and paste the “Clustal Distance Matrix” table into a WORD document. This is also data!! You
will examine it and turn it in.
3. The last piece of data that could be helpful in the future (so you should include) is a BOXSHADE.
You can also copy and paste this into a WORD document.
Repeat the alignment, phylogram, and matrix after including the dog and raccoon, then again
after adding the cow and the skink.
Analysis Questions. Answer these questions after analyzing the data you obtained from
alignments, matrices, and phylograms..
1. What are we trying to show by constructing phylograms, matrices and sequence alignments?
2. As you examine the phylogenetic tree of the bear species’ 12s rRNA gene sequences, which
two are most closely alike? Which “pair of bears” seems to be the most distantly related?
3. Which are closer relatives; “American Brown and American Black Bears” or “American Black
and Asiatic Black Bears”? Explain your answer referring to your data.
4. Is the giant panda more of an “American” relative or “Asiatic” relative as compared to the bear
group? Explain.
5. Further research the spectacled bear. Where is it found today? Explain its possible
evolutionary origin.
6. Does the Giant Panda “belong” in the bear family at all? Explain your answer.
7. Where does the Red Panda fit into the phylogenetic tree? Who are its closest relatives?
8. Why did we include a cow and a crocodile skink in the final phylogram? What other organisms
could have served the same purpose?
9. Finally, if you were a taxonomist, how would you answer this question: What is a bear? Is a
bear a dog or is a dog a bear?
Include with your answers, your data, phylograms and any outside research that you used to
answer these questions.
3
TEACHERS GUIDE
Examining Phylogeny of Bears using Biology Workbench.
Sequences Used to prepare this key.
>American Black-gi|1871572|emb|Y08520.1|MIUA12SR U.americanus mitochondrial 12S rRNA gene
CAAAGGTTTGGTCCTGGCCTTCCTATTAGCTGCTAACAAGATTACACATGTAAGTCTCCGCGCCCCAGTG
AAAATGCCCTTTGGATCTTAAAGCGGTTCGAAGGAGCGGGCATCAAGCACACCTCTCCCCGGTAGCTTAT
AACGCCTTGCTTAGCCACACCCCCACGGGATACAGCAGTGATAAAAATTAAGCTATGAACGAAAGTTTGA
CTAAGCTATGTTGATCAAAGGGTTGGTTAATTTCGTGCCAGCCACCGCGGTTATACGATTGACCCAAGTT
AATAGGCCCACGGCGTAAAGCGTGTGAAAGAAAAATTTTTTCCCCACTAAAGTTAAAGTTTAACCAAGCT
