This analysis identifies gene pairs evolving under functional divergence, where one duplicate is highly conserved while the other evolves in a highly variable manner [ 39].
The first method is Gu's method (Gu99) that is implemented in the software DIVERGE2 [ 50], which detect specific amino acid sites under functional divergence between two paralog lineages after gene duplication [ 73, 74].
When looking at the spatial localization of all sites previously identified as under positive selection and/or under functional divergence, two sites, Ser 156 and Cys 157, were oriented toward the active site.
We found that these sites are those positively selected sites detected by the branch-site model of PAML 4.8 with PP ≥ 95%% and those under functional divergence predicted by FunDi and GroupSim with PP ≥ 99%%.
We also studied the pattern of functional divergence and found three different profiles represented by the amino acid sites under functional divergence.
Examination of the distribution of sites under functional divergence when we compared GroEL1 to GroEL2 only yielded three amino acid positions under functional divergence using the posterior probability (PP) threshold of PP= 0.75.
We adjusted for the differential evolutionary rate and divergence time to define CNSs that are under functional constraint.
To assess whether the coding sequences are under functional constraint, the ratio of non-synonymous (dN) to synonymous (dS) divergence was calculated for all pairwise sequence comparisons between 6 representative streptophytes (Table 1).
Otherwise, amino acid positions candidates to be under type I functional divergence are more homogeneously distributed along the 3D structure (results not shown): two of the tree positions with high PP values, however, are also in the functionally important predicted protein domains.
Interestingly, six of the seven sites (all but 74G) were also identified to have been under Type-I functional divergence, while 70R, 88 L, 106A were under positive selection and under Type-I and Type-II functional divergence.
