How To Do A Phylogenetic Tree

Phylogenetic trees, also known as evolutionary trees, are diagrams that depict the evolutionary relationships among different species, genes, or other entities. These trees are essential tools in understanding the history of life, tracing the origins of diseases, and making predictions about the evolution of organisms. Constructing a phylogenetic tree involves a combination of data collection, analysis, and interpretation, making it a fascinating and complex process.

Understanding the Basics of Phylogenetic Trees

Before diving into the steps of constructing a phylogenetic tree, it's crucial to understand the fundamental components and terminology.

• Root: The root represents the common ancestor of all taxa (the entities being compared) in the tree. It is the starting point of the evolutionary relationships depicted.
• Branches: Branches represent the evolutionary lineage over time. The length of a branch can sometimes indicate the amount of evolutionary change or the time elapsed.
• Nodes: Nodes represent common ancestors. Each node signifies a point where an ancestral lineage diverged into two or more descendant lineages.
• Leaves (Tips): Leaves, or tips, represent the taxa being studied. These can be species, genes, or any other entities for which you have data.
• Topology: The topology of a tree refers to the branching pattern, showing the relationships among the taxa.
• Scale: Phylogenetic trees can be scaled or unscaled. Scaled trees have branch lengths that represent the amount of evolutionary change or time, while unscaled trees only show the relationships without indicating the magnitude of change.

Step 1: Data Collection and Alignment

The first step in constructing a phylogenetic tree is to gather the data that will be used to infer evolutionary relationships. This data can come from various sources, including morphological characteristics, DNA sequences, RNA sequences, and protein sequences. The most common and reliable data source is molecular data, particularly DNA sequences.

• Choosing the Data: Select a gene or a set of genes that are present in all the taxa you want to include in your tree. Commonly used genes include ribosomal RNA genes (such as 16S rRNA for prokaryotes and 18S rRNA for eukaryotes) and protein-coding genes. Ensure that the selected gene has enough variability to provide meaningful phylogenetic information but is conserved enough to be easily aligned across all taxa.
• Data Acquisition: Obtain the sequences for the selected gene from each taxon. This can be done through laboratory techniques such as PCR amplification and DNA sequencing or by retrieving sequences from online databases like GenBank, EMBL, or DDBJ.
• Sequence Alignment: Once you have the sequences, the next step is to align them. Sequence alignment is the process of arranging the DNA, RNA, or protein sequences to identify regions of similarity and difference. This is a critical step because phylogenetic inference relies on the assumption that similarities reflect common ancestry.
  • Alignment Tools: Several software programs are available for sequence alignment, including:
    • ClustalW: A widely used tool for multiple sequence alignment.
    • MAFFT: Known for its speed and accuracy, particularly with large datasets.
    • MUSCLE: Another popular choice for multiple sequence alignment, balancing speed and accuracy.
  • Alignment Process:
    1. Input Sequences: Load your sequences into the alignment software.
    2. Parameter Settings: Adjust the alignment parameters, such as gap opening and extension penalties, to optimize the alignment.
    3. Run Alignment: Execute the alignment algorithm.
    4. Manual Adjustment: Inspect the alignment and manually adjust it if necessary. This is particularly important in regions with high variability or complex patterns of insertions and deletions (indels).

Step 2: Choosing a Phylogenetic Method

After aligning the sequences, the next step is to choose a method for constructing the phylogenetic tree. There are several methods available, each with its own assumptions and strengths. The main categories of phylogenetic methods include:

• Distance-Based Methods: These methods calculate a distance matrix based on the differences between sequences and then use this matrix to construct the tree.
  • UPGMA (Unweighted Pair Group Method with Arithmetic Mean): A simple method that assumes a constant rate of evolution. It is generally not recommended for phylogenetic inference but can be useful for clustering closely related sequences.
  • Neighbor-Joining: A more sophisticated method that does not assume a constant rate of evolution. It is computationally efficient and often used for large datasets.
• Character-Based Methods: These methods directly analyze the characters (i.e., the individual nucleotides or amino acids) in the sequence alignment to infer the phylogenetic tree.
  • Maximum Parsimony: This method seeks the tree that requires the fewest evolutionary changes to explain the observed data. It is conceptually simple but can be computationally intensive for large datasets.
  • Maximum Likelihood: This method evaluates the probability of the observed data given a particular tree and a model of sequence evolution. It is statistically rigorous but computationally demanding.
  • Bayesian Inference: This method uses Bayes' theorem to calculate the posterior probability of a tree given the data and a prior probability distribution. It is considered one of the most accurate phylogenetic methods but requires significant computational resources.

Step 3: Building the Phylogenetic Tree

Once you have chosen a phylogenetic method, you can use software to build the tree. Here are some popular software packages for phylogenetic analysis:

• MEGA (Molecular Evolutionary Genetics Analysis): A user-friendly software package with a graphical interface that supports various phylogenetic methods, including distance-based, maximum parsimony, maximum likelihood, and Bayesian inference.
• PhyML: A software package specifically designed for maximum likelihood phylogenetic inference. It is known for its speed and accuracy.
• MrBayes: A software package for Bayesian phylogenetic inference. It uses Markov chain Monte Carlo (MCMC) methods to estimate the posterior probability of trees.
• RAxML (Randomized Axelerated Maximum Likelihood): Another software package for maximum likelihood phylogenetic inference. It is optimized for large datasets and can handle thousands of taxa.
• BEAST (Bayesian Evolutionary Analysis Sampling Trees): A powerful software package for Bayesian phylogenetic inference that can incorporate various types of data, including morphological and molecular data. It is particularly useful for estimating divergence times.

