The molecular clock was also used to compute the date of duplication by calculating the number of substitutions per silent site (Ks).
The molecular clock was not rejected in any case.
The molecular clock was rejected for very few datasets (Table 1).
The uncertainty in the scale bar, which represents uncertainty in the calibration of the molecular clock, was (2719 9194 years) for a scale bar of length 5000 years.
To investigate further whether the molecular clock was different for synonymous or non-synonymous substitutions, we performed the molecular-clock analysis using only 1st+2nd nonsynonymous codon positions or only synonymous 3rd codon positions (see Methods).
Based on the results of the LRTs, a global molecular clock was rejected for all seven WRKY loci, however HyPhy detected evidence of local clock-rate rates in portions of each tree.
Tajima's Neutrality test for the mtDNA sequence data [50] was performed with DNAsp [51] and the assumption of a local or global molecular clock was tested with PHYHYP v. 1 [52].
A global molecular clock was rejected in favor of lineage-specific rates of molecular evolution (LRT: unconstrained model –ln L = 14940.81; constrained model –ln L = 15209.60; χ288 = 537.58; p<0.001).
The relaxed molecular clock was favored over the strict clock, as the 95% highest posterior density interval (HPD) of the standard deviation of the lognormal marginal posterior distribution excluded zero (mean 0.764; 95% HPD 0.232 1.383).
Deviation from a constant rate of molecular evolution within the PCWDE data sets (ie a "molecular clock") was first assessed using the likelihood ratio test (LRT) implemented in the program HYPHY [33].
No molecular clock was assumed.
