In the protein coding genes, both the second and the third positions had negative AT-skew and GC-skew, with the first position having positive AT-skew and GC-skew; overall, PCGs had a negative AT-skew and a positive GC-skew (Additional file 8).
The same test was also performed on the third positions of CGN codons.
The codon substitution rate matrix of KHG [14] indicates that some types of double nucleotide mutations at the first and the third positions frequently occur.
Namely, CG1998 contain 6 exons, while CG11162 contains only 2. However, the last intron of both genes interrupts the coding sequence at very similar positions (i.e. between the second and the third positions of a Lys codon, Figure 3).
With Hsp70 HSEs excluded, overall HSEs vary among species (Tamura-Nei gamma distance: mean = 0.410, SE = 0.089; transversions only) as much as do the third positions of codons within the coding sequence of the genes in which they occur.
However, overall base compositions are not highly skewed because the third positions for these codons in the L. hesperus MaSp1 and MaSp2 are extremely biased towards adenine and also strongly biased, but less dramatically, towards thymine (86% of MaSp1, 91% of MaSp2 glycine, alanine, and proline codons end with adenine or thymine; Table 1).
In the Singlet-Doublet-Triplet (SDT) mutation model [20], single-nucleotide, doublet and triplet mutations spanning codon boundaries are taken into account, but double nucleotide mutations at the first and the third positions in a codon were not taken into account.
We used only the third positions of the codons.
Only the third positions of some codons may be fourfold degenerate.
Table 1 also presents the data for the third positions of codons only.
This process is associated with perturbations in the expected pattern of sequence evolution such that substitutions are concentrated in the third positions of the original reading frame rather than in the third positions of the new frame.
