Corrected placement of Mus-Rattus fossil calibration forces precision in the molecular tree of rodents

Time calibration derived from the fossil record is essential for molecular phylogenetic and evolutionary studies. Fossil mice and rats, discovered in the Siwalik Group of Pakistan, have served as one of the best-known fossil calibration points in molecular phylogenic studies. Although these fossils have been widely used as the 12 Ma date for the Mus/Rattus split or a more basal split, conclusive paleontological evidence for the nodal assignments has been absent. This study analyzes newly recognized characters that demonstrate lineage separation in the fossil record of Siwalik murines and examines the most reasonable nodal placement of the diverging lineages in a molecular phylogenetic tree by ancestral state reconstruction. Our specimen-based approach strongly indicates that Siwalik murines of the Karnimata clade are fossil members of the Arvicanthini-Otomyini-Millardini clade, which excludes Rattus and its relatives. Combining the new interpretation with the widely accepted hypothesis that the Progonomys clade includes Mus, the lineage separation event in the Siwalik fossil record represents the Mus/Arvicanthis split. Our test analysis on Bayesian age estimates shows that this new calibration point provides more accurate estimates of murine divergence than previous applications. Thus, we define this fossil calibration point and refine two other fossil-based points for molecular dating.