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. 2023 Feb 4;14(2):414. doi: 10.3390/genes14020414

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

Jingjing Li 1,2, Bin Yan 1,2, Hongli He 1,2, Xiaoli Xu 1,2, Yongying Ruan 3, Maofa Yang 1,2,*
Editor: Zhiteng Chen
PMCID: PMC9957443  PMID: 36833341

Abstract

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.

Keywords: leaf beetles, mitochondrion, monophyly, next-generation sequencing, phylogeny

1. 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.

2. Materials and Methods

2.1. 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].

2.2. 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].

2.3. 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-synonymous substitutions (Ka) and synonymous substitutions (Ks) of 13 PCGs of 3 species (A. zanthoxylumi, A. planipennis, and A. ornatus) were calculated using DnaSP v 5 [25].

2.4. 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 ‘automated1’ to eliminate the gap-only and ambiguous-only locations, and was concatenated using FASconCAT g v1.04 [27].

Table 1.

Mitochondrial genomes were used for the phylogenetic analyses in this research.

Subfamily Tribe Species Length (bp) G + C% A + T% AT-Skew GC-Skew GenBank No.
Galerucinae Alticini Agasicles hygrophila 15,917 24.9 75.2 0.05 −0.19 NC_028332
Altica cirsicola 15,864 22.2 77.8 0.03 −0.21 NC_042876
Altica fragariae 16,220 22.0 78 0.03 −0.22 NC_042875
Altica viridicyanea 16,706 22.3 77.8 0.04 −0.23 NC_048472
Argopistes tsekooni 16,552 20.5 79.5 0.04 −0.19 NC_045929
Chaetocnema pelagica 16,331 23.1 76.9 0.06 −0.20 NC_041170
Macrohaltica subplicata 15,840 22.0 78 0.02 −0.22 NC_041169
Phyllotreta striolata 15,689 24.2 75.8 0.05 −0.26 NC_045901
Psylliodes chlorophana 14,561 23.7 76.3 0.05 −0.22 NC_053362
Luperomorpha hainana 16,081 22.2 77.8 0.05 −0.25 MF960124
Luperomorpha xanthodera 16,021 20 80 0.03 −0.16 ON631248
Incertae sedis Paleosepharia posticata 15,729 20.2 79.8 0.02 −0.17 NC_033532
Podagricomela nigricollis 16,756 21.3 78.8 0.05 −0.18 NC_041423
Galerucini Diorhabda carinata 16,232 22.1 77.9 0.03 −0.19 NC_042945
Diorhabda carinulata 16,298 21 78.9 0.03 −0.18 NC_042946
Galeruca daurica 16,615 21.8 78.1 0.04 −0.20 NC_027114
Ophraella communa 16,553 22.6 77.4 0.06 −0.18 NC_039710
Luperini Aulacophora indica 16,246 21.4 78.6 0.03 −0.20 NC_047467
Aulacophora lewisii 15,691 21.1 78.9 0.03 −0.18 NC_039712
Diabrotica barberi 16,366 20.8 79.2 0.04 −0.18 NC_022935
Hoplasoma unicolor 14,568 22.2 77.8 0.05 −0.23 NC_041168
Monolepta hieroglyphica 15,963 19.7 80.3 0.02 −0.18 NC_057489
Monolepta occifluvis 15,998 20.7 79.2 0.03 −0.20 NC_045838
Monolepta quadriguttata 16,130 19.7 80.3 0.02 −0.19 NC_039711
Oidini Oides livida 16,127 20.0 79.9 0.05 −0.14 MF960098
Chrysomelinae Chrysomelini Chrysomela vigintipunctata 17,474 21.7 78.2 0.04 −0.21 NC_050933
Chrysolina aeruginosa 16,335 24.5 75.5 0.07 −0.25 NC_052915
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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.

3. Results and Discussion

3.1. 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 protein-coding 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 (tRNAGln, tRNACys, tRNATyr, tRNAPhe, ND5, tRNAHis, ND4, ND4L, tRNAPro, ND1, tRNALeu, 16S rRNA, tRNAVal, 12S rRNA) were on the minority strand (R-strand) (Table 2).

