Lab 2/Phylogenetics/September 16, PHYLOGENETICS

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1 Lab 2/Phylogenetics/September 16, Read: Tudge Chapter 2 PHYLOGENETICS 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

2 Lab 2/Phylogenetics/September 16, 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 Figure 1 Carnivora Mammalia Chordata 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

3 Lab 2/Phylogenetics/September 16, 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: 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:

4 Lab 2/Phylogenetics/September 16, 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.

5 Lab 2/Phylogenetics/September 16, 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 Starr C. and Taggart, R. Evolution of Life (Eighth Edition). Belmont, CA: Wadsworth Publishing Co.

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