3.2 | Nuclear microsatellite genotypes
Among the 32 ncEST-SSR loci, we found three loci (CcC00610, FcC03095,
and QmC01794), which had ≥ 0.173 estimated frequency of null alleles and
showed significant (P < 0.001) deviation from the
Hardy-Weinberg equilibrium in ≥ 8 populations (Data S1). Thus, we used
remaining 29 out of the 32 loci in the following genetic analysis.
In PCA for allele frequencies of the 24 populations, the first and
second PCs (PC1 and PC2) contributed to 22.5% and 17.0%, respectively,
of variation among populations. The coordinates of PC1 and PC2 indicated
that 13 Qc populations and eight Qch populations were
grouped separately, except for three populations 05Hx, 06Hx, and 07Hx
(Figure 3b). The three populations were identified as Qch in the
field observation but were grouped to Qc in the ncEST-SSR
variation. However, the separation of the genetic group of Qc (13Qc populations and three Qch populations 05Hx, 06Hx, and
07Hx) and the genetic group of Qch (eight Qch populations)
was continuous, and three northern and southern marginal Qchpopulations 02H1, 02H2, and 11H, one Qc population 11C, and oneQch population 06Hx were located at intermediate positions in the
PC coordinates (Figures 2, 3b). PCA of individual genotypes indicated
that individuals of the genetic group of Qch tended to have
higher values of PC1, which contributed to only 3.8% of variation among
individuals, than individuals of the genetic group of Qc (Figure
3a). The PC1 values of individuals were overlapped between the genetic
groups (Figure 3a).
The allelic richness (AR [32]) in the 24
populations was lower in higher latitudes (P = 0.011), was not
related to elevation (P = 0.172), and was not different between
the genetic groups of Qc and Qch (P = 0.449; Table
2, Figure S3a, b). The expected heterozygosity
(H E) was neither dependent on latitude nor
elevation (P ≥ 0.341) and was not different between the genetic
groups of Qc and Qch (P = 0.416; Table 2, Figure
S3c, d). The inbreeding coefficient (F IS) was not
significantly (P < 0.05) positive, except for three
populations 06C, 08C, and 06H (0.054 ≤ F IS ≤
0.066), and were not different between the genetic groups of Qcand Qch (P = 0.259; Table 2).
The log probability of data (LnPD) in Bayesian clustering of individual
genotypes showed a substantial increase from K = 1 to K =
2 and additional increases to K = 3 and to K = 4 (Figure
S4). At K = 5, LnPD was unstable (Figure S4). At K = 2,
two clusters were likely to represent the genetic groups of Qcand Qch (Figure 4a). Intermediate ancestry proportions of both
clusters were frequently found, especially in some populations (for
example, 11C and 11H; Figure 4a). At K = 3, the Qc cluster
was divided into two clusters, one of which was dominant in population
05Hx (Figure 4b). At K = 4, the Qch cluster was divided
into two clusters, one of which was frequent in northern populations
02H1, 02H2, and 04H of the genetic group of Qch (Figures 2, 4c).
The Weir and Cockerham’s pairwise F ST tended to
be higher between the genetic groups (median F ST= 0.046, 0.026 ≤ F ST ≤ 0.107) than within the
genetic group of Qc (median F ST = 0.019,
0.005 ≤ F ST ≤ 0.055) and the genetic group ofQch (median F ST = 0.039, 0.015 ≤F ST ≤ 0.079; Data S1). A neighbor-joining (NJ)
tree of populations based on the Nei’s distanceD A showed divergence between the genetic groups,
although the length of a branch between the genetic groups was
relatively short (Figure 5). Jackknifing over loci (removing one out of
the 29 loci) generated 29 NJ trees, and seven populations of the genetic
group of Qch , except for population 11H, were always grouped in
the generated NJ trees (Figure 5).