2.6 Molecular dating of chlorotypes and population historical
dynamics
Divergence dating of the chlorotypes in a Bayesian framework was
performed in BEAST v.1.7.5 (Drummond et al. , 2012). Owing to the
absence of fossil records of Gesneriaceae with known ages, we usedP. pteropoda (with an estimated age of c. 1.31 Myr; Gao et
al. , 2015; Genbank accession number KF498219) to calibrate a
molecular clock, on the grounds that it has the closest phylogenetic
relationship with P. heterotricha (Gao et al. , 2015). Four
independent Markov Chain Monte Carlo (MCMC) runs were analyzed for 80
million generations and sampled every 1000 steps with the nucleotide
substitution model GTR+I+G, as selected by MRMODELTEST 2.3 (Nylander,
2004) based on the Akaike Information Criteria (AIC). The Yule
speciation tree priors and the relaxed clock model with an uncorrelated
lognormal distribution were followed. The resulting trees from the four
runs were combined by LOGCOMBINER (Bouckaert et al. , 2014),
discarding the first 25% of generations as burn-in. We specified a
normal distribution with standard deviation of one million years for all
the time priors. TRACER 1.5 (Rambaut & Drummond, 2007) was used to
access the chain convergence and ensuring ESS values was well above 200.
The maximum clade credibility (MCC) tree was summed up using
TREEANNPTATOR in the BEAST package (Bouckaert et al. , 2014). The
averages and PP of age estimates were visualized by FIGTREE v.1.4.2
(Rambaut, 2009).
The probable ancestral biogeographical scenarios in the phylogeny ofP. heterotricha chlorotypes were reconstructed separately by a
Statistical Dispersal-Vicariance Analysis (S-DIVA) by using the S-DIVA
(Yu et al. , 2010), which is parsimony-based and favours
vicariance events without any prior assumptions about area relation (Yu
et al. , 2010). We chose to be cautious in our definition of
regions, and arranged three areas based on the geographic barriers and
contiguities: (1) NW-Northwest of Changhua River, including the
populations BW, YJ, EX, WX and YG; (2) SE-Southeast of Changhua River,
including the populations WZ, QX, XA and QL; (3) SW-Southwest of
Changhua River, consisting only of the population JF. The chlorotype
trees for S-DIVA analysis were generated in BEAST v.1.7.5, including the
outgroup P. pteropoda (see above).
In order to detect possible recent range expansions, Tajimaʼs D(Tajima, 1989) and Fuʼs F s (Fu, 1997) were calculated for testing
the deviations from the null hypothesis of constant population size and
neutral evolution for each DNA fragment, pairwise mismatch distribution
and neutrality tests for all populations and clades (NW, SE and SW) were
conducted in DNASP v. 6.12.01 based on nrITS and cpDNA separately. These
tests show a unimodal shape in the mismatch distribution of populations
that experienced historical expansion.