Figure 1.

Figure 1

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Complete mitogenome map of Luperomorpha xanthodera. The majority strand (F-strand) encodes 14 genes, while the minority strand (R-strand) encodes 23 genes. Protein and rRNA genes are denoted using standard abbreviations. tRNA genes are represented by a single letter for each amino acid, with two leucine tRNAs and two serine tRNAs separated by numbers.

Table 2.

Organization of the Luperomorpha xanthodera mitochondrial genome. IGN: intergenic nucleotides. Negative values refer to overlapping nucleotides. dots (•) indicate no value, hyphens (-) represent the size of overlapping regions.

Gene Direction Location (bp) Size (bp) Codon Anti-Codon IGN
from to Start Stop
tRNAIle F 1 62 62 • • GAT
tRNAGln R 63 131 69 • • TTG 0
tRNAMet F 131 199 69 • • CAT -1
ND2 F 200 1210 1011 ATT TAA • 0
tRNATrp F 1211 1274 64 • • TCA 0
tRNACys R 1267 1328 62 • • GCA -8
tRNATyr R 1329 1391 63 • • GTA 0
COⅠ F 1384 2926 1543 ATT T • -8
tRNALeu F 2927 2991 65 • • TAA 0
COⅡ F 2992 3676 685 ATG T • 0
tRNALys F 3677 3746 70 • • TTT 0
tRNAAsp F 3747 3809 63 • • GTC 0
ATP8 F 3810 3965 156 ATC TAA • 0
ATP6 F 3959 4633 675 ATG TAA • -7
COⅢ F 4633 5421 789 ATG TAA • -1
tRNAGly F 5424 5488 65 • • TCC 2
ND3 F 5489 5842 354 ATT TAG • 0
tRNAAla F 5841 5904 64 • • TGC -2
tRNAArg F 5905 5968 64 • • TCG 0
tRNAAsn F 5966 6033 68 • • GTT -3
tRNASer F 6034 6100 67 • • TCT 0
tRNAGlu F 6102 6165 64 • • TTC 1
tRNAPhe R 6165 6227 63 • • GAA -1
ND5 R 6228 7932 1705 ATT T • 0
tRNAHis R 7933 7995 63 • • GTG 0
ND4 R 8005 9325 1321 ATG T • 9
ND4L R 9319 9600 282 ATG TAA • -7
tRNAThr F 9603 9665 63 • • TGT 2
tRNAPro R 9666 9729 64 • • TGG 0
ND6 F 9732 10,235 504 ATT TAA • 2
CYTB F 10,235 11,374 1140 ATG TAG • -1
tRNASer F 11,373 11,439 67 • • TGA -2
ND1 R 11,457 12,407 951 TTG TAG • 17
tRNALeu R 12,409 12,473 65 • • TAG 1
16S rRNA R 12,474 13,753 1280 • • • 0
tRNAVal R 13,754 13,821 68 • • TAC 0
12S rRNA R 13,822 14,633 812 • • • 0
A + T rich region F 14,634 16,021 1388 • • • 0
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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 tRNATrp and tRNACys and tRNATyr 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 tRNASer 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.

