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As such, a number of studies of amphibian diversification utilize paleobiogeographic calibrations based on the timing of separation of landmasses as estimates of the minimum divergence time between two taxa or clades.
In addition, to allow the possibility that stratigraphic assignment of fossil taxa might be incorrect, we relaxed the strict minimum divergence time calibration by using a normal distribution around the minimum date for a particular stratigraphic interval.
To obtain a minimum divergence time estimate of L. helicina from Arctic and Antarctic regions we also analysed the data within BEAST v1.4.8 (using a SRD06 nucleotide model and an uncorrelated log-normal relaxed clock model) using a fixed calibration date of 58.7 Ma on the divergence of Limacina (Limacinidae) and Hyalocylis (Creseinae) [29].
While the removal of fossil calibration points did not affect the divergence times of interest (i.e., Barbourula Bombina and within Barbourula), this analysis serves to underscore the need to critically evaluate calibration points as well as the importance of divergence estimation methods that incorporate uncertainty, including "relaxed" minimum divergence time calibrations [47].
Each fossil calibration point provided a "relaxed" minimum divergence time estimate (see above) and assigned a prior with a normal distribution around a mean that was set to a time corresponding to the most recent age of the stratigraphic level estimated for the fossil based on the relevant literature.
The Rhynie chert oomycete fossils [ 21] were used to define a minimum divergence time of 408 Ma between oomycetes and ochrophytes (5-95% quantiles = 418 550 Ma).
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We must emphasize that all of these rate estimates are approximate, based on rough estimates of minimum divergence times.
Also to accommodate more recent information, the minimum divergence times of F2 and F3 were changed from the original values used by Van Bocxlaer et al. (from 306.1 and 245.0 to 312 and 230, respectively).
For a maximum-divergence-time r8s run the horse/ass split was fixed at 3.5 mybp, and for the minimum-divergence-time r8s run, it was fixed to 1.0 mybp.
Specifically, divergence in a given SO cluster should range between minimum and maximum divergence time of the species included in this cluster.
Since no estimates of COI mutation rate are available for bats, we did not use a fixed substitution rate but estimated minimum and maximum divergence times using two substitution models, 2% and 5% per million years, based on estimates for cyt b in small-bodied mammals [ 49].
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