The First Three Mitochondrial Genomes for the Characterization of the Genus Egeirotrioza (Hemiptera: Triozidae) and Phylogenetic Implications

(1) Background: Mitochondrial genomes are important markers for the study of phylogenetics and systematics. Triozidae includes some primary pests of Populus euphratica. The phylogenetic relationships of this group remain controversial due to the lack of molecular data. (2) Methods: Mitochondria of Egeirotrioza Boselli were sequenced and assembled. We analyzed the sequence length, nucleotide composition, and evolutionary rate of Triozidae, combined with the 13 published mitochondrial genomes. (3) Results: The evolutionary rate of protein-coding genes was as follows: ATP8 > ND6 > ND5 > ND2 > ND4 > ND4L > ND1 > ND3 > APT6 > CYTB > COX3 > COX2 > COX1. We reconstructed the phylogenetic relationships of Triozidae based on 16 triozid mitochondrial genomes (thirteen ingroups and three outgroups) using the maximum likelihood (ML) and Bayesian inference (BI) approaches. The phylogenetic analysis of the 16 Triozidae mitochondrial genomes showed that Egeirotrioza was closely related to Leptynoptera. (4) Conclusions: We have identified 13 PCGs, 22 tRNAs, 2 rRNAs, and 1 control region (CR) of all newly sequenced mitochondrial genomes, which were the mitochondrial gene type in animals. The results of this study provide valuable genomic information for the study of psyllid species.


Introduction
Mitochondrial genomes, as one of the important molecular markers, are usually used for studies on speciation, phylogeography, evolutionary history, and phylogeny in insect groups [1][2][3], benefiting from easy availability, maternal inheritance, and high substitution rates [3,4].Meanwhile, insect mitochondrial genomes' structural characteristics are conserved, and they can also offer additional evidence of morphological classification.The insect mitochondrial genome ranges mostly from 14,000 to 20,000 bp in length [3], containing 2 ribosomal RNAs (rRNAs), 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), and 1 non-coding control region (CR) [4].Mitochondrial genomes are often referred to as the secondary genetic information system due to their distinctive genetic characteristics [5].
Psyllids (Psylloidea), or jumping plant lice, are an important group within the suborder Sternorrhyncha (Hemiptera), containing more than 4000 described species [6].All species of Sternorrhyncha are primarily pests, such as whiteflies, aphids, and scales, and they directly inflict damage on host plants through feeding or gall development [7].During the immature stages, species of psyllids exhibit highly host-specific behavior [8].These pests not only cause direct damage by siphoning plant sap but also act as vectors for the Genes 2024, 15, 842 2 of 14 transmission of plant viruses, inflicting substantial economic losses on agriculture and forestry crops.
Triozidae is a family within Psylloidea.This family comprises about 1000 described species and 70 genera [9].There are many synonymous genera and species of psyllids, and most phylogenetic studies of Psyllioidea have been based on morphological research, but there has been no evidence of molecular correlation.As Egeirotrioza has never been reported as a molecular marker, the genus was not included in previous phylogenetic studies based on molecular morphology.Some species of this group are the main pests of P. euphratica.Some psyllid species, in order to create a good, stable growth and development environment, can make galls, but psyllid galls are mainly deposited on the leaves of the host plant.A gall is the abnormal tissue formed by the plant when stimulated through psyllid feeding or egg-laying; the nymphs live in the galls as adults [10].P. euphratica is an important forest resource in desert areas, playing a crucial role in windbreak and sand fixation, regulating the climate of oases, and stabilizing the ecological balance in desert zones [10,11].Investigations into the endangered situation of P. euphratica have revealed significant damage caused by psyllids in northern and southern Xinjiang [12].
