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The highly rearranged mitochondrial genomes of three economically important scale insects and the mitochondrial phylogeny of Coccoidea (Hemiptera: Sternorrhyncha)

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Biodiversity and Conservation

Introduction

The scale insects (Coccoidea) are well-known sap-sucking hemipterans which are economically important pests causing severe damage to native crops and plants (Kondo, Gullan & Williams, 2008). Adult males of Coccoidea are hyperpaurometamorphosis, whereas the adult females are paurometamorphosis and resemble their nymphs (Gullan & Kosztarab, 1997). These insects are usually smaller than 5 mm and often appear similar color with their host plants. Most scale insects can produce waxy secretion covering their bodies as a protection armature (Gullan & Kosztarab, 1997), which also causes difficulty in using chemical control methods.

When compared with other superfamilies of the monophyletic suborder Sternorrhyncha: Aphidoidea (aphids), Aleyrodoidea (whiteflies) and Psylloidea (jumping plant lice), the superfamily Coccoidea possess a higher biodiversity and morphological variety (Gullan & Martin, 2003; Gullan & Cook, 2007). Despite the previous morphological and molecular contributions (Koteja, 1974; Von Dohlen & Moran, 1995; Gullan & Cook, 2007; Cook, Gullan & Trueman, 2002; Hodgson & Hardy, 2013), the scale insect systematics especially the family-level classification still remains unresolved.

Morphology of scale insects has apparent limits when used for resolving the higher-level phylogeny of scale insects, which is expected to be improved by the DNA sequence data. Mitochondrial genome (mitogenome) usually contains a typical set of 37 genes: 13 protein-coding genes (PCG), 22 transfer RNA genes (tRNA), two ribosomal RNA genes (rRNA) and a non-coding control region (CR) and has become one of the most popular molecules used in insect phylogenetic studies (Cameron, 2014). Recently, Deng, Lu & Huang (2019) and Lu, Huang & Deng (2020) respectively sequenced the mitogenomes of the two scale insects, Ceroplastes japonicus (Green, 1921) and Saissetia coffeae (Walker, 1852) and investigated the efficiency of using mitogenome data in the phylogeny of Sternorrhyncha. Mitochondrial gene rearrangement and truncation of tRNA genes have been found in the two mitogenomes. To facilitate the resolution of phylogeny and molecular evolution of Coccoidea, we sequenced the complete mitogenomes of Unaspis yanonensis (Kuwana, 1923), Planococcus citri (Risso, 1813) and Ceroplastes rubens (Maskell, 1893), which includes the first representatives of Pseudococcidae and Diaspididae. The mitogenomic organizations, gene rearrangements, nucleotide compositions, codon usages of PCGs, secondary structures of tRNA genes and CR were analyzed for the three mitogenomes. In addition, the phylogenetic relationships of four species of Coccoidea were reconstructed to evaluate the validity of the newly obtained molecular data.

Materials & Methods

Sample preparation and DNA extraction

The specimens of U. yanonensis, P. citri and C. rubens were collected from Chengdu, Sichuan Province of China in October of 2019. The specimens were reliably identified by experts of Sichuan Academy of Agricultural Sciences, and were preserved in 100% ethanol. The total genomic DNA of the three scale insects was isolated using the E.Z.N.A.® Tissue DNA Kit (OMEGA, America) and preserved at −20 °C before the sequencing process.

Sequencing, assembly and annotation

The Illumina TruSeq short-insert libraries (insert size = 450 bp) were constructed using 1.0 µg of purified DNA fragments and sequenced by Illumina Hiseq 4000 (Shanghai BIOZERON Co., Ltd). Prior to assembly, raw reads were filtered and high-quality reads were retained and assembled into contigs by SOAPdenovo2.04 (Luo et al., 2012). Then the assembled contigs were aligned to the reference mitogenome of C. japonicus (GenBank accession number MK847519) using BLAST. The aligned contigs (≥80% similarity and query coverage) were arranged according to the reference mitogenome. Finally, the clean reads were mapped to the assembled draft mitogenome to fix the wrong bases; gaps were filled using GapFiller v2.1.1 (https://sourceforge.net/projects/gapfiller/). The mitogenome sequences of U. yanonensis, P. citri and C. rubens were deposited in GenBank under the accession numbers MT611525, MT611526 and MT677923, respectively.

