Characterization of the Complete Mitochondrial Genome of a Flea Beetle Luperomorpha xanthodera (Coleoptera: Chrysomelidae: Galerucinae) and Phylogenetic Analysis

In this study, the mitochondrial genome of Luperomorpha xanthodera was assembled and annotated, which is a circular DNA molecule including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and 1388 bp non-coding regions (A + T rich region), measuring 16,021 bp in length. The nucleotide composition of the mitochondrial genome is 41.3% adenine (A), 38.7% thymine (T), 8.4% guanine (G), and 11.6% cytosine (C). Most of the protein-coding genes presented a typical ATN start codon (ATA, ATT, ATC, ATG), except for ND1, which showed the start codon TTG. Three-quarters of the protein-coding genes showed the complete stop codon TAR (TAA, TAG), except the genes COI, COII, ND4, and ND5, which showed incomplete stop codons (T- or TA-). All the tRNA genes have the typical clover-leaf structure, except tRNASer1 (AGN), which has a missing dihydrouridine arm (DHU). The phylogenetic results determined by both maximum likelihood and Bayesian inference methods consistently supported the monophyly of the subfamily Galerucinae and revealed that the subtribe Luperina and genus Monolepta are polyphyletic groups. Meanwhile, the classification status of the genus Luperomorpha is controversial.


Introduction
The insect mitochondrial DNA (mtDNA) is a double-stranded circular DNA molecule, with the maternal inheritance features of a relatively small molecular mass, a relatively conservative gene arrangement, and a rapid rate of gene evolution, etc. [1]. The mitochondrial genes have been widely used in species identification, the estimation of evolutionary relationships, and the recognition of population structure and phylogeography [2]. The mitogenome contains 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 1 A + T-rich region [3]. Nowadays, mitochondrial DNAs (mtDNAs) have been commonly used as molecular markers for reconstructing phylogenetic relationships, revealing population genetic structures, estimating divergence times, identifying relatedness between recently diverged species, etc. [4,5].
The invasive flea beetle, Luperomorpha xanthodera (Coleoptera: Chrysomelidae: Galerucinae), was originally described by Fairmaire [6] in Jiangxi Province of China [7]. It has previously been recorded in 16 provinces in China, distributing throughout Jilin, Gansu, Shanxi, Shandong, Jiangsu, Zhejiang, Hubei, Jiangxi, Hunan, Fujian, Taiwan, Guangdong, Guangxi, Sichuan, Guizhou, and Yunnan. It has also been found in many other countries, such as Korea, Japan, Italy, France, the Netherlands, and Poland [8][9][10][11][12]. This flea beetle is a crucial pest of economic crop, such as roses (Rosa) and Zanthoxylum spp. Their larvae are commonly found concealed in the roots of plants, while the adults feed on grasses, shrubs, and trees flowers. Luperomorpha xanthodera has been reported to feed on 21 different plants, including white mustard (Sinapis alba L., Brassicaceae), hyssop (Hyssopus officinalis L.), and Monarda spp. (Lamiaceae), etc. [8]. To our knowledge, in the natural population of Luperomorpha xanthodera, the females constituted the vast majority [13]. This means that this species is highly susceptible to outbreaks.
In the past, the species of the genus Luperomorpha Weise were placed among the Alticinae (currently known as Alticini) due to the presence of the metafemoral spring. However, some scholars [14,15] studied the morphological characters of Galerucinae and Alticinae (currently known as Galerucini and Alticini), and they discovered that the metafemoral spring could not be the sole trait that divides the two subfamilies. In a later paper, it was noted that although Luperomorpha has a metafemoral spring, it only has one basal patch at the base of the ventral side of the sheath wing and should be transferred to Galerucinae. However, the classification and taxonomic position of the genus Luperomorpha has continued to prove problematic. Nowadays, only one species of Luperomorpha has been reported for a complete mitogenome.
For the present study, we sequenced and annotated Luperomorpha xanthodera's complete mitogenome and studied its characteristics. This study aimed to acquire the data on mitochondrial genome, additional analyses of the composition of the Luperomorpha xanthodera mitochondrial genome, the values of the relative synonymous codon usage (RSCU), the evolutionary rate of Luperomorpha and the phylogenetic relationship, etc. We believe such results will be helpful in studies of the population genetics and phylogenetic relationships of this species, as well as studies on other flea beetles in the future. We adopted Nie's classification system [16] to conduct the subsequent analyses.

