Relaxed molecular clock analysis of cyprinids based on complete cyt b nucleotide sequence data. Relaxed molecular clock for cyprinids based on penalized likelihood (PL) and a calibration using a mean substitution rate of 0.0082 substitutions per site per million year (see text for details). A) ML phylogram based upon the complete cytb nucleotide sequence data. B) Relationship between root age and inferred mean substitution rate for the cyprinid tree. The tree was calibrated by iteration of the age of the root  until the mean rate equaled the cyprinid cytb clock  For each root age the optimal smoothing value for PL was assessed independently using the cross validation procedure implemented in r8s. A root age of 33.2 MY resulted in a mean substitution rate of 0.0082 substitutions per site per million year . C) Resulting chronogram using a fixed root age of 33.2 MY. The nodes labelled 1–4 are mentioned in the text and in Table 1. The Rasborinae clade A (see Figure 1) is highlighted in both A and C.