Complete Mitochondrial Genome of Scolytoplatypodini Species (Coleoptera: Curculionidae: Scolytinae) and Phylogenetic Implications

The complete mitochondrial genomes (mitogenomes) of beetles in the tribe Scolytoplatypodini (genus Scolytoplatypus) were sequenced and annotated. These included Scolytoplatypus raja (15,324 bp), Scolytoplatypus sinensis (15,394 bp), Scolytoplatypus skyliuae (15,167 bp), and Scolytoplatypus wugongshanensis (15,267 bp). The four mitogenomes contained 37 typical genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes (rRNAs). The gene orientation and arrangement of the four mitogenomes were similar to other Coleoptera mitogenomes. PCGs mostly started with ATN and terminated with TAA. The Ka/Ks ratio of 13 PCGs in the four species revealed that cox1 had the slowest evolutionary rate and atp8 and nad6 had a higher evolutionary rate. All tRNAs had typical cloverleaf secondary structures, but trnS1 lacked dihydrouridine arm. Partial tRNAs lost the discriminator nucleotide. The trnY did not possess the discriminator nucleotide and also lost three bases, showing a special amino-acyl arm. Bayesian inference (BI) and maximum likelihood (ML) methods were conducted for phylogenetic analyses using 13 PCGs. Scolytoplatypodini was clustered with Hylurgini and Hylastini, and the monophyly of Scolytoplatypodini was supported. The four newly sequenced mitogenomes increase understanding of the evolutionary relationships of Scolytoplatypodini and other Scolytinae species.


Introduction
The Scolytoplatypodini is a tribe of wood-boring ambrosia beetles in the subfamily Scolytinae (Curculionidae). They farm fungi as the food source for larvae and adults in their gallery systems [1,2]. The females of most species have a unique mycangial structure located on the pronotum for carrying fungal spores [3]. However, this mycangial structure is absent in some species [4]. Except for Scolytoplatypus bombycinus, which is host-specific, other Scolytoplatypodini species have no selection propensity for hosts [4]. Scolytoplatypodini species are mostly secondary borers and usually attack small branches of dead trees. They do not usually attack healthy trees, but some species will attack healthy living trees [5]. There are two genera in Scolytoplatypodini: Scolytoplatypus Schaufuss 1891 and Remansus Jordal 2013, including 60 species. Scolytoplatypus occur mainly in the Afrotropical and Oriental regions, with a few occurrences in temperate regions of Japan and India, and Remansus are only found in Madagascar [6]. Phylogenetic analysis studies on Scolytoplatypodini have mostly focused on selected gene fragments. Jordal [6] conducted a phylogenetic analysis of the Scolytoplatypodini in Africa and Asia using four gene fragments and found that the Asian species of Scolytoplatypus clearly differ from the African species of Scolytoplatypus. Additional molecular data are needed to explore the taxonomic status and systematic relationship of Scolytoplatypus and Scolytoplatypodini in Scolytinae. Among various animal groups, complete mitochondrial genomes (mitogenomes) phylogenetic analyses were credible [7]. Mitogenomes also have been applied to evaluate population genetics, phylogeography, and systematics at different taxonomic levels [8]. Therefore, the mitogenome can be a powerful tool for determining the relationship of Scolytoplatypodini within the Scolytinae.
DNA sequencing technology has allowed the sequencing of many insect mitogenomes [9,10]. The typical mitogenome of insect has closed-circular and double-stranded DNA, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and an A+T-rich region (D-loop), totaling 37 genes [8,11,12]. Mitogenomes are commonly used in phylogenetic analysis of insect lineages because of their maternal inheritance, rare recombination, relatively high evolutionary rate, and conserved gene components [11,13]. The mitogenome was used to reconstruct the phylogeny of weevils (Curculionoidea) and show that Scolytinae forms a separate lineage [7,14]. Representative bark and ambrosial beetles of Scolytinae have also been studied for phylogeny and taxonomy based on mitogenomes [15].
In this study, the mitogenome of S. raja (Blandford, 1893); S. sinensis (Tsai and Huang, 1965); S. skyliuae (Liao, Lai, and Beaver, 2022); and S. wugongshanensis (Liao, Lai, and Beaver, 2022) were provided and analyzed. These complete mitogenomes are first reported in the Scolytoplatypodini. We also explored the genome structure, nucleotide compositions, codon usage, gene overlaps, intergenic spacers, tRNA secondary structure, and the D-loop. Reconstructing the phylogenetic tree of Scolytoplatypodini based on 13 PCGs by Bayesian Inference (BI) and Maximum Likelihood (ML) methods.