Sample Collection and DNA Extraction
The invasive flea beetle, Luperomorpha xanthodera, was collected on the Zanthoxylum planispinum from Zhenfeng county, Guizhou, China (25 • 24 N, 105 • 35 E), on 23 June 2021. The total DNA was extracted from the entire body without the abdomen using the DNeasy Blood & Tissue Kit (Cat. No. 69504) (Qiagen, Hilden, Germany), as per the manufacturer's protocol. The voucher specimen's genome DNA and male genitalia were deposited at the Institute of Entomology, Guizhou University, Guiyang, China (GUGC). The identification of adult specimens was based on morphological characteristics [8,10].

Genome Sequencing, Assembly and Annotation
Illumina TruSeq libraries with an average insert size of 300 bp were prepared and sequenced on the Illumina NovaSeq 6000 platform (Beijing Berry Bioinformatics Technology Co., Ltd., Beijing, China), obtaining 150 bp paired-end reads. The mitogenome was preliminarily annotated by Mitoz 2.4-α [17], with the invertebrate mitochondrial genetic codes. The MITOS web server (http://mitos.bioinf.uni-leipzig.de/index.py, accessed on 14 December 2021) [18] and the tRNAscan-SE search server (http://lowelab.ucsc.edu/tRNAscan-SE/, accessed on 14 December 2021) [19,20] were used to reconfirm and predict the locations and secondary structures of the tRNA genes, and then the tRNA secondary structures were visualized using Adobe Illustrator. The circular mitogenome maps were visualized using Geneious Prime [21].

Mitogenome Sequence Analyses
MEGA 7.0 [22] was used to obtain the nucleotide composition statistics, relative synonymous codon usage (RSCU) of all the protein-coding genes (PCGs), and the base composition. The following formula was used to calculate the strand asymmetry: AT skew = [A − T]/[A + T] and GC skew = [G − C]/[G + C] [23]. In the A + T rich region, the tandem repeat elements were verified using the Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html, accessed on 23 December 2021) [24]. The non-

Phylogenetic Analysis
For the phylogenetic analysis, 24 additional mitogenome sequences were downloaded from GenBank, and we selected Chrysomela vigintipunctata and Chrysolina aeruginosa as the outgroups ( Table 1). The L-INS-I strategy in MAFFT software was used for the sequences of amino acids of 13 PCGs, trimmed using trimAl v1.4.1 [26] with the heuristic method 'au-tomated1' to eliminate the gap-only and ambiguous-only locations, and was concatenated using FASconCAT g v1.04 [27]. Using the amino acid sequence with 13 PCGs in the phylogenetic analysis, the maximum likelihood (ML) and Bayesian inference (BI) were used to create phylogenetic trees. The ML analyses were carried out using IQ-TREE V1.6.3 [28] and 1000 ultrafast bootstrap [29] and 1000 SHaLRT replicates [30]. The best model was determined through the AIC and BIC scores. The PMSF model [31] was used for the amino acid sequence matrix with 13 PCGs by specifying the profile mixture model with the option "-mtInv + C60 + FO + R". Under the CAT + GTR model [32], the amino acid sequence matrix with 13 PCGs was further analyzed with Bayesian inference using PhyloBayes MPI v1.8b [33] and independently run for 10,000 generations for two separate chains. We calculated the largest discrepancy (maxdiff) across all the bipartitions using the bpcomp program. When the maxdiff was <0.3, runs were considered to achieve a good convergence. The tracecomp program was also used to evaluate the discrepancy between two independent runs. The minimum effective size >50 was recognized to be a good run. In this study, values greater than 98 percent (SH-aLRT, UFBoot2) and 0.99 (posterior probability) are considered to be of a "high" support; values of 80 percent to 98 percent for SH-aLRT, 95 percent to 98 percent for UFBoot2, and 0.95 percent to 0.99 percent for the posterior probability are considered to be of a "moderate" support; and values of 95 percent for UFBoot2 and 0.95 percent for the posterior probability are considered to be of a "low" support. The resulting trees were visualized and edited using FigTree v.1.4.2.

