Before investigating the change of C.
faberi in population size, we estimated the divergence time of
haplotypes firstly. About the origin of the Orchidaceae andCymbidium , previous analysis suggested that the ancestor of
orchids lived in 76 – 84 million years ago (Ma), and diversification
in cymbidium is estimated to have begun 33.9 – 50 Ma (Ramírez,
Gravendeel, Singer, Marshall, & Pierce, 2007); other analysis suggested
that major diversification of the largest orchid subfamilies perhaps
occurring during the cooler period at the end of the Eocene and into the
Oligocene, an age for cymbidium clade is 28 or 31Ma (Gustafsson,
Verola, & Antonelli, 2010); Givnish et al. (2016) suggested that
Orchidaceae have arisen in Australia 112 Ma, and Cymdibiumoriginated about 10 – 20 Ma. Ultimately, 33.9 – 50 Ma was chosen as
the originated time of cymbidium .
The divergence time of C. faberi in the Qingling Mountains was
estimated using BEAST Version 2.3.2 (Bouckaert et al., 2014); nucleotide
substitution model (TVM+I+G) was chosen as the best fit to our data sets
by AIC in Modeltest (Posada & Crandall, 1998) combined with PAUP*
Version 4.0b10 (Swofford, 2002). Because the substitutions were expected
to be constant and the rate of molecular evolution in the plastome is
notably high in Orchidaceae, strict clock and a rate of
2.1×10−9 substitutions per site per year were
implemented to calibrate the tree (Gaut, 1998). Markov chain Monte Carlo
(MCMC) chain was run 100 million generations, then we combined the three
independent runs using LogCombiner and check the effective sampling size
values by Tracer; the first 25% of trees were discarded as burn-in
using TreeAnnotator and the maximum credibility tree was generated by
FigTree. Finally, we generated an Extended Bayesian Skyline Plot using
BEAST.