Sample Collection and DNA Extraction
The samples for genome sequencing of S. raja, S. sinensis, S. skyliuae, and S. wugongshanensis were collected in China (Table 1) and identified according to the corresponding key [1]. All of the fresh samples were placed in absolute alcohol, preserved at −20 • C, and stored at the School of Agricultural Sciences, Jiangxi Agricultural University, Nanchang, Jiangxi 340045, China. The total genomic DNA was extracted from the leg muscle tissue of individual specimens using the Universal Genomic DNA Kit (Jiangsu, China), following manufacturer instructions. DNA was stored for sequencing at −20 • C.

Mitogenome Sequencing and Assembly
Following manufacturer recommendations, NEB Next ® Ultra™ DNA Library Prep Kit was used to generate the sequencing library for Illumina (Lincoln, NE, USA) and add index code to each sample. Using Illumina PE Cluster Kit (Illumina, Lincoln, NE, USA), we performed the clustering of the index-coded samples on a cBot Cluster Generation System, according to manufacturer instructions. When the cluster was generated, we used the Illumina platform (NovaSeq 6000) to sequence the DNA libraries and generated 150 bp paired-end reads. After removing low-quality sequences, the reads were assembled into a complete mitogenome by MitoFlex [16].

Sequence Annotation and Analyses
MITOS [17] web server was used to determine 37 genes and all tRNA secondary structures under default parameters. According to the MITOS predictions, secondary structures for tRNAs were manually drawn with Microsoft PowerPoint 2017. The remaining PCGs and rRNA were manually corrected in Geneious 8.1.3 [18]. To draw the mitogenome circular map, we used OrganellarGenomeDRAW (OGDRAW) version 1.3.1 [19]. The nucleotide composition and relative synonymous codon usage (RSCU) were calculated by MEGA X [20] and PhyloSuite v 1.2.2 [21], respectively. Strand asymmetry was calculated by the formulas: GC-skew = [G − C]/[G + C] and AT-skew = [A − T]/[A + T] [22]. DnaSP v6.12.03 was used to calculate the nucleotide diversity (Pi) and nonsynonymous (Ka)/synonymous (Ks) mutation rate ratios of 13 PCGs [23].

Phylogenetic Analysis
A total of 34 mitogenomes ( Table 2) were used to construct the phylogenetic tree. Phy-loSuite v 1.2.2 [21] was used to download the data onto mitogenomes from GenBank (except S. raja, S. skyliuae, S. sinensis, and S. wugongshanensis). All sequences were standardized and extracted 13 PCGs by PhyloSuite v 1.2.2 [21]. The 13 PCGs (excluding the stop codons) of the 34 beetle species were aligned individually using codon-based multiple alignments with MAFFT v7.313 software with default settings [24]. Gblocks v 0.91b software was used to remove the intergenic gaps and ambiguous sites [25], and all PCGs genes were concatenated in PhyloSuite v 1.2.2 [21]. The best partitioning scheme and evolutionary models for constructing BI and ML trees were selected by PartitionFinder2 [26], with a greedy algorithm, BIC criterion, and the gene and codon model. The results are presented in Supplementary Table S1.  MrBayes v 3.2.6 [31] and IQ-TREE v.1.6.8 [32] software were employed in Phylo-Suite v 1.2.2 to construct the BI and ML phylogenetic trees [21] (refer to Du et al. [15]). Sitophilus oryzae and Sitophilus zeamais were used as the out-group. ML phylogeny was inferred using IQ-TREE v.1.6.8 [32] under the Edge-linked partition model for 10,000 ultrafast bootstraps [33]. BI phylogeny was inferred using MrBayes 3.2.6 [31] under the partition model (two parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. A PSRF close to 1.0 and a standard deviation of split frequencies below 0.01 were accepted.