Most tRNA genes were predicted and depicted by MITOS (Bernt et al., 2013); structures of several tRNA genes of C. rubens were predicted manually. PCGs and rRNA genes were identified by homology alignments. Gene boundaries of PCGs were confirmed in ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The graphic view of the mitogenomes were computed using CGView Server (http://stothard.afns.ualberta.ca/cgview_server/) (Grant & Stothard, 2008). The probable mitochondrial rearrangement scenarios during the evolution of U. yanonensis, P. citri and C. rubens were predicted by the CREx (Common Interval Rearrangement Explorer) online server (Bernt, 2007) using Drosophila yakuba as a reference (Clary & Wolstenholme, 1985). Nucleotide composition of each gene and codon usage of PCGs were calculated by MEGA v.6.0 (Tamura et al., 2013). The composition skew analysis was conducted by AT-skew = [A−T]/[A +T] and GC-skew = [G−C]/[G +C] formulas (Perna & Kocher, 1995). The software DnaSP v. 5.10 (Librado & Rozas, 2009) was used to calculate the synonymous substitution rate (Ks) and the nonsynonymous substitution rate (Ka). Presumed secondary structures in the control region were predicted by the online tool Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.advanced.submit.html) and DNAMAN v6.0.3.

Phylogenetic analysis

Nucleotide sequences of PCGs derived from four species of Coccoidea, including U. yanonensis, P. citri and C. rubens sequenced in this study, were used in the phylogenetic analysis (Table 1). The species S. coffeae was not included in the dataset due to the unannotated and unreliable status of its sequence as noted in Genbank. The two aphids, Aphis glycines and Diuraphis noxia were used as the outgroups. The 13 PCGs were aligned by MAFFT and concatenated as a combined dataset using SequenceMatrix v1.7.8 (Katoh & Standley, 2013). PartitionFinder v2.1.1 was used to determine the optimal nucleotide substitution models and partitioning schemes by using the Bayesian Information Criterion (BIC) and a greedy search algorithm (Lanfear et al., 2016). Two phylogenetic inferences were conducted with the partition schemes, including Bayesian inferences (BI) and Maximum likelihood (ML) analysis. BI analysis was conducted by MrBayes v3.2.7, with 10 million generations sampling every 1,000 generations, running one cold chain and three hot chains with a burn-in of 25% trees (Ronquist & Huelsenbeck, 2003). Stability of the results of BI analysis was examined by Tracer v.1.5. ML analysis was performed by RAxML v8.2.12 with 1,000 bootstrap replicates (Stamatakis, 2014). Tree files generated by both BI and ML trees were adjusted and visualized in FigTree v1.4.2.

Table 1:
Species of Hemiptera used in this study.
Superfamily Family Species Accession number
Coccoidea Coccidae Ceroplastes japonicus MK847519
Ceroplastes rubens MT677923
Diaspididae Unaspis yanonensis MT611525
Pseudococcidae Planococcus citri MT611526
Aphidoidea Aphididae Aphis glycines MK111111
Diuraphis noxia KF636758
DOI: 10.7717/peerj.9932/table-1

Results

Mitogenome annotation and nucleotide composition

The complete mitogenomes of U. yanonensis, P. citri and C. rubens were all typical double-strand circular molecules with a length of 15,220 bp, 15,549 bp and 15,387 bp, respectively (Fig. 1), which were similar to other mitogenomes of Coccoidea (Deng, Lu & Huang, 2019; Lu, Huang & Deng, 2020). The standard set of 37 genes (13 PCGs, 22 tRNA genes and two rRNA genes) were all found in the mitogenome of U. yanonensis (Table 2), whereas trnV was lost in P. citri (Table 3); C. rubens lacked five tRNA genes, trnC, trnR, trnS2, trnL1 and trnV (Table 4). In U. yanonensis, there were nine overlapping nucleotides located in four pairs of neighboring genes (Table 2); while in P. citri, there were 36 overlapping nucleotides in nine gene boundaries (Table 3). In C. rubens, there were only seven overlapping nucleotides in four gene boundaries (Table 4). The longest overlap was 18-bp long and located between trnS2 and ND1 in P. citri. There were 227 intergenic nucleotides (IGNs) dispersed in 20 locations for U. yanonensis, 126 IGNs in 19 locations for P. citri and 478 IGNs in 19 locations for C. rubens, indicating a loose structure of the three scale insect mitogenomes.