GTAAAAAGCTATCGGTAACACTAAAATAAACTACGAAAGTGACTTTAATACTCTCAACCACACGACAGCT
AAGATCCAAACTGGGATTAGATACCCCACTATGCTTAGCCTTAAACATAAGTAATTTATTAAACAAAATT
ATTCGCCGGAGAACTACTAGCAACAGCTTAAAACTCAAAGGACTTGGCGGTGCTTTAAACCCCCCTAGAG
GAGCCTGTTCTATAATCGATAAACCCCGATAGACCTCACCACCTCTTGCTAATCCAGTCTATATACCGCC
ATCTTCAGCTAACCCTTAAAAGGAGTAAAAGTAAGCACAATCATCCCACATAAAAAAGTTAGGTCAAGGT
GTAACCCATGGGGTGGGAAGAAATGGGCTACATTTTCTATTCAAGAACAACCTACGAAAGTTTTTATGAA
ACTAAAAACTAAAGGTGGATTTAGCAGTAAACCAAGAATAGAGAGCTTGGTTGAATAGGGCAATGGAGCA
TGCACACACCGCCCGTCACCCTCCTCAAGTGGCACAAGTCAAATATAACCTATTGAAATTAAATAAAACG
CAAGAGGAGACAAGTCGTAACAAGGTAAGCATACTGGAAAGTGTGCTTGGATAAAC
>American Brown Bear gi|418058|gb|L21889.1|URSMTRG12S Ursus arctos mitochondrial 12S ribosomal
RNA (12S rRNA) gene fragment
AAATAATTCATTAAACAAAATTATTCACCAGAGAACTACTAGCAACAGCTTAAAACTCAAAGGACTTGGC
GGTGCTTTAAACCCTCCTAGAGGAGCCTGTTCTATAATCGATAAACCCCGATAGACCTCACCACCTCTTG
CTAATTCAGTCTATATACCGCCATCTTCAGCAAACCCTTAAAAGGAACAAGAGTAAGCACAATCATCTTG
CATAAAAAAGTTAGGTCAAGGTGTAACCCATGGGGTGGGAAGAAATGGGCTACATTTTCTATTCAAGAAC
AACTTACGAAAGTTTTTATGAAACTAAAAACTAAAGGTGGATTTAGTAGTAAATCAAGAATAGAGAG
>Spectacled Bear gi|418052|gb|L21883.1|TMCMTRG12S Tremarctos ornatus mitochondrial 12S
ribosomal RNA (12S rRNA) gene fragment
AGATAATTTACCAAACAAAATTATTCGCCAGAGAACTACTAGCAATCGCTTAAAACTCAAAGGACTTGGC
GGTGCTTTAAACCCCCTAGAGGAGCCTGTTCTATAACCGATAAACCCCGATAAACCTCACCACCCCTTGC
TAATCCAGTCTATATACCGCCATCTTCAGCGAACCCTTAAAAGGAAAAAAAGTAAGCATAATTATCTCAC
ATAAAAAAGTTAGGTCAAGGTGTAACCTATGGGATGGGAAGAAATGGGCTACATTTTCTACTCAAGAATA
GTCTACGAAAATTTTTATGAAACTAAAAACTAAAGGCGGATTTAGTAGTAAACTAAGAATAGAGAG
>Asiatic black bear gi|418045|gb|L21890.1|SNAMTRG12S Selenarctos thibetanus mitochondrial 12S
ribosomal RNA (12S rRNA) gene fragment
AAATAATTTATCAAACAAAATTATTCGCCAGAGAACTACTAGCAACGGCTTAAAACTCAAAGGACTTGGC
GGTGCTTTAAACCCCCCTAGAGGAGCCTGTTCTGTAATCGATAAACCCCGATAGACCTCACCACCCCTTG
CTAATCCAGTCTATATACCGCCATCTTCAGCGAACCCTTAAAAGGAAAAAGAGTAAGCACAATCATCTTG
CATAAAAAAGTTAGGTCAAGGTGTAACCCATGGGATGGGAAGAAATGGGCTACATTTTCTATTCAAGAAC
AACCTACGAAAGTTTTTATGAAACTAAAAACTAAAGGTGGATTTAGTAGTAAACCAAGAATAGAGAG
>Polar bear gi|418048|gb|L22164.1|TLTMTRG1SA Thalarctos maritimus mitochondrial 12S ribosomal
RNA gene fragment AGATAATTTACCAAACAAAATTATTCGCCAGAGAACTACTAGCAACCGCTTAAAACTCAAAGGACTTGGC
GGTGCTTTAAACCCCCTAGAGGAGCCTGTTCTATAACCGATAAACCCCGATAAACCTCACCACCCCTTGC