Protein-Coding Genes
The total lengths of the 13 PCGs of S. raja, S. sinensis, S, skyliuae, and S. wugongshanensis were 11,070 bp, 11,062 bp, 11,023 bp, and 11,025 bp, respectively (Table S2). Of these 13 PCGs, 9 PCGs are located at the J-strand, the other 4 PCGs were encoded by the N-strand ( Figure 1, Table S3). The whole 13 PCGs AT-skew and GC-skew were all negative; the ATskew were −0.13 (S. raja and S. skyliuae) and −0.14 (S. sinensis and S. wugongshanensis), the GC-skew were −0.04 (S. raja and S. sinensis) and −0.07 (S. skyliuae and S. wugongshanensis). All PCGs used ATN as the initiation codons, except for nad1 (S. raja and S. sinensis), which begins with TTG, and atp6 (S. raja), which begins with GTG. Only atp6 and cox2 (S. skyliuae) had incomplete stop codon with T residue; other PCGs terminated with TAA/TAG. The incomplete termination codons are presumed to be filled by polyadenylation during the mRNA maturation process [34].
The RSCU of the four Scolytoplatypodini species was calculated ( Figure 2). The codons that were most commonly used were UUA-Leu, UUU-Phe, AUU-Ile, and AUA-Met. This result indicated that UUA is the most preferred codon. Additionally, there was a strong A/T bias in the PCGs.

Protein-Coding Genes
The total lengths of the 13 PCGs of S. raja, S. sinensis, S, skyliuae, and S. wugongshanensis were 11,070 bp, 11,062 bp, 11,023 bp, and 11,025 bp, respectively (Table S2). Of these 13 PCGs, 9 PCGs are located at the J-strand, the other 4 PCGs were encoded by the N-strand ( Figure 1, Table S3). The whole 13 PCGs AT-skew and GC-skew were all negative; the ATskew were −0.13 (S. raja and S. skyliuae) and −0.14 (S. sinensis and S. wugongshanensis), the GC-skew were −0.04 (S. raja and S. sinensis) and −0.07 (S. skyliuae and S. wugongshanensis). All PCGs used ATN as the initiation codons, except for nad1 (S. raja and S. sinensis), which begins with TTG, and atp6 (S. raja), which begins with GTG. Only atp6 and cox2 (S. skyliuae) had incomplete stop codon with T residue; other PCGs terminated with TAA/TAG. The incomplete termination codons are presumed to be filled by polyadenylation during the mRNA maturation process [34].
The RSCU of the four Scolytoplatypodini species was calculated ( Figure 2). The codons that were most commonly used were UUA-Leu, UUU-Phe, AUU-Ile, and AUA-Met.

Nucleotide Diversity (Pi) and Nonsynonymous (Ka)/Synonymous (Ks) Mutation Rate Ratios
The Pi of the four Scolytoplatypodini species based on 13 PCGs was computed (Figure 3), and it ranged from 0.15 to 0.32. Among the PCGs, atp8 (0.32) had the highest Pi values, followed by nad6 (0.26) and nad2 (0.25). The cox1 (0.15) had the lowest Pi values, which implies that cox1 is the most conserved gene in Scolytoplatypus.
The ratios of Ka/Ks for every gene of the 13 PCGs were also computed ( Figure 3). The values of Ka/Ks ranged from 0.05 to 0.52; 13 PCGs displayed low evolutionary rates (0 < ω < 1), indicating evolutions of 13 PCGs under the purification option [35]. Cox1 (0.05) exhibits the lowest evolutionary rate, suggesting that it experienced the strongest purifying selection. The atp8 (0.52) and nad6 (0.38) exhibited a faster rate of evolution.
This result indicated that UUA is the most preferred codon. Additionally, there was a strong A/T bias in the PCGs.

Nucleotide Diversity (Pi) and Nonsynonymous (Ka)/Synonymous (Ks) Mutation Rate Ratios
The Pi of the four Scolytoplatypodini species based on 13 PCGs was computed (Figure 3), and it ranged from 0.15 to 0.32. Among the PCGs, atp8 (0.32) had the highest Pi values, followed by nad6 (0.26) and nad2 (0.25). The cox1 (0.15) had the lowest Pi values, which implies that cox1 is the most conserved gene in Scolytoplatypus.
The ratios of Ka/Ks for every gene of the 13 PCGs were also computed ( Figure 3). The values of Ka/Ks ranged from 0.05 to 0.52; 13 PCGs displayed low evolutionary rates (0 < ω < 1), indicating evolutions of 13 PCGs under the purification option [35]. Cox1 (0.05) exhibits the lowest evolutionary rate, suggesting that it experienced the strongest purifying selection. The atp8 (0.52) and nad6 (0.38) exhibited a faster rate of evolution.
Intergenic spacers were identified in the four mitogenomes, including 14 intergenic spacers in S. raja, 15 in S. sinensis, 17 in S. skyliuae, and 13 in S. wugongshanensis. The length of intergenic spacers ranged from 1 bp to 68 bp (Table S3). The longest intergenic spacer was located between trnS2 and nad1 in S. raja.
Intergenic spacers were identified in the four mitogenomes, including 14 intergenic spacers in S. raja, 15 in S. sinensis, 17 in S. skyliuae, and 13 in S. wugongshanensis. The length of intergenic spacers ranged from 1 bp to 68 bp (Table S3). The longest intergenic spacer was located between trnS2 and nad1 in S. raja.

