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）对于利用分子定年（molecular dating）方法获取准确的分歧时间估计值至关重要，这一点已得到学界广泛认可，但鲜有研究评估不同校准策略所导致的结果差异。根据可获取的研究资料，研究者通常会选用两类校准点：一类是来自化石记录的一级校准点（primary calibrations），另一类是来自既往分子定年研究的二级校准点（secondary calibrations）。在被子植物（flowering plants）的分子定年分析中，一级校准数据可来源于大化石与中化石（macro- and mesofossils，如叶片、花及果实）或微化石（microfossils，如花粉）。化石数据的准确性与精确性差异显著，这使得研究者在选择合适的校准点时往往难以抉择。本研究以南山毛榉属（Nothofagus，南山毛榉科（Nothofagaceae），壳斗目（Fagales））为研究对象——该属拥有极为丰富且被充分研究的化石记录，我们测试了8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壳斗目其他类群的38个化石类群的系统发育位置与地质年代学数据，并确定了壳斗目系统发育最多18个节点的最小年龄约束。针对每个校准场景，我们分别采用最大似然法（maximum likelihood，RAxML + r8s）与贝叶斯法（Bayesian，BEAST），基于叶绿体与核基因组6个区域的序列数据开展分子定年分析。单独使用内群（ingroup）约束、外群（outgroup）约束，或同时使用二者，除了在影响较强的校准节点附近之外，均得到了相似的年龄估计结果。结合使用“早期但存在风险”的化石约束与“保守但偏晚”的约束，或是以隔离分化（vicariance）假设替代化石约束，会得到更古老的年龄估计值。与之相反，使用二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，分歧时间估计值仍可能存在显著的不确定性。例如，在所有校准场景下，南山毛榉冠群年龄的估计值跨度为13至113百万年（Ma）。我们建议，在校准流程的所有阶段都应加强基础研究，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进行批判性重新评估。