Adult specimens of M. matsumurae (Fig. 1) were collected from Pinus massoniana in Guizhou Province, China (Suiyang County, 107°2′52.06″E, 27°52′10.02″N, September 2021). All fresh specimens were preserved in 100% ethyl alcohol immediately after collection in the field and deposited at -20°C in the laboratory of Guizhou Academy of Forestry, Guiyang, Guizhou. Identification of the specimens was based on morphological characteristics (Li 2016). The total DNA was extracted from thoracic muscles using the Biospin Insect Genomic DNA Extraction Kit (BioFlux) following the manufacturer’s instructions. Voucher specimens are stored in the entomological collection of the Guizhou Academy of Forestry.

Figure 1.  

An adult male of Matsucoccus matsumurae on Pinus massoniana (Guizhou Province, China).

The whole genomic DNA of M. matsumurae was sequenced using next-generation sequencing (Illumina HiSeq X10, Biomarker Technologies Corporation, Beijing, China). About 2.13 Gb clean data were assembled into a complete circular mitogenome using NOVOPlasty v.4.3.1 (Dierckxsens et al. 2017) with the cox1 gene of Saissetia coffeae (Lu et al. 2020) as the seed sequence.

The annotation of mitogenome was conducted using Geneious Prime v.2022.0.1 (Biomatters, Auckland, New Zealand). The locations and sequences of tRNA genes were determined by the MITOS Web Server (http://mitos2.bioinf.uni-leipzig.de/index.py) (Donath et al. 2019) and comparison with homologous mitogenome sequences. Secondary structures of tRNAs were plotted using Adobe Illustrator CC2017 according to MITOS results and manual predictions (following the expected cloverleaf secondary structure of tRNA genes). Two rRNA genes and 13 PCGs were identified, based on their alignments with the other scale insect (Coccoidea) mitogenome sequences. The mitogenomic circular map was depicted with the help of OrganellarGenomeDRAW (OGDRAW) (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) (Greiner et al. 2019).

The organisation tables, nucleotide composition and relative synonymous codon usage (RSCU) of the mitogenomes of Coccoidea species were calculated and produced using PhyloSuite v.1.2.2 (Zhang et al. 2020). The nucleotide diversity (Pi) analyses of 13 PCGs and two rRNA genes of the Coccoidea species and a sliding window analysis (a sliding window of 200 bp and step size of 20 bp), were conducted by DnaSP v.6.0 (Rozas et al. 2017). The non-synonymous substitution rates (Ka), synonymous substitution rates (Ks) and ratios of non-synonymous to synonymous substitution (Ka/Ks) for each of the concatenated 13 PCGs of the Coccoidea mitogenomes were also calculated by DnaSP v.6.0. Tandem repeats of the control region were identified with the Tandem Repeats Finder server (https://tandem.bu.edu/trf/trf.html) (Benson 1999).

A total of 34 mitogenomes from five superfamilies of Hemiptera were used for the phylogenetic analyses (Table 1). Of these, 32 species belong to all four superfamilies of Sternorrhyncha (the ingroup), while the remaining two species from the superfamily Fulgoroidea were chosen as outgroups. Nucleotide sequences (without stop codons) for the 13 PCGs were aligned using MAFFT v.7 (Katoch and Standley 2013) with the L-INS-i (accurate) strategy and codon alignment mode (Code table: Invertebrate mitochondrial genetic codon), rRNA and tRNA gene sequences were aligned using MAFFT v.7 (Katoch and Standley 2013) with the G-INS-I algorithm (which takes account of the secondary structure of rRNA and tRNA genes) and normal alignment mode and amino acid sequences of 13 PCGs were aligned using the -auto strategy and normal alignment mode. Ambiguously aligned areas were removed using Gblocks v.0.91b (Talavera and Castresana 2007), respectively. Individual gene alignments were concatenated using PhyloSuite v.1.2.2 (Zhang et al. 2020). For amino acid sequence data, Bayesian Inference (BI) phylogenetic analysis were conducted using PHYLOBAYES MPI v.1.5c (Lartillot et al. 2013) in the CIPRES Science Gateway (Miller et al. 2010) which employs the site-heterogeneous model CAT + GTR. Two independent Markov Chain Monte Carlo (MCMC) chains were run and the analysis was stopped when the two runs had satisfactorily converged (maxdiff. fell below 0.3). A consensus tree was computed from the remaining trees combined from two runs after the initial 25% trees from each MCMC chain run were discarded as burn-in. For nucleotide sequence data, the best partitioning scheme and nucleotide substitution models for Maximum Likelihood (ML) and BI phylogenetic analyses were selected with PartitionFinder v.2 (Lanfear et al. 2017) using the Bayesian Information Criterion (BIC) (Suppl. materials 1, 2). ML analyses were conducted using IQ-TREE v.1.6.3 (Nguyen et al. 2015) under the ultrafast bootstrap (UFB) approximation approach (Minh et al. 2013) with 5,000 replicates. BI analysis was performed using MrBayes v.3.2.7a (Ronquist et al. 2012) in the CIPRES Science Gateway server (Miller et al. 2010) with four chains (one cold chain and three hot chains). Two independent runs of 5,000,000 generations were carried out with sampling every 1,000 generations. The first 25% of trees were discarded as burn-in. After the average standard deviation of split frequencies fell below 0.01, stationarity was assumed.

