The specimen of X. (H.) maculatus was collected at Jianfengling in Hainan, China in June 2017 and stored in 100 % ethanol at −4 °C. Genomic DNA was extracted from the leg muscle tissue of a single adult male species of using a DNeasy Blood & Tissue Kit (Qiagen, USA) according to the manufacturer’s instruction and sent to a company for library prep and sequencing (Genesky Biotechnologies Inc., Shanghai). The library was prepared using a TruSeq DNA sample Preparation kit (Vanzyme, China) and sequenced with 150 bp pair-end reads on the Illumina Hiseq 2500 sequencing platform (Illumina, USA).

13,799,778 raw reads were sequenced by the Illumina Hiseq 2500 platform. The raw paired-end reads were filtered to obtain high-quality clean reads by using CLC Genomics Workbench 8 (CLC Bio, Aarhus, Denmark) with default parameters. Then the filtered reads were aligned to the mitochondrial genome of X. (X.) fascipes (JQ326212) as a reference using MITObim v1.8 (Hahn et al. 2013) and Mira 4.0.2 (Chevreux et al. 2004) to assembly. All of 32,608 clean mitochondrial reads yielded an average coverage of 255.4 X. The complete mitochondrial genome sequence was annotated using the software Geneious v 10.1.2 (Biomatters Ltd., Auckland, New Zealand) by comparing with the mitochondrial genome of X. (X.) fascipes (JQ326212). The tRNA genes were predicted using online software MITOS (Bernt et al. 2013). The nucleotide relative synonymous codon usage (RSCU) values of PCGs were analysed with MEGA v7 software (Kumar et al. 2016).

Phylogenetic analyses were performed on the concatenated datasets of PCGs and rRNAs of the newly sequenced mitogenome and 24 Tettigoniidae species downloaded from GenBank, with two Phaneropteridae taxa (Ducetia japonica and Phyllomimus sinicus) selected as the outgroups. Alignment of each protein-coding gene inferred from the amino acid alignment was performed using MEGA v7.0 (Kumar et al. 2016), and the alignment results were then concatenated. Bayesian inference (BI) analysis was used for phylogenetic reconstruction with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) under the partitioned models chosen by PartitionFinder 2 (Lanfear et al. 2017). In the BI analysis, 10,000,000 generations were run, with four MC chains, and the trees were sampled every 1000 generations with a burn-in step. The confidence values for the BI tree were expressed as the Bayesian posterior probabilities in percentages. A maximum likelihood (ML) tree was constructed using RAxML 8.0 (Stamatakis 2014) and the optimal partitions and best models were also selected by PartitionFinder 2 and the robustness of the phylogenetic results was tested through bootstrap analysis with 1000 replicates in RAxML and the bootstrap support values were printed on the best ML tree.

The complete mitogenome of X. (H.) maculatus is 16,358 bp in length and has been deposited in GenBank under accession no. MG779499. Xizicus (H.) maculatus mtDNA is larger than that observed in other species in Meconematinae, which typically ranged from 16,044 bp (Zhou et al. 2017) to 16,166 bp (Yang et al. 2012).

The mitochondrial genome structure is detailed in Table 1. It contained a typical gene content found in metazoan mitogenomes: 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal (rRNA) genes, and one control region (Table 1, Fig. 1). Gene order and arrangement was identical to the X. (X.) fascipes mitogenome. The X. (H.) maculatus mitochondrial genes were separated by a total of 65 bp intergenic spacer sequences, which spreaded over ten regions and range in size from one to 17 bp. There were 13 overlaps amongst all 48 bp and the longest overlaps of 8 bp were located between tRNA^Trp-tRNA^Cys and tRNA^Tyr-COI. The overall base composition of the whole mitochondrial genome was 37.0 % A, 32.2 % T, 20.2 % C, and 10.6 % G, exhibiting obvious anti-G and AT bias (69.2 %) which was slightly lower than X. (X.) fascipes (70.2 %) and X. (E.) howardi (71.0 %).

Figure 1. 

Circular visualization of the mitogenome of Xizicus (Haploxizicus) maculatus.

The total length of all 13 PCGs was 11,224 bp, and the overall A+T content of X. (H.) maculatusPCGs was 68.3 %. The initiation codons of all PCGs were typical with ATN (COII, ATP6, COIII, ND4, ND4L, and Cytb with ATG, ND2, COI, ATP8, ND5 with ATT; ND6, ND1 with ATA, Only ND3 with ATC). The ATN codon was prevalently regarded as initiation codons for mitogenome PCGs in insects. Seven genes (ND2, COII, ATP8, ATP6, ND3, ND4L, ND6) used TAA as the termination codons, and two genes (Cytb, ND1) were stopped with TAG. COI, COII, ND5, and ND4 had an incomplete termination codon T (Table 1). The incomplete termination codon is common in metazoan mitochondrial genomes and exhibit function after post-transcription polyadenylation converts into full stop codon (Ojala et al. 1981).

