NEXUS file preparation

1. Open the NEXUS file with MacClade 4.03 or higher.
2. Select characters for one locus by shift-clicking on characters for that locus.
3. Under the Characters menu, choose "Character Set..." then "Store Character Set".
4. Name this set by the name of the locus (omit spaces)
5. Do the same for other loci.
6. Make sure that the genetic code is correct for your loci by going to "Genetic Code..." in the Characters menu. For mixed nuclear and mitochondrial loci, choose Drosophila mtDNA.
7. For protein-coding loci, select all the characters in one exon, then go to Characters -> "Codon Positions" -> "Calculate Positions...". Select the option "Choose to minimize stop codons".
8. Select non-protein-coding regions (i.e., introns, rDNA) and go to Characters -> "Codon Positions" -> "Non-coding".
9. Go to Taxa -> "Taxon list".
10. Move the outgroup taxa so that they are next to each other by dragging the number to the left of the taxon name.
11. Select the whole group of outgroup taxa by shift-clicking on their numbers.
12. Go to Taxa -> "Taxon sets" -> "Store taxon set".
13. Name this taxon set "outgroup".
14. Do the same for other taxa you would like in a set. Feel free to change the order of the taxa to do this.
15. When you are done, click on the icon that looks like a Mayan pyramid, then click on one of the taxa. This alphabetizes the taxa, making later steps in PAUP easier.
16. Go to File -> "Print list" to print the list, along with taxon numbers.
17. Save the file and quit MacClade.
18. Open PAUP 4.0b8 or higher. PAUP often has updates, so check its website to make sure you are using a recent version. Also check for bug reports.
19. From within PAUP, open the file you just saved with MacClade. Choose "Edit", not "Execute".
20. Scroll down to below the data matrix.
21. NEXUS files are arranged in blocks, starting with "BEGIN ###;" and ending with "END;". Go to the SETS block [skip this and the next 2 instructions if you have only one locus or no coding regions].
22. Start a new line. Type "CHARSET", a space, then "###first = ", replacing ### with the name of your first coding locus.
23. Look for the CODONS block. You should see something within this block that looks like "1: 1-1300\3 1400-2700\3", with the numbers changed. If your first coding locus is 1302 long, for example, copy the "1-1300\3"and place it after the equals sign in the line you just created. If your first coding locus extends all the way to 2700 in this example, just with an intron in the middle, copy the entire "1-1300\3 1400-2700\3" to after the equals sign in the line you just created.
24. Type a semicolon at the end of this line after pasting in the numbers.
25. You have just created a character set containing all the bases with the first codon position from your first coding locus. Repeat the same process for other loci, all three positions. The general format is "CHARSET [charset name] = [character numbers];".
26. You can do the same thing with sets of taxa. You should already have a taxset named "outgroup". You can make other taxsets using the format "TAXSET [taxset name] = [taxon numbers];". The taxon number is the same as the number on the taxon's left in MacClade's taxon list window, which you have printed out. It's also the order of taxa within the DATA block.
27. Create a PAUP block. Make sure this is after one of the "end;" lines but before the next "Begin ###;" lines. The format is "Begin PAUP;" skip a few lines, then "end;". Don't forget the semicolons.
28. Within the PAUP block, create a character partition. The general format is "CHARPARTITION [partition name] = [charset name 1]:[charset name 1], [charset name 2]:[charset name 2], [charset name 3]:[charset name 3];" For example, if the data consists of 28S and COI sequence, you would have a charset called 28s, another charset called coI, and the charparition command would be "CHARPARTITION locus = 28s:28s, coI:coI;" This is used in ILD tests.
29. Within the PAUP block, paste the following text:
    
    outgroup outgroup /only; set maxtrees=5000 increase=auto autoclose=no torder=right tcompress=yes taxlabels=full storebrlens=yes storetreewts=yes outroot=paraphyl;
    
    This sets the outgroup to be whatever you determined in the outgroup taxset, sets a reasonable starting maxtrees number, and sets other PAUP preferences to useful ones for starting a search. Other PAUP commands can be included in the PAUP block as well.
30. You may find it useful to keep a running log of all analyses done. This can be helpful in checking what p-values were of various tests, how many replicates were done in a particular search, etc. To do this, paste the following text within the PAUP block:
    
    log file=[name] append=yes replace=no;
    
    If you do this, delete the log commands from other batch files (see below) so that everything is logged to one file