Protocol: Creating a Phylogenetic Tree Based on a Conserved Gene

This protocol outlines the steps to create a phylogenetic tree using the conserved rbcL gene from various red algae species as an example.

Step 1: Selecting the Conserved Gene and Species Group

1. Choose a Species Group:
   • in this case red algae.
2. Select a Conserved Gene:
   • in this casee rbcL, which is conserved in red algae and plants.

Step 2: Gathering Gene Sequences from NCBI

1. Access NCBI:
   • Visit NCBI.
2. Search for the Gene in Different Species of your selected group:
   • Enter the search term: red algae + rbcL.

   

3. Select Species and Download Sequences:

   • Choose the species you want to include in the tree. Here are the selected species for this example:
     • Gracilaria dura strain BOR4
     • Gracilaria gracilis from United Kingdom
     • Solieria sp. isolate LUCXXXX
     • Ptilophora sp. voucher Ptilo4
     • Chondria sp. isolate PD1961
4. Download the FASTA Sequences:
   • Go to the gene’s NCBI page for each species (click on the page).
   • Click on FASTA to download the nucleotide sequence.
   • Save these sequences in a Word or Notepad file, including the labels starting with >.

   

Step 3: Aligning Sequences in Mega11

1. Open Mega11 Software:
   • If not installed, download Mega11.
2. Create a New Alignment:
   • Navigate to ALIGN -> Edit/Build Alignment -> Create a New Alignment.
   • Select DNA (or Protein depending on your study).

   

   

   

3. Input Sequences:
   • Copy the FASTA sequences, including titles starting with > for all your species together, and paste them into the alignment window in Mega11.

   

4. Align the Sequences:
   • Select all sequences by holding Shift and clicking.
   • Click Alignment -> Align by ClustalW in the toolbar.

   

5. Save the Alignment:
   • Save the aligned sequences to a known location on your computer.

Step 4: Constructing the Phylogenetic Tree

1. Open the Tree Construction Tool:
   • In Mega11, go to PHYLOGENY -> Construct/Test Neighbor-Joining Tree.

1. Select the Alignment File:
   • Choose the alignment file saved in the previous step (ensure you check “All files” to view all file types).
2. View the Tree:
   • To see the branch lengths, select the Branch Length option.
3. Customize the Tree View:
   • Use Mega11’s tools to adjust the appearance and labels of the tree as required. —

This protocol provides a step-by-step guide for constructing a phylogenetic tree using the conserved rbcL gene from red algae species. By following these steps, you can visualize the evolutionary relationships between the chosen species based on their gene sequences.