2. Materials and methods
2.1.Sample collection and genomic DNA extraction
A total of 72 Velarifictorus micado populations contain 346
individuals were collected, 57 populations from China, one from Korea,
eight from Japan, one from Vietnam, one from Cambodia and four from
America (Fig. 1). All materials were presented in 100% ethanol and
stored in a freezer at -20°C, genomic DNA was extracted from the leg of
the cricket using AxyPrepTM Multisource Genomic DNA
Miniprep kit.
2.2.PCR amplification and sequencing
Universal primers for cytochrome c oxidase unit 1 (COI), and cytochrome
b (CytB) genes were designed in the present study (Table 1). The PCR
procedure for the three genes included an initial denaturation at 94°C
for 4 min, followed by 35 cycles of 30s at 94°C, 30 s at 45°C and 30 s
at 72°C, ending with a final extension at 72°C for 5 min. Sequencing was
performed on 3730xl DNA Analyzer, sequencing was proofread and
aligned in ATGC Ver 7.0.2 (Genetyx Corporation, Tokyo, Japan).
2.3.Genetic analysis
2.3.1. Genetic polymorphism
Base substitution saturation test was performed in DAMBE (Xia and Xie,
2001) to make sure ISS < ISS.cSym. Different haplotype,
Haplotype diversity (Hd) and nucleotide diversity (π) were calculated in
DNASP 5.10.01 (Librado and Rozas, 2009), A median joining (MJ) haplotype
network was constructed in Popart (Leigh and Bryant, 2015), each
population has its own color. Genetic differentiation among different
populations and sets that defined groups was calculated in ARLEQUIN
3.5.2.2 (Excoffier and Lischer, 2010). Analyses of PairwiseFST were performed in two ways. The first
comprising data sets divided by the different modes of life cycle mainly
and the second sets divided by the main zoogeographic regions (Zhang,
1983) comprising NEC (northeastern China), NC (northern China), SWC
(southwestern China), CC (central China), SC (southern China), VK
(Vietnam and Cambodia), KJ (Korea and Japan) and USA (The United States)
data (Table 2).
2.3.2.Historical demographic changes
Haplotype diversity, nucleotide diversity and neutrality test were used
to speculate historical demographic change. Neutrality tests were
implemented in ARLEQUIN 3.5.2 (Excoffier and Lischer, 2010), including
Tajima’ D (Tajiama, 1989) and Fu’ Fs (Fu, 1997).
2.3.3.Divergence time estimation
Estimating divergence times were based on mitochondrial markers of COI
in BEAST 2.5.0 (Bouckaert et al ., 2014). A set of 15 specimens
comprising 6 Velarifictorus specimens and 9 ancient haplotypes ofV. micado was employed for the estimation of divergence time
(Table 3). The substitution models were selected under JModeltest v.2
(Darriba et al ., 2012). The Bayesian information criterion (BIC)
was preferentially used to compare substitution models, and HKY+I+G
model of sequence evolution was selected. An uncorrelated lognormal
relaxed clock was applied with 1.7% per site per lineage per million
years for COI (Pons and Vogler, 2005; Kiyoshi and Sota, 2006; Sharipo,et al ., 2006; Papadopoulou et al ., 2010; Allegrucciet al ., 2011; Kaya et al ., 2016). Divergence time was
estimated using a Yule model. After running the chains for 500 million
generations in BEAST, the stability on the log-likelihood curves and the
split-frequencies were checked in Tracer v1.7.1. The parameter estimates
with ESS > 200 were accepted. Discarding the first 20% as
burn-in and summarize trees in Tree Annotator. Trees were visualized
with the FigTree v1.1.2 (These software are included in the BEAST
package).