The complete mitogenome of M. matsumurae is a closed circular double-stranded DNA molecule (Fig. 2, Suppl. material 3), 15,360 bp in length. This is medium-sized amongst the available mitogenomes of Coccoidea: from 15,143 bp for Didesmococcus koreanus to 15,389 bp for Saissetia coffeae (Lu et al. 2020, Xu et al. 2021). The newly-sequenced mitogenome encodes 37 genes (13 PCGs, 22 tRNAs and two rRNAs) and an A+T-rich region (control region), which is a typical architecture for bilaterian animal mitogenomes (Boore 1999). The gene order of M. matsumurae (Matsucoccidae) differs considerably from that of the other two Coccidae species (Lu et al. 2020, Xu et al. 2021), but it is comparatively similar to gene orders of most Cimicomorpha, Fulgoroidea, Membracoidea, Psylloidea and Aphidoidea species (only the tRNA cluster of trnW–trnC–trnY was rearranged to trnW–trnY–trnC) (Fig. 3). Amongst the three scale insect species, twenty-three genes (nine PCGs and 14 tRNAs) are encoded on the majority strand (H strand) and the remaining 14 genes (four PCGs, eight tRNAs and two rRNAs) are encoded on the minority strand (L strand) (Fig. 3). A total of 85 overlapping nucleotides were found in nine pairs of neighbouring genes, with the longest identified overlap between the trnN and trnS1 (22 bp). Furthermore, there are 193 intergenic nucleotides dispersed across 16 gene boundaries. The longest intergenic region (103 bp) is located between cox3 and trnG.

Figure 2.  

Circular map of the mitogenome of Matsucoccus matsumurae. The outer circle shows the gene map of M. matsumurae, with the genes outside the map encoded on the major strand (H-strand), whereas genes on the inside of the map are encoded on the minor strand (L-strand). Genes are represented by different colour blocks.

Figure 3.  

Gene orders in Hemiptera mitogenomes. With the exception of Coccoidea (Didesmococcus koreanus, Matsucoccus matsumurae and Saissetia coffeae), each superfamily (plus the infraorder Cimicomorpha) is represented by one species. Genes shown with “-“ signs are located on the minor strand (L-strand), while others are located on the major strand (H-strand).

The overall nucleotide composition of the M. matsumurae mitogenome is 47.2% A, 6.4% C, 0.6% G and 45.8% T. It therefore exhibits a strong AT bias of 91.1%, which is higher than in other Coccoidea insects (82.5% for D. koreanus and 84.7% for S. coffeae) (Table 2) (Lu et al. 2020, Xu et al. 2021). The PCGs have the lowest AT content (90.3%) and the control region has the highest (92.9%), which differs from all previously-sequenced scale insects (Table 2) (Lu et al. 2020, Xu et al. 2021). The new mitogenome exhibits negative GC-skews (-0.267) and positive AT-skews (0.056), which is common for scale insects (Table 2) and the Hemiptera in general (Wang et al. 2015, Chen et al. 2019).

Nucleotide diversity (Pi value) of 13 PCGs and two rRNAs amongst the three scale insect species is shown in Fig. 4. Nucleotide diversity values, calculated for individual genes, ranged from 0.249 (cox1) to 0.429 (nad6). The results indicated that atp8 (Pi = 0.418) and nad6 (Pi = 0.429) had comparatively high nucleotide diversity; atp6, cox2, cox3, cytb, nad1, nad2, nad3, nad4l, nad4, nad5 and rrnS (Pi = 0.300-0.384) exhibited an intermediate nucleotide diversity; and cox1 (Pi = 0.249) and rrnL (Pi = 0.286) were highly conserved genes, with low nucleotide diversity (Fig. 4). The lowest nucleotide diversity in cox1 is in agreement with observations in most sequenced insect mitogenomes (Ma et al. 2019, Ge et al. 2021, Li et al. 2021, Zhang et al. 2022), which presumably accounts for its ability to provide reliable species identification.