TAATCCAGTCTATATACCGCCATCTTCAGCGAACCCTTAAAAGGAAAAAAAGTTAGCATAATTATCTCAC
ATAAAAAAGTTAGGTCAAGGTGTAACCTATGGGATGGGAAGAAATGGGCTACATTTTCTACTCAAGAACA
GTCTACGAAAATTTTTATGAAACTAAAAACTAAAGGTGGATTTAGTAGTAAACTAAGAATAGAGAG
>Giant Panda gi|1871553|emb|Y08521.1|MIAM12SR A.melanoleuca mitochondrial 12S rRNA gene
TAAAGGTTTGGTCCTAGCCTTCCTATTAGCCATTAACAAGATTACACATGTAAGTCTCCACGCTCCAGTG
AAAATGCCCCTTAAGTCCTCTTAGACGACCTAAAGGAGCGGGTATCAAGCACACCTTATGGTAGCTCACA
ACGCCTTGCTTAGCCACACCCCCACGGGAAACAGCAGTGATAAAAATTAAGCTATGAACGAAAGTTCGAC
TAAGCTATGTTAATACTAGGGTTGGTAAATCTCGTGCCAGCCACCGCGGTCATACGATTAACTCGAGTTA
ATAGGCCTACGGCGTAAAGCGTGTAAAAGAAAAAATCTCCTCTACTAAAGTTAAAGTATGATTAAGCTGT
AAAAAGCTACCATTAATACTAAAATAAACTACGAAAGTGACTTTAAAATTTCTGATTACACGATAGCTAG
GGCCCAAACTGGGATTAGATACCCCACTATGCCTAGCTCTAAACATAGATATTTTACTAAACAAAACTAT
TCGCCAGAGAACTACTAGCAACAGCTTAAAACTCAAAGGACTTGGCGGTGCTTTATATCCCCCTAGAGGA
GCCTGTTCTGTAATCGATAAACCCCGATAGACCTCACCATCCCTTGCTAATTCAGTTTATATACCGCCAT
CTTCAGCAAACCCTTAAAAGGAAAAAAAGTAAGCATAACTACCCTACATAAAAAAGTTAGGTCAAGGTGT
AACCTATGGGCTGGGAAGAAATGGGCTACATTTTCTATTCAAGAACAACTTCTACGAAAACTTTTATGAA
ACTAAAAGCTAAAGGCGGATTTAGTAGTAAATTAAGAATAGAGAGCTTAATTGAACAGGGCAATGAAGCA
CGCACACACCGCCCGTCACCCTCCTCGAGTGATATAATTTAATTATAACCTATTTAAACTAAGCAAAGCA
TAAGAGGAGACAAGTCGTAACAAGGTAAGCATACTGGAAAGTGTGCTTGGATGAGC
>Red Panda
gi|1911545|gb|S80939.1| 12S rRNA [Ailurus fulgens=red pandas, Mitochondrial, 349
nt] AAAACTATTCGCCAGAGAACTACTAGCAATAGCCTAAAACTCAAAGGACTTGGCGGTGCTTTACACCCCT
CTAGAGGAGCCTGTTCTATAATCGATAAACCCCGATAAACCTTACCACTTCTAGCTACTTCAGTCTATAT
ACCGCCATCTTCAGCAAACCCTCAAAAGGAAGCAAAGTAAGCATAATAATCCCTGCATAAAAAAGTTAGG
TCAAGGTGTAACCCATGAAGTGGAAAGAAATGGGCTACATTTTCTAAACAAGAACACTATACGAAAATTT
TTATGAAATTAAAACCTAAAGGTGGATTTAGTAGTAAATTAAGAATAGAGAGCTTAGTTGAATTGGGCT
4
>Dog gi|1871555|emb|Y08507.1|MICF12S Canis familiaris mitochondrial 12S rRNA gene
TAAAGGTTTGGTCCTAGCCTTCCTATTAGTTTTTAGTAGACTTACACATGCAAGCCTCCACGCCCCAGTG
AGAATGCCCTTAAAATCACCAGTGATCTAAAGGAGCAGGTATCAAGCACACTCTTAAGTAGCTCATAACA
CCTTGCTAAGCCACACCCCCACGGGATACAGCAGTGATAAAAATTAAGCCATAAACGAAAGTTTGACTAA
GCCATACTAAATAGGGTTGGTAAATTTCGTGCCAGCCACCGCGGTCATACGATTAACCCAAACTAATAGG
CCTACGGCGTAAAGCGTGTTCAAGATATTTTTACACTAAAGTTAAAACTTAACTAAGCCGTAAAAAGCTA
CAGTTATCATAAAATAAACCACGAAAGTGACTTTATAATAATCTGACTACACGATAGCTAAGACCCAAAC
TGGGATTAGATACCCCACTATGCTTAGCCCTAAACATAGATAATTTTACAACAAAATAATTCGCCAGAGG
