Phylogenetics Using MEGA

For today’s lab we are going to work with the Molecular Evolutionary Genetic Analysis program (https://www.megasoftware.net/home) to examine the phylogentic relationship of the cytochrome oxidase I (COI) gene in several ants. These 10 ants were collected by amateur entomolgists in south Florida, five were collected from one location (ants 1-5) and five were collected from another location (6-10). The DNA was extracted from all of these ants and the COI gene was amplified and sequenced in order to compare the two populations. We were also supplied with sequence from Formica rufa a close relative of the ants being sequenced. Your goal is to identify each of these ants (using BLAST) (Camponotus atriceps_1) and create two distance matrices and two phylogenetic trees (using MEGA) to compare these populations and identify if these two populations are actually one giant population. In order to proceed, you will need to download the MEGAX program from https://www.megasoftware.net/ . You will then make a MEGA alignment for the 10 ant species and the outgroup species (Formica rufa) using the sequence files provided. You will need to trim the sequences and correct any errors in the sequences by using the editing functions in MEGAX.
In order to visualize your sequences, you can open the .abi files in and use the trace files in MEGAX. Or you can use Chromas Lite for PC users (http://technelysium.com.au/wp/) or 4Peaks for MAC users (https://nucleobytes.com/4peaks/index.html). Or alternatively you can use the .pdf files provided to correct the sequence. You will need to edit your sequences to get rid of bad sequence at both the 5′ and 3′ end. Your alignment can be made either with CLUSTAL W or Muscle. These are both available in MEGAX. Once you have made your alignment, save it as a MEGA file type (.meg) for use in later work. 
Answer the following questions using the MEGA manual (under the documentation tab of the MEGA website) and the following resources found in your BlackBoard shell including: the MEGA Tutorial Modified for this class; the presentation Creating Phylogenetic Trees With MEGA; the Creating Phylogenetic Trees video; the T. Ryan Gregory paper and any other resources. 
1. For your assignment you will need to make a distance matrix looking at the number of differences between each taxa and a distance matrix looking at p-distance. Which two ants (not including the outgroup) are the most different? Take a screenshot of each and explain the difference between the two types of matrices. 
2. Answer the question What is an outgroup? 
3.You will use your alignment file (saved as a .meg) to make a neighbor-joining tree that has been bootstrapped 500 times and using Formica rufa as an outgroup. Make a .pdf of the tree and submit it labeled Phylogenetic tree for various ant species for Cytochrome Oxidase I using the Neighbor-joining algorithm and bootstrapped 500x. Make sure your taxa are clearly identified with their species name (ie. Camponotus atriceps_1). 
4.You will use your alignment file (saved as a .meg) to make a maximum parsimony tree that has been bootstrapped 500 times and using Formica rufa as an outgroup. Make a .pdf of the tree and submit it labeled Phylogenetic tree for various ant species for Cytochrome Oxidase I using the Maximum Parsimony algorithm and bootstrapped 500x. Make sure your taxa are clearly identified with their species name (ie. Camponotus atriceps_1).
 5. What were the differences between your two trees? Are these a single population of ants? What method(s) is best for making a tree that compares two different populations? Explain your answer.

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