Figure 4.  

Sliding window analysis of 13 PCGs and two rRNAs of three Coccoidea species. The blue line shows the value of nucleotide diversity Pi (window size = 200 bp, step size = 20 bp). The Pi value for each gene is shown in the graph.

The non-synonymous/synonymous (Ka/Ks) substitution ratio can be used to estimate whether a sequence is undergoing purifying, neutral or positive selection. The rates of non-synonymous (Ka) and synonymous substitutions (Ks) and their ratio (Ka/Ks) were calculated for the 13 PCGs of each of the three scale insect species using Aphis craccivora as the reference sequence (Fig. 5). A value of Ka/Ks greater than 1 implies that a gene is evolving predominantly under positive selection. This indicates that non-synonymous mutations are favoured by the Darwinian selection, i.e. that they are retained at a rate greater than synonymous mutations. All Ka/Ks values were above 1, which strongly suggests the presence of positive selection in these species.

Figure 5.  

Evolutionary rates of mitochondrial genomes in the three scale insects. The rate of non-synonymous substitutions (Ka), synonymous substitutions (Ks) and the ratio of Ka/Ks were calculated for the PCGs of each mitogenome, using Aphis craccivora as the reference sequence.

The 13 PCGs (length: 10,626 bp) account for 69.2% of the complete mitogenome of M. matsumurae. All PCGs were initiated by the typical start codon ATN (ATA/T/G/C) and ended with the TAA/G stop codon or their incomplete form T-. This is almost identical to D. koreanus (Xu et al. 2021) and S. coffeae (Lu et al. 2020). Such incomplete stop codons are common in insects and believed to be completed by post-transcriptional polyadenylation (Ojala et al. 1981). The AT-skews of PCGs were similar (-0.115 to -0.072) amongst the three available scale insects (Table 2). In M. matsumurae, the 13 PCGs encode a total of 3,531 amino acids, amongst which the most frequently used are Leu, Met, Ile and Phe (accounting for 56.65% of the total amount), whereas Cys is the least frequently used (0.34%) (Fig. 6). These amino acid usage patterns are very consistent in Coccoidea species (Lu et al. 2020, Xu et al. 2021). Relative synonymous codon usage (RSCU) is summarised in Fig. 7. Results indicate that the four most frequently used codons are UUA (Leu), AUU (Ile), UUU (Phe) and AUA (Met). All of them are composed solely of A or U, which is reflected in the high A+T content of PCGs. Excluding TAA and TAG, 61 codons were observed in D. koreanus (no CGC), 46 in M. matsumurae (no CUG, GUC, GUG, CCC, CCG, ACG, GCC, GCG, CAG, UGC, CGC, CGG, AGC, AGG, GGC and GGG), and 56 in S. coffeae (no CUC, ACG, GCG, UGC, CGC and GGC) (Lu et al. 2020, Xu et al. 2021).

Figure 6.  

Percentages of each amino acid within the three scale insects. The stop codon is not included.

Figure 7.  

Relative synonymous codon usage (RSCU) in mitogenomes of the three scale insects. The stop codon is not shown.

All three sequenced scale insect mitogenomes encode 22 tRNA genes (Suppl. material 3) (Lu et al. 2020, Xu et al. 2021). The AT content of tRNA genes is slightly higher than that of the PCGs, ranging from 87.9% (D. koreanus) to 92.3% (M. matsumurae) (Table 2). Furthermore, the AT-skew values of tRNAs were all greater than zero (0.043 to 0.054). The length of the 22 tRNA genes ranged from 46 bp (trnS1 of D. koreanus) to 78 bp (trnS1 of M. matsumurae) (Suppl. material 3) (Lu et al. 2020, Xu et al. 2021). Most tRNAs lack either the DHU or TψC arm and some even lost both DHU and TψC arms (Fig. 8) (Lu et al. 2020, Xu et al. 2021). Only ten tRNAs of D. koreanus, nine of M. matsumurae and 11 of S. coffeae could be folded into the common clover-leaf secondary structures (Fig. 8) (Lu et al. 2020, Xu et al. 2021). This suggests that the reduction of DHU or TψC arms of tRNA genes could be a very common phenomenon in the mitogenomes of scale insects. The anticodons of tRNA genes are identical amongst the three scale insects (Fig. 8) (Lu et al. 2020, Xu et al. 2021), except for the trnE of D. koreanus and trnK of S. coffeae, which employ UCG and UUU instead of UUC and CUU respectively.