ACTACTAGCAATAGCTTAAAACTCAAAGGACTTGGCGGTGCTTTATATCCCTCTAGAGGAGCCTGTTCTA
TAATCGATAAACCCCGATAAACCTCACCACCTTTCGCTAATTCAGTCTATATACCGCCATCTTCAGCAAA
CCCTCAAAAGGTAGAACAGTAAGCACAATCATTTTACATAAAAAAGTTAGGTCAAGGTGTAACTTATGAG
GTGGGAAGAAATGGGCTACATTTTCTACCCAAGAACATTTCACGAATGTTTTTATGAAATTAAAAACTGA
AGGAGGATTTAGTAGTAAATTAAGAATAGAGAGCTTAATTGAATAGGGCCATGAAGCACGCACACACCGC
CCGTCACCCTCCTCAAGTAATAAGACACAACCATAACCATATTAACTTAACTAAAACACAAGAGGAGACA
AGTCGTAACAAGGTAAGCATACCGGAAGGTGTGCTTGGATTAAT
>Raccoon gi|1871568|emb|Y08510.1|MIPL12SR P.lotor mitochondrial 12S rRNA gene
TAAAGGTTTGGTCCTGGCCTTCCTATTAGTTCTTGACAAATTTACACATGCAAGTCTCCACATCCCAGTG
AAATATGCCCTCCAAATCACTCCAATGATTAAAAGGAGCGGGTATCAAGCACACTAATACTAGTAGCTCA
CGACACCTTGCTCAGCCACACCCCCACGGGATACAGCAGTGATAAAAATTAAGCCATGAACGAAAGTTCG
ACTAAGTTATGTCAATGAGGGTTGGTAAATTTCGTGCCAGCCACCGCGGTCACACGATTAACCCAAACTA
ATAGGCCCACGGCGTAAAACGTGTCAAAAACCTCCACACTAAAGTTAAAACCTGGCCAGGCCGTAAAAAG
CAATTGCCAGCATAAAATAAACTACGAAAGTGACTTTATTACCCTAATTACACGATAGCTAAGATCCAAA
CTGGGATTAGATACCCCACTATGCCTAGCCCTAAACATAAATAATTAACGTAACAAAATTATTTGCCAGA
GAACTACTAGCAACAGCTTAAAACTCAAAGGACTTGGCGGTGCTTTACATCCCTCTAGAGGAGCCTGTTC
TATAATCGATAAACCCCGATAAACCTCACCATCTCTAGCTAAATCAGTCTATATACCGCCATCTTCAGCA
AACCCTTAAAAGGAAGAGCAGTAAGCACAATAATAATACATAAAAAAGTTAGGTCAAGGTGTAACCTATG
AGGTGGGAAGAAATGGGCTACATTTTCTAATAAATAAGAATATATACCACGGAAATTTTTATGAAACTAA
AAATCAAAGGTGGATTTAGTAGTAAATTAAGAATAGAGAGCTTAGTTGAATTGGGCCATAAAGCACGCAC
ACACCGCCCGTCACCCTCCTCAAGCAGTAGTAATTCAACCACAATATATTAACGGATAAATTAATGTAAG
AGGAGACAAGTCGTAACAAGGTAAGCATACTGGAAGGTGTGCTTGGATTAAT
>Cow Bos Taurus AY676873 isolate 32027 mitochondrion, complete genome
CATAGGTTTGGTCCCAGCCTTCCTGTTAACTCTTAATAAACTTACACATGCAAGCATCTACACCCCAGTG
AGAATGCCCTCTAGGTTATTAAAACTAAGAGGAGCTGGCATCAAGCACACACCCTGTAGCTCACGACGCC
TTGCTTAACCACACCCCCACGGGAAACAGCAGTGACAAAAATTAAGCCATAAACGAAAGTTTGACTAAGT
TATATTAATTAGGGTTGGTAAATCTCGTGCCAGCCACCGCGGTCATACGATTAACCCAAGCTAACAGGAG
TACGGCGTAAAACGTGTTAAAGCACCATACCAAATAGGGTTAAATTCTAACTAAGCTGTAAAAAGCCATG
ATTAAAATAAAAATAAATGACGAAAGTGACCCTACAATAGCCGACGCACTATAGCTAAGACCCAAACTGG
GATTAGATACCCCACTATGCTTAGCCCTAAACACAGATAATTACATAAACAAAATTATTCGCCAGAGTAC
TACTAGCAACAGCTTAAAACTCAAAGGACTTGGCGGTGCTTTATATCCTTCTAGAGGAGCCTGTTCTATA
ATCGATAAACCCCGATAAACCTCACCAATTCTTGCTAATACAGTCTATATACCGCCATCTTCAGCAAACC
CTAAAAAGGAAAAAAAGTAAGCGTAATTATGATACATAAAAACGTTAGGTCAAGGTGTAACCTATGAAAT
