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).