Phylogenetic analysis using a relaxed molecular clock based on mitochondrial protein sequences to estimate the age of divergence between Demodex species. Traditional groupings within the Acari were recovered in all phylogenies constructed. This phylogeny represents the results for one particular fossil calibration density, which is based on the minimum age of 410 mya for the time to the most recent common ancestor of the Acariformes (as inferred based on the four oldest Acariforme fossils identified in ); the particular fossil calibration density used in this analysis is depicted at the bottom of the tree, along with dashed arrow pointing to the common ancestor of the Acariformes. Amino acid sequences were used for all age-estimation analyses to minimize the effects of mutation saturation. For the particular analysis depicted in this phylogeny, the resulting estimated mean time to the most recent common ancestor of the two Demodex lineages was 134 mya, with a 95% highest probability density interval of 86 – 180 mya. Similar age estimates were obtained for two other fossil calibration densities, one that placed greater probability density near 410 mya, and one with a uniform distribution between 410–510 mya (Table 1). Furthermore, these estimates were not altered substantially when two species of Solifugae were included in the analysis (Table 1 and Additional file 1: Figure S1). Finally, these results are broadly overlapping with the results obtained based on an alignment of 18S sequences. Overall, these results suggest that Demodex have probably been coevolving with their mammalian hosts since before the placental radiation.