Phylogenetic analyses
The whole chloroplast genome matrix was aligned using MAFFT version 7.1
(Yamada et al. 2016). Only one inverted repeat region was used in the
phylogenetic analyses. Two matrixes were prepared, one including all
gaps, and another removed gaps using trimAl (Capella-Gutiérrez et al.
2009) by “-gt 0.4 -st 0.001 -cons 40”. Maximum Likelihood (ML) and
Bayesian Inference (BI) methods were used to reconstruct phylogenetic
trees. No nucleotide positions were excluded from analyses. The ML tree
analyses and bootstrap estimation of clade support were conducted with
RAxML version 8.2.10 (Stamatakis et al. 2008). These analyses used the
GTR substitution model with gamma-distributed rate heterogeneity among
sites and the proportion of invariable sites estimated from the data.
Support values for the node and clade were calculated from 1000
bootstrap replicates. Bootstrap support (BS) ≥ 70 is considered as well
supported (Hillis & Bull 1993). The BI analyses were performed using
MrBayes version 3.2.6 (Ronquist & Huelsenbeck 2003), with DNA
substitution models selected for each gene partition by the Bayesian
information criterion (BIC) using jModeltest version 2.1.10 (Darriba et
al. 2012; Guindon & Gascuel 2003). Markov Chain Monte Carlo (MCMC)
analyses were run in MrBayes for 10,000,000 generations, with two
simultaneous runs, and each run comprising four incrementally heated
chains. The BI analyses were started with a random tree and sampled
every 1000 generations. The number of generations for the three datasets
was sufficient because the average standard deviation of split
frequencies for the datasets was lower than 0.005, and Potential Scale
Reduction Factor (PSRF) of Convergence Diagnostic (Gelman & Rubin 1992)
for the datasets was 1.00. The first 25% of the trees were discarded as
burn-in, and the remaining trees were used to generate a majority-rule
consensus tree. Posterior probability values (PP) ≥ 0.95 were considered
as well supported (Alfaro et al. 2003; Erixon et al. 2003; Kolaczkowski
& Thornton 2007). Both ML and BI analyses, as well as jModeltest, were
performed
at
the CIPRES Science
Gateway
(http://www.phylo.org).