We conducted a field survey of poplar forests in Luntai County and Shaya County, Xinjiang, China, from 2021 to 2023.Through field research, we found that infestation rates on new shoots of saplings often exceed 80% (unpublished), with a high propensity for rapid dissemination, leading to widespread infestations that result in seedling mortality and wilting and thereby causing catastrophic impacts on the growth and development of P. euphratica.In Xinjiang, both natural and artificially planted P. euphratica forests are affected to varying extents by psyllids, including Egeirotrioza rufa and Egeirotrioza gracilis, leading to poor growth and abnormal development in young trees.During psyllid outbreaks, when pest density is high, P. euphratica leaves are covered with galls on both sides, and severe infestations lead to premature leaf yellowing, resulting in weakened tree vigor and slow growth (Figure 1).These pests not only harm P. euphratica but also threaten surrounding species such as tamarisk and Haloxylon, posing a threat to the desert forests of Xinjiang and the ecological environment of western China's desert regions.
Genes 2024, 15, 842 2 of 14 pests not only cause direct damage by siphoning plant sap but also act as vectors for the transmission of plant viruses, inflicting substantial economic losses on agriculture and forestry crops.
Triozidae is a family within Psylloidea.This family comprises about 1000 described species and 70 genera [9].There are many synonymous genera and species of psyllids, and most phylogenetic studies of Psyllioidea have been based on morphological research, but there has been no evidence of molecular correlation.As Egeirotrioza has never been reported as a molecular marker, the genus was not included in previous phylogenetic studies based on molecular morphology.Some species of this group are the main pests of P. euphratica.Some psyllid species, in order to create a good, stable growth and development environment, can make galls, but psyllid galls are mainly deposited on the leaves of the host plant.A gall is the abnormal tissue formed by the plant when stimulated through psyllid feeding or egg-laying; the nymphs live in the galls as adults [10].P. euphratica is an important forest resource in desert areas, playing a crucial role in windbreak and sand fixation, regulating the climate of oases, and stabilizing the ecological balance in desert zones [10,11].Investigations into the endangered situation of P. euphratica have revealed significant damage caused by psyllids in northern and southern Xinjiang [12].
We conducted a field survey of poplar forests in Luntai County and Shaya County, Xinjiang, China, from 2021 to 2023.Through field research, we found that infestation rates on new shoots of saplings often exceed 80% (unpublished), with a high propensity for rapid dissemination, leading to widespread infestations that result in seedling mortality and wilting and thereby causing catastrophic impacts on the growth and development of P. euphratica.In Xinjiang, both natural and artificially planted P. euphratica forests are affected to varying extents by psyllids, including Egeirotrioza rufa and Egeirotrioza gracilis, leading to poor growth and abnormal development in young trees.During psyllid outbreaks, when pest density is high, P. euphratica leaves are covered with galls on both sides, and severe infestations lead to premature leaf yellowing, resulting in weakened tree vigor and slow growth (Figure 1).These pests not only harm P. euphratica but also threaten surrounding species such as tamarisk and Haloxylon, posing a threat to the desert forests of Xinjiang and the ecological environment of western China's desert regions.Despite Triozidae being important pests, molecular data, and especially mitochondrial genome resources, on Triozidae are still deficient.To date, only a few mitochondrial genomes in this group have been made available on GenBank and other databases.The lack of molecular data seriously restricts our understanding of biological characteristics' evolution.Despite Triozidae being important pests, molecular data, and especially mitochondrial genome resources, on Triozidae are still deficient.To date, only a few mitochondrial genomes in this group have been made available on GenBank and other databases.The lack of molecular data seriously restricts our understanding of biological characteristics' evolution.
In this study, we newly sequenced, assembled, and annotated three species of Egeirotrioza within Triozidae and also analyzed the features of their mitochondrial genome structure.Combined with 10 previously published mitochondrial genomes among Tri-ozidae, we compared the substitution, main features, and evolutionary rates.Finally, we reconstructed the phylogenetic relationships of Triozidae based on 16 mitochondrial genomes (13 ingroups and 3 outgroups) via maximum likelihood (ML) and Bayesian inference (BI) approaches.

