Calibration of a molecular clock in tits (Paridae)--do nucleotide substitution rates of mitochondrial genes deviate from the 2% rule?

The ongoing debate on the reliability of avian molecular clocks is actually based on only a small number of calibrations carried out under different assumptions with respect to the choice and constraints of calibration points or to the use of substitution models. In this study, we provide substitution rate estimates for two mitochondrial genes, cytochrome b and the control region, and age estimates for lineage splits within four subgenera of tits (Paridae: Parus, Cyanistes, Poecile and Periparus). Overall sequence divergence between cytochrome b lineages covers a range of 0.4-1.8% per million years and is thus consistent with the frequently adopted approximation for a sequence divergence between avian lineages of 1.6-2% per my. Overall rate variation is high and encompasses the 2% value in a 95% CI for model corrected data. Mean rate estimates for cytochrome b range between 1.9 and 8.9 x 10(-3) substitutions per site per lineage. Local rates differ significantly between taxonomic levels with lowest estimates for haplotype lineages. At the population/subspecies level mean sequence divergence between lineages matches the 2% rule best for most cytochrome b datasets (1.5-1.9% per my) with maximum estimates for small isolated populations like those of the Canarian P. teneriffae complex (up to 3.9% per my). Overall rate estimates for the control region range at similar values like those for cytochrome b (2.7-8.8 x 10(-3), 0.5-1.8% per my), however, within some subgenera mean rates are higher than those for cytochrome b for uncorrected sequence data. The lowest rates for both genes were calculated for coal tits of subgenus Periparus (0.04-0.6% per my). Model-corrected sequence data tend to result in higher rate estimates than uncorrected data. Increase of the gamma shape parameter goes along with a significant decrease of rate and partly age estimates, too. Divergence times for earliest deep splits within tit subgenera Periparus and Parus were dated to the mid Miocene at 10-14my bp. Most recent splits between east and west Palearctic taxa of blue, willow and great tits were dated to the Pliocene/Pleistocene boundary with the earliest estimates based on model-corrected trees. Relaxation of the Messinian calibration point leads to more recent divergence times for North African coal and blue tit populations during the mid Pliocene. Despite a relatively broad age constraint for the split between Nearctic and Palearctic Poecile due to the Pliocene re-opening of the Bering Strait, the split between chickadees and willow tits is dated considerably earlier than in former studies to the upper bound of the age constraint at 7.4 my BP.