Creating and comparing phylogenies using morphological data and molecular data
Bioinfomatics Tools
The purpose of this exercise is to develop a basic understanding of bioinfomatics and learn some of the skills
involved in constructing and interpreting phylogenetic trees and cladograms. The basic tools that we will use consist
of banks of data (DNA sequences and your character matrix), and programs to compare the similarities and
differences between sequences or the character states in your matrix. These programs display the results in the form
of a tree. Simply generating the tree is not the end of the story. Interpretation is a time-consuming and extensive
process and we will spend most of our time in this lab interpreting our trees.
Creating a phylogeny based on morphological data
The first step is to create a data file using Word. The format is very precise, so follow the directions very carefully.
On the first line
 space three times
 enter the number of taxa
 space three times
 enter the number of characters for each taxon
 press enter
There must be a code (character state) for each character and phylum.
On the second line
 Put a three-letter capital code to represent the phylum
 space 10 times
 enter the codes for each character state. They can wrap to the next line if necessary
 press enter
 repeat for each phylum
 save the file as a text file. This is very important. PhyLip can’t read word files.
Go to:
http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html
or http://portal.litbio.org/Registered/Option/phylip.html
Scroll down and choose PARS.
You should now be at: http://bioweb.pasteur.fr/seqanal/interfaces/pars-simple.html
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Enter you e-mail
cut and paste your character matrix into the box provided
Press “Run PARS”
You should now be at: http://bioweb.pasteur.fr/cgi-bin/seqanal/pars.pl
 In the pulldown box select “Drawgram”
 Press “Run the selected program on OUTTREE”
You should now be at: http://bioweb.pasteur.fr/cgi-bin/seqanal/lib/connect.pl
 Click on “Advanced drawgram form”
 Scroll down until you see “Drawgram options”
 Select “W:MS-Windows Bitmap from the pulldown menu
 Go back to the top of the page and select “Run drawgram”
You should not be at: http://bioweb.pasteur.fr/cgi-bin/seqanal/drawgram.pl
 Double click on “plotfile”
 You should see your tree.
 Save your tree as a file and print a copy by right-clicking and selecting the appropriate option. Attach
your tree to the end of this lab calling it Figure 1 and adding an appropriate title. 10 pts.
Creating a phylogeny based on molecular data
1.
2.
3.
4.
5.
Get on to the internet and type in http://workbench.sdsc.edu/
Click on “Set up a free account” and follow the instruction to set up an account OR use the account that you set
up several weeks ago. Click on “Enter the Biology Workbench 3.2” and all you have to do is to type your name
and password.
Once you are in, click on “Nucleic Tools”.
Highlight “Ndjinn – Multiple Database Search”. Click “Run”.
There many databases available. For our project we will use GBPLN (GenBank Plant Sequences, which
includes the fungi and algae.) We will be working on the plants in Table 1.
Group
Group 1
(flowering plants)
Group 2
(bryophytes)
Group 3
(algae)
Group 4
(ferns)
Group 5
(seedless
vascular plants)
Group 6
(ancestral
gymnosperms)
Group 7
(derived
gymnosperms)
Group 8
GBPLN #
GBPLN:7595531
GBPLN:19515
GBPLN:547571
GBPLN:987712
GBPLN:2463094
GBPLN:515787
GBPLN:7242518
GBPLN:2564932
GBPLN:2358227
GBPLN: 511872
GBPLN: 7981618
GBPLN: 5825256
GBPLN:3401953
Different database!!
GBBCT: 1668771
GBPLN:603755
GBPLN:7670254
GBPLN:10765099
GBPLN:861086
GBPLN:861139
18s rRNA gene sequence
Salix reticulata
Lapageria rosea
Marchantia polymorpha
Anthoceros agrestis
Hypnum cupressiforme
Siphonocladus tropicus
Geosiphon pyriforme
Batrachospermum turfosum
Vaucheria bursata
Prymnesium patelliferum
Ceratium furca
Euglena anabaena
Fucus distichus
Nostoc sp. – 16S rRNA
Phylum
Angiosperm
Angiosperm
Hepatophyta
Anthoceratophyta
Bryophyta
Chlorophyta
Bacillariophyta
Rhodophyta
Chrysophyta
Prymnesiophyta
Dinophyta
Euglenophyta
Phaeophyta
Cyanobacteria
Tmesipteris tannensis
Azolla pinnata
Dicksonia antarctica
Psilotum nudum
Selaginella umbrosa
Pteridophyta
Pteridophyta
Pteridophyta
Psilophyta
Lycopodophta
GBPLN:19131
GBPLN:860933
GBPLN:403026
GBPLN:2588905
Lycopodium annotinum
Equisetum robustum
Pinus wallichiana
Pinus luchuensis
Lycopodophyta
Equisetophyta
Gymnosperms
Gymnosperms
GBPLN:2588904