Taxon Sampling and Sequencing
In this study, we analyzed 16 taxa of Triozidae.We newly sequenced three species within Egeirotrioza which were collected from Luntai County, Xinjing, China (84.3081 • E, 41.5911 • N), by sweeping (detailed information shown in Table 1).In addition, the mitochondrial genomes of 13 Triozidae species were downloaded from GenBank for phylogenetic and comparative mitogenomic analyses.We selected three species that are closely related to Triozidae as outgroups.All species were identified by Xin-yu Luo, and the voucher specimen was deposited at the College of Life Science and Technology, Xinjing University, Xinjiang, China.The samples were stored in 95% ethanol at −20 • C until morphological examination and DNA extraction.Legs were used to extract the whole genomic DNA using a Qiagen DNeasy Blood & Tissue Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's protocol.The Qubit ® DNA Assay Kit in a Qubit ® 2.0 Flurometer (ThermoFisher, Waltham, MA, USA) was applied to measure the concentration of the DNA (Berry Genomics, Beijing, China).The Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) platform was used to generate sequencing libraries of 150 bp paired-end reads with an insert size of 350 bp.Trimmomatic v0.32 [13] was applied to remove short reads, adapters, and low-quality reads of raw data.

Assembly, Annotation, and Composition Analyses
Two methods were used for de novo assembly: (1) NOVOPlasty v3.8.3 (Brussels, Belgium) [14] was utilized to assemble Illumina reads and k-mer sizes of 23-39 bp; (2) IDBA-UD v1.1.3(Boston, MA, USA) [15] was implemented for mitochondrial genome assembly ("-mink 40 -maxk 120").Mitochondrial genome sequences were compared, which were obtained by the aforementioned methods, using Geneious 2020.2.1 [16] and merged into a single sequence.Then, tRNAscan SE 2.0 [17] and MITOS WebServer were utilized to annotate and analyze the secondary structure of tRNAs.Clustal Omega in Geneious was applied to annotate rRNAs and PCGs based on Bactericera cockerelli.The boundaries of rRNAs and PCGs were manually proofread using MEGA X software [18].SeqKit v0.16.0 (Chongqing, China) [19] was used to examine the bias of the nucleotide composition and the nucleotide composition of each gene.AT-skew and GC-skew were calculated by two formulas: AT-skew = (A − T)/(A + T); GC-skew = (G − C)/(G + C).The base composition, AT-and GC-skew, and relative synonymous codon usage (RSCU) of 10 species of Nitidulidae were calculated using MEGA X.The nucleotide diversity (Pi) of 13 PCGs and 2 rRNAs of three Egeirotrioza species was calculated using DnaSP v6.0 with a sliding window and a step size window of 200 bp and 20 bp, respectively.DnaSP 6.0 [20] was utilized to calculate the non-synonymous substitution rate (Ka)/synonymous substitution rate (Ks) for each PCG.CGview (https://cgview.ca/,accessed on 28 February 2024), an online server, was used to generate the visual se-quence features of the mitochondrial genomes.Finally, all newly sequenced mitochondrial genomes were submitted to GenBank (for accession number).
Here, we used the ML and BI methods to reconstruct the phylogenetic relationship within Triozidae.In the ML analysis, ModelFinder [25] in IQ-TREE 2 (Canberra, ACT, Australia) [26] was used to select befitting substitution models.To minimize long-branch attraction artifacts, we also used the posterior mean site frequency (PMSF) [27] model ('−m − mtART + C60 + FO + R') in IQ-TREE for the cds_faa matrix.A BI tree was generated via PhyloBayes-MPI (Montréal, QC, Canada) [28] with the site-heterogeneous mixture model (−m CAT + GTR).An available online website, iTOL, was utilized to fine-tune the final phylogenetic tree (https://itol.embl.de/upload.cgi,accessed on 15 February 2024).

Mitogenomic Organization
Here, we sequenced approximately 6 GB of raw data for each species.A total of three mitochondrial genomes of Triozidae were obtained, of which two were complete mitochondrial genomes (E.xingi and E. rufa), while that of E. gracilis was a linear mitochondrial genome; all of them have been deposited in GenBank with the accession numbers PP471964-PP471966.The whole lengths of the newly sequenced genomes are as follows: E. rufa, 15,830 bp; E. gracilis, 15,355 bp; E. xingi, 15,301 bp (Table 2).The unstable size of the CR was the primary reason for the whole length, as previously observed from other triozid species [6].We identified 13 PCGs, 22 tRNAs, 2 rRNAs, and 1 CR, which were the typeset mitochondrial genes in animals (Figure 2), and the three mitochondrial genomes exhibit a high degree of conservation.Our newly assembled mitochondrial genomes are similar to those of previously published species of Hemiptera and other insects in length and gene order [2, 3,6,[29][30][31].The mitochondrial features of the represented species are depicted in Figure 2. The trnS1 secondary structures of the three mitochondrial genome sequences of Egeirotrioza species lack stem-loop structures with dihydrouracil, and the other 21 tRNA secondary structures are typical clover structures.The stem-loop structure with dihydrouracil deletion of trnS1 is a typical feature in insects' mitochondrial genomes [6].The newly sequenced mitochondrial genomes were found to have similar nucleotide compositions (Table 2), revealing the characteristic AT-biased composition in Triozidae and other insects [3,31,32].The AT contents (%) of the three newly reported genomes are 76.92 (E.rufa), 74.46 (E.gracilis), and 76.40 (E.xingi) (Table 2).All the newly reported mitochondrial genomes have a positive AT-skew, while the GC-skew is negative.In most hemipteran species, there are fewer T than A and fewer G than C bases.The AT-skew and GC-skew values of the three newly reported genomes are 0.010 and −0.154 for E. rufa, −0.239 and 0.033 for E. gracilis, and 0.031 and −0.243 for E. xingi, respectively (Table 2), suggesting that the chain asymmetry was reversed [33].