3.2. Protein-Coding Genes and Codon Usage

The PCGs include seven NADH dehydrogenases (ND1-ND6 and ND4L), three cytochrome c oxidases (COⅠ-COⅢ), two ATPases (ATP6 and ATP8), and one cytochrome b (CYTB). The total length of the 13 PCGs of Luperomorpha xanthodera was 11,116 bp, with an A + T content of 78.2%. Additionally, nine of its genes (ND2, COⅠ, COⅡ, ATP8, ATP6, COⅢ, ND3, ND6, CYTB) are located on the major strand (F-strand), and the others (ND5, ND4, ND4L, ND1) are located on the minor strand (R-strand). The ND5 gene was the longest at 1705 bp, and the smallest was the ATP8 gene at 156 bp. The base compositions of the 13 PCG genes were as follows: 40.2% A, 38.0% T, 9.4% G, and 12.5% C (Table 3). Most PCGs of Luperomorpha xanthodera were initiated with the typical ATN codon (except ND1 with TTG). Most PCGs (ND2, ATP8, ATP6, COⅢ, ND3, ND4L, ND6, CYTB, and ND1) had the complete stop codon of TAA or TAG, whereas four PCGs (COⅠ, COⅡ, ND4, and ND5) ended with the incomplete stop codon (T-). Some scholars believe that incomplete termination codons will be repaired by polyadenylation after transcription [38,39]. Based on 3698 codons, the 13 PCGs’ relative synonymous codon usage (RSCU) values were determined. UUU (Phe), UUA (Leu2), AUU (Ile), and AUA (Met) were the four most common codons. The preferred codons all ended in A or U, contributing to the overall A + T bias of the mitogenomes. (Table 4; Figure 2).

Table 3.

Nucleotide composition of the Luperomorpha xanthodera mitogenomes.

Genes Size (bp) Nucleotides Composition ATskew GCskew
A (%) T (%) G (%) C (%) A + T (%) G + C (%)
Complete mitogenome 16,021 41.3 38.7 8.4 11.6 80 20 0.032 −0.160
PCGs 11,125 33.9 44.3 11.1 10.7 78.2 21.8 −0.133 0.018
1st codon position 3711 35.0 38.2 16.7 10.1 73.2 26.8 −0.043 0.247
2nd codon position 3707 21.1 47.9 13.4 17.5 69.0 29 −0.388 −0.135
3rd codon position 3707 45.5 46.7 3.2 4.6 92.2 7.8 −0.014 −0.181
tRNA 1432 41.9 38.8 8.7 10.6 80.7 19.3 0.038 −0.098
rRNA 2092 44.6 39.6 5.5 10.3 84.2 15.8 0.059 −0.304
16S rRNA 1280 45.1 38.7 5.7 10.5 83.8 16.2 0.076 −0.296
12S rRNA 812 43.8 41.0 5.2 10.0 84.8 15.2 0.033 −0.316
A + T rich region 1388 44.1 43.2 5.0 7.7 87.3 12.7 0.010 −0.213
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Table 4.

Relative synonymous codon usage (RSCU) of Luperomorpha xanthodera mitochondrial PCGs. The asterisk (*) represent Termination codon.

Amino Acid Codon Count RSCU Amino Acid Codon Count RSCU
Phe UUU 325 1.86 Tyr UAU 162 1.79
UUC 24 0.14 UAC 19 0.21
Leu2 UUA 524 5.22 His CAU 59 1.66
UUG 21 0.21 CAC 12 0.34
Leu1 CUU 22 0.22 Gln CAA 57 1.84
CUC 3 0.03 CAG 5 0.16
CUA 27 0.27 Asn AAU 190 1.83
CUG 5 0.05 AAC 18 0.17
Ile AUU 391 1.87 Lys AAA 102 1.87
AUC 27 0.13 AAG 7 0.13
Met AUA 241 1.85 Asp GAU 62 1.8
AUG 20 0.15 GAC 7 0.13
Val GUU 66 1.8 Glu GAA 69 1.86
GUC 5 0.14 GAG 5 0.14
GUA 69 1.88 Cys UGU 30 1.88
GUG 7 0.19 UGC 2 0.13
Ser2 UCU 107 2.58 Trp UGA 88 1.91
UCC 7 0.17 UGG 4 0.09
UCA 88 2.12 Arg CGU 19 1.36
UCG 3 0.07 CGC 1 0.07
Pro CCU 66 2.06 CGA 34 2.43
CCC 8 0.25 CGG 2 0.14
CCA 53 1.66 Ser1 AGU 27 0.56
CCG 1 0.03 AGC 3 0.07
Thr ACU 90 2.03 AGA 85 2.05
ACC 11 0.25 AGG 12 0.29
ACA 76 1.72 Gly GGU 50 1.05
ACG 0 0 GGC 6 0.13
Ala GCU 63 1.81 GGA 110 2.3
GCC 16 0.46 GGG 25 0.52
GCA 59 1.7 * UAA 0 0
GCG 1 0.03 UAG 0 0
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Figure 2.

