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PHYLOGENETICS Required: Title Page, Introduction, Results (no stats required), Literature Cited.

Note: This assignment will be used to produce a Discussion assignment in Biology 103 in the winter term. Polymerase Chain Reaction: PCR enables the rapid synthesis of millions of copies of a given piece of DNA from a small sample. It is a required step for most genetic research because it provides adequate quantities of DNA for experimentation. Sequence Analyses: This step allows us to see the sequence of DNA bases in the piece magnified by PCR. See Sanger Method in the textbook or online. The information in the mitochondria autoradiograph accompanying this lab would have been produced by Sanger sequence analysis. Phylogenetics involves the study of evolutionary relationships between groups of organisms. These relationships can be based on shared characteristics, including information derived from molecular sequencing analysis. The relationship is usually presented as a hypothesis since we are never exactly sure about events that have occurred in the past. A family tree or phylogeny is often used as a visual representation of this hypothesis. A B C In this sample phylogeny, species A & B share a node, and would be considered to be more closely related than to C. A clade is a group of species that share a node, and so A&B are a clade. A&B&C can also be considered a clade because they also share a node. Clades are sometimes given names like genus, family, order, and class, and so in this example A&B might be in the same genus, and A&B&C might be in the same family.

Appearances can be deceiving. Organisms can appear different from their divergent ancestor, but unrelated species can appear similar through convergent evolution. Genetic Clock: Mitochondrial DNA is haploid, and maternally inherited, thus it not expected to undergo recombination with other DNA as nuclear chromosomes do. Therefore, changes in a mitochondrial DNA gene sequences are assumed to be a result of point mutations, which can be factored against time to produce a type of genetic clock. However, over millions of years internal and external events can influence a phylum and may influence the reliability of the clock. For this lab we will not use the clock as a direct quantitative measure, but instead will infer that the number of nucleotide differences between species is an indicator of a relative amount of divergence. For example, it will be assumed that two species with three nucleotide differences between them for a specific sequence will be more closely related than two species with 10 nucleotide differences between them. In this lab we will be examining the possible evolutionary relationship between four sampling populations of Eastern Garter Snake (Thamnophis sirtalis sirtalis). The four populations are MD for Main Duck Islands, located south of Kingston in Lake Ontario; CA for the Cataraqui Region Conservation Area,
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located within the City of Kingston just north of Hwy. 401; GL for the Gould Lake Conservation Area located about a half hour drive north of Kingston; and AP for Algonquin Provincial Park, about a two hour drive north of Kingston. Mitochondrial DNA analysis was performed on individuals from each population using the mitochondrial control region. The mitochondrial DNA sequence was isolated, sequenced using the Sanger dideoxy sequencing method, electrophoresed on a sequencing gel and autoradiographed. A representation of that autoradiograph needs to be examined for nucleotide differences among each of the four populations.

Methods 1. Read the base pair sequences for each individual (starting from the bottom of the page). Determine the number of base pair differences between each of the four samples. This translates to six comparisons. 2. Set up a table of differences (i.e., mutations), similar to the one below, but instead of letters, use the four population codes. 3. When finished, your table might look like the following. These mutational differences can be used to create a phylogenetic tree to reflect a possible evolutionary relationship of these four species. This example table provides a sample comparison of mitochondrial DNA differences between A, B, C, and D. A is different from B, C, and D by 12 differences (mutations), B is different from C and D by 8, and D and C are different by 6 . A A B C D 12 12 12 B 8 8 C D These tabulated differences were used to produce the following phylogenetic tree, which suggests that A diverged from the ancestor of B,C, and D first, then B diverged from the ancestor of C and D, and then C and D diverged.

6 A B C D

The math in your table might not work out as nicely as the one above and there may be some challenges in developing your phylogenetic tree. Keep in mind the notion of parsimony, in which the simplest explanation is likely the best. In other words, more divergent species are likely going to have more mutational differences between them than closely related species. Your results should appear as in the previous assignments, with a phylogenetic tree and accompanying text. Do not submit the table. There is no statistical analysis for this assignment.

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CA A

A P A

MD A

G L A

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