Lab 2/Phylogenetics/September 16, 2002
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PHYLOGENETICS
Read: Tudge Chapter 2
Objective of the Lab: To understand how DNA and protein sequence information can be used
to make comparisons and assess evolutionary relationships between species.
Introduction
Evolutionary theory is central to a clear understanding of the biology of all organisms.
One important area in the study of evolution is phylogeny, the study of evolutionary
relationships among organisms. For example, how do we know that birds are closely related to
reptiles (i.e. dinosaurs), or that of the five types of great apes; gibbons, orangutans, gorillas,
chimpanzees and humans, the gorillas and chimpanzees are the most closely related to man in an
evolutionary sense? The study of gene sequences from these different organisms allows for a
different mode of comparison, which provides for a clearer understanding of evolutionary
relationships.
In the past relationships between organisms were made based upon phenotype, are the
structures and traits of two species closely related, compared to a third species. With the advent
of recombinant DNA technology, systematic biologists (those scientists who study relationships
between species) moved into the realm of genetics and molecular biology.
This process of making comparisons between species, whether it is based upon
phenotypic traits or genetic sequences, is a way of using large-scale evolutionary changes, thus
firmly putting it in the realm of macroevolution. We can then define macroevolution as the
large-scale trends or patterns in evolution, and the rates of change among those closely related
species in which we are interested. This would mean a scientist studying the macroevolution of
a related group of species would need to consider such factors as global or regional
environmental changes when evaluating macroevolutionary effects on the species under study.
Phylogeny is a branch of Systematics, or more specifically Taxonomics. This means an
organized way of grouping and understanding relationships between species, based upon the
named taxonomic unit at any level this taxonomic unit is known as a taxon. Historically
Aristotle was one of the first to organize and classify life based upon physical appearance. This
system was more clearly defined and organized by Carolus Linneaus as a system of taxonomy,
which we call binomial nomenclature. This means that in the process of identifying individual
species, a two-part name is assigned to each unique species, with the first word being the genus
to which the species belongs, and the second word being the scientific epithet or name of the
species in question. For example the domestic cat is Felis silvestris, while the mountain lion or
puma is Felis concolor, indicating these two species belong to the same genus, and thus are
closely related in an evolutionary sense, but are not identical. Additionally broader phylogenetic
groups indicate other levels of relationships between species, usually based upon a defined set of
phenotypic characters. These broader areas of classification for taxonomy in which a genus is
placed include the family, with families being placed into orders, orders into classes, classes into
phyla (phylum being the singular use of the term) and phyla into kingdoms. So using our
example of the genus Felis, these cats are related to the genus Panthera (lions, tigers, leopards
and jaguars) in the family Felidae. This family resides in the order Carnivora, which also
includes the Canidae (dogs), Ursidae (bears) and a few others. All of these are part of the class
known as Mammalia, which belongs to the phylum Chordata in the kingdom Animalia (see
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Figure 1). So the use of phylogenetic diagrams allows the systematic biologist to make
comparisons about the relationships between species, based upon the traits that are used to place
that species into these taxonomic categories. These comparisons can be made based upon
phenotypic traits, or with the advent of recombinant DNA technology and gene sequencing,
these comparisons can be made at the level of the gene.
A Phylogenetic Tree for Cats
Felis silvestris Felis concolor
Felis
Panthera
Felidae
Canidae Ursidae
Carnivora
Mammalia
Chordata
Figure 1
Animalia
In this lab we will use online databases of gene sequences to test hypotheses about the
evolutionary relationships within different classes of organisms. This is in truth creating an
evolutionary history for these organisms, expressed in the form of a phylogenetic tree, a
branching diagram that indicates the points in time in which the currently extant species diverged
from one another. The phylogenetic tree thus created represents a genealogy of probable
evolutionary relationships between the species under study, and the higher taxonomic groups to
which they belong.
The fact that sequences of nucleotides in DNA are inherited and these sequences in the
form of genes program the construction of proteins fro amino acids, use of DNA sequencing
provides a very powerful tool for making these species to species comparisons. The methods
usually employed for these include, DNA-DNA hybridization, restriction mapping and DNA
sequencing. The hybridization method uses the complementarity of DNA to determine the level
of hydrogen bonding between two single strand pieces of DNA, thus indicating the similarity of
the two base sequences. Restriction mapping uses restriction enzymes to cleave DNA and
compare the size of the DNA fragments made through restriction mapping. Finally DNA
sequencing determines the precise sequence of the nucleotides for genes for a very precise
comparison of their make-up. It is information generated by this last method, in the form of
Internet databases that you will use in today’s lab to make comparisons based upon a
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predetermined hypothesis, and then to assess a hypothesis, which you and your partners will
develop.
The Activity
In the introduction, you were exposed to taxonomy. Felines, canines and bears are all
mammals-they all shared a common evolutionary ancestor. Over times (millions of years),
animals belonging to each of these groups migrated to different parts of the globe. In order to
survive, these animals needed to adapt (evolve) to their physical environment. For example,
polar bears live in the Arctic and seem to have white fur (actually it is colorless and they have
black skin). Why would this be advantageous? First of all, camouflage for hunting. The darker
skin allows for greater absorption of heat from the sun’s ray’s.
Polar bears, located in the northern regions of the Northern hemisphere, share the
continent with American brown and black bears. What would you hypothesized about their
evolutionary relationship. Did the polar bears evolve from a population of brown bears that were
isolated far north?
In the first part of the lab, you will compare the DNA sequence of the 12s rRNA
(ribosomal RNA) gene. Remember, genes that are important for essential metabolic functions
(protein synthesis, in this case) are conserved within species and even larger taxonomic groups.