GGGAAGAAATGGGCTACATTCTCTACACCAAGAGAATCAAGCACGAAAGTTATTATGAAACCAATAACCA
AAGGAGGATTTAGCAGTAAACTAAGAATAGAGTGCTTAGTTGAATTAGGCCATGAAGCACGCACACACCG
CCCGTCACCCTCCTCAAATAGATTCAGTGCATCTAACCCTATTTAAACGCACTAGCTACATGAGAGGAGA
CAAGTCGTAACAAGGTAAGCATACTGGAAAGTGTGCTTGGATAAAT
>Crocodilian skink (outgroup)
CCCAAAAACACAAAGTTTTGGTCCTAAACTTGCTCTTGTTTTTTATCAAAATTATACATGCAAGCCTCAA
CACACCAGTGAGAATGCCCACAAAACCCTAAAAAGATTGTTGGAGCGGGCATCAGGAAGCAACACTAATT
AGCCAAAGACGCCTTGCACACGCCACACCCACACGGGCCCTCAGCAGTAATTAACATTAAGAATGAGTGA
AAGCTCGACTTAGTTATGATAAACACGGTCGGTAAATTTCGTGCCAGCCACCGCGGTTATACGAAAGACC
AAAAACAAGAGCTACCGGCGTAAAGCGTGACTAAAGACACAAATACTAAAGGGGCACAACTGCCAAGTCG
TGAAACACTACTGCAAGTTAGAACAACAACAAAATGCCTTTAAAACCACTTTACCTCACGAAAGCTAAGA
AACAAACTGGGATTAGATACCCCACTATGCTTAGCCATAAACATAGATAGAACACAACACAAACCTATCC
GCCAGAGAACTACAAGCGAAAAGCTAAAAACTCCAAGGACTTGGCGGTGCTTCAAATCACCCTAGAGGAG
CCTGTCCTATAATCGATACTCCACGTTCCACCTTACCACCCCTAGCCAACCAGCCTATATACCGCCGTCG
TCAGCCTACCTTGTGAGAGAAGCAAAGCAGGCAAAAGAGTCAACAACTAATACGTCAGGTCAAGGTGTAG
CACACGGGGCGGAAGAGATGGGCTACATTTTTTCAAAGGAAAAACACGAACAGTCTACTGAAACACACAC
TCAAAGGCGGATTTAGCAGTAAAATAAACAAGAGAGTTTACTTTAAACCTGCTCTGAAGCGCGCACACAC
CGCCCGTCACCCTCATCA
5
DNA Distances
(DRAWGRAM or CLUSTALDISTANCE give same data)
(1)
(2)
(1) American_Black
0.0000
0.0474
(2) American_Brown
0.0474
0.0000
(3) Asiatic_black
0.0383
0.0444
(4) Polar_Bear
0.0820
0.0726
(5) Spectacled Bear
0.0882
0.0788
(3)
0.0383
0.0444
0.0000
0.0537
0.0598
(4)
0.0820
0.0726
0.0537
0.0000
0.0117
(5)
0.0882
0.0788
0.0598
0.0117
0.0000
NOTE 1: Students often choose Rooted Trees from ClustalW. However this tool does not have an
algorithm for determining the root of the tree so these are not rooted. They are only squared of
presentations of the unrooted tree. Most of the trees in their textbooks are rooted - this is a good
opportunity to review the criteria for an outgroup (which provides a way to denote a root) – a species that
is related to the monophyletic or polyphyletic group of interest but is more distantly related than the group
of interest are to each other. A good outgroup for this group under study might be non-canid mammals
or reptiles. If students would like to modify the “rooted” tree (not really rooted) they can hand draw in the
outgroup. For this key, the “rooted” tree (which is unrooted) is not shown.
