Data from: Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales)

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 re-assessment of their phylogenetic position.

尽管时间校准（temporal calibration）被广泛认为是利用分子定年方法（molecular dating methods）获取准确分歧时间估计值的关键环节，但鲜有研究评估不同校准策略所带来的差异。根据可获取的研究资料，研究者通常会选用来自化石记录的初级校准点，或是既往分子定年研究得到的次级校准点。
在被子植物（flowering plants）的分析中，初级校准数据可来自大型与中型化石（macro- and mesofossils，例如叶片、花与果实）或是微型化石（microfossils，例如花粉）。化石数据在准确性与精密度上存在显著差异，这使得研究者在选择合适的校准点时面临艰难抉择。
本研究以南山毛榉属（Nothofagus，南山毛榉科（Nothofagaceae），壳斗目（Fagales））——一个拥有极为丰富且研究充分的化石记录的植物类群——为研究对象，测试了八种合理校准场景对分子定年结果的影响。为此，我们梳理了南山毛榉属及其他壳斗目共38个化石类群的系统发育位置与地质年代学数据，并为壳斗目系统发育的至多18个节点确定了最小年龄约束。
针对每个校准场景，我们分别采用最大似然法（RAxML + r8s）与贝叶斯法（BEAST），对叶绿体与核基因组六个区域的序列数据开展分子定年分析。无论仅使用内群约束、外群约束，或是同时使用二者，除了受校准节点影响极强的区域外，得到的年龄估计值均较为相似。
此外，结合“早期但存在风险”的化石约束与“保守但偏晚”的约束，或是基于生物地理隔离（vicariance）而非化石约束进行分析，得到的年龄估计值均偏老。与之相反，使用次级校准点得到的年龄估计值则显著偏年轻。这项实证研究凸显了校准环节对分子定年分析的关键影响。即便在最佳场景下，即拥有大量经过严格审核的化石数据，分歧时间的估计值仍可能存在显著的不确定性。例如，南山毛榉属冠群年龄的估计值在所有校准场景下跨度为13至113 Ma。
我们建议，在校准流程的所有阶段都应加强背景研究，尽可能降低误差——从验证化石的地质年代学数据，到对其系统发育位置开展批判性重新评估。