Estimation of species divergence time of a phylogenetics tree This tutorial will guide the users for step by step procedure for the generation of phylogenetic tree with species divergence time. In general, we generate the simple phylogenetic tree using DNA/protein sequence alignment with multiple alignment softwares, followed by NJ, UPGMA or distance based methods for generation of tree. Evolutionary tree generated by these methods lack the accurate branch length. Requirement: ClustalW, Phylip, PAML, FigTree 1. Download the mitochondrial genome of Anopheles species from ncbi. Combine all the fasta files into a single fasta file (example: All.fasta). 2. Run the multiple sequence alignment software like ClustalW to generate alignment file (example: All.aln) and convert to phylip format All.phylip using clustal2phylip tool 3. Exceute Phylip dnaml program from terminal using the input file generated in previous step i.e. All.phylip, this will generate output file outtree. 4. Execute Phylip reroot tree using input from step 3 for the generation of mid-point rooted tree rooted tree. At this step, we are ready with our tree file and alignment file. 5. Download the mcmctree control file mcmc tree.cntrl and place in the same folder where all your input files are present. 6. Run the command "mcmctree mcmc-tree-1.ctl" and this will produce output file out.BV. Now rename the file from out.BV to in.BV . 7. Download the mcmctree control file mcmc-tree-2.cntrl and place in the same folder where all your input files are present. 8. Run the command "mcmctree mcmc-tree-2.ctl" and this will produce output in the form of Figtree that can be visualized by Figtree software.
Phylogenetic tree of Anopheles species with divergence time in Ma (eg. D. melanogastor is 214 Ma)