CLADOGRAMS & GENETIC PHYLOGENIES INTRODUCTION Taxonomists since Linnaeus have used relative similarities and differences to group species into a taxonomic hierarchy of genera, families, orders, etc. Darwin did not change the way species were classified, but he did change the interpretation of the taxonomic hierarchy. He considered species in the same genus to share a relatively recent common ancestral species, species in the same family to have a more distant common ancestor, and species in the same order to have a more distant common ancestor. The development of modern gene sequencing has led to a revolution in the way species are currently classified. Recall that DNA consists of a sequence of A, G, C and T bases that code for specific amino acids in a protein. For example, the following sequence of sixty nucleotide bases codes for the first twenty amino acids of beta-hemoglobin, a part of the protein that carries oxygen in human red blood cells: ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAAC Not only has the human genome been sequenced, but all or part of the genome of many other species has also been sequenced. These sequences can be accessed by anyone and searched using powerful search engines to find similar genes and sequences in other species. Similarities and differences in the sequence of bases of a particular gene are now used to classify species into taxonomic groups. Just as in traditional taxonomy, the similarities in a gene like beta hemoglobin are assumed to be due to inheritance from a common ancestral species with bata hemoglobin. Any differences are assumed to be due to mutations that can change one base into another, or add a base into a sequence, or delete a base from a sequence. It is assumed that the longer the time since a common ancestor of two species, the more time there has been to accumulate random mutations. For example, if two species differ in 5% of the bases in their beta hemoglobin gene, they share a more recent common ancestor than if they differ in 10% of their bases. Molecular biologists construct trees of similarities and differences between species that are termed cladograms. Cladograms are assumed to represent the phylogenetic (evolutionary) history of the species. Your textbook has examples and illustrations of cladograms. In this lab you will compare a part of the beta-hemoglobin gene of several species and construct a cladogram depicting the degree of similarity and difference between species. OBJECTIVES 1. Determine the similarities and differences between two nucleotide sequences. 2. Explain how evolutionary biologists interpret the similarities and differences between nucleotide sequences. 3. Construct a phylogenetic tree of several species using a set of nucleotide sequences. 4. Explain the assumptions upon which the phylogenetic tree is based.
PROCEDURE 1. Your instructor will provide you with a sheet of paper with the nucleotide sequences of a portion of the same gene (beta-hemoglobin) from several species. The sequence of each species is fifty nucleotides long. Note that nucleotides are arranged in groups of ten, with spaces separating each group. A - may appear in a sequence where a nucleotide has been deleted by mutation (or where an extra nucleotide has been inserted in another species. 2. Your instructor will assign each person or group one or more pairs of species to compare. 3. Use a pair of scissors to cut out each species sequence to form long strips of paper. Set the pair your assigned species nucleotide sequences next to each other so the first through fiftieth nucleotides of each species align. 4. Compare each pair of nucleotides to determine if they match or if they differ. Count the number of differences between the two species. Record this number in the table below and on the class table on the whiteboard at the front of the room. Chic Chim Dog Gorr Huma Mars Monk Mous Rat Chicken 0 Chimp 0 Dog 0 Gorilla 0 Human 0 Marsupial 0 Monkey 0 Mouse 0 Rat 0 5. Repeat this procedure for all pair of species you have been assigned. 6. When all of the information on the board has been recorded, transcribe the class results onto your table. You now have the information necessary to construct your phylogenetic tree. 7. Observe the pairs of nucleotide differences in the table. Find the pair with the least number of differences. 8. Place the names of these two species next to each other at the top of a sheet of paper. Draw a shallow V below the two species and write the number of differences between the species below the V. Your instructor will illustrate how this is done on the board. 9. Determine from the table the pair of species with the next fewest differences. Write the names of these two species at the top of the page and connect them with another, deeper V. If one of the species is already on the page, do not rewrite the name, but connect the existing species with the new species with the deeper V. 10. Continue this process until all of the species are connected together into one large cladogram. FOR THOUGHT AND DISCUSSION
1. How do evolutionary biologists account for the similarities and differences between the sequences of these species? 2. Does the cladogram seem to accurately reflect how the species are classified by taxonomists? Why or why not? 3. What are some of the assumptions that must be made if the cladogram is to be accepted as a depiction of the evolutionary history of these species?
Mouse Rat Human Chimp Gorilla Monkey Dog ATGGTGCACC TGACTGATGC TGAGAAGTCT GCTGTCTCTT GCC-TGTGGG ATGGTGCACC TAACTGATGC TGAGAAGGCT GCTGT-TAAT GCCCTGTGGG ATGGTGCATC TGACTCCTGA GGAGAAGTCT GCCGT-TACT GCCCTGTGGG ATGGTGCACC TGACTCCTGA GGAGAAGTCT GCCGT-TACT GCCCTGTGGG ATGGTGCACC TGACTCCTGA GGAGAAGTCT GCCGT-TACT GCCCTGTGGG ATGGTGCATC TGACTCCTGA GGAGAAGACT GCCGT-TACC ACCCTGTGGG ATGGTGCATT TTACTGCTGA GGAGAAGGCT GCTGT-TATT AGCCTGTGGG Marsupial ATGGTGCATC TGACTGCTGA AGAGAAGAGT CTTGTCT-CC GGCCTGTGGG Chicken ATGGTGCACT GGACTGCTGA GGAGAAG-CA GCTCATCACC GGCCTCTGGG
Chic Chim Dog Gorr Huma Mars Monk Mous Rat Chicken 0 Chimp 0 Dog 0 Gorilla 0 Human 0 Marsupial 0 Monkey 0 Mouse 0 Rat 0