Testing the Impact of Calibration on Molecular Divergence Times Using a Fossil-Rich Group: The Case of Nothofagus (Fagales)

Abstract

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.

A well-sampled phylogenetic analysis of the polystichoid ferns (Dryopteridaceae) suggests a complex biogeographical history involving both boreotropical migrations and recent transoceanic dispersals

“…Selecting and calibrating temporal constraints, as well as models of rate variation, are critical steps in estimating divergence times (Rothfels and Schuettpelz, 2014;Sauquet, 2013;Sauquet et al, 2012). The number of fossils attributed to Dryopteridaceae remains scarce, and to date no fossils of polystichoid ferns have been described (Collinson, 2001).…”

Evolutionary events in Lilium (including Nomocharis, Liliaceae) are temporally correlated with orogenies of the Q–T plateau and the Hengduan Mountains

“…In an ideal situation, divergence time dating should be conducted using multiple fossil calibration points including both ancient and recent fossils (Sauquet et al, 2012). Thus, we began by reviewing the fossil record of Liliales, with particular emphasis on Liliaceae and its sister family Smilacaceae (comprised of Smilax L. including Heterosmilax Kunth).…”

Fossil calibration of Magnoliidae, an ancient lineage of angiosperms

“…These calibration points are not only used to convert the time scale of relative ages into one of absolute ages, but they also serve as anchors for modeling molecular rate variation across the tree (e.g., Sanderson, 1997;Sauquet et al, 2012). In this context, calibration is being recognized as a critical point and can have a drastic influence on results (Inoue et al, 2010;Meredith et al, 2011;Sauquet et al, 2012). Because of re-evaluation of the boundaries of geological time units, and sometimes re-dating of fossil beds, the accepted age of a fossil can change through time (Gandolfo et al, 2008).…”

“…Therefore, an exhaustive geological review is essential in order to provide an accurate minimum age (Parham et al, 2012). Except for recently developed total evidence dating approaches that combine the phylogenetic analysis of extant and fossil taxa with the estimation of divergence times (Pyron 2011;Ronquist et al, 2012), a review of the phylogenetic position of a fossil is a second crucial point in the calibration process (Gandolfo et al, 2008;Parham et al, 2012;Sauquet et al, 2012). Molecular scaffold approaches (analyzing morphological data with the topology of living taxa fixed by a backbone constraint tree; Springer et al, 2001) or total evidence approaches (using both molecular and morphological data; Kluge, 1989) can provide a genuine test for the phylogenetic position of extinct taxa.…”