Mitogenome Organization and Nucleotide Composition
In this study, the complete mitogenome of Luperomorpha xanthodera was successfully obtained and uploaded to GenBank under the accession number ON631248 (Table 1; Figure 1). It has a total length of 16,021 bp and includes 37 genes, including 13 proteincoding genes (PCGs), 2 ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes, as well as a noncoding control region (A + T-rich region). The gene order of the Luperomorpha xanthodera mitogenome was consistent with the ancestral gene order of Drosophila yakuba, this is thought to be the ground pattern for insect mitogenomes. As with most galerucine species, 23 genes were on the majority strand (F-strand), and the other 14 genes (tRNA Gln , tRNA Cys , tRNA Tyr , tRNA Phe , ND5, tRNA His , ND4, ND4L, tRNA Pro , ND1, tRNA Leu , 16S rRNA, tRNA Val , 12S rRNA) were on the minority strand (R-strand) ( Table 2).   The Luperomorpha xanthodera mitogenome contained 41 bp overlapping regions in 11 pairs of neighboring genes ranging in length from 1 to 8 bp. The longest overlapping region of 8 bp was located between tRNA Trp and tRNA Cys and tRNA Tyr and COI, respectively. A total of 34 bp intergenic nucleotides (IGN) ranging in size from 1 to 17 bp were distributed across seven locations. The longest intergenic spacer was found between the tRNA Ser and ND1 (Table 2). These overlapping and intergenic regions are very frequent in flea beetle mitochondrial genomes [34][35][36]. The start codons and termination codons of all the PCGs genes coincided with the typical coleopteran [37] (truncated termination codons).
The overall nucleotide composition of Luperomorpha xanthodera is 41.3% A, 38.7% T, 11.6% C, and 8.4% G and includes a high A + T content (80%) and positive AT skew (0.032) and negative GC skew values (−0.160), this is comparable to other flea beetles. For instance, all the 27 flea beetles we analyzed had positive AT skews and negative GC skews, indicating that the base compositions of the flea beetle mitogenomes were, overall, biased towards A and C (Table 1). Furthermore, the A + T content of the third codon position was the highest (92.2%), while the A + T content of the second codon position was the lowest (69.0%). The AT skews of all the codon positions are positive, indicating a slightly higher occurrence of a likeness to T nucleotides. Meanwhile, the GC skews of the first codon position are positive, but the other two codon positions are negative.

Genes Size (bp) Nucleotides Composition ATskew GCskew A (%) T (%) G (%) C (%) A + T (%) G + C (%)
Complete mitogenome 16   We calculated the nonsynonymous substitution rate (Ka), synonymous substit rate (Ks), and the ratio of Ka/Ks for each PCG in three species (Figure 3). In all PCG highest Ks value was exhibited by ATP6, whereas the Ka value of ATP8 was disti We calculated the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and the ratio of Ka/Ks for each PCG in three species (Figure 3). In all PCGs, the highest Ks value was exhibited by ATP6, whereas the Ka value of ATP8 was distinctly higher than others (Figure 3). The average Ka/Ks ranged from 0.181 (ATP6) to 1.037 (ND4). It is noteworthy that with the exception of ND4, all other PCGs of Ka/Ks values are lower than 1, demonstrating that the mutations were exchanged out by synonymous substitutions (Figure 3). The ND4 gene reached 1.037; this result provides an indication that positive selection had a dominating impact on the evolution of ND4 gene. The smallest Ka/Ks -values were 0.181 for COX1 and ATP6, which was considered to be a strong purifying selection.

Transfer and Ribosomal RNA Genes
The length of the 22 tRNA genes of Luperomorpha xanthodera range from 62 bp (tRNA Ile ) to 70 bp (tRNA Lys ), comprising 8.94% of the complete mitogenome (Table 2), of which all the tRNA genes can be folded into the typical cloverleaf structure, except for trnS1(AGN), whose dihydrouridine (DHU) arm is converted to a simple loop ( Figure 4)-this characteristic is frequent in flea beetle mitogenomes [40]. The anticodon of tRNA Lys mutations is TTT, and when we identified the insects of Chrysomelidae, using this would be one of the quickest methods [16].
The Luperomorpha xanthodera of ribosomal RNA (rRNA) is composed of 16S rRNA (situated between tRNA Leu and tRNA Val ) and 12S rRNA (situated between tRNA Val and the A + T rich region). The size of the two genes was 1280 bp (16S rRNA) and 812 bp (12S rRNA), respectively. The A + T content reached 84.2% in Luperomorpha xanthodera. Furthermore, the two rRNAs contained a positive AT skew and negative GC skew (Table 3).

