The complete mitochondrial genome of Triplax ainonia Lewis, 1877 (Coleoptera: Erotylidae)

Abstract The beetle Triplax ainonia Lewis, 1877 is a serious pest of cultivated the mushroom Pleurotus ostreatus in China. The complete mitochondrial genome of this species was reported for the first time in this study. The mitogenome was 17,555 bp in length and had a base composition of 39.4% A, 36.1% T, 8.7% G and 15.3% C, which indicated that the base composition was AT-biased. Similar to other species of Coleoptera, the mitogenome of T. ainonia contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA unit genes, and a large noncoding region. Phylogenetic analysis based on mitogenomes suggested that the family Erotylidae was a monophyletic group.


Introduction
The genus Triplax Fabricius belongs to the tribe Tritomini of the subfamily Erotylinae, and the species of this genus are widely distributed in East Asia such as China, Korea and Japan (Jung and Park 2017;Jung 2019). Most species of the genus Triplax are fungivorous (Goodrich and Skelley 1993;Jung 2019). For example, we found that both the adults and the larvae of the species Triplax ainonia Lewis, 1877 can feed on the fruit bodies of the Pleurotus ostreatus and become a serious pest of this cultivated mushroom in China. This beetle species has the following typical morphological features: body elongate oval, mostly shiny and black; body length 2.82-4.86 mm, and width 1.63-2.67 mm; elytra bright black, head black; pronotum with two circular and black markings; antennae brown, but 9-11antennomeres dark brown; legs and abdomen orange. In this study, we report the complete mitochondrial genome of T. ainonia, representing the first mitogenome of the genus Triplax, and the results of this study will contribute to the species identification of this pest based on molecular data.

Materials and methods
The specimens of the Triplax ainonia were collected from Baijin Town, Huishui County, South Guizhou Autonomous Prefecture, Guizhou Province, China (26 6 0 32 00 N, 106 50 0 9 00 E) on 16 November 2021. The insect specimens used in this study were collected and identified by the author (Ben Hong). A specimen was deposited at the Institute of Entomology, Guizhou University (http://www.gzu.edu.cn/, Changqing Luo, luochangqing13@163.com) under the voucher number GZU IE2021100101. The sample was preserved in 100% ethanol at À20 C ( Figure 1).
Genomic DNA of T. ainonia was extracted from one leg using the CTAB method (Reineke et al. 1998). DNA samples were stored at the Institute of Entomology, Guizhou University (http://www. gzu.edu.cn/, Changqing Luo, luochangqing13@163.com) under the voucher number GZU E2021112801. A total amount of 1 mg DNA per sample was used as input material for the DNA library preparations. The DNA libraries were sequenced on Illumina NovaSeq 6000 platform (Illumina, USA), and 150 bp paired-end (PE) reads were generated. After quality assessment, a total of 38,710,978 raw reads were used to assemble the mitochondrial genome, and the assembly was annotated and visualized using MitoZ (Meng et al. 2019). Circularity was checked using the MITOS Web Server (Bernt et al. 2013, http://mitos.bioinf.uni-leipzig.de/), and manually revised based on the mitogenome of the species Neotriplax arisana which is closely related to T. ainonia. Thirteen protein-coding genes (PCGs) of the mitogenomes of five species, including the mitogenome of the species T. ainonia and all other known mitogenomes of the species belonging to the family Erotylidae of the Cucujoidea, and the mitogenomes of the species of four families (each family with two representative species) which also belong to the superfamily Cucujoidea were used for the phylogenetic analysis. One species of another superfamily (i.e. Sphaerioidea) of the Coleoptera was employed as the outgroup taxon. Except for the species T. ainonia, the complete mitochondrial sequences of the other 13 beetle species were downloaded from the NCBI (www.ncbi.nlm.nih.gov). Multiple sequence alignment was carried out using Clustal X 1.81 (Thompson et al. 1997). The maximum likelihood tree was reconstructed by MEGA X (Kumar et al. 2018) with 1000 bootstrap replications. The best model of evolution for the ML analyses was selected with MEGA X (Kumar et al. 2018), and the GTR þ GþI was chosen as the final model of nucleotide substitutions.

Results
The complete mitochondrial genome of Triplax ainonia is a typical circular double-strand DNA molecule of 17,555 bp (GenBank accession no. OM084946). The mitogenome of this species contains 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), the small and large ribosomal RNA unit genes (rrnS and rrnL), and a large noncoding region (putative control region) (Figure 2). The mitogenome of T.  Twelve protein-coding genes use ATN (ATG, ATT, and ATA) as the start codons. Among those genes, six PCGs (cox2, cox3, nad4, nad6, cob, and atp6) start with ATG, five PCGs (cox1, atp8, nad3, nad5, and nad4l) start with ATT, and one PCG (nad2) starts with ATA. For the proteincoding gene nad1, TTG is used as a start codon. Eleven protein-coding genes stops with TAA, and the gene nad5 uses the TAG as a stop codon. However, the gene nad4 stop with an incomplete stop codon TA. The large noncoding region is 2,742 bp long, and located between rrnS and trnl. The 22 tRNAs are interspersed among ribosomal RNAs and protein-coding regions, and the size of these tRNAs varies from 64 bp (trnC, trnH, and trnT) to 70 bp (trnK and trnV). The rrnS is located between trnV and the noncoding region, and the rrnL is located between trnL and trnV. The rrnS and rrnL are 811 bp and 1329 bp long, respectively.
Phylogenetic relationships for 13 beetle species belonging to five families of the Cucujoidea were reconstructed ( Figure  3). The inferred phylogeny tree showed that the family Erotylidae which comprises the beetle species T. ainonia was a monophyletic group with 99% bootstrap probability ( Figure  3). Furthermore, the other four families formed a well-supported monophyletic clade, respectively (BP ¼ 100%) (Figure 3).

Discussion and conclusions
This study determined the main features of the mitochondrial genome of the beetle Triplax ainonia. The gene composition of the mitogenome of this species resembles the mitogenomes of other beetle species belonging to Coleoptera Xing et al. 2022). In addition, similar to the mtDNA sequences of other insects, the base composition of T. ainonia mitogenome is biased toward A þ T bases (Yang et al. 2009). In the beetle T. ainonia, the gene nad4 stops with an incomplete stop codon, and this phenomenon of mitochondrial genes with immature stop codons have also been reported in other beetles Xing et al. 2022).
The phylogenetic analysis showed that the Erotylidae formed a monophyletic clade, and the monophyly of this family was also confirmed by previous phylogenetic studies based on morphological and molecular characters (Lawrence and Newton 1982;Leschen et al. 2005;McElrath et al. 2015;. In our results, the family Erotylidae was the sister to the other three families: Cucujidae þ Silvanidae þ Nitidulidae, and this topology was strongly supported. However, clarification of the phylogenetic relationships among families of the Cucujoidea need further phylogenetic studies which should be based on a much broader taxon sampling of this superfamily.

Ethical approval
The study was approved by the Guizhou University Subcommittee of Experimental Animal Ethics (approval no. EAE-GZU-2021-E015).

Authors' contributions
Changqing Luo designed the study. Ben Hong and Yunxue Xiao performed the data analysis. Changqing Luo and Ben Hong drafted the manuscript. Yunxue Xiao critically reviewed the article. All authors approved the final version of the manuscript to be published and agreed to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).