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To start off, you need to perform an alignment of all your sequences. This can be done using MUSCLE. The output file (.txt) then needs to be converted to a .nex file, which can be done using ALTER.


  1. Go to the MUSCLE website
  2. Paste your sequences in FASTA format with two returns between each one
    • To generate FASTA format from Strider, select File and then Export as…; when the new window pops up, set the Format bar to FASTA
  3. Set the output to "Pearson/FASTA"
  4. Do NOT change any of the other parameters
  5. Hit Submit
  6. When you have the alignment, click on the Result Summary tab
  7. Right click on the link beneath Alignments in FASTA format and select Save link as…
  8. Give the file the name that you want and then add the extension .txt

NOTE: At this point you can view your aligned sequences in MacClade.


  1. Go to the ALTER website
  2. Under 1. Select format
    • Choose "MUSCLE" for program
    • Choose "FASTA" for format
  3. Under 2. Paste or Upload MSA hit upload…
  4. Navigate to the .txt file that you created using MUSCLE, select it, and hit open
  5. Under 3. Select output format and convert
    • Choose "PAUP" for program
    • Choose "NEXUS" for format
  6. Hit Convert!
  7. Under 4. Save converted MSA hit save… and then Okay
  8. Open the .nex file with a text editor
  9. At the top of the file, change
    interleave datatype=NUCLEOTIDE gap=-;
    interleave datatype=NUCLEOTIDE gap=-;options gapmode=missing;
  10. Scroll to the bottom and change
    to the following[1]:
    begin paup;
    set autoclose=yes;
    set criterion=parsimony;
    set root=outgroup;
    set storebrlens=yes;
    hsearch addseq=random nreps=1000 swap=tbr hold=1;
    savetrees file=yourfilename.tree.nex format=altnex brlens=yes;
  11. Save

NOTE: If you're doing this with multiple alignments, you'll have to refresh the ALTER page before working on a new .txt file.



NOTE: PAUP has a command-line interface. A .pdf user manual is available here and a .pdf command line reference manual is available here.

  1. Open PAUP
  2. Move the file(s) you intend to analyze into the same folder as the PAUP executable file
    • e.g. if PAUP is on the desktop, then move your .nex file(s) to the desktop, too
  3. Use the cd ("change directory") command to navigate to PAUP
    • e.g. cd Desktop
  4. Use the execute command to analyze your .nex file
    • e.g. execute yourfilename.nex

If PAUP generates more than one tree, do not close the Terminal shell! You need to generate a consensus tree. Enter the following:

contree all/majrule save file=yourfilename.consensus.nex;

This will give you both a majority rule consensus tree[2] and a strict consensus tree.[3]


  1. Open MacClade
  2. Select the alignment file
    • e.g. yourfilename.nex
  3. Click on the Windows menu
  4. Select Tree Window
  5. Select Open Treefile…
  6. Load your saved tree file
    • e.g. yourfilename.tree.nex


  1. Open FigTree
  2. Load your saved tree file
    • e.g. yourfilename.tree.nex


  1. See this.
  2. A majority rule consensus tree only shows branches which appear in at least 50% of the "rival" trees.
  3. A strict consensus tree only shows branches which appear in 100% of the "rival" trees.