Here’s how to build a phylogenetic tree using some of these software packages:

Using MEGA

1. Input Alignment: Open MEGA and load your sequence alignment file. MEGA supports various alignment formats, including FASTA, Clustal, and MEGA.
2. Phylogenetic Analysis: Go to the "Phylogeny" menu and select the method you want to use (e.g., "Construct Maximum Likelihood Tree").
3. Parameter Settings: Adjust the parameters for the chosen method. This may include selecting a substitution model, specifying the number of bootstrap replicates, and setting other options.
4. Compute Tree: Click the "Compute" button to start the phylogenetic analysis. MEGA will generate the phylogenetic tree based on your settings.
5. Tree Visualization: MEGA provides tools for visualizing and manipulating the phylogenetic tree. You can change the tree layout, branch colors, and other visual settings.

Using PhyML

1. Input Alignment: Prepare your sequence alignment file in a suitable format (e.g., PHYLIP or FASTA).
2. Run PhyML: Open PhyML and specify the input alignment file. You can run PhyML from the command line or through a graphical interface if available.
3. Parameter Settings: Set the parameters for the maximum likelihood analysis, such as the substitution model, tree search algorithm, and bootstrap replicates.
4. Compute Tree: Run PhyML to generate the phylogenetic tree. The output will include the tree file in Newick format.
5. Tree Visualization: Use a tree visualization program (e.g., FigTree, TreeView) to view and manipulate the phylogenetic tree.

Using MrBayes

1. Input Alignment: Prepare your sequence alignment file in NEXUS format.
2. Create MrBayes Block: Write a MrBayes block that specifies the parameters for the Bayesian analysis, including the substitution model, prior distributions, and MCMC settings.
3. Run MrBayes: Open MrBayes and execute the MrBayes block. The program will perform MCMC sampling to estimate the posterior probability of trees.
4. Convergence Diagnostics: Monitor the convergence of the MCMC chains to ensure that the analysis has reached a stable state. This can be done by examining the trace plots and calculating the potential scale reduction factor (PSRF).
5. Summarize Results: Summarize the results of the Bayesian analysis to generate a consensus tree. This tree represents the most probable phylogenetic relationships based on the data.
6. Tree Visualization: Use a tree visualization program to view and manipulate the consensus tree.

Step 4: Evaluating the Tree

Once you have constructed a phylogenetic tree, it is essential to evaluate its reliability and robustness. This can be done using several methods:

• Bootstrapping: Bootstrapping is a resampling technique that involves creating multiple pseudo-replicates of the original alignment by randomly sampling columns with replacement. A phylogenetic tree is constructed for each pseudo-replicate, and the percentage of trees in which a particular clade (a group of taxa sharing a common ancestor) appears is recorded. Bootstrap values range from 0 to 100, with higher values indicating stronger support for the clade. Generally, bootstrap values of 70% or higher are considered significant.
• Bayesian Posterior Probabilities: In Bayesian phylogenetic inference, the posterior probability of a clade is a measure of the probability that the clade is real given the data and the model. Posterior probabilities range from 0 to 1, with higher values indicating stronger support for the clade. Generally, posterior probabilities of 0.95 or higher are considered significant.
• Likelihood Ratio Test: The likelihood ratio test (LRT) compares the likelihood of the best tree to the likelihood of alternative trees. It can be used to test specific hypotheses about phylogenetic relationships.
• Consensus Tree: A consensus tree is a tree that summarizes the agreement among a set of trees. It can be constructed using various methods, such as the majority-rule consensus, which includes clades that appear in more than 50% of the trees.

Step 5: Interpreting the Phylogenetic Tree

The final step in constructing a phylogenetic tree is to interpret the results and draw conclusions about the evolutionary relationships among the taxa.

• Rooting the Tree: If the tree is unrooted, you will need to root it to infer the direction of evolution. This can be done by using an outgroup, which is a taxon that is known to be distantly related to the taxa of interest. The outgroup is placed at the base of the tree, and the root is located on the branch connecting the outgroup to the rest of the tree.
• Identifying Clades: Identify the clades in the tree, which are groups of taxa that share a common ancestor. Clades can be nested within each other, forming a hierarchical structure.
• Inferring Evolutionary Relationships: Use the topology of the tree to infer the evolutionary relationships among the taxa. Taxa that are closely related on the tree are assumed to have diverged more recently from a common ancestor.
• Estimating Divergence Times: If the tree is scaled, you can use the branch lengths to estimate the divergence times among the taxa. This requires calibrating the tree using fossil data or other sources of information about the timing of evolutionary events.
• Testing Evolutionary Hypotheses: Use the phylogenetic tree to test hypotheses about the evolution of particular traits or the biogeographic history of the taxa.

Practical Tips for Constructing Phylogenetic Trees

• Choose the Right Data: The choice of data is crucial for constructing accurate phylogenetic trees. Select genes or other characters that are informative for the taxa you are studying.
• Align Sequences Carefully: Sequence alignment is a critical step in phylogenetic inference. Pay attention to the alignment quality and manually adjust it if necessary.
• Select an Appropriate Method: The choice of phylogenetic method depends on the data and the research question. Consider the assumptions and strengths of each method before making a decision.
• Evaluate Tree Reliability: Evaluate the reliability of the tree using bootstrapping, Bayesian posterior probabilities, or other methods.
• Interpret Results Cautiously: Interpret the results of the phylogenetic analysis cautiously and consider alternative explanations for the observed patterns.

Conclusion

Constructing a phylogenetic tree is a complex but rewarding process that can provide valuable insights into the evolutionary history of life. By following these steps and considering the practical tips, you can build accurate and reliable phylogenetic trees that will help you answer your research questions. Whether you are studying the origins of diseases, tracing the evolution of genes, or understanding the relationships among species, phylogenetic trees are an essential tool for exploring the diversity and history of the natural world.