Mitochondrial maps of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Figure 1: Mitochondrial maps of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

(A) Unaspis yanonensis; (B) Planococcus citri; (C) Ceroplastes rubens. Genes outside the map are transcribed clockwise, whereas those inside the map are transcribed counterclockwise. The inside circles show the GC content and the GC skew. GC content and GC skew are plotted as the deviation from the average value of the entire sequence.
Table 2:
Mitochondrial genome structure of Unaspis yanonensis.
Gene Position (bp) Size (bp) Direction Intergenic nucleotides Anti- or start/ stop codons A+T%
Control region 1–78 260 + 78 81.9
ATP8 261–428 168 + 0 ATT/TAA 93.5
ATP6 429–1121 693 + 0 ATT/TAA 86.0
trnL2 (UUR) 1127–1193 67 + 5 TAA 85.1
trnMet (M) 1196–1260 65 + 2 CAT 89.2
trnVal (V) 1261–1326 66 + 0 TAC 90.9
rrnS 1327–2133 807 + 0 90.6
trnAla (A) 2134–2202 69 + 0 TGC 72.5
rrnL 2203–3516 1314 + 0 89.6
trnLeu1 (CUN) 3517–3581 65 + 0 TAG 86.2
nad1 3582–4517 936 + 0 ATT/TAA 84.5
trnSer2 (UCN) 4516–4584 69 −2 TGA 88.4
CYTB 4587–5759 1173 2 ATA/TAA 81.8
ND6 5760–6300 541 0 ATG/T– 93.0
trnPro (P) 6306–6373 68 + 5 TGG 91.2
trnThr (T) 6374–6437 64 0 TGT 95.3
ND4L 6441–6728 288 + 3 ATT/TAA 89.9
ND4 6731–8062 1332 + 2 ATT/TAA 87.8
trnHis (H) 8062–8121 60 + –1 GTG 93.3
ND5 8131–9810 1680 + 9 ATA/TAA 88.2
trnPhe (F) 9818–9882 65 + 7 GAA 95.4
trnGlu (E) 9890–9957 68 7 TTC 95.6
COX1 9959–11515 1557 + 1 TTG/TAA 78.4
COX3 11554–12288 735 + 38 ATT/TAA 82.3
trnGln (Q) 12339–12407 69 50 TTG 89.9
trnGly (G) 12417–12479 63 + 9 TCC 88.9
ND3 12480–12830 351 + 0 ATT/TAA 88.3
trnArg (R) 12831–12883 53 + 0 TCG 86.8
trnCys (C) 12885–12954 70 + 1 GCA 94.3
trnSer1 (AGN) 12956–13014 59 + 1 GCT 89.8
trnAsn (N) 13013–13080 68 −2 GTT 83.8
ND2 13082–14107 1026 + 1 ATT/TAA 92.1
COX2 14104–14793 690 −4 ATT/TAA 83.6
trnIle (I) 14795–14861 67 1 GAT 83.6
trnTrp (W) 14862–14928 67 + 0 TCA 94.0
trnLys (K) 14929–14997 69 + 0 CTT 91.3
trnAsp (D) 15001–15069 69 3 GTC 92.8
trnTyr (Y) 15074–15142 69 4 GTA 85.5
DOI: 10.7717/peerj.9932/table-2
Table 3:
Mitochondrial genome structure of Planococcus citri.
Gene Position (bp) Size (bp) Direction Intergenic nucleotides Anti- or start/ stop codons A+T%
trnIle (I) 1–70 70 0 GAT 84.3
ND2 76–1089 1014 + 5 ATT/TAA 87.4
trnTrp (W) 1088–1156 69 + −2 TCA 89.9
trnTyr (Y) 1167–1232 66 10 GTA 84.8
trnAsn (N) 1232–1295 64 + −1 GTT 84.4
trnSer1 (AGN) 1295–1359 65 + −1 GCT 80.0
trnCys (C) 1368–1432 65 + 8 GCA 92.3
trnArg (R) 1434–1497 64 1 TCG 79.7