Transfer RNA, Ribosomal RNA Genes, and Non-Coding Regions
The position and secondary structures of tRNA genes were identified by the MITOS server. The mitogenomes of the four species each contained 22 typical tRNAs, and 14 tRNAs were encoded by the J-strand; the others were encoded by the N-strand ( Figure 1 and Table S3). The length of the four Scolytoplatypodini mitogenomes ranged from 59 bp (trnS1, trnY) to 71 bp (trnQ) in S. raja, from 59 bp (trnY) to 71 bp (trnQ) in S. sinensis, from 55 bp (trnS1) to 68 bp (trnQ, trnM, trnK) in S. skyliuae, and from 57 bp (trnS1) to 70 bp (trnK) in S. wugongshanensis (Table S3).
A typical tRNA consists of a discriminator nucleotide, amino-acyl (AA) arm, TΨC (T) arm, variable (V) arm, anticodon (AC) arm, and dihydrouridine (DHU) arm. All tRNAs exhibit a canonical cloverleaf structure, except for trnS1 lacking the DHU arm, it is common in most metazoan mitogenomes for trnS1 to lack the DHU arm [8,11,36]. Such abnormal tRNAs may sustain their function through a posttranscriptional RNA editing

Transfer RNA, Ribosomal RNA Genes, and Non-Coding Regions
The position and secondary structures of tRNA genes were identified by the MITOS server. The mitogenomes of the four species each contained 22 typical tRNAs, and 14 tRNAs were encoded by the J-strand; the others were encoded by the N-strand ( Figure 1 and Table S3). The length of the four Scolytoplatypodini mitogenomes ranged from 59 bp (trnS1, trnY) to 71 bp (trnQ) in S. raja, from 59 bp (trnY) to 71 bp (trnQ) in S. sinensis, from 55 bp (trnS1) to 68 bp (trnQ, trnM, trnK) in S. skyliuae, and from 57 bp (trnS1) to 70 bp (trnK) in S. wugongshanensis (Table S3).
A typical tRNA consists of a discriminator nucleotide, amino-acyl (AA) arm, TΨC (T) arm, variable (V) arm, anticodon (AC) arm, and dihydrouridine (DHU) arm. All tRNAs exhibit a canonical cloverleaf structure, except for trnS1 lacking the DHU arm, it is common in most metazoan mitogenomes for trnS1 to lack the DHU arm [8,11,36]. Such abnormal tRNAs may sustain their function through a posttranscriptional RNA editing mechanism [37,38]. Nine mismatched base pairs (UU, GG, AA, UG, UC, AG, AC, two A, and single U) of tRNAs were found in the four Scolytoplatypodini mitogenomes. The trnY not only lacks the discriminator nucleotide but also lost three bases, so the three bases (GGU) of the 5' end are exposed (Figure 4). The trnA (S. raja) and trnK (S. sinensis and S. skyliuae) also lack the discriminator nucleotide.
The rrnL genes of the four Scolytoplatypodini mitogenomes are located at the intergenic region between trnL1 and trnV, with lengths that range from 1290 bp to 1313 bp. The rrnS genes were located between trnV and the D-loop, with sizes ranging from 753 bp to 760 bp. The two rRNAs are all encoded on the N-strand and have a high A/T bias that reached 81.5% in S. raja, 80.9% in S. sinensis, 80.3% in S. skyliuae, and 80.5% in S. wugongshanensis. Since rRNAs do not have functional annotation features like PCGs, it is so hard to establish their boundaries [11,39]. mechanism [37,38]. Nine mismatched base pairs (UU, GG, AA, UG, UC, AG, AC, two A, and single U) of tRNAs were found in the four Scolytoplatypodini mitogenomes. The trnY not only lacks the discriminator nucleotide but also lost three bases, so the three bases (GGU) of the 5' end are exposed (Figure 4). The trnA (S. raja) and trnK (S. sinensis and S. skyliuae) also lack the discriminator nucleotide. The rrnL genes of the four Scolytoplatypodini mitogenomes are located at the intergenic region between trnL1 and trnV, with lengths that range from 1290 bp to 1313 bp. The rrnS genes were located between trnV and the D-loop, with sizes ranging from 753 bp to 760 bp. The two rRNAs are all encoded on the N-strand and have a high A/T bias that reached 81.5% in S. raja, 80.9% in S. sinensis, 80.3% in S. skyliuae, and 80.5% in S. wugongshanensis. Since rRNAs do not have functional annotation features like PCGs, it is so hard to establish their boundaries [11,39].
The D-loop acts in the initiation and regulation of replication and transcription in insects [12,40]. The D-loops of the four Scolytoplatypodini mitogenomes are all located between the rrnS and trnI. The full lengths of the D-loops are 586 bp in S. raja, 621 bp in S. sinensis, 495 bp in S. skyliuae, and 670 bp in S. wugongshanensis. The AT content ranged from 81.2% (S. raja) to 85.3% (S. sinensis). There were different lengths of repeat sequences in the D-loop.