GBPLN:7670259
GBPLN:7595579
Pinus elliottii
Gnetum montanum
Welwitschia mirabilis
Gymnosperms
Gnetophyta
Gnetophyta
GBPLN: 556508
GBPLN:1777635
Ginkgo biloba
Amborella
Ginkgophyta
Angiosperm
(ancestral)
Table 1. List of the organisms.
Click on “GBPLN”. (You have to scroll down for this option.)
Scroll up and type in the number for one of the species on the list in Group 1. Click on “Show 10 hits”. Make
sure that every team chooses different combinations of species. i.e. don’t always take the first species in each
group.
8. Click on “Search”. You will see a list of choices. Scroll down until you see the number you are searching for in
the GBPLN column. Highlight this line and click on the “Import Sequence(s)” button located at the end of the
first line of the interactive box. If you get too many sequences, then search by species name and then look for
the specific GBPLN number.
9. Now we need to repeat the process to retrieve more sequences from the gene banks. Highlight “Ndjinn” and
click “Run.”
10. Search and import the sequence of one organism from each group in Table 1.
6.
7.
11. Now we are ready to generate a tree. Select all the sequences EXCEPT Group 8. Click “Run”. All the boxes in
front of these organism names should be checked.
12. Using the scroll box and scroll down and highlight “CLUSTALW – Multiple Sequence Alignment”. Click
“Run”.
13. A new screen will appear. You can choose to make a rooted or unrooted tree by clicking on the arrow next to the
box labeled “Guide tree display” and choosing rooted or unrooted. Then, click “Submit”. The screen will go
blank and you may have to wait several minutes. Wait until a screen titled “CLUSTALW” with “Sequence
alignment” appears. Scroll down to examine the DNA sequences and how they align with each other. Scroll
further down and you will see your tree! Right click on your treee and select “Copy” from the menu.
14. Open Microsoft Word. Go to “Edit” and “Paste” and your tree will reappear. Adjust the size of your tree by
selecting the tree image and resize from the lower right corner so two trees can fit on a page. Type in a label for
each tree using consecutive figure numbers and adding appropriate titles.
15. Look up each number and write the corresponding phylum beside the number by hand. (Consult Table 1.) You
can also label each one by pulling down the “Insert” menu. Selecting and releasing on “TextBox”. Click where
you want the textbox to be located and type the name of the species. Drag the textbox next to the corresponding
number. Attach your tree to the lab and label it “Phylogeny of Kingdom Plantae”.
Time to think:
A. Compare your phylogeny from the character matrix with the molecular data tree. Where are there
differences/similarities? Why do you think this might be the case? 3 pts.
B. Compare your phylogeny from the character matrix and the molecular data tree with information from your
textbook. Where are there differences/similarities? What might account for any differences you see? 3 pts.
C. Re-examine your character matrix and the sequences you’ve chosen from GenBank. Make any adjustments you
think are desirable. State the changes you make here and why you made them:
D. Rerun the programs to generate new trees. Number them and add appropriate titles and attach them to this lab
report. What changes occurred? Was this what you expected? Why or why not?
E. Using the information above, and information from our work in class, draw a cladogram and clearly label at
least one, preferably two characteristics that separate each phylum we have studied. Indicate using sequential
numbers where the evidence for this cladogram is in your phylogenetic trees and character matrix. . 5 pts.
Turn over for last page of lab.
Reflection (4 pts.): As a result of the series of labs we have completed on the evolution of photosynthetic organisms,
what is the most important idea/concept that you have learned?
What is the most important understanding you have gained about systematics and phylogeny construction?
For this lab turn in:

This worksheet with the answers completed. (7 answers at 3 pts. each)

Phylogenetic trees as indicated in the lab with each numbered, titled and all representative phyla labeled.
(at least 4 trees depending on how many revisions you make) 16 pts.

Your character matrix in table form with an additional table including the character, coding for the
character states, and importance of the character in determining phylogenetic relationships. 9 pts.
Revised 10-29-07 BJB