ND3 1504–1854 351 6 ATT/TAA 84.3
trnGly (G) 1855–1918 64 0 TCC 92.2
COX3 1928–2716 789 9 ATG/TAA 76.6
ATP6 2721–3395 675 4 ATG/TAA 80.1
ATP8 3389–3550 162 −7 ATT/TAA 85.8
trnAsp (D) 3551–3616 66 0 GTC 90.9
trnLys (K) 3629–3695 67 + 12 CTT 86.6
COX2 3700–4380 681 4 ATT/TAA 78.6
trnLeu2 (UUR) 4384–4451 68 3 TAA 85.3
COX1 4460–5989 1530 8 ATA/TAA 74.4
trnGlu (E) 5991–6057 67 + 1 TTC 94.0
trnPhe (F) 6057–6124 68 −1 GAA 94.1
ND5 6130–7803 1674 5 ATT/TAA 84.3
trnHis (H) 7822–7886 65 18 GTG 84.6
ND4 7889–9199 1311 2 ATA/TAA 83.5
ND4L 9220–9507 288 20 ATT/TAA 86.8
trnThr (T) 9510–9575 66 + 2 TGT 90.9
trnPro (P) 9575–9641 67 −1 TGG 83.6
ND6 9645–10212 568 + 3 ATG/T − 86.6
CYTB 10210–11349 1140 + −3 ATT/TAA 77.3
trnSer2 (UCN) 11348–11412 65 + −2 TGA 81.5
ND1 11395–12333 939 −18 ATA/TAA 80.2
trnLeu1 (CUN) 12334–12402 69 0 TAG 87.0
rrnL 12403–13798 1396 0 86.9
trnAla (A) 13799–13873 75 0 TGC 84.0
trnGln (Q) 13879–13946 68 + 5 TTG 91.2
rrnS 13947–14802 856 0 88.4
trnMet (M) 14803–14871 69 0 CAT 84.1
Control region 14872–15549 678 + 0 84.4
DOI: 10.7717/peerj.9932/table-3
Table 4:
Mitochondrial genome structure of Ceroplastes rubens.
Gene Position (bp) Size (bp) Direction Intergenic nucleotides Anti-or start/ stop codons A+T%
COX1 1–1527 1527 + 42 ATA/TAA 80.4
trnLeu2 (UUR) 1532–1600 69 + 4 TAA 88.4
COX2 1601–2261 661 + 0 ATA/T − 83.4
trnLys (K) 2262–2328 67 + 0 CTT 83.6
trnAsp (D) 2325–2383 59 + −4 GTC 93.2
ATP6 2411–3091 681 27 ATA/TAA 89.7
COX3 3118–3891 774 + 26 ATA/TAA 86.3
trnGly (G) 3894–3950 57 + 2 TCC 94.7
ND3 3951–4286 336 + 0 ATA/TAA 90.8
trnAla (A) 4291–4350 60 4 TGC 91.7
trnAsn (N) 4370–4424 55 + 83 GTT 87.3
trnSer1 (AGN) 4424–4469 46 + −1 GCT 80.4
trnGlu (E) 4469–4522 54 + −1 TTC 94.4
trnTrp (W) 4527–4577 51 + 4 TCA 94.1
ND5 4579–6189 1611 56 ATT/TAA 88.3
trnHis (H) 6264–6320 57 74 GTG 89.5
ND4 6325–7605 1281 4 ATA/TAA 89.4
ND4L 7619–7963 345 13 ATT/TAG 92.2
ND6 7980–8375 396 + 16 ATA/TAA 89.6
trnPro (P) 8375–8433 59 −1 TGG 89.8
ATP8 8435–8524 90 1 ATA/TAA 90.0
trnIle (I) 8546–8612 67 + 21 GAT 86.6
ND2 8613–9551 939 + 0 ATT/TAA 91.5
trnTyr (Y) 9558–9606 49 6 GTA 87.8
trnThr (T) 9608–9659 52 + 1 TGT 90.4
CYTB 9660–10736 1077 + 0 ATC/TAA 85.0
trnGln (Q) 10745–10796 52 8 TTG 92.3
ND1 10823–11728 906 86 ATT/TAG 86.5
rrnL 11729–12991 1263 0 90.7
rrnS 12992–13578 587 0 87.9
Control region 1 13579–14408 830 + 0 85.4
trnPhe (F) 14409–14476 68 0 GAA 79.4
Control region 2 14477–15276 800 + 0 88.4
trnMet (M) 15277–15345 69 + 0 CAT 82.6
DOI: 10.7717/peerj.9932/table-4

