This value is higher than the mean substitution rate values observed by others [22] [26].
Table 3 shows the correlation of mean substitution rate with biosynthetic cost for all amino acid cost types.
Analyses were run with both strict clock and uncorrelated log-normal relaxed clock [24] models, with the mean substitution rate fixed to 1.0.
Substitution rates at each site were divided by the estimated tree length across the entire gene to control for the mean substitution rate of the encoding gene.
The mean substitution rate was calculated for each amino acid across all sites where it was inferred in the ancestral protein and then compared with the S. cerevisiae biosynthetic cost for that amino acid.
Following Fagundes et al. [13], we used the HKY+Γ evolutionary model, a log-normal relaxed molecular clock with a mean substitution rate of 1.26×10−8 mutations/site/year [17] for the complete coding sequence.
For both data sets, the mean substitution rate was set to one, the base frequencies were estimated from the data, and six gamma categories as well as a UPGMA starting tree were used.
Similarly, CRF01_AE presented a mean substitution rate per site twice as high as that observed for subtype B. Significant differences in adaptive selection and substitution rate between HIV-1 subtypes have been reported before [59], [63] and were attributed to differences in immune selective pressure from the host and in mutation rate or generation time of the virus.
The estimated mean substitution rate in HBV was 4.2×10−5 nucleotide substitutions/site/year. Applying this rate to the phylogenetic analysis, we estimated that the origin of 'CD1' may have occurred 520 years ago and 'CD2' may have occurred 410 years ago.
