Estimation of divergent times and phylogeographical history
Dating analyses were conducted using Markov Chain Monte Carlo (MCMC) methods in BEAST version 2.4 (Bouckaert et al. 2014), which was performed at the CIPRES Science Gateway (http://www.phylo.org). The setting parameters in BEAUti, we chose “BEAST model test” for “Site model”, “Relaxed Clock Log Normal” for “Clock model”, and “Yule Model” for speciation. Meanwhile, we selected two crown nodes for calibrations from published data using the CladeAge package (Matschiner et al. 2016). (1) The Lobelia thuliniana – L. columnaris clade was 8.0 Mya (sigma 1.0, offset: 0.0), and (2)Lobelia laxiflora - L. columnaris clade was 21 Mya (sigma 2.0, offset: 0.0) (Chen et al. 2016; Knox 2014). For reconstructing the phylogeographical history of L. columnaris , a plastome phylogeny of the L. thuliniana – L. columnaris clade including 22 samples of L. columnaris and five outgroups was estimated in BEAST2 with “beast-classic package” of BEAUti. For each dataset, MCMC ran 200,000,000 generations and sampled every 20,000 generations. The first 5,000 generations were removed as “Pre Burnin”. Log output of the BEAST analysis was evaluated using Tracer version 1.6. Effective sample sizes (ESS) of all parameters were more than 200, indicating that the estimations were confident. Maximum clade credibility (MCC) tree was generated using TreeAnnotator by setting “Mean heights” for the “Node heights”. The MCC tree was visualized using FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).