For Consideration in Systematic Biology Evaluating fossil calibrations for dating phylogenies in light of rates of molecular evolution: a comparison of three approaches

Evolutionary and biogeographic studies increasingly rely on calibrated molecular clocks to date key events. While there has been significant recent progress in development of the techniques used for molecular dating, many issues remain. In particular, controversies abound over the appropriate use and placement of fossils for calibrating molecular clocks. Several methods have been proposed for evaluating candidate fossils, however, few studies have compared the results obtained by different approaches. Moreover, no previous study has incorporated the effects of nucleotide saturation from different data types in the evaluation of candidate fossils. In order to address these issues, we compared three approaches for evaluating fossil calibrations: the single-fossil cross-validation method of Near et al. (2005); the empirical fossil coverage method of Marshall (2008); and the Bayesian multi-calibration method of Sanders and Lee (2007), and explicitly incorporate the effects of data type (nuclear vs. mitochondrial DNA) for identifying the most reliable or congruent fossil calibrations. We used advanced (Caenophidian) snakes as a case study however our results are applicable to any taxonomic group with multiple candidate fossils, provided appropriate taxon sampling and sufficient molecular sequence data are available. We found that data type strongly influenced which fossil calibrations were identified as outliers, regardless of which method was used. Despite the use of complex partitioned models of sequence evolution and multiple calibrations throughout the tree, saturation severely compressed basal branch lengths obtained from mitochondrial DNA compared with nuclear DNA. The effects of mitochondrial saturation were not ameliorated by analysing a combined nuclear and mitochondrial dataset. While removing the third codon positions from the mitochondrial coding regions did not ameliorate saturation effects in the single-fossil cross-validations, it did in the Bayesian multi-calibration analyses. Saturation significantly influenced the fossils that were selected as most reliable for all three methods evaluated. Our findings highlight the need to critically evaluate the fossils selected by data with different rates of nucleotide substitution and how data with different evolutionary rates affect the results of each method for evaluating fossils. Our empirical evaluation demonstrates that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages.