Phylogenetic Analysis

There are several software that are needed to be downloaded.

1. BioEdit Sequence Alignment Editor was used to align forward and reverse sequences for sequence clean-up. The reverse sequences was set to reverse complement, grouped together with the forward sequences, align together using the pairwise alignment (optimal GLOBAL alignment), delete sequence where appropriate based on chromatogram and saved as FASTA files. Then, all the cleaned sequences for tetragnathid species with the downloaded selected tetragnathid species and two out-group sequences from genbank were saved as into one file as FASTA. 

2. Clustal X program was used for multiple sequences alignment and saved as NEXUS. Phylogenetic Analysis Using Parsimony (PAUP) software was used to elucidate the phylogenetic relationships between the tetragnathid species.

3. The PAUP software was keyed as follows:

a.       Neighbor-joining (NJ):

begin paup; log start file=NJ.log; outgroup 50;  set criterion=distance increase=auto;  dset distance=K2P; NJ treefile=NJ.tre; set autoclose=yes; bootstrap nreps=1000 search=NJ treefile=NJBt.tre; log stop; end

b.      Maximum Parsimony (MP):

begin paup; log start file=MP.log;  set criterion=parsimony; set autoclose=yes; set root=outgroup;  outgroup 50; set increase=auto; hsearch swap=tbr start=stepwise addseq=random nreps=100 hold=1; contree; set storebrlens=yes; bootstrap nreps=1000 treefile=COI/18S.tree search=heuristic / swap=tbr start=stepwise addseq=random nreps=1 hold=1; savetrees from=1 to=1 file=COI/18S.cb.pa.tree.nex format=altnex brlens=yes savebootp=nodelabels maxdecimals=0; pscores / tl ci ri rc hi; log stop; end;

c.       Maximum Likelihood (ML):

For COI: begin paup; log start file=ML.log; set autoclose=yes; set criterion=likelihood; set root = outgroup; outgroup 50; set storebrlens=yes; set increase=auto; Lset  Base=(value)  Nst=value  Rmat=(value)  Rates=type Shape=value  Pinvar=value; bootstrap nreps=100 search=heuristic/ addseq=random swap=tbr hold=1; contree; savetrees from=1 to=1 file=COI/ML.cb.mlb.tree.nex format=altnex brlens=yes savebootp=NodeLabels MaxDecimals=0; log stop; end;

d.      d. Genetic distance

Begin paup; dset distance=p; showdist; savedist format=nexus file=P_dist.nex; dset distance=p; showdist; savedist format=onecolumn file=P_dist1.txt; end;