6
Distance matrix after adding the Giant Panda:
(1)
(2)
(1) American_Black
(2) American_Brown
(3) Asiatic_black
(4) Polar_Bear
(5) Spectacled bear
(6) Giant_Panda
0.0000
0.0474
0.0383
0.0820
0.0882
0.0882
0.0474
0.0000
0.0444
0.0726
0.0788
0.1345
7
(3)
(4)
(5)
(6)
0.0383
0.0444
0.0000
0.0537
0.0598
0.0817
0.0820
0.0726
0.0537
0.0000
0.0117
0.0791
0.0882
0.0788
0.0598
0.0117
0.0000
0.0789
0.0882
0.1345
0.0817
0.0791
0.0789
0.0000
Distance matrix after the addition of another supposed panda (red panda), dog, and raccoon
(could do these one at a time). What does this additional information suggest? First of all one can see
that the canids are all pretty closely related. Also shows that these three critters are not as closely related
to the previous 5. That is, we have introduced a new clade.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(1)Asiatic_black
0.0000 0.0444 0.0383 0.0537 0.0598 0.0849 0.1234 0.1188 0.1166
(2)American_Brown 0.0444
0.0000
0.0474
0.0726
0.0788
0.1357
0.1895
0.1201
0.1741
(3)American_Black
0.0383
0.0474
0.0000
0.0851
0.0914
0.0914
0.1068
0.1016
0.1035
(4) Polar_Bear
0.0537
0.0726
0.0851
0.0000
0.0117
0.0728
0.1141
0.1127
0.1174
(5) Spectacled
Bear
(6) Giant_Panda
0.0598
0.0788
0.0914
0.0117
0.0000
0.0726
0.1173
0.1124
0.1138
0.0849
0.1357
0.0914
0.0728
0.0726
0.0000
0.1909
0.1259
0.1653
(7) Raccoon
0.1234
0.1895
0.1068
0.1141
0.1173
0.1909
0.0000
0.0991
0.1439
(8)Red_Panda
0.1188
0.1201
0.1016
0.1127
0.1124
0.1259
0.0991
0.0000
0.1127
(9) Dog
0.1166
0.1741
0.1035
0.1174
0.1138
0.1653
0.1439
0.1127
0.0000
(10) Crocodilia
(outgroup)
0.3586
0.4128
0.3595
0.3337
0.3236
0.3860
0.3780
0.3256
0.3966
8
To confirm a hypothesized relationship, include an outgroup. For mammal studies – some scientists
choose a reptile since they are evolutionarily related to mammals. However, this study involves
some very closely related groups so a noncanid mammal, like Bos Taurus (cow) would also be
appropriate. You might ask students to find other appropriate outgroups and compare the data.
Here are the data (I did not include the matrix) when including Bos taurus (cattle, left) or a
Crocodilian skink (reptile, right) as an outgroup. Both clearly show that 2-3 major clades (circled), yet
also show how closely related the canids are to each other.
9
Answers to Possible Follow-up Questions for Evaluation of Understanding.
1. What are we trying to show by constructing phylograms, matrices and sequence alignments?
Mutations are random and occur rarely (because organisms have repair mechanisms to correct them). So if a
mutation occurs, for ex., once in 10,000 generations and two mutations have occurred, we can infer that 20,000
generations separate the two DNAs under study. So, the number of mutations can indirectly tell us the relative
time since two organisms shared an ancestor. The distances of the branches on the trees is indicative of the
accumulated differences between two or more species. The more differences, the less related they are.
2. As you examine the phylogenetic tree of the bear species’ 12s rRNA gene sequences, which two are
most closely alike? Which “pair of bears” seems to be the most distantly related? Explain. The most
closely related species in the group under study are the polar and spectacled bears. Among the bears, the
Giant Panda is least related to the American Black Bear (be sure to look at total “distance”). The Red panda is
not clustering with the bears at all – the similarity in anatomy is presumably convergent evolution.