Figure 8.  

Secondary structures of 22 tRNAs in the mitogenome of Matsucoccus matsumurae. Lines (-) indicate Watson-Crick base pairings, whereas dots (·) indicate unmatched base pairings.

The AT nucleotide content of rrnS and rrnL genes of the three scale insects ranges from 86.2% to 92.7% (Table 2). Two rRNAs in these three mitogenomes show a negative AT-skew (-0.099 to -0.027) (Table 2). The rrnL gene, located between trnL1 and trnV, ranged in length from 1,161 bp to 1,177 bp and rrnS gene, located between trnV and trnI (M. matsumurae) or trnM (D. koreanus and S. coffeae), ranged from 713 bp to 804 bp in length (Fig. 3, Suppl. material 3) (Lu et al. 2020, Xu et al. 2021). Therefore, the length and AT content of rRNAs are conserved in the Coccoidea.

The AT-rich region is believed to be involved in regulating the transcription and replication of DNA in insects (Clayton 1982, Cameron 2014). The AT-rich region of M. matsumurae is located between rrnS and trnI, whereas the AT-rich regions of D. koreanus and S. coffeae are located between rrnS and trnM and the size of three scale insects ranges from 1,271 bp to 2,261 bp (Fig. 3, Suppl. material 3) (Lu et al. 2020, Xu et al. 2021). Analyses of the AT-rich regions by the Tandem Repeats Finder indicated that M. matsumurae and S. coffeae have different numbers of absolute tandem repeat units. Two types of absolute tandem repeats were present in M. matsumurae (nucleotide positions: 7 to 1,023 and 1,026 to 1,373 of the AT-rich region). The AT-rich region of S. coffeae had only one kind of absolute tandem repeat, located between the nucleotide positions 1,096 to 1,533 (Fig. 9). Like in most insect mitogenomes, tandem repeats are common and the size of tandem repeat regions varies depending on the number of copies of the repeating units (Garey and Wolstenholme 1989). Tandem repeats are thought to play an important role in the control of DNA methylation, gene transcription and replication (Zhang et al. 1995, Huang et al. 2013).

Figure 9.  

Structures of AT-rich regions in mitogenomes of the three scale insects. The location and copy number of absolute tandem repeat units are represented by blue and red ovals. Green boxes indicate non-repeat regions.

Phylogenetic analyses of 32 species of Sternorrhyncha, including two outgroups, based on ML and BI analyses of nucleotide sequence data of 37 mitochondrial genes, yielded largely congruent topologies, with most branches receiving strong support (Figs 10, 11). As proposed by previous studies (Lu et al. 2020, Xu et al. 2021), the relationships amongst sternorrhynchan superfamilies are inferred as ((Coccoidea + Aphidoidea) + (Aleyrodoidea + Psylloidea)).

Figure 10.  

ML phylogenetic tree for Sternorrhyncha, based on the nucleotide sequence data of 37 mitochondrial genes from Matsucoccus matsumurae and other 33 species belonging to five superfamilies of Hemiptera. Bootstrap support values (BS) are indicated on the branches.

Figure 11.  

BI phylogenetic tree for Sternorrhyncha, based on the nucleotide sequence data of 37 mitochondrial genes from Matsucoccus matsumurae and other 33 species belonging to five superfamilies of Hemiptera. Bayesian posterior probabilities (PP) are indicated on the branches.

All analyses consistently recovered the monophyly of Sternorrhyncha and its four superfamilies (Coccoidea, Aphidoidea, Aleyrodoidea and Psylloidea) with strong support (BS = 86; PP = 1.00) (Figs 10, 11Suppl. material 4). Furthermore, M. matsumurae clustered with the other two coccoid species, D. koreanus and S. coffeae, with strong support (BS = 100; PP = 1.00) (Figs 10, 11Suppl. material 4), thus confirming its affiliation with the superfamily Coccoidea. Within the clade of Coccoidea, M. matsumurae is positioned as the earliest branching taxon in both nucleotide and amino acid trees, which suggests that Matsucoccidae is a more ancient taxon than Coccidae.