Figure 2

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Relative synonymous codon usage (RSCU) of mitogenome of Luperomorpha xanthodera. Codon families are depicted in boxes below the x-axis; the box colors represent the stacked columns; and the values of RSCU are shown on the y-axis.

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.

Figure 3.

Figure 3

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Evolutionary rates of 13 PCGs in between Luperomorpha xanthodera and Luperomorpha hainana. A blue boxes, green boxes, and orange line indicate the non-synonymous substitutions rate (Ka), the synonymous substitutions rate (Ks), and the ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions (Ka/Ks), respectively.

3.3. Transfer and Ribosomal RNA Genes

The length of the 22 tRNA genes of Luperomorpha xanthodera range from 62 bp (tRNAIle) to 70 bp (tRNALys), 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 tRNALys mutations is TTT, and when we identified the insects of Chrysomelidae, using this would be one of the quickest methods [16].

Figure 4.

Figure 4

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Predicted secondary structures of 22 tRNAs in Luperomorpha xanthodera. Lines (-) indicate Watson–Crick base pairings, whereas dots (•) indicate unmatched base pairings. The uppercase letter indicate the identity of each tRNA genes. Structural elements in tRNA arms and loops are illustrated as for trnV.

The Luperomorpha xanthodera of ribosomal RNA (rRNA) is composed of 16S rRNA (situated between tRNALeu and tRNAVal) and 12S rRNA (situated between tRNAVal 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).

3.4. 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 tRNAIle 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).

Figure 5.

Figure 5

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A + T-rich region of Luperomorpha xanthodera mitogenome. Gray blocks represent the A + T rich region. The numbers at the beginning and end of the gray blocks show the position of the control region in the Luperomorpha xanthodera mitogenome. R stands for repeat unit, and the number denotes the number of repeats. The black and green blocks represent the structures of poly-A and poly-T, respectively.

3.5. 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 (Figure 6 and Figure 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.

Figure 6.

Figure 6

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ML analysis was conducted using IQ-TREE with the dataset of amino acids of 13 PCGs. Note: Name in black bold show the phylogenetic position of Luperomorpha xanthodera that we sequenced in this research. The numbers on the nodes are the SH-aLRT support (%) and ultrafast bootstrap support (%).

Figure 7.

Figure 7

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BI analysis was conducted using PhyloBayes with the dataset of amino acids of 13 PCGs. Note: Name in black bold show the phylogenetic position of Luperomorpha xanthodera that we sequenced in this research. The numbers on the nodes are the posterior probability.

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].

4. 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.

Author Contributions

Conceptualization: J.L., B.Y. and M.Y.; methodology: J.L. and B.Y.; software: J.L., B.Y., H.H. and X.X.; data curation: J.L. and B.Y.; writing—original draft preparation: J.L. and B.Y.; writing—review and editing: J.L., Y.R., B.Y. and M.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The Luperomorpha xanthodera sequenced mitogenome was submitted to the GenBank database (ON631248), the multiple sequence alignment used for phylogentic analysis are avalable on FigShare at this weblink: https://doi.org/10.6084/m9.figshare.21957440.v1, accessed on 23 December 2021.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This study was supported by the Guizhou Province Science and Technology Plan Project (Qian Ke He Zhicheng [2021] Yiban221) and Guizhou Province Science and Technology Innovation Talent Team Project (Qian Ke He Pingtai Rencai-CXTD [2021]004).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The Luperomorpha xanthodera sequenced mitogenome was submitted to the GenBank database (ON631248), the multiple sequence alignment used for phylogentic analysis are avalable on FigShare at this weblink: https://doi.org/10.6084/m9.figshare.21957440.v1, accessed on 23 December 2021.

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