The four most-used amino acids in X. (H.) maculatus were Leu (16.1 %), Ser (8.9 %), Phe (8.7 %), and Ile (8.4 %), whose proportions were similar to those observed in other Tettigoniidae species. All codons were present in the protein-coding genes of this mitogenome. Excluding incomplete termination codons, there were 3,735 codons in the X. (H.) maculatus protein-coding genes. The codon usage in X. (H.) maculatus appeared to be typical of other insect mitochondrial sequences. The RSCU analysis indicated that codons including A or T at the third position were always overused compared with other synonymous codons in Xizicus (Fig. 2). The codon usage could also reflect nucleotide bias.

Figure 2. 

Relative synonymous codon usage of X. (X.) fascipes, X. (E.) howardi, X. (H.) maculatus mitochondrial protein-coding genes. Condon families are provided on the x-axis.

The length of tRNA genes ranged from 63 to 71 bp and the relative locations for each tRNA are shown in Table 1. All tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN). The secondary structures of tRNA^Ser(AGN) was completely identical with X. (X.) fascipes and showed a lengthened anticodon stem (9 bp) with a bulging nucleotide in the middle, an unusual 6 bp length T-stem, a mini DHU arm (2 bp), and no connector nucleotides (Yang et al. 2012).

The lrRNA and srRNA were 1304bp and 785bp in length, respectively. They were located between tRNA^Leu(CUN) and A+T-rich region, being separated by tRNA^Val.

The control region was 1570 bp in length and located between srRNA and tRNA^Ile in X. (H.) maculatus mitogenome, and was composed of 64.4 % A and T nucleotides (Fig. 1; Table 1). The control region is characterized by a high AT content and is thought to be involved in the regulation of mtDNA transcription and replication (Bae et al., 2004), whose size differences are not only because of high rates of nucleotide substitution, insertion or deletion, but also due to the length of tandem repeat unit and the number of tandem repetitions (Yang et al. 2012). The sequence analysis revealed four tandem repeats size from 26 to 162 bp, contributing 738 bp to the length of the region.

Bayesian analyses and maximum likelihood produced identical topologies using the best-fit partitioning scheme and site-homogeneous models, excepting the location of Lipotactes tripyrga (Lipotactinae) and genera relationships in Meconematinae (Fig. 3). Bayesian inference recovered each Tettigoniinae, Bradyporinae, Conocephalinae, and Meconematinae as a monophyletic group with strongly supported (PP ≥ 0.93), while the monophyly of Conocephalinae and Meconematinae (only include the tribe Meconematini) was not well supported in ML topology. The position of L. tripyrga was at basal in the ML tree while it formed the most basal clade together with Hexacentrinae in the BI analysis. The relationship of Lipotactinae with other subfamilies was unascertainable may due to only one taxa used in analyses.

Figure 3. 

Phylogenetic reconstruction of Tettigoniidea using mitochondrial PCGs and rRNA concatenated dataset. A Bayesian result, applicable posterior probability values are shown B Maximum likelihood result with applicable bootstrap values shown.

In present study, regardless of the position of Lipotactinae, the relationships among the subfamilies of Tettigoniidae were as follows: ((((Tettigoniinae, Bradyporinae) Meconematinae) Conocephalinae) Hexacentrinae), which was congruent with the phylogenetic results using site-homogeneous both in ML and BI analyses (Zhou et al. 2017). The relationships among subfamilies within Tettigoniidae were sensitive to the methods used for tree reconstruction and the molecular maker used. Previous phylogenetic studies based on multi-molecular makers (28SrDNA, 18SrDNA, COII, Histone and Wingless genes) inferred a striking difference results that placed Hexacentrinae and Meconematinae as more ‘advanced’ groups sister to the clade consisting of Tettigoniinae and Lipotactinae, and Conocephalinae as the more ‘primitive’ group diverging at an earlier node (Mugleston et al. 2013). The present topologies placed Tettigoniinae as an apical node sister to Bradyporinae and then assembled with Meconematinae, which was congruent with the phylogenetic results using site-homogeneous both in ML and BI analyses (Zhou et al. 2017), while differed with the site-heterogeneous CAT-GTR model tree (Zhou et al. 2017). Recent studies suggest that analysis model used in mitochondrial phylogenetic reconstruction potentially impact phylogenies result when genomic data existing lineage compositional heterogeneity and saturation due to accelerated substitution rates causing homoplasy (Li et al. 2015; Zhou et al. 2017).

The generic relationships within the subfamily Meconematinae were not identical in the BI and ML trees. The relationships between the four genera were as follows: (((Pseudocosmetura, Decma) Pseudokuzicus) Xizicus) in the BI tree (Fig. 3A); (((Xizicus, Pseudokuzicus) Pseudocosmetura) Decma) in the ML tree (Fig. 3B). Xizicus (H.) maculatus, X. (X.) fascipes and X. (E.) howardi clustered into one clade in both analyses, and X. (H.) maculatus was closer to the nominate species X. (X.) fascipes than X. (E.) howardi. Additional taxon sampling will be needed to clarify the relationships of genera in Meconematinae.