Neutrality analysis
Neutrality plot analysis was used to assess codon usage bias caused by mutational pressure and natural selection. Codon neutrality is expressed through the relationship between GC1,2 (average GC content between GC1 and GC2) and GC3, represented by a regression line (neutrality plot)34,35. The slope ε of this regression line, calculated using least squares regression analysis, is a neutrality value that ranges from 0 when there is no effect of mutation pressure (full selective constraints) to 1 when mutation bias is the main force affecting the codon usage (complete neutrality of codon usage). The equilibrium point Ep was also defined. This point is at the intersection of the neutrality plot (regression line) and the line of complete equilibrium between the average GC content and the directional mutation pressure with neutrality equal to one (ε = 1). The Ep value was computed as\(\frac{\mathrm{A}_{\mathrm{0}}}{\left(\mathrm{1-\varepsilon}\right)}\), where A0 is the value calculated from the regression of GC1,2 and GC3 at GC3 = 034. The direction of the mutational pressure in the gene was defined according to the position of this gene on the neutrality plot relative to the equilibrium point.