Making Phylogenies and Identifying Low and High Branching Confidence

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Lab Assignment 2
Before attempting to begin this assignment it is absolutely imperative that you ensure you have access to a PC or MAC computer (a requirement for taking this course); you will need to download free software in order to complete this assignment.
Follow all steps given in the next chapters before attempting to answer assignment questions.
(for example, how to download MEGA-X, how to download a .FASTA file to your desktop, how to do a screen capture of your tree, how to insert an image into a word processing document).
Lab Assignment 2
Background information
Introduction to phylogenies
Evolution, i.e. the change in heritable characteristics of biological populations over multiple generations, is the fundamental concept underlying the entire field of biology. The implication of this concept is that the range of life on Earth can be traced back to common ancestors from which they arose, however, understanding which organisms share more recent common ancestors, and are therefore more closely related, is a daunting challenge.
To investigate these questions, humans require a method of categorizing and organizing living things. A phylogeny (also called a phylogenetic tree) is a representation of the evolutionary history of an organism that is used by biologists to classify living things. However, it is important to remember that an individual phylogeny is an evolutionary hypothesis and therefore may not accurately reflect the true evolutionary history of a given organism. Organisms in nature often have messy and complicated evolutionary histories, and phylogenies are just our best way of making sense of these complex histories of organisms for our purposes. How a phylogeny is constructed is extremely important as the classic adage “garbage in, garbage out” strongly applies. If you supply phylogenetic tree software with too few organisms, poor quality sequences, or include organisms that don’t make sense to build a tree from, you will end up with results that have no resemblance to true evolutionary relationships.
What do phylogenetic trees tell us?
A phylogeny is a powerful genetic tool that scientists use to understand the relationships between organisms. Much like a family tree, a phylogeny shows the relationships between organisms or groups of organisms back to a common ancestor. The closer a common branch point between two organisms, the closer the genetic relationship those two organisms have.
In the past -- and as you did in previous lab exercises -- a phylogeny was made using only the phenotypic characteristics (obvious, observable, and measurable traits) of the organism being studied. Characteristics included general appearance, behaviour, molecular features, motility, etc. When using these characteristics, past phylogenies frequently grouped organisms that looked similar but were not actually closely related, resulting in phylogenetic trees that often did not reflect true evolutionary history. Thanks to advances in genetics, scientists now have a more accurate way to reconstruct the evolutionary history of living things by constructing phylogenetic trees using the genes of the organisms they are studying. Because genes are heritable (passed from parents to offspring), they tell us far more about the evolution of an organism than assuming relatedness from physical characteristics.
How to interpret a phylogeny
A phylogeny is read by looking at its branches. The branching pattern shows the most recent common ancestor between all the organisms of interest, and can tell the reader which of the organisms are more genetically related. The points where branches come together represent common ancestors and are called nodes. A diagram showing the parts of a phylogeny can be found in Figure 1. An explanation of the relationships between organisms in a phylogeny can be found in Figure 2.
Bootstrapping
In most phylogenies, there is also a set of numbers located at each node. These numbers tell the reader how confident the software is in the relationship as shown in Figure 3. The software can calculate this confidence a number of different ways, however, one of the most common ways is by doing something called bootstrapping.
Bootstrapping is a complicated statistical process, but for our purposes can be understood as the software running the same analysis a number of times on the exact same genetic input. If you were to ask your phylogeny building software to do 1000 bootstrap “iterations”, it would generate 1000 phylogenies with the genetic information you’ve given it. The software then takes those 1000 phylogenies and counts the number of times a relationship exists.
In the figure below, the branch point between A + B is marked with a confidence of 0.90. This means that of the 1000 iterations the software ran on this phylogeny, 900 showed the relationship between A + B as pictured in Figure 3. In addition, branch lengths represent the number of changes in the gene or protein sequence over evolutionary time. Longer branches mean more changes and therefore more differences compared to other organisms in the phylogeny.