Phylogenetic Analysis
The phylogeny of Scolytinae, based on the 13 PCGs, was constructed (Figures 5 and S5) using 32 Scolytinae species and two out-groups (Sitophilus oryzae and Sitophilus zeamais). The ML tree has a similar topology to the BI tree, and their support values are reported above and below the nodes. Compared to the ML tree, the BI tree had higher confidence values, and the monophyly of most tribes and genera of the species studied was well supported. The node support values of BI trees were always higher than ML trees, especially in low-valued branches in the ML tree. The D-loop acts in the initiation and regulation of replication and transcription in insects [12,40]. The D-loops of the four Scolytoplatypodini mitogenomes are all located between the rrnS and trnI. The full lengths of the D-loops are 586 bp in S. raja, 621 bp in S. sinensis, 495 bp in S. skyliuae, and 670 bp in S. wugongshanensis. The AT content ranged from 81.2% (S. raja) to 85.3% (S. sinensis). There were different lengths of repeat sequences in the D-loop.

Phylogenetic Analysis
The phylogeny of Scolytinae, based on the 13 PCGs, was constructed ( Figures 5 and S5) using 32 Scolytinae species and two out-groups (Sitophilus oryzae and Sitophilus zeamais). The ML tree has a similar topology to the BI tree, and their support values are reported above and below the nodes. Compared to the ML tree, the BI tree had higher confidence values, and the monophyly of most tribes and genera of the species studied was well supported. The node support values of BI trees were always higher than ML trees, especially in low-valued branches in the ML tree. The clades consisting of Hylurgini, Hylastini, and Scolytoplatypodini had a high support value. However, within each of the close clades, Xyloterini and Cryphalini, the internal relationship among the included group was not strongly supported in the ML results. The monophyly of Scolytoplatypodini was confirmed. All Scolytoplatypus were clustered on one branch, and the nodes received high support. The Scolytoplatypus split into two branches; one branch was composed of S. skyliuae and S. wugongshanensis, and, despite The clades consisting of Hylurgini, Hylastini, and Scolytoplatypodini had a high support value. However, within each of the close clades, Xyloterini and Cryphalini, the internal relationship among the included group was not strongly supported in the ML results. The monophyly of Scolytoplatypodini was confirmed. All Scolytoplatypus were clustered on one branch, and the nodes received high support. The Scolytoplatypus split into two branches; one branch was composed of S. skyliuae and S. wugongshanensis, and, despite their morphological similarities, the molecular data were diagnostic. The other branch was composed of S. raja and S. sinensis. The branch of S. skyliuae and S. wugongshanensis formed a sister lineage with S. raja and S. sinensis. These results support those of previous morphological and phylogenetic studies [1]. The Scolytini was the first diverging lineage in the subfamily Scolytinae lineage and was highly supported. This result was consistent with previous studies [41][42][43]. The Ipini, Dryocoetini, and Xyleborini were clustered in a branch and had high support values. This was consistent with the results of Du et al. [15]. The Corthylini and Polygraphini had low support values, although they were clustered together. This is the first analysis of the relationship between Scolytoplatypodini in Scolytinae based on the mitogenome. However, the relationship between the Scolytoplatypodini and the other partial groups remains unclear. This is primarily due to the limited number of published mitogenomes within the Scolytinae. This issue could be resolved when the mitogenomes of additional Scolytinae species are obtained.