The whole mitogenomes of U. yanonensis, P. citri and C. rubens were strongly biased toward A and T nucleotides (86.6%, 82.7% and 87.5%, respectively). The U. yanonensis mitogenome had negative AT-skew and positive GC-skew, whereas P. citri and C. rubens exhibited positive AT-skew and negative GC-skew. The A+T contents were also rich in the mitochondrial genes, showing the highest in trnF of U. yanonensis and P. citri, and trnG of C. rubens.

Gene rearrangement

The mitochondrial genes of U. yanonensis, P. citri and C. rubens were highly rearranged, being different from the two sequenced scale insects, C. japonicus and S. coffeae (Deng, Lu & Huang, 2019; Lu, Huang & Deng, 2020). When compared with D. yakuba, U. yanonensis and P. citri both showed a conserved gene cluster trnE-trnF- ND5-trnH- ND4- ND4l-trnT-trnP- ND6-CYTB-trnS2- ND1-trnL1-rrnL; C. rubens had three shorter conserved gene clusters, COX1-trnL-COX2-trnK-trnD, COX3-trnG-ND3 and ND5-trnH-ND4-ND4L (Fig. 2). The mitogenome of U. yanonensis exhibited the rearrangement of three cytochrome c oxidase subunit genes (COX1, COX2, COX3), two NADH dehydrogenase subunit genes (ND2 and ND3) and many tRNA genes. Despite the multiple tRNA gene rearrangements, the mitogenome of P. citri also had a reversal of the ancestral gene cluster COX1- COX2-ATP8-ATP6- COX3- ND3. The mitogenome of C. rubens showed fewer rearrangements than U. yanonensis and P. citri, including two PCGs (ND2 and ATP8) and multiple tRNA genes.

Gene arrangements of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens in comparison with Drosophila yakuba.

Figure 2: Gene arrangements of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens in comparison with Drosophila yakuba.

(A) Unaspis yanonensis; (B) Planococcus citri; (C) Ceroplastes rubens. Conserved gene arrangements are covered in grey areas.

The CREx analysis predicted the alternative scenarios how the three scale insect mitogenomes rearranged from the ancestral type of mitogenome of D. yakuba (Figs. 35). The mitochondrial gene order of U. yanonensis changed from D. yakuba by nine steps of rearrangement events, including the transposition of trnV and rrnS, the subsequent reverse transposition of trnK and trnD, the reversal of trnS1, and additional three reversal events and three tandem duplication and random loss (TDRL) events (Fig. 3). In P. citri, the first step is the reversal of trnK, followed by two alternative scenarios: the first one contained two reversal events, one TDRL event and one transposition event; the second one included three reversal events, two TDRL events and one transposition event (Fig. 4). Fewer rearrangement events were predicted in C. rubens, including the first step of transposition, the subsequent three reversal events, and final three TDRL events (Fig. 5). Considering the similarly rearranged mitochondrial genes of C. japonicus and S. coffeae, extensive mitochondrial rearrangement events are expected to occur very frequently in other unsequenced scale insects.

Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Unaspis yanonensis.

Figure 3: Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Unaspis yanonensis.

The tRNA genes are represented by the amino acid abbreviations.
Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Planococcus citri.

Figure 4: Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Planococcus citri.

The tRNA genes are represented by the amino acid abbreviations.
Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Ceroplastes rubens.

Figure 5: Reconstruction of mitochondrial gene rearrangement scenarios in the evolution of Ceroplastes rubens.

The tRNA genes are represented by the amino acid abbreviations.