Sample sizes
Just like most statistical analyses, phylogenies are made stronger by increasing the sample size. Generally, this can be accomplished by including additional genes or proteins, which will provide more information for comparison. Using a sample size that is too small can lead to unsupported and ambiguous results. Selection of appropriate organisms can provide greater evolutionary context and better illustrate the relationships between all the included organisms. However, there are also negatives to choosing a sample size too large. A very large sample size can waste resources, cost a lot of money, and take a long time to calculate.
In our assignment, the Cox1 dataset we use is comparatively small; however, the analysis can still take substantial time depending on your computer. Ensure you begin this assignment well before the due date to provide enough time to complete the analysis.
Using the MEGA-X software
To make the phylogenetic tree you will use to answer the assignment questions, follow these steps to install and use the MEGA-X software.
NOTES on known issues:
All files have been tested on a variety of Windows-based OS and the most recent version of MEGA-X. The only issue that arose during testing was that some Windows-based machines produce error messages at Step 8 for some databases. If this happens, delete any "@" symbols in the FASTA file as described at the bottom of this page.
All files have been tested, and run without error, using the Mac 64-bit GUI version and Mac OSX Catalina (using a Mac running OSX Mojave will not work). If you are using a MacBook (especially if you have iCloud enabled), and it crashes at Step 4, see Alternate Step 4 instructions at the bottom of this page. There have been issues with alignment runtime for 2020 and newer MacBooks with an M1 chip (use phylogeny.fr instead).
MEGA-X does not run on Chromebooks, as far as we know. If you have a Chromebook or are running Windows or Mac OS and cannot get MEGA-X to work, you will need to use this site (use the "One Click" method and follow the on-screen instructions)
INSTRUCTIONS ARE PROVIDED IN THE IMAGES HOW TO INSTALL THE SOFTWARE AND HOW TO PROCCEED THE ASSIGNMENT
Database files used for AssignmenT
Created by Craig Soutar and Emily Haidl (2020).
As instructed in the previous chapter (called Using the MEGA-X software), you will download one of ten the .fasta files listed to your computer's desktop (or download folder).
Database 1 files: .fasta and .txt
Database 2 files: .fasta and .txt
Database 3 files: .fasta and .txt
Database 4 files: .fasta and .txt
Database 5 files: .fasta and .txt
Database 6 files: .fasta and .txt
Database 7 files: .fasta and .txt
Database 8 files: .fasta and .txt
Database 9 files: .fasta and .txt
Database 10 files: .fasta and .txt
To download a .fasta file, right click (or control + click on a Mac), save to your computer's Desktop or Downloads folder, and open it from within the MEGA-X program. The .txt files are there only if you want to see the information in the database.
NOTE:
All files have been tested, and run without error, using the Mac 64-bit version.
Some PCs produce error messages at Step 8 for some databases. If this happens, delete any "@" symbols in the FASTA file.
9 QUESTIONS TO ANSWER
Questions to answer:
Which Cox1 database (of the 10 provided) did you use? How many genes in total were in the database you chose?
Which model did the MEGA software decide was the best fit for your data? What do you think would happen if you decided to use a model that was not the ‘best fit’ for your data?
Look at your phylogeny and note down three nodes that have low branching confidence and three nodes that have high branching confidence.
What do we know about the relationships that have low confidence? What about the ones with high confidence?
Does your phylogeny show protists grouping in with other Eukaryotes? Why do you think these organisms are (or are not) grouping in with other Eukaryotes?
Do the plant-like protists seem to group with plants? What about the animal-like? Fungal-like?
Do you think your phylogeny shows the true relationships between the organisms that were included? Explain.
If you were going to make another phylogeny, how would you change what you did to make it better? (Hint: re-read the background in the Assignment 2 “chapter” to help answer this question).
Do you think you can make any concrete conclusions about the relationships between the organisms that were in your database from your phylogeny? Why or why not?