LABORATORY EXERCISE 12: BUILDING PHYLOGENIES WITH MOLECULARCHARACTERSGoals1. Understand how the structure of DNA can change over evolutionary time within aphylogeny2. Access the NCBI database to extract information on the structure of DNA indifferent taxa.3. Use the program ClustalX to align DNA sequences (and understand why this isimportant)4. Use the program PAUP to create a phylogeny based on molecular data obtainedwhile achieving goals 1 and 2.5. Create a phylogenetic hypothesis for a group of real organisms based onmolecular data.Introduction.Last week you learned how to create phylogenies using morphological traits oforganisms. This is the traditional type of data used in the construction of phylogenies andfor many years it was usually the only type of data available. In recent decadessystematists have begun to use molecular information to construct phylogenies. Thesequence of bases in DNA or in RNA or the sequences of amino acids in proteins can beused 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 allorganisms have DNA. The number of characters is potentially enormous. And if acharacter is accurately identified then the character state should be unambiguous. Manycharacters are also thought to be neutral with respect to selection as discussed later in thecourse. However there are also a number of difficulties in using molecular data .You willexperience some of these difficulties in the exercises below.Molecular data can be used to construct phylogenies because as a lineage evolves inaccumulates changes in the structure of its DNA. Many changes in the structure of DNAare neutral and have no effect on the phenotype of the organism. Therefore selectiondoes not act against these neutral mutations. The program Forensic EA mimics thisprocess in a micro-organism. When speciation occurs each new species inherits themutations of the ancestor but then begins to accumulate its own new mutations.An example. The following table gives DNA sequences for several species.SpeciesSequenceOutgroupGCTTAGGCATAACTSpecies AGCTTAGACATAACTSpecies BGTTTAGACATAAGTSpecies CGTTTAGACATAAGGSpecies DGTTTAGACACCACTSpecies EGTTTAGACACAACTThese data generate the following tree.Outgroup A B C D E y z x wThe thick bars crossing the tree indicate where particular changes in the DNA sequencehave 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 morecomplicated and statistical software will be needed to calculate trees.Activities.1. Run a short simulation showing molecular divergence within a phylogeneticframework. The program we will use is called Forensic EA and it was created byone of the authors of your evolution text.2. Perform all the steps involved in using molecular data to create a phylogenetichypothesis using an example provided for you. Work in groups of 2 or 3 for thisactivity. This activity will have three stages: A) downloading the molecularinformation from Gen Bank (the NCBI website - http://www.ncbi.nih.gov/)), B)aligning the sequences using the ClustalX, software, and C) using PAUP to createa 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 ontothe phylogeny.Assignment. Complete the sample molecular phylogeny exercise and show it to yourTA. Each group will present their aligned sequences and their phylogeny for their groupof organisms at the beginning of lab 13.Acitivty 1: Forensic EA. This software is designed to show you how to use molecularevolution to solve a forensics problem by looking at the evolution of a virus. You willuse it for a far more basic purpose – to simulate the divergence of DNA strands indifferent organisms (in this case a virus but the principle is the same). Click on theForensics EA icon in the IB 151/201 folder. When the program opens you will see acouple 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 seeboth the generation number and the sequence. Now hit the ‘run’ button. The remainingdots (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 coloreddots into the right hand box. The table should indicate that this virus is from generation 1and 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 underSimulation. 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 – theSequence should appear to the right. Now click on fast forward. The virusesshould begin living and evolving very quickly. Hit pause after around 50generations (exact number doesn’t matter) and drag another dot into the othersource box. How many differences do you see?Now hit the fast fwd button again and click on the box of viruses. It should nowsplit into two independently evolving groups. Keep doing this every 50-100generations (follow any pattern you like) until you have 6 boxes and at least 400generations have passed. Now compare each of the six taxa to the originalancestor sequence (to remove an old sequence just drag the old dot out of the boxinto some other part of the screen). Then compare one box at the top (a terminaltaxon) to all of the others in turn. Do you see any pattern in similarity ofsequence and the structure of the tree?Activity 2. GENBANK AND PAUPWe will now move on to analyzing some actual DNA data from real organisms . Thedata 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 themorphological exercises we