In our previous studies, the evolutionary rate (substitutions/site/million years) of mitochondrial DNA (mtDNA) in rodents and moles was estimated to be approximately 0.11 in more recent divergence times of a few tens of thousands of years and declined quickly to approximately 0.03 in more distant divergence times. However, the modes and mechanisms associated with the time dependency have not been well documented.

We addressed the abundance of nonsynonymous substitutions that had appeared in the protein-coding region of mitochondrial cytochrome b gene sequences in rodents and moles, using sequences obtained from the nucleotide database. We collected 23 haplogroups with signals of late Quaternary population expansion events and categorized them into three groups. The expansion events were predicted to have started during the last 15,000 years (Group I), ca. 53,000 years ago (Group II), and 130,000-230,000 years ago (Group III). We counted the numbers of nonsynonymous and synonymous substitutions in all haplogroups. The nonsynonymous substitution ratios were exclusively low (0.06–0.20) in Groups II and III. By contrast, the values in Group I varied markedly, and Group I could be further classified into two subgroups based on high (0.28–0.43) and low (0.09–0.20) values of the nonsynonymous substitution ratio, which were roughly associated with the time expansion started, suggesting that the shift from high to low values of the nonsynonymous substitution ratio occurred within the last 15,000 years. The Group II and III networks exhibited two- or three-step star-shaped structures and a trend of frequent nonsynonymous substitutions on exterior branches, which were less frequent on interior branches, supporting the idea that processing to remove nonsynonymous substitutions occurs over a short evolutionary time. 

Based on temporal dynamics, nonsynonymous mtDNA substitutions in small mammals accounted for 40% of all substitutions during the very early evolutionary stage and then rapidly declined, dropping to approximately 15%. This process appears to have been completed in ca. 15,000 years. This is a good explanation of the time-dependent trend in the rate of mtDNA evolution predicted by previous studies.