Background

Phylogenetic inference is a cornerstone of evolutionary biology because it provides explicit, testable hypotheses of evolutionary relationships from molecular sequence data [1]. Molecular dating extends phylogenetic inference by placing divergences on an absolute timescale, enabling evolutionary events to be compared with geological history and the fossil record [2]. In the phylogenomic era, molecular clock analyses must often accommodate complex patterns of rate variation and other challenges that become more prominent as data scale increases. Molecular dating is increasingly challenged by both model fit and analysis efficiency for genome-scale datasets. Genome-scale data commonly exhibit substantial rate heterogeneity, making divergence-time estimates sensitive to model specification, calibration strategy, and prior settings [3]. Moreover, because Bayesian dating relies on repeated likelihood calculations throughout Markov chain Monte Carlo (MCMC), phylogenomic datasets often impose substantial computational demands, motivating practical strategies to improve efficiency and stability [4].

A comprehensive molecular dating workflow typically involves interdependent steps, including complex multi-gene data preparation (e.g., sequence alignment, trimming, and concatenation), optimal model selection, rigorous application of clock models, and fossil calibrations [5]. In practice, this process relies heavily on specialized software such as MCMCtree and r8s [6,7]. MCMCtree uses a Bayesian framework to estimate divergence times by integrating sequence data with clock models and fossil calibration priors, whereas r8s uses a penalized likelihood approach that infers divergence times by smoothing rate variation across branches. Accordingly, MCMCtree is generally preferred when prior information and calibration uncertainty need to be modeled more explicitly, whereas r8s may be useful for relatively simpler or computationally lighter analyses. These tools typically operate via command-line interfaces, where parameter configuration is complex and error-prone, thereby representing a significant technical barrier [8]. For example, fossil calibration choices can strongly influence posterior time estimates, and best-practice guidelines stress that calibrations should be explicitly justified and that calibration-derived priors should be evaluated before analysis [9,10]. In addition, because Bayesian dating relies on MCMC sampling, routine convergence assessment (e.g., trace inspection and ESS summaries) is recommended before interpreting posterior estimates [11].

To reduce hands-on time and lower operational barriers for routine analyses, PhyloSuite was developed as an integrated GUI platform for streamlining sequence data management and phylogenetic workflows for multi-gene and genomic datasets [12]. Building on this framework, a major update in PhyloSuite v2 is the introduction of a molecular dating suite, comprising MDGUI (molecular dating analysis), TimeTreeAnno (Timetree annotation and visualization), and MCMCTracer (convergence diagnostics), which were not available in the previous version of the suite [13]. By integrating core programs such as MCMCtree, r8s, and IQ-TREE, this suite aims to provide users with a unified and visualized workflow for molecular dating analyses [14].

In the PhyloSuite v2 interface, the molecular dating workflow described here is mainly implemented through the modules under the Phylogeny menu, whereas other menus such as Flowchart, File, Alignment, and Mitogenome are primarily used for workflow organization, file management, sequence alignment-related operations, and mitogenome-oriented analyses, respectively. A more detailed introduction to these general functions can be found in our previous paper [15].

Using a dataset of fish nuclear genomes as a case study, this protocol provides a detailed tutorial demonstrating how to leverage PhyloSuite v2 to efficiently and accurately execute the entire analysis pipeline—from molecular dating to visualization—offering a user-friendly solution for the research community.

Users can locate three plugins—MDGUI, TimeTreeAnno, and MCMCTracer—by hovering over the Phylogeny menu in the main interface of PhyloSuite v2 (Figure 1).

Figure 1. Entry point of the molecular dating analysis suite in PhyloSuite v2. Screenshot showing the location from which users can access the integrated molecular dating analysis suite within the PhyloSuite v2 main interface: (1) phylogeny option in the main interface; (2) entry point of MDGUI; (3) entry point of TimeTreeAnno; (4) entry point of MCMCTracer.