2.4 Phylogenetic analysis and divergence time estimation
To reconstruct phylogenetic relationships among the 11 populations ofC. chuniana , we employed a maximum likelihood (ML) analysis to
generate phylogenetic trees using the SNPs extracted based on the GBS
dataset. Cercis chingii was used to root the trees, as based on
the results of Fritsch & Cruz (2012). Analyses were performed on the
high-performance computer cluster available in the CIPRES Science
Gateway 3.3 (www.phylo.org; Miller et al. , 2015). The ML analyses
were performed simultaneously with 1000 ML bootstrap pseudoreplicates in
RAxML v8 (Alexandros, 2014). The model of nucleotide substitution was
selected with the Akaike information criteria (AIC; Akaike, 1974) in
PhyML-SMS
(http://www.atgc-montpellier.fr/phymL/;
Lefort, Longueville, & Gascuel, 2017).
Divergence times within Cercis based on the fossil record
suggested that C. chuniana originated 2.4 Ma (Fritsch et
al. , 2018; Liu et al. , unpublished data, 2020). Therefore,
to estimate the divergence time within C. chuniana , we used BEAST
v2.4.7 (Bouckaert et al. , 2014) and applied the age of 2.4 Ma as
the secondary calibration point with a normal prior distributions and
standard deviations of 0.2 Ma, which covered the 95% HPD range. The
divergence time analyses were conducted with the GTR + G + I model and
four rate categories, a Coalescent Constant Population prior, and the
Strict Clock setting with uncorrelated and log-normal-distributed rate
variation across the branches. We ran the MCMC simulations in BEAST for
10 million generations with parameters sampled every 1000th generation.
We used Tracer v1.6 (Rambaut, Suchard, Xie, & Drummond, 2014) to assess
convergence and to check that the effective sample size (ESS) was
> 200 for each parameter. We discarded the first 10% of
trees as burn-in with the mean node heights option, and then generated
the maximum clade credibility (MCC) chronogram from the remaining trees
with nodal mean heights and 95% confidence time intervals with
TreeAnnotator v2.4.7 (Bouckaert et al. , 2014) in BEAST. The final
trees were edited with FigTree v.1.4.3
(http://tree.bio.ed.as.uk/software/figtree/;
Rambaut, 2014).