3. Results
3.1.Genetic polymorphism and haplotype network
COI gene (658 bp) was successfully obtained from 346 individuals. The
variable sites included 12 singleton variable sites and 33 parsimony
information sites. 36 unique haplotypes were derived from 346
individuals. The distribution of different haplotypes based on COI was
showed in Fig.2. The haplotype distributions were dividing three starry
shapes in the network. According to the haplotype network, the
geographical populations were further divided into 85 populations and
this was done for further analysis of individuals from different
lineages in the same collection site. 85 populations belonged to three
groups, which included NE, SE and SN group. NE contained the individuals
that produced with egg diapause and mainly lived in the northern
regions. SE included the populations with egg diapause and mainly lived
in the southern regions. SN group was consisted with individuals that
produced with nymph diapause and mainly lived in the southern regions
(Table 2). The location of three groups was showed in the Fig.1. There
was no sharing haplotype among three groups. Hap24, Hap 2, Hap 4 and Hap
11 haplotypes were the most frequent haplotypes, characterizing 25.72%,
25.14%, 17.92%, 4.05% individuals, respectively. Hap 2 and Hap 11
were the ancestral haplotypes of NE group, and Hap11 was disjoint from
Hap 2, which suggested was differentiated due to the long distance. Hap
4 and Hap 24 were the ancestral haplotypes of SE and SN groups,
respectively. There were two lineages in populations of SDDY, but the
haplotypes of the residents of SDDY belonged to the SE were the tips of
Hap 2, thus the SE lineage may immigrate lately. In additional, the
haplotypes from the United States were the tips of the gene tree, Hap 5
and Hap 8 were originated from the NE group. Populations of USAGA3 and
USATN3 shared hap 5 with JPDB3, JPSH3 and JPSS3 in Japan. Hap 7 was from
SE group, populations of USAMO1 and USAVA1 shared hap 7 with SDDY1,
ZJTM1 and SHBS1 in China (Fig.2 and Table 2).
Pairwise FST among SE, NE and SN groups range
from 0.87072 to 0.95084 (P < 0.001 Table 4), which suggested
that the three groups were differentiated significantly. Isolations
between SE, SN and NE group were consistent with results of AMOVA. The
partitioning of total genetic variation in three clades using AMOVA
indicated 93.23% diversity among groups, 2.78% within populations, and
4.00% among populations within groups (Table 5), suggesting thatV. micado has significant genetic differentiation at the
different mode of life cycle and geographical separation of northern and
southern regions. According to the zoogeographic regions, 85 populations
were divided into 8 regions, including NEC, NC, SWC, CC, VK, KJ and USA.
The partitioning of total genetic variation in nine clades using AMOVA
indicated 24.94% diversity among groups, 69.56% among populations
within groups, and 5.49% within populations (Table 6). The results were
consistent with the network based on COI, which meant the different
modes of life cycle accounted for the most of genetic variation.
Based on the CytB gene, 579 bp fragments were successfully obtained from
162 individuals. The variable sites included 18 singleton variable sites
and 31 parsimony information sites. 36 unique haplotypes were derived
from 162 individuals. The distribution of different haplotypes was
showed in Fig.3. All individuals were divided into two groups
significantly. Hap 12 and Hap 13 were the ancestral haplotypes of
northern China (NC), and Hap 4 was the ancient haplotypes of southern
China (SC). Coincidentally, all the individuals in the NC group were the
members of the NE group defined by the COI, and others were in the SE
and SN group. There were two lineages in SDDY and SHBS, which mainly
including Hap1, Hap15 and Hap12. While Hap1 and Hap 15 were the tips of
Hap 4, thus the NE lineage may be the native in SDDY and SHBS (Fig.3).
This result was consisted with the assumption based on COI.
The partitioning of total genetic variation in different regions
indicated 41.11% among populations within groups, 30.23% diversity
among groups, and 28.66% within populations (Table 7). While the AMOVA
result showed that the source of variation among groups categorized by
three clades based on COI accounts 84.78% (Table 8).
3.2.Historical demographic changes
Based on COI, the negative of values of Tajima’ D suggested a relative
constant population size. In SHBS3, SDDY3 and SDTA3, the current
population size is stable. The NE lineage may be the native in SDDY and
SHBS, which is consistent to the results of haplotype network (Table 2).
Based on CytB, the negative and significant values of Tajima’ D
indicated past population expansion in populations of GZXY (Guizhou
province). Additionally, Hap 4 was the ancient haplotypes of populations
of egg and nymph diapause. Individuals produced with nymph diapause in
Zhejiang, Hainan and Guangxi (ZJTT, HNBS, HNJFL, HNWZ, HNCJ, HNXA, GXPM,
GXSL and GXJX), whereas those were with egg and nymph diapause in Yunnan
and Guizhou (YNKM and GZXY), which suggested the ancient haplotypes of
SE group distributed there (Fig.3), and the positive Tajima’ D value
based on COI and CytB suggested there may be a colonization of SE group
in Guizhou (Table 2 and 9). Thus, it is possibility that SE group was
originated from Yunnan and colonize to the other regions.
3.3.Divergence time estimation
The chronogram reconstructed with BEAST was based on COI (Fig. 4). The
most recent common ancestor for all V. micado was dated at
approximately 0.79 Myr (95% HPD: 0.46-1.13Myr). The diversification
time between SN and SE clades was 0.50 Myr (95% HPD: 0.25-0.71Myr).