The complete mitochondrial genome and phylogenetic analysis of Tipula (Yamatotipula) nova Walker, 1848 (Diptera, Tipulidae) from Qingdao, Shandong, China.

The genus Tipula Linnaeus, 1758 is a large group of crane flies with more than 2400 known species from 41 subgenera. In this study, we report the first complete mitochondrial (mt) genome sequence of the subgenus Tipula (Yamatotipula), which is a circular molecule of 15,668 bp with an AT content of 77.2%. The mt genome contains 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a long non-coding region. Three conserved overlapping regions, 8 bp between tRNATrp and tRNACys , 7 bp between ATP8 and ATP6, and 7 bp between ND4 and ND4L, are found. Phylogenetic analysis reveals that the Tipulomorpha includes the family Trichoceridae and the Trichoceridae is sister-group to the remaining Tipulomorpha.

The genus Tipula Linnaeus, 1758 (Linnaeus 1758) is a large group of crane-flies with more than 2400 known species, of which 1018 were known from the Palaearctic Region, 496 were known from the Nearctic Region, 464 were known from the Neotropic Region, 352 were known from the Oriental Region, 112 were known from the Afrotropic Region, and 30 were known from the Australasian (Oceanian) Region (Oosterbroek 2019). Forty-one subgenera are recognized within the genus Tipula. However, only two complete or nearly complete mitochondrial (mt) genome sequences representing two subgenera, Acutipula Alexander, 1924 (Alexander 1924) and Nippotipula Matsumura, 1916 (Matsumura 1916), were available in GenBank. Here, we report the first complete mt genome sequence of the subgenus Yamatotipula Matsumura, 1916.

The specimen of Tipula (Yamatotipula) nova Walker, 1848 (Walker 1848) used in this study was collected from Mt. Lao in Shandong province of China (36°13′N, 120°36′E) and stored in the Entomological Museum of Qingdao Agricultural University, China (No. TIP0002). Genomic DNA was extracted from thoracic muscle using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China). The DNA concentration was measured by the NANODROP 2000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA). Qualified DNA samples were pooled for next-generation sequencing library construction following the method proposed by Gillett et al. (2014). The mixed DNA library was sequenced with Illumina HiSeq by Berrygenomics Co., Ltd. (Beijing, China). The fragment COI was amplified as a ‘bait’ sequence to identify our target mt genome assembly in the pooled contigs. The standard PCR reactions were sequenced by primers designed by Simon et al. (2006). BLAST searches were conducted with BioEdit 7.0.5.3 for the bait sequence against mt genome assemblies. The sequence was annotated following the method of Cameron (2014). Maximum-likelihood analysis was conducted using RAxML-HPC2 v8.1.11 (Stamatakis 2006).

The whole mt genome of T. (Y.) nova (MN583330) is 15,668 bp in length. It contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a long non-coding region that shows a conserved arrangement pattern with 23 genes transcribed on the majority strand and 14 genes encoded on the minority strand. The overall base composition is A (38.4%), T (38.8%), C (13.4%), and G (9.4%), with an AT content of 77.2%. The 16S rRNA was 1321 bp in length with AT content of 80.7%, and the 12S rRNA was 780 bp in length with AT content of 78.2%. The longest non-coding intergenic region in the mt genome is 861 bp in length with AT content of 92.1%. Three conserved overlapping regions are also found: 8 bp between tRNA and tRNA, 7 bp between ATP8 and ATP6, and 7 bp between ND4 and ND4L. Furthermore, one 16 bp long non-coding conserved intergenic region which often existed in dipteran insects located between tRNA and ND1 is also found.

The phylogenetic tree in our study (Figure 1) shows a strong support for the monophyly of the Tipulomorpha and indicates that the family Trichoceridae is sister-group to the remaining Tipulomorpha, which was accepted by Hennig (1973), Oosterbroek and Courtney (1995), Bertone et al. (2008), Zhang et al. (2016), and Kang et al. (2017).

Figure 1.

Phylogenetic tree of Nematocera based on whole mitochondrial genomes using maximum-likelihood analysis. Numbers above the branches are bootstrap percentages. GenBank accession numbers of each species were listed in the tree.