Protein-coding genes

The 13 PCGs of U. yanonensis were similar in size to those of P. citri, without truncated or duplicated PCGs (Tables 2 and 3). However, most PCGs of C. rubens were shorter than U. yanonensis and P. citri, especially for ATP8 and ND6 (Fig. 6). Most PCGs of the three mitogenomes utilized the standard ATN start codon (ATA, ATT, ATC and ATG). However, the special start codon TTG was used by COX1 of U. yanonensis (Table 2). Twelve PCGs of each mitogenome had the complete termination codon TAN (TAA or TAG), whereas ND6 of U. yanonensis and P. citri and COX2 of C. rubens ended with an incomplete stop codon T. In the previously sequenced scale insect, C. japonicus, COX2 also ended with an incomplete T (Deng, Lu & Huang, 2019). The relative synonymous codon usage (RSCU) values were calculated for the three mitogenomes (Fig. 7). In U. yanonensis, the most frequently used codon was TTA (Leu) whereas CTG(Leu), TCC(Ser), ACC(Thr), ACG(Thr), GCC(Ala), CAG(Gln), TGC(Cys), CGG(Arg) and AGC(Ser) were not used. In P. citri, the mostly used codon was also TTA (Leu), but CTC (Leu), AGC (Ser) and CGC (Arg) were the least. In C. rubens, TTA (Leu) was the most frequently used codon.

Comparison of the length for each PCG and rRNA gene in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Figure 6: Comparison of the length for each PCG and rRNA gene in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Relative synonymous codon usage (RSCU) of PCGs in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Figure 7: Relative synonymous codon usage (RSCU) of PCGs in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

(A) Unaspis yanonensis; (B) Planococcus citri; C: Ceroplastes rubens. Full codon families are indicated below the X-axis.

To evaluate the evolutionary rates of the PCGS, the average ratio of Ka/Ks was calculated for each PCG of the three mitogenomes (Fig. 8). The results showed that ND4L had the highest evolutionary rate, followed by ATP8 and ND5, while COX1 and CYTB appeared to be the lowest. The ratios of Ka/Ks were above 1 for most PCGs except for COX1 and CYTB, suggesting that these genes are evolving under positive selection. However, the ratios of Ka/Ks for COX1 and CYTB were below 1, indicating the purifying selection in these genes. The two genes, COX1 and CYTB which with relatively slow evolutionary rates have already been used as efficient phylogenetic markers in insects.

Average evolutionary rates of PCGs in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Figure 8: Average evolutionary rates of PCGs in Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

The bar indicates each gene’s Ka/Ks value.

Transfer RNA genes

The typical set of 22 tRNA genes were all detected in the mitogenome of U. yanonensis, but trnV was absent from the mitogenome of P. citri (Figs. 9 and 10). In C. rubens, only 17 tRNA genes were recognized and the three tRNA genes trnA, trnQ and trnW were manually predicted (Fig. 11). Length and A+T content of the tRNA genes were subequal between U. yanonensis and P. citri, whereas the lengths of tRNA genes of C. rubens were generally shorter than U. yanonensis and P. citri. Individual tRNA gene of the three mitogenomes ranged in size from 49 to 75 bp; the longest tRNA gene was trnA in P. citri (Table 3); the shortest tRNA gene was trnY in C. rubens (Table 4). In the mitogenomes of U. yanonensis and P. citri, most of the tRNA genes could fold into cloverleaf secondary structures, but the dihydrouridine (DHU) arms of trnR and trnS1 were consistently lost. In C. rubens, most tRNA genes exhibited reduced DHU arms or T ψC arms. Such reductions of DHU arms were also reported in the tRNA genes of S. coffeae (Lu, Huang & Deng, 2020), suggesting that tRNA gene reduction could be a very common phenomenon in the mitogenomes of scale insects. The anticodons of the tRNA genes were identical among the three scale insects. In the tRNA genes of U. yanonensis and P. citri, a total of 12 and 19 mismatched base pairs were respectively identified and all of them were G-U pairs. In C. rubens, only four mismatched G-U pairs were identified.

Secondary structures of tRNA genes in the mitogenome of Unaspis yanonensis.

Figure 9: Secondary structures of tRNA genes in the mitogenome of Unaspis yanonensis.

(A) trnA (Alanine); (B) trnN (Asparagine); (C) trnD (Aspartic acid); (D) trnR (Arginine); (E) trnC (Cystine); (F) trnQ (Glutamine); (G) trnE (Glutamic acid); (H) trnG (Glycine); (I) trnH (Histidine); (J) trnI (Isoleucine); (K) trnL1(CUN) (Leucine); (L) trnL2(UUR) (Leucine); (M) trnK (Lysine); (N) trnM (Methionine); (O) trnF (Phenylalanine); (P) trnP (Proline); (Q) trnS1(AGN) (Serine); (R) trnS2(UCN) (Serine); (S) trnT (Threonine); (T) trnW (Tryptophan); (U) trnY (Tyrosine); (V) trnV (Valine). The tRNA genes are labelled with their corresponding amino acids.
Secondary structures of tRNA genes in the mitogenome of Planococcus citri.