A + T Rich Region
The A + T-rich region is an important noncoding region in insect mitogenome, which regulates the mitochondrial DNA (mtDNA) transcription and replication [41][42][43]. The A + T rich region of Luperomorpha xanthodera is located between 12S rRNA and tRNA Ile and is 1388 bp in length (Table 2). Meanwhile, the A + T content was 87.3%, with positive AT skews (0.010) and negative GC skews (−0.213) ( Table 3). Of note, we found four tandem repeat units in the control region, ranging from 13 to 35 bp. Furthermore, a poly-A region and three poly-T regions were found in the control region ( Figure 5).

Phylogenetic Relationships
In the past two decades, molecular systematic approaches are widely used for disclosing unsettled classification and phylogenetic relationships in Insecta [44,45]. The phylogenetic trees of 37 mitochondrial genes (including one newly generated sequence, 24 other Chrysomelidae sequences from GenBank and two outgroup sequences) were generated from a single dataset (amino acids sequence with the 13 PCGs) using maximum likelihood (ML) and Bayesian inference; the BI tree and ML tree are shown in this paper (Figures 6 and 7). The two phylogenetic trees produced consistent results: (1) the Galerucinae is represented as a monophyletic group; (2) the Alticini were clustered into a monophyletic clade and formed sister groups with other subtribes of the Galerucini; (3) the Luperina was the polyphyletic group, which was consistent with the results of Gillespie et al. [46]; and (4) Monolepta occifluvis does not cluster with the other two species of the genus Monolepta but is instead sister to Paleosepharia posticata with high support values (0.99/98.7/100). These findings are consistent with earlier mitogenome-based research [47], which indicates that Monolepta is a polyphyletic genus. There are distinctions between the two trees as well. In the BI tree, the Hoplasoma unicolor is recovered as the sister group of the Oises livida, and their combined clade is grouped with Luperomorpha Weise. In the ML tree, Luperomorpha and Luperina showed a closer relationship.
According to our phylogenetic tree, the two Luperomorpha species are nested in Galerucini. The cladistic taxonomic position of several genera (including Luperomorpha Weise) has been rotating between Galerucini and Alticini in evolutionary studies of Chrysomelidae [48][49][50], and these genera are known as the 'problematic' genera [15]. As research on these two tribes has progressed, their taxonomic basis has shifted from a single character analysis (metafemoral spring) to a combination of characters (metafemoral spring, hind wing venation, female spermatheca, male aedeagus, etc.), resulting in Luperomorpha Weise being removed from Alticinae (currently Alticini) and added to Galerucinae (currently Galerucini) [51]. Some phylogenetic studies also support the Luperomorpha Weise being attributed to Galerucini [16,52].

Conclusions
At present, we sequenced and analyzed Luperomopha xanthodera's complete mitogenome. The complete mitochondrial genome of Luperomopha xanthodera has a final size of 16,021 bp, with 80% AT content. The circular mitogenome encoded a typical set of 37 genes (13 PCGs, two rRNAs, 22 tRNAs, and one control region). The majority of PCGs use ATN (except ND1 with TTG) as their start codon and TAA/TAG/T-as the stop codons. For the analysis of the selective pressure, we discovered that most of the 13 PCGs of Luperomorpha were less than one, particularly COX1 and ATP6 genes, which had the lowest value, indicating a high conservation. This revealed that PCGs were subjected to purifying selection in the genus, while the ND4 gene has a high value, demonstrating that it may have been mutated during the evolution process. Phylogenetic trees based on the mitogenomes of 27 species contributed to the scientific classification of Luperomopha xanthodera. Our study identified a close relationship between Luperomopha Weise and Luperini (currently Luperina). Overall, our results provide a hint for the phylogenetic position of Luperomorpha, and they have provided basic genetic information for understanding the phylogeny and evolution of leaf beetles.

Conflicts of Interest:
The authors declare no conflict of interest.