3. Which are closer relatives” American Brown and American Black or American Black and Asiatic Black?
Explain your answer and why this might be the case. Based on sharing of a common ancestor (at the
branch point) the American Black Bear is more closely related to the Asiatic Black Bear than to the American
Brown Bear. Several hypotheses can be proposed for this – ancestors of Asiatic and American Black bears
were dispersed via the Bering Strait setting the stage for allopatric speciation is a popular idea.
4. Is the giant panda more of an “American” relative or “Asiatic” relative as compared to the bear group?
Explain. If one considers the polar bear to be an American bear, then the Giant Panda is indeed more of an
American bear relative. Also notice that the distance from Giant Panda to Brown bear is less than the distance
from Giant Panda to Asian Black bear.
5. Further research the spectacled bear; where is it found today? Epxlaln its possible evolutionary origin.
The last of the lineage of short-faced bears, the spectacled bear lives as the sole bear species in the Andes of
South America. Their early ancestors are thought to have crossed the Bering Strait Bridge from eastern Asia
some 15 million years ago.
6. Does the giant panda “belong” in the bear family at all? Explain your answer. On the basis of this data,
once might hypothesize that the Giant Panda is not a bear at all – that Pandas and Bears are a polyphyletic
group.
7. Where does the Red Panda fit into the phylogenetic tree? Who is its closest relative? It appears that the
Red Panda is more closely related to the raccoon and not at all closely related to the Giant Panda.
8. Why did we include a cow and a crocodile skink in the final phylogram? These animals were example of
a herbivore mammal and a reptile so they provided us with a comparison with an outgroup or a distant or nonrelative.
9. From your research and data, how would you define a bearl? Your students will ikely come up with many
possible answers, none definitive. This poses an example of a current debate among biologists. Evolutionary
biologists have presented data that supports dogs, dog relatives ,and bears being a monophyletic group – that
is, bears and dogs are one group (the canids). It is important to remember that these phylogenetic trees, based
on sequence data, are only as good as the data used to construct them. It would be useful to compare other
sequences besides 12s rRNA to see if the results are consistent. It would also be helpful to add other bear and
canid species to get a clearer picture of the canid relationships before concluding that pandas are not
bears…or that bears are dogs!! If time allows, your students may be able to extend this study to include these
additional analyses. If skeletal samples are available, these might be informative as well. Some students
readily see the similarity in morphology in the heads of wolves and bears.
Additional References:
•
James P. Noonan, Michael Hofreiter, Doug Smith, James R. Priest, Nadin Rohland, Gernot Rabeder, Johannes Krause, J.
Chris Detter, Svante Pääbo, and Edward M. Rubin. Genomic Sequencing of Pleistocene Cave Bears Science 22 July 2005
309: 597-599 (shows how closely related bears are to dogs).
•
Morris, R. and D. Morris (revised by Jonathan Barzdo). 1981. The Giant Panda. New York:. Penguin Press.
•
Molecular clockwork and related theories http://www.athenapub.com/molclock.htm
•
Cann, Rebecca L., Stoneking, Mark & Wilson, Allan C. Mitochondrial DNA and Human Evolution," Nature, 325 (1987), 316. (http://www.artsci.wustl.edu/~landc/html/cann/)
•
The Spectacled Bear www.grizzlybear.org/bearbook/spectacled_bear.htm
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Flow Chart
Go to: www.ncbi.nlm.nih.gov
Locate needed nucleotide sequences by accession numbers
Save in WORD document; SAVE AS text document
Go to Biology Workbench, log in
Create and name a new session
Upload Sequence file (.txt file) to enter into WORKBENCH
Edit labels
NUCLEIC TOOLS – Perform Clustal W
Import alignment
ALIGNMENT Tools – CLUSTALDIST
Copy and paste distance matrix into WORD document
ALIGNMENT TOOLS – BOXSHADE
Copy and paste box shaded alignment into WORD document.
Draw Conclusions
Engage in Discussion
Answer questions.
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