Figure 10: Secondary structures of tRNA genes in the mitogenome of Planococcus citri.

(A) trnA (Alanine); (B) trnN (Asparagine); (C) trnD (Aspartic acid); (D) trnR (Arginine); (E) trnC (Cystine); (F) trnQ (Glutamine); (G) trnE (Glutamic acid); (H) trnG (Glycine); (I) trnH (Histidine); (J) trnI (Isoleucine); (K) trnL1(CUN) (Leucine); (L) trnL2(UUR) (Leucine); (M) trnK (Lysine); (N) trnM (Methionine); (O) trnF (Phenylalanine); (P) trnP (Proline); (Q) trnS1(AGN) (Serine); (R) trnS2(UCN) (Serine); (S) trnT (Threonine); (T) trnW (Tryptophan); (U) trnY (Tyrosine). The tRNA genes are labelled with their corresponding amino acids.
Secondary structures of tRNA genes in the mitogenome of Ceroplastes rubens.

Figure 11: Secondary structures of tRNA genes in the mitogenome of Ceroplastes rubens.

(A) trnA (Alanine); (B) trnN (Asparagine); (C) trnD (Aspartic acid); (D) trnE (Glutamic acid); (E) trnQ (Glutamine); (F) trnG (Glycine); (G) trnH (Histidine); (H) trnI (Isoleucine); (I) trnL2(UUR) (Leucine); (J) trnK (Lysine); (K) trnM (Methionine); (L) trnF (Phenylalanine); (M) trnP (Proline); (N) trnS1(AGN) (Serine); (O) trnT (Threonine); (P) trnW (Tryptophan); (Q) trnY (Tyrosine). The tRNA genes are labelled with their corresponding amino acids.

Ribosomal RNA genes

There were two rRNA genes identified in in each mitogenome. The length and A+T content of each rRNA gene were subequal between U. yanonensis and P. citri, but the lengths of rRNA genes were much shorter in C. rubens (Tables 24). In U. yanonensis, the large ribosomal RNA (rrnL) gene was 1314 bp with an A+T content of 89.6%; the small ribosomal RNA (rrnS) gene was 807 bp with a high A+T content of 90.6%. In P. citri, the rrnL gene was 1396 bp with an A+T content of 86.9%; the rrnS gene was 856 bp with an A+T content of 88.4%. In C. rubens, the rrnL gene was 1,263 bp with a high A+T content of 90.7%; the rrnS gene was 587 bp with an A+T content of 87.9%. Locations of the two rRNA genes were similar to D. yakuba, being neighbored with the CYTB-ND1 PCG cluster (Fig. 2). Instead of the commonly found trnV between the rrnL and rrnS genes in other insects, the intermediate tRNA gene between the two rRNA genes was trnA in U. yanonensis, trnA and trnQ in P. citri, and completely absent in C. rubens.

Control region

Control region (CR), also known as A+T rich region, was the longest and most variable non-coding area in the three mitogenomes (Fig. 12). The CR of U. yanonensis was short with only 260 bp, being located between trnY and ATP8 and with a relatively high A+T content of 81.9% (Table 2). The CR of P. citri was much longer than U. yanonensis (678 bp), being located between trnM and trnI and with an A+T content of 84.4% (Table 3). Two putative CRs were found in the mitogenome of C. rubens: the 830-bp long CR1 between rrnS and trnF and the 800-bp long CR2 between trnF and trnM (Table 4). A+T content of the two CRs was 85.4% and 88.4%, respectively, higher than U. yanonensis and P. citri. The CR of C. japonicus and S. coffeae was 507 bp and 1454 bp, respectively, indicating the highly variable length of CRs in scale insects (Deng, Lu & Huang, 2019; Lu, Huang & Deng, 2020).

Predicted structural elements in the control regions of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Figure 12: Predicted structural elements in the control regions of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens.

Tandem repeat units are indicated by orange boxes. Poly-[TA]n stretch is indicated with purple ellipse. Stem-loop structure is indicated by its shape and base pairs.

