LABORATORY EXERCISE 12 BUILDING PHYLOGENIES WITH MOLECULAR CHARACTERS Goals 1 Understand how the structure of DNA can change over evolutionary time within a phylogeny 2 Access the NCBI database to extract information on the structure of DNA in different taxa 3 Use the program ClustalX to align DNA sequences and understand why this is important 4 Use the program PAUP to create a phylogeny based on molecular data obtained while achieving goals 1 and 2 5 Create a phylogenetic hypothesis for a group of real organisms based on molecular data Introduction Last week you learned how to create phylogenies using morphological traits of organisms This is the traditional type of data used in the construction of phylogenies and for many years it was usually the only type of data available In recent decades systematists have begun to use molecular information to construct phylogenies The sequence of bases in DNA or in RNA or the sequences of amino acids in proteins can be used as characters just as we used the presence of absence of gills limbs etc last week Molecular data has a number of advantages It can be used across all organisms as all organisms have DNA The number of characters is potentially enormous And if a character is accurately identified then the character state should be unambiguous Many characters are also thought to be neutral with respect to selection as discussed later in the course However there are also a number of difficulties in using molecular data You will experience some of these difficulties in the exercises below Molecular data can be used to construct phylogenies because as a lineage evolves in accumulates changes in the structure of its DNA Many changes in the structure of DNA are neutral and have no effect on the phenotype of the organism Therefore selection does not act against these neutral mutations The program Forensic EA mimics this process in a micro organism When speciation occurs each new species inherits the mutations of the ancestor but then begins to accumulate its own new mutations An example The following table gives DNA sequences for several species Species Sequence Outgroup GCTTAGGCATAACT Species A Species B Species C Species D Species E GCTTAGACATAACT GTTTAGACATAAGT GTTTAGACATAAGG GTTTAGACACCACT GTTTAGACACAACT These data generate the following tree Outgroup A B C D E y z x w The thick bars crossing the tree indicate where particular changes in the DNA sequence have occurred in evolutionary history Not all changes are indicated on the diagram W G changes to A 7th postion X C changes to T 2nd position Y C changes to G 13th position Z T changes to C 10th position Of course just as with morphological traits real examples are going to much more complicated and statistical software will be needed to calculate trees Activities 1 Run a short simulation showing molecular divergence within a phylogenetic framework The program we will use is called Forensic EA and it was created by one of the authors of your evolution text 2 Perform all the steps involved in using molecular data to create a phylogenetic hypothesis using an example provided for you Work in groups of 2 or 3 for this activity This activity will have three stages A downloading the molecular information from Gen Bank the NCBI website http www ncbi nih gov B aligning the sequences using the ClustalX software and C using PAUP to create a phylogeny from the aligned sequences 3 Repeat this process using a group of organisms assigned to you by your TA Your assignment will include some biological information for you to map onto the phylogeny Assignment Complete the sample molecular phylogeny exercise and show it to your TA Each group will present their aligned sequences and their phylogeny for their group of organisms at the beginning of lab 13 Acitivty 1 Forensic EA This software is designed to show you how to use molecular evolution to solve a forensics problem by looking at the evolution of a virus You will use it for a far more basic purpose to simulate the divergence of DNA strands in different organisms in this case a virus but the principle is the same Click on the Forensics EA icon in the IB 151 201 folder When the program opens you will see a couple of boxes on the screen Drag a black dot from the left box into the right hand box While you are dragging you will see the viral sequence Once in the left box you can see both the generation number and the sequence Now hit the run button The remaining dots viruses will slowly start to grow and divide as they go through their life cycle Wait for one full generation to complete Notice that some dots have changed color This is due to random changes mutations in the sequence Now drag one of the colored dots into the right hand box The table should indicate that this virus is from generation 1 and has one difference in its sequence from the original sequence See if you can find it Go up to the menu at the top of the screen and choose Tree from the list under Simulation There is now one much smaller box at the bottom of the big green box Drag a dot out of that box and put it into the small Source box underneath the Sequence should appear to the right Now click on fast forward The viruses should begin living and evolving very quickly Hit pause after around 50 generations exact number doesn t matter and drag another dot into the other source box How many differences do you see Now hit the fast fwd button again and click on the box of viruses It should now split into two independently evolving groups Keep doing this every 50 100 generations follow any pattern you like until you have 6 boxes and at least 400 generations have passed Now compare each of the six taxa to the original ancestor sequence to remove an old sequence just drag the old dot out of the box into some other part of the screen Then compare one box at the top a terminal taxon to all of the others in turn Do you see any pattern in similarity of sequence and the structure of the tree Activity 2 GENBANK AND PAUP We will now move on to analyzing some actual DNA data from real organisms The data has been extracted from the NCBI database and placed in the proper format for you You will use the software PAUP to analyze the data and produce a tree Just as with the morphological exercises we will use several different groups of organisms and each group of students will work on a different set of taxa As part of this exercise we will also practice accessing DNA data from Gen Bank NCBI database and the