You can compare the 12s rRNA gene sequence for the polar bear, black and brown American
bears, the Asiatic black bear and the spectacled bear and determine their evolutionary
relationship.
In the second activity, you will compare the 12s rRNA gene sequence of the five bears,
above, with the giant panda. At a quick glance, the giant panda resembles the bear family. But
if diet and behavior of the giant panda is compared with the bear family, a different situation
comes to light.
For both exercises, you will be accessing the 12s rRNA gene sequences from a national
data bank and importing the sequences to a program to analyze the data (Workbench) that will
allow you to construct: 1) a distance matrix that compares the genetic differences between
species (the lower the number, the closer the evolutionary match) and 2) a phylogenetic tree,
showing the relationship between the bears, and the bear family and the giant panda.
INVESTIGATING POLAR BEAR AND GIANT PANDA ANCESTRY
USING BIOLOGY WORKBENCH AND GENBANK
INSTRUCTIONS FOR IMPORTING GENE SEQUENCES:
1. Enter GenBank at: www.ncbi.nlm.nih.gov
2. In the “Search GenBank” box, type in the accession number of the sequence you want to
find. Click Go. (You will need to do each sequence separately.)
3. Click on the highlighted accession number and information about the species will appear.
4. Next to the “Display” button, pull down the menu and highlight FASTA. Click the
Display button.
5. Highlight the entire FASTA sequence including the initial “>” symbol. Copy the
highlighted sequence (pull down the Edit menu and click on copy).
6. Enter Biology Workbench at: http://workbench.sdsc.edu
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7. Set up a free account (you will have to create a username and password).
8. Click on Nucleic Tools at the bottom of the webpage.
9. On the Nucleic Tools page, click on Add New Nucleic Sequence. Click run.
10. A new screen will appear. You will be able to save the FASTA information under a new
name in the Label Box. Name the sequence according to the bear it represents (EX:
American Black Bear).
11. Under the Label Box is a box for the Sequence. Paste (pull down the Edit menu and
click on paste) the saved sequence in the box.
12. Place the cursor behind the > in the FASTA identification line, and replace the FASTA
identification line (>gi…..) with the label you gave the sequence (EX: American Black
Bear). The line will now read: >American Black Bear. Click on Save.
13. Return to GenBank. Repeat steps 2-12 for each of the remaining accession numbers.
Accession Numbers for Species:
American Black Bear
American Brown Bear
Spectacled Bear
Asiatic Black Bear
Polar Bear
Y08520
L21889
L21883
L21890
L22164
Make sure you have all 5 bear sequences imported BEFORE moving on!
INSTRUCTIONS FOR GENE SEQUENCE ANALYSIS AND TREE CONSTRUCTION:
1. In Biology Workbench, select “Nucleic Tools,” and select the 5 bear sequences you
imported.
2. In the Nucleic Tools menu box, scroll down and highlight CLUSTALW. Click Run.
Click Submit.
3. Click on Import Alignments.
4. Scroll down through the Import Alignments window and highlight CLUSTALDIST.
Under the Import Alignments window, select the group of sequences you want to
analyze. Click Run. Click Submit.
5. A data matrix will appear on the next screen. Print this screen so you can examine the
data later.
6. Close the screen with the data matrix.
7. Click the Back button once.
8. Scroll down through the Import Alignments window and highlight DRAWGRAM.
Once again, make sure you choose the sequences you want to analyze. Click Run. Click
Submit.
9. The phylogenetic tree will appear on the next screen. Print this screen so you can
examine the data later.
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GIANT PANDA BEAR RELATIONSHIP TO THE PREVIOUSLY DETERMINED DATA:
In GenBank, access the giant panda 12s rRNA gene sequence (accession number Y08521) and
import it into Biology Workbench. Run a new alignment using all 5 of the previous bear
sequences and the panda bear sequence. Import the alignment into Alignment Tools. Use
CLUSTALDIST to create a distance matrix and DRAWGRAM to develop the phylogenetic tree.
Print the matrix and the tree.
The Assignment:
A. Evolutionary relationship of five bears species using 12s RNA gene sequence.
1. Distance matrix
2. Phylogenetic tree
B. Evolutionary relationship of the five bears species and the giant panda using 12s RNA gene
sequence.
1. Distance matrix
2. Phylogenetic tree
C. Questions
1. Hypothesis for bear’s phylogenetic tree. What would you state as a hypothesis for the
relationships between the five bear species?
2. Reevaluate your hypothesis. Were you correct? Why? What is the true relationship?
3. Hypothesis for bear’s phylogenetic tree. What would you state as a hypothesis for
the relationships between the five bear species and the giant panda?
4. Reevaluate your hypothesis. Were you correct? Why? What is the true relationship?
5. Briefly discuss genotypic analysis vs. phenotypic analysis. What would be the
advantages and disadvantages to both?
EACH PERSON SHOULD GENERATE THEIR OWN DISTANCE MATRICES AND
PHYLOGENETIC TREES. ANSWERS TO ALL QUESTIONS SHOULD BE
INDIVIDUAL WORK.
References:
Campbell, N.A. (1996) Biology (Fourth Edition). Redwood City, CA: Benjamin/Cummings
Publishing Co.
Maier, C.A. (2001) “Building Phylogenetic Trees from DNA Sequencing Data: Investigating
Polar Bear and Giant Panda Ancestry”. The American Biology
Teacher 63(9)
Nov./Dec. 2001.
Starr C. and Taggart, R. Evolution of Life (Eighth Edition). Belmont, CA: Wadsworth
Publishing Co.