Relationships among species
Using MEGA
Introduction:
DNA provides an essentially limitless source of information. Some of this information is stored in online data bases that can be accessed by everyone with an internet connection. In this exercise we will use the program MEGA to access the GenBank database to search for genetic sequences and to build a phylogenetic tree that represents the evolutionary relationships among those species. This will be a self-guided exercise in that it is up to you, the student, to choose a group of organisms to research.
Materials required:
MEGA which is installed on your computer.
List of 5 species that you have an interest in and that are from widely separated taxa e.g. different genera, orders, or classes. The second requirement is to make the data set conducive to applying a molecular clock. For some of the species you will also need an estimate of when they diverged (e.g. an online search could give you an estimate of when the evolutionary branches of birds and crocodiles were split).
Objectives:
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1.To familiarize yourself with searching for sequence data using MEGA and GenBank. Following that to align your sequences.
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2.To conduct basic phylogenetic analysis on the data set, including calculating sequence divergence, building trees, and calibrating a molecular clock.
Steps:
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3.Open MEGA. Using the ‘Query databanks’ option under the alignment tab, and search for sequence data for a single gene from each of five species (e.g. cytochrome oxidase I Pinus banksiana; cytochrome oxidase I Oryza sativa, and so on). Add each sequence to your alignment (click on ‘add to alignment’ option under the ‘Data’ tab).
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4.When you have found your 5 species, go to the alignment explorer window and align the sequences using clustal W. Examine the alignment – is it good (do most bases match among sequences)?
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5.Export the alignment as a .meg (Mega) file and then open this new file
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6.Determine the pairwise distances among species.
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7.Build a neighbour-joining tree, and a maximum parsimony tree (using the Mega window that appeared when you first opened the program).
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8.When you have built a tree choose the node for which you have found a divergence date (if you have been unable to find a divergence date, it’s okay to make one up for this exercise so that you can see how the calibration would be done). For example, if you may know from the fossil record that species A and B diverged 10 million years ago you would select the most recent common ancestor of these two species. When the node is selected use the ‘set divergence time’ button to enter the divergence date. The tree will then be modified to show a scale.
Questions:
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1.Which species have the lowest sequence divergence between them (see Step 6)? From your knowledge, are these species the most taxonomically similar (e.g. are they members of the same genus or family)?
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2.Do the various trees that you built using different methods and models all give the same pattern of evolution (see Step 7)? Why might they give differing results?
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3.Why might the speciation events on your tree disagree with evidence from the fossil record (see Step 8)?
