3.4 Molecular dating of cpDNA and demographic history
The BEAST-derived chronogram of 19 chlorotypes was supported by maximum
PP, and revealed three diverging genetic lineages (Figure 3A), which
correspond to the NW, SE and SW clades revealed by the chlorotype
Median-Joining network (Figure 3B). The time-calibrated phylogenetic
tree placed the split of P. heterotricha at c. 0.83 Myr
(Figure 3A). The splitting time of the remaining chlorotypes from clades
NW and SE was estimated at around 0.48 Myr, roughly at the end of early
Pleistocene, and most other chlorotypes were dated between c.0.26-0.05 Myr (Figure 3). And two vicariance events were detected among
three clades, first events gave rise to the genetic split in SW and the
rest clades, second events offered opportunity to the genetic divergence
in SE and NW clades (Figure S3).
The results of Tajimaʼs D test and Fuʼs Fs test were
presented in Table 2 with the associated simulated P -values. The
values for D was negative and Fs was positive for nrDNA
sequences (D = -0.00585, P > 0.10; Fs= 0.538, P = 0.132), but all were positive for cpDNA sequences
(D = 0.98381, P > 0.10; Fs = 9.119,P < 0.01) (Table 2), indicating an insignificant less
and excess of rare nucleotide site variants for nrDNA and cpDNA
sequences separately compared to the expectation under a neutral model
of evolution, and an significant excess of rare haplotypes for nrDNA and
cpDNA sequences over what would be excepted under neutrality. The
Hierarchical mismatch analysis showed the distributions of differences
for all populations, clade NW, SE and SW were ragged or multimodal
(Figure S4), which rejected the hypothesis of demographic population
expansion of these population clades.