Figure 5. Neutrality plots (GC1,2 vs. GC3) for Hsp60 genes from 17 phyla. The GC1,2 values represent the average GC content at the first and second positions of codon (GC1 and GC2), while GC3 values represent the GC content at the third synonymous codon position. The solid line represents the linear regression of GC1,2 versus GC3, the correlation of which is described by the regression coefficient R and its p-value. The correlation coefficient R reflects the strength of the impact of GC3 on GC1,2. The p-value characterizes the significance of R. Changes in GC3 values actually affect the GC1,2 values when the p-value of R is less than 0.05. In turn, changes in GC3 are considered random, and the R coefficient is not irrelevant when the p-value is greater than 0.05, i.e. GC3 and GC1,2 values are not correlated. The slope ε of the regression line indicates the neutrality of the codon usage. Neutrality values were determined by equation [ε × 100, %]. Slope values ranging from 0 to 1 were calculated using the least-squares regression analysis. The dashed line is a complete neutrality plot, which reflects the complete equilibrium of the nucleotide composition of the gene/genome with directional mutation pressure. The equilibrium point Ep was defined as the intersection point of the neutrality plot (regression line) and the complete neutrality plot. The Epvalue reflects the GC3 content of the gene/genome when the mutation frequencies (AT→GC and GC→AT) are equal. The direction of the mutational pressure, indicating an imbalance in the frequencies of the AT→GC and GC→AT mutations, was determined in accordance with the following conditions: the average GC content value less than theEp value reflects the AT mutational pressure; the average GC content value greater than the Ep value reflects the GC mutational pressure. Phyla were sorted by average total GC content of Hsp60 genes.
The results of the neutrality analysis reflect statistically significant correlations between the GC1,2 and GC3values of the Hsp60 genes from 15 phyla (Figure 5). Taking into account the results of neutrality analysis and difference between the average GC3 values and the corresponding values at equilibrium points, 17 phyla were divided into three groups according to the direction of mutational pressure (Table 2).
Table 2. The results of neutrality plot analysis