Conclusions
In this study, we sequenced and analyzed the mitogenomes of S. raja, S. sinensis, S. skyliuae, and S. wugongshanensis, in which mitogenomes had common and similar structures. Gene rearrangements are unusually rare in the mitogenomes in Coleoptera, especially PCGs. [44]. The complete mitogenomes of the four Scolytoplatypodini species, like other Scolytinae, revealed high A/T bias [15,45,46]. Most PCGs start with ATN except for nad1 in S. raja and S. sinensis, which begin with TTG, and for atp6 in S. raja, which begins with GTG. Only atp6 and cox2 (S. skyliuae) are terminated with an incomplete T residue, other PCGs terminated with TAA/TAG. By analysis of KA/KS, cox1 was determined to be the most conserved gene. In contrast, atp8 and nad6 had relatively higher evolutionary rates that differed from several bark beetles [15]. Most tRNA molecules have a typical cloverleaf structure, but trnS1 lacks the DHU and trnA (S. raja) and trnK (S. sinensis and S. skyliuae) lack the discriminator nucleotide. The trnY lacks not only the discriminator nucleotide but also the three bases. While the cox1 gene is the most conserved, the atp8 and nad6 have relatively higher evolutionary rates, and this differs from other bark beetles [15]. The phylogenetic tree using 13PCGs showed the monophyly of Scolytoplatypodini. Compared to the results of Du et al. [15], our use of 13 PCGs to construct the ML tree yielded the same results as obtained with the BI tree. This implies that the phylogenetic tree that was constructed using 13 PCGs obtained more stable results.
On the issue of whether Scolytoplatypodini belongs to Scolytinae or Platypodinae, there is no doubt that Scolytoplatypodini belongs to Scolytinae. However, Scolytinae and Platypodinae have the same convergent evolution as resemblant wood-boring. Nevertheless, current molecular data show that Dryophthorinae and Platypodinae are indeed sister groups, refuting the close relationship between Scolytinae and Platypodinae [7,14,47]. Remansus was the second genus in Scolytoplatypodini. Although no samples of Remansus were obtained in this study, Pistone et al. [41] showed Scolytoplatypus as a sister group to Remansus with high node support, then both clustering with other Scolytinae, for example, genera Scolytodes and Gymnochilus. Jordal [6] suggested that the African Scolytoplatypus and the Asian Scolytoplatypus might represent two unique genera. In males, the Asian Scolytoplatypus differ from the African Scolytoplatypus by having a strongly modified prosternum with nodules or hooked projections, by the longer and more triangular antennal club, and in all but two species by the large fovea on the anterolateral angle of the male pronotum [6]. African Scolytoplatypus and Asian Scolytoplatypus have previously been divided into two major phylogenetic branches [1,6]. However, few representative specimens were sequenced in this study, making it difficult to provide a more detailed discussion. The four new mitogenomes were from species collected in Asia. It is necessary to sequence more Scolytoplatypus species to explore the relationship of African Scolytoplatypus with the Asian Scolytoplatypus.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/genes14010162/s1, Figure S1: Secondary structures for tRNA genes from the mtDNA of S. raja; Figure S2: Secondary structures for tRNA genes from the mtDNA of S. sinensis; Figure S3: Secondary structures for tRNA genes from the mtDNA of S. skyliuae; Figure S4: Secondary structures for tRNA genes from the mtDNA of S. wugongshanensis; Figure S5: Phylogenetic tree of Scolytinae using the BI method based on 13 PCGs. Table S1: The best-fit partitioning scheme and corresponding models used on the phylogenetic tree; Table S2: Nucleotide compositions in regions of the S. raja, S. sinensis, S. skyliuae, and S. wugongshanensis mitochondrial genomes; Table S3  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The mitogenomes were deposited at NCBI, with accession numbers OP719285, OP719283, OP719284, and OP712675.