Alignment (ClustalW in MEGA)

Format Data (DnaSP)

Define Sequence Set (DnaSP)

Generate Haplotype Data File (DnaSP)

- Arlequin Haplotype List (.hap & .arp)

Look at your haplotypes (window blue background). Your unique haplotypes are your individual samples. Your
number of haplotypes and haplotype diversity are also given there. For haplotype pie charts, use MS Excel in
creating them using your sample count per haplotype.

Find Best DNA/Protein Model using your aligned .fas file (MEGA>Models)

Your best-fit model is the one on the top (usually based on the criteria, i.e. highest AICc and BIC, and lowest
parameter). You will be using this in your Arlequin runs; however, Arlequin has limited models that you may use so
make sure to pick the best-fit model available and its corresponding gamma value if applicable.

Construct NJ tree (MEGA>Phylogeny).

You also need the best-fit model available + gamma value when constructing your tree to address the substitution
trends of your samples.

Arlequin

Open your .arp file by clicking Open Project in the Arlequin software. If it does not open, it might mean that you have
deleted something when you were editing/renaming your .hap and .arp files. Go back to that and find what you did
wrong.

In AMOVA settings, also check the Compute minimum spanning network and use distance matrix. Use the best-fit
model available + gamma value based on the model test you did in the previous Best DNA model test in MEGA.

In Population Comparisons, check Pairwise FSTs and compute pairwise differences. Use best-fit model available +
gamma value.

In Molecular Diversity Indices, check Standard Diversity Indices. Also check Molecular Diversity Indices and compute
minimum spanning tree among haplotypes. In molecular distance, use the best-fit model available + gamma value.
These settings will get you your basic and molecular indices like nuclear diversity, expected heterozygosity,

In your results page, you may find your indices in within-samples summary folder. Under molecular diversity indices,
you may find the header NEXUS notation for MST. Copy the item under that (beginning with #NEXUS), paste in
Notepad and save as a .nex or .nexus. You may view that MST using the software Treeview which you may google
since its a free software.