The CR of U. yanonensis was composed of 2.9 copies of tandem repeats; the first two copies had a consensus size of 91 bp, whereas the third repeat was 78 bp in length. The CR of P. citri contained three types of secondary structures that might function in regulating the replication and transcription of the mitogenome, including 2.3 copies of 110-bp long tandem repeats, one 40-bp long poly-[TA]n stretch, and a 21-bp long stem-loop (SL) structure. The SL structure was initiated by a “TAA” motif and ended with a “GTA” motif. The longer tandem repeats and extra secondary structures of P. citri resulted in the longer CR than that of U. yanonensis. The CR1 of C. rubens contained 3.6 copies of 33-bp long tandem repeats but had no SL structures. The CR2 of C. rubens included 5 copies of 24-bp long tandem repeats and a combined SL structure. The length, nucleotide composition, number and types of structural elements in CRs of the three mitogenomes were found highly variable, which implied that the scale insect mitogenomes were likely to be regulated in different ways during the mitogenomic replication and transcription processes.

Discussion

To test the reliability of the three sequenced mitogenomes and investigate the mitochondrial phylogenetic relationships within Coccoidea, nucleotide sequences of available scale insects were obtained from GenBank and used in the phylogenetic analyses (Table 1). The two phylogenetic trees using BI and ML analyses generated identical topological structures for Coccoidea (Fig. 13). The three families of Coccoidea were grouped together, suggesting the probable monophyly of Coccoidea as found in Von Dohlen & Moran (1995), which used the small-subunit (18S) ribosomal DNA in the phylogenetic analysis. The monophyly of Coccidae was supported with high values, indicating the efficiency of mitogenome data in grouping members of the same family and partially supporting the correctness of the tree topologies. Pseudococcidae was recovered as the sister group of Diaspididae and the phylogenetic position of their combined clade was supported basal to Coccidae. However, in previous molecular and morphological studies (Gullan & Cook, 2007; Cook, Gullan & Trueman, 2002; Hodgson & Hardy, 2013), Pseudococcidae was supported basal to Coccidae and Diaspididae. The insufficient mitogenome data of Coccoidea, and the selection of different taxa and different molecular markers in the phylogenetic analysis were very likely to cause different phylogenetic results especially for the family levels (Chen et al., 2018). The new mitogenome data obtained in this study provided a basis for the accurate reconstruction of mitochondrial phylogeny in Coccoidea. The sequencing of more scale insects in future can also provide new data for our understanding of the highly rearranged mitogenomes and evolutionary history of these enigmatic insects. Sufficient representatives and molecular data will furtherer resolve the inner relationship of Coccoidea.

Phylogenetic relationships within Coccoidea inferred by Bayesian inference and maximum likelihood analysis.

Figure 13: Phylogenetic relationships within Coccoidea inferred by Bayesian inference and maximum likelihood analysis.

Numbers at the nodes are posterior probabilities and bootstrap values. The family names are listed after the species.

Conclusions

The complete mitochondrial genomes of U. yanonensis, P. citri and C. rubens were sequenced and analyzed. The mitochondrial genes of the three scale insects were highly rearranged and different from other scale insects. The phylogenetic reconstructions with BI and ML methods generated identical phylogenetic topology and supported the inner relationship of Coccoidea as Coccidae + (Pseudococcidae + Diaspididae). More mitogenomes should be obtained in future works to resolve the phylogeny of scale insects.

Supplemental Information

Mitogenome sequence of Unaspis yanonensis

DOI: 10.7717/peerj.9932/supp-1

Mitogenome sequence of Planococcus citri

DOI: 10.7717/peerj.9932/supp-2

Mitogenome sequence of Ceroplastes rubens

DOI: 10.7717/peerj.9932/supp-3

Mitogenomic annotations of iUnaspis yanonensis downloaded from Bankit submission tool of Genbank

DOI: 10.7717/peerj.9932/supp-4

Mitogenomic annotations of Planococcus citri downloaded from Bankit submission tool of Genbank

DOI: 10.7717/peerj.9932/supp-5

Mitogenomic annotations of Ceroplastes rubens downloaded from Bankit submission tool of Genbank

DOI: 10.7717/peerj.9932/supp-6