To investigate whether rates of molecular evolution fit with a strict molecular clock model, the likelihood of the ML phylogeny was recalculated with the constraint of global molecular clock using the Rambaut parameterization for clock optimization implemented in PAUP4b10 [58].
The relaxed molecular clock model also had a larger marginal likelihood according to the Bayes factors comparison (−2696.62 for the strict molecular clock compared to −2690.78 for the relaxed molecular clock).
The relaxed molecular clock model chosen was uncorrelated lognormal.
For example, H5N1 is the most recent lineage and originated between 12 and 16 years ago depending on the molecular clock model used, while the H4 and H6 subtypes have a tMRCA of at least 50 years but do not predate 1900 as suggested by the previous local clock analysis (Table 2).
Thus, for molecular sequence data, a molecular clock model must be applied to calibrate the tree.
Prior, the relaxed uncorrelated lognormal molecular clock model [ 106] was implemented to assess the clock-like nature of the data.
Uncorrelated lognormal relaxed clock and strict molecular clock model (Additional file 1: Figure S1) were studied to estimate divergence times.
The overall rate for the cox1 data (all codon positions) is 2.13%; this compares well with published rates for molluscan cox1 molecular clocks; for example 1.21% for Arcidae (Bivalvia) [ 89], and 1.18% for freshwater tropical Ampullariidae [ 90].
These TMRCA measures should be interpreted with great caution, however, given the untested assumptions underlying these particular molecular clock analyses – for example, that mtDNA mutation rates are the same for C. elegans and Panagrolaimus and that the rate of mutation equals the rate of silent-site substitution.
For example molecular clock analyses of plant virus genomes sampled over the last century have indicated that whereas the genera Sobemovirus, Luteovirus and Potyvirus are probably less than 10 000 years old [1], [3], [55], the genus Tobamovirus is probably no more than 100 000 years old [2].
