Does drive toward canonic exonic splicing sites exist in mammals?

About 2/3 of introns are inserted between G and G/A, which has previously been explained by codon usage frequencies existing during the period of intron insertions. However, less is known about the evolution of exonic splicing sites. Exonic nucleotides that frame introns are involved in both protein coding and splicing. While a compromise between protein coding and splicing constraints is achieved differently in each intron phase, AG|G is the most common site in all phases comprising about one quarter of all such sites. There is also a great variety of other splicing sites. Here we examine evolutionary changes in exonic nucleotides located at positions -2 -1|+1 which occurred after the beginning of eutherian radiation using comparisons of orthologous splicing sites from five mammalian species. AG|G accumulated fewer substitutions and was more conservative than less frequent exonic splicing sites. Such trend could potentially increase frequencies of AG|G during mammalian evolution and cause a decline of less common sites which had higher substitution rates. However, there is a limit to this process determined by the dynamic equilibrium of substitution rates and the frequencies of different splicing sites. It seems that this equilibrium was already achieved at the time of eutherian radiation and a moderate increase in AG|G frequency was observed only in the human genome.