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A Step-by-Step GUI-Based Protocol for Molecular Dating Analysis Using PhyloSuite v2
Dong Zhao1,2, Ivan Jakovlić2, Xiantong Liu2
1Key Laboratory of Biodiversity and Environment on the Qinghai-Tibetan Plateau, Ministry of Education, School of Ecology and Environment, Xizang University, Lhasa, China.
None:
In current genomic research, molecular dating is challenged by both imperfect substitution modeling and analysis efficiency, as genome-scale datasets often exhibit substantial rate heterogeneity and complex patterns of sequence evolution, which can make divergence-time estimation sensitive to modeling assumptions and computational settings. Meanwhile, commonly used molecular dating workflows remain operationally demanding; preparing correctly formatted inputs, implementing model settings, configuring fossil calibrations, and performing basic diagnostics and visualization frequently require multiple tools and extensive manual steps, resulting in high hands-on time and avoidable operational errors. To facilitate the practical implementation of molecular dating analyses and lower the operational barrier for users, this protocol describes a GUI-based workflow in PhyloSuite v2 for molecular dating analysis. Using a dataset of fish nuclear genomes as an example, the tutorial covers multi-format data import, visual configuration of fossil calibrations, automatic selection and implementation of substitution models, automation of complex analytical procedures, and assessment of Markov chain Monte Carlo (MCMC) convergence, along with data visualization. Through this protocol, users can quickly master the full workflow-from input preparation and molecular dating to MCMC sample statistical assessment and timetree visualization-thus significantly enhancing the efficiency of molecular dating analysis and result verification. Key features • Guides users through a complete molecular dating workflow in PhyloSuite v2, from input preparation to final output. • Specifies required inputs, intermediate files, and final outputs for molecular dating analyses. • Provides a practical visual procedure for setting and checking fossil calibrations before analysis. • Helps users configure reproducible runs and interpret diagnostic outputs to assess convergence and dating performance.
