The first complete mitogenome of skin beetles Omorgus chinensis (Coleoptera: Trogidae) with the phylogenetic implications

Abstract The first complete mitogenome of Trogidae, Omorgus chinensis (Coleoptera: Trogidae) is sequenced using the next generation sequencing. The genomic structure is a circular molecule with 18682 bp in length, comprising 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNAs (rRNAs) and a control region. The nucleotide composition is A (39.44%), C (13.82%), T (36.78%), and G (9.96%) with an AT content of 76.22%. The phylogenetic analysis of 18 insects in Scarabaeoidae show that Omorgus chinensis shares a close ancestry with Lucanidae and Geotrupidae.


Introduction
Omorgus chinensis (Coleoptera: Trogidae) is an important necrophagous insect with an oval shape and tan color. Like most members in Trogidae, this species has a marked engraved and dimpled area on the anterior thoracic plate and a strong arch on the back but a flattened ventral surface (Ren 2003). In recent years, with the development of molecular biology, especially the advancement of next-generation sequencing technology, mitochondrial genome (mitogenome) have been widely used as a typical marker for the phylogenetic study of coleopteran insects (Cameron 2014;Li et al. 2015;Timmermans et al. 2015). Fagua et al. 2018; However, there are only a few molecule phylogenetic researches in Trogidae (Str€ umpher et al. 2014), and no complete mitogenome data has yet been sequenced.
In this study, we sequenced the complete mitogenome of Omorgus chinensis for the first time using the next-generation methods and reconstructed the phylogenetic relationships within other species in Scarabaeoidae, which provides reliable data for subsequent further phylogenetic studies.

Sample collection and DNA extraction
The specimens of O. chinensis were collected by Yongjing Chen and Shiju Zhou (2289788957@qq.com) from Yaoluoping National Nature Reserve, Anhui province, P. R. China, in July 2017. For adult specimens, the muscle tissues were placed in 100% ethanol. Total genomic DNA was extracted from the muscle of a single O. chinensis using the Qiagen DNAeasy Kit.
The specimen deposited in the Museum of Anhui university and the sequence was submitted to GenBank with the accession number MK937809.

Polymerase chain reaction amplification, sequencing and assembly
Complete mitogenome was assembled by overlapping highthroughput fragment sequences, the amplification of the three primers were used as markers for tandem highthroughput sequencing fragment sequences. The primers used for amplification listed in Table 1. PCR amplification reactions were carried out in 25 lL volumes containing 10 lM of each primer (forward and reverse) 1 lL, 2 lL template DNA, 12.5 lL 2 Â EasyTaq SuperMix (þdye), and 8.5 lL sterile double-distilled water to make up a final volume of 25 lL. The polymerase chain reaction amplifications were performed under the following conditions: an initial denaturation at 94 C for 2 min, followed by 35-37 cycles of denaturation at 94 C for 40 seconds, annealing at 52-58 C for 50 seconds, and elongation at 70 C for 1 min, and then a final extension step at 72 C for 7 min. The temperature of annealing was determined by the length of fragments. An Illumina TruSeq library was prepared with an average insert size of 450 bp and was sequenced using the Illumina Hiseq2500 platform with 250 bp paired-end reads. Raw reads were trimmed of adapters using Trimmomatic. Low quality and short reads were removed with Prinseq. High quality reads were used in denovo assembly using IDBA-UD with minimum and maximum k values of 80 and 240 bp, respectively. To investigate the accuracy of the assembly, clean reads were mapped onto the obtained mt contig using Geneious Prime 2019.1.1 (http://www.geneious.com/), allowing for mismatches of up to 2%, maximum gap size of 3 bp and minimum overlap of 100 bp. Finally, we obtained the mitogenome of O. chinensis with the average sequencing depth of 242Â.

Phylogenetic analyses
A total of 18 mitochondrial genomes of Scarabaeoidae were used for phylogenetic analysis (Table 2). Each PCGs was aligned individually based on codon-based multiple alignments using the MEGA-X (Kumar et al. 2018). Models of nucleotide substitution were selected according to the Akaike Information Criterion (AIC) with jModelTest v2.1.4 (Posada 2008). Phylogenetic trees were generated from ML analysis using RAxML (Stamatakis 2014) and Bayesian inference (BI) with MrBayes v3.2.5 (Ronquist et al. 2012), both under the GTR þ I þ G model. Node support in the ML tree was estimated through bootstrap analysis with 1,000 replicates. The BI was conducted with two simultaneous Markov chain Monte Carlo runs of 2 Â 10 9 generations, sampled every 1,000 steps, with the first 25% discarded as burn-in.

Results and discussion
The mitogenome of O. chinensis is 18,682 bp in length, with the typical gene content as other known Coleoptera mitogenomes: 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (16S rRNA and 12S rRNA), 13 protein-coding genes (PCGs) and 1 non-coding AT-rich region (Sheffield et al. 2009;Kim and Farrell 2015;Wu et al. 2016;Yang et al. 2016;Du et al. 2017). The base compositions of the mitogenome nucleotide composition were A (39.44%), C (13.82%), T (36.78%), and G (9.96%) with an AT content of 76.22%. ATskew and GC-skews were calculated as 0.035 and À0.162. ATbiased were found in the protein-coding region (75.66%), rRNA (80.09%), tRNAs (78.11%) and control region (75.16%). As a typical invertebrate mitogenome, 12 PCGs of the sequence employ same initiation codons ATN, except the start codon of COI is AAT. 10/13 PCGs shared the typical termination codons TAA and TAG, while others use TA residue or a single T as the terminator codons. All of the 22 tRNAs range from 63 to 70 bp in length, 14 of the 22 tRNA-coding genes were located on the J-strand and others were located on the N-strand (Table 3). Secondary structures predicted by the tRNA scan-SE suggested that all the tRNA genes in O. chinensis adopted a typical clover-leaf structurewith the exception of tRNA-Ser (AGN), tRNA-Ser (AGN) has the deficiency of the dihydrouridine arm. which is a typical feature of metazoan mitochondrial genomes (Cameron 2014). The control region is 4006 bp long and is located between 12S rRNA and tRNA-Ile. ML and BI analyses obtained the consistent topology. The resolution of maximum likelihood and Bayesian analyses (Figure 1) both indicates that the newly sequenced species O. chinensis (Coleoptera: Trogidae) shared a close ancestor with Anoplotrupes stercorosus (Coleoptera: Geotrupidae) and closely related to Lucanidae.
In conclusion, the first complete mitogenome of Trogidae, O. chinensis is a typical metazoan mitogenome. Phylogenetic  analysis show that Trogidae shares a close ancestry with Lucanidae and Geotrupidae.