Protein-Coding Genes, Composition, and Evolutionary Rates
There are no remarkable differences in the sizes of tRNAs, PCGs, and rRNAs among each species when compared.The PCG lengths of the three newly obtained species are 10,825 (E.rufa), 10,822 (E.gracilis), and 10,818 bp (E.xingi).All newly obtained mitochondrial genomes exhibit a negative GC-skew and AT-skew; the GC-skew values are −0.08 (E.rufa), −0.11 (E.gracilis), and −0.09 (E.xingi), while the AT-skew values are −0.13(E.rufa), −0.14 (E.gracilis), and 0.13 (E.xingi) (Table 2).The AT contents (%) are 78.34 (E.rufa), 73.55The newly sequenced mitochondrial genomes were found to have similar nucleotide compositions (Table 2), revealing the characteristic AT-biased composition in Triozidae and other insects [3,31,32].The AT contents (%) of the three newly reported genomes are 76.92 (E.rufa), 74.46 (E.gracilis), and 76.40 (E.xingi) (Table 2).All the newly reported mitochondrial genomes have a positive AT-skew, while the GC-skew is negative.In most hemipteran species, there are fewer T than A and fewer G than C bases.The AT-skew and GC-skew values of the three newly reported genomes are 0.010 and −0.154 for E. rufa, −0.239 and 0.033 for E. gracilis, and 0.031 and −0.243 for E. xingi, respectively (Table 2), suggesting that the chain asymmetry was reversed [33].

Protein-Coding Genes, Composition, and Evolutionary Rates
There are no remarkable differences in the sizes of tRNAs, PCGs, and rRNAs among each species when compared.The PCG lengths of the three newly obtained species are 10,825 (E.rufa), 10,822 (E.gracilis), and 10,818 bp (E.xingi).All newly obtained mitochondrial genomes exhibit a negative GC-skew and AT-skew; the GC-skew values are −0.08 (E.rufa), −0.11 (E.gracilis), and −0.09 (E.xingi), while the AT-skew values are −0.13(E.rufa), −0.14 (E.gracilis), and 0.13 (E.xingi) (Table 2).The AT contents (%) are 78.34 2).Compared with published data, we found that the AT content of the third codon positions was higher than that of the first and second positions in PCGs (Figure 3).2).Compared with published data, we found that the AT content of the third codon positions was higher than that of the first and second positions in PCGs (Figure 3).Similar to insect mitochondria, the three newly obtained mitochondrial genomes' PCGs started with ATN [2, 3,31].However, diverse start codons were discovered: COI used ATG as the start codon in three species; the ND3 gene used ATT in two species and ATA in one species; ND4L used TTG in three species; ND5 used ATC in one species and ATT in two species; ND2 used ATG in one species and ATA in two species, etc.The codon sizes of the three newly sequenced species were 1764 (E.rufa), 1303 (E.gracilis), and 1290 (E.xingi).Most PCGs in this group used TAA or TAG as the stop codon.However, ND5, ND1, and COX2 in Egeirotrioza have an incomplete termination codon of TA or T; for example, that for ND1 in E. xingi was TA and in E. rufa was T, and that for COX2 in E. xingi and E. rufa was T. Incomplete termination codons of PCGs are frequently observed in insects and are typically completed through polyadenylation following the excision of the downstream tRNA gene [34][35][36].
The Ka/Ks value (ω) is commonly employed to gauge the rate of sequence evolution driven by natural selection [37].The Ka/Ks analysis results showed that all 13 PCGs have a value of less than one, ranging from 0.07 (COX1) to 0.55 (ATP8) (Figure 4), and the evolution rate is as follows: This indicates that each gene has undergone purifying selection, and some genes such as ATP8 and ND6 experienced relatively relaxed selection Similar to insect mitochondria, the three newly obtained mitochondrial genomes' PCGs started with ATN [2, 3,31].However, diverse start codons were discovered: COI used ATG as the start codon in three species; the ND3 gene used ATT in two species and ATA in one species; ND4L used TTG in three species; ND5 used ATC in one species and ATT in two species; ND2 used ATG in one species and ATA in two species, etc.The codon sizes of the three newly sequenced species were 1764 (E.rufa), 1303 (E.gracilis), and 1290 (E.xingi).Most PCGs in this group used TAA or TAG as the stop codon.However, ND5, ND1, and COX2 in Egeirotrioza have an incomplete termination codon of TA or T; for example, that for ND1 in E. xingi was TA and in E. rufa was T, and that for COX2 in E. xingi and E. rufa was T. Incomplete termination codons of PCGs are frequently observed in insects and are typically completed through polyadenylation following the excision of the downstream tRNA gene [34][35][36].
The Ka/Ks value (ω) is commonly employed to gauge the rate of sequence evolution driven by natural selection [37].The Ka/Ks analysis results showed that all 13 PCGs have a value of less than one, ranging from 0.07 (COX1) to 0.55 (ATP8) (Figure 4), and the evolution rate is as follows: This indicates that each gene has undergone purifying selection, and some genes such as ATP8 and ND6 experienced relatively relaxed selection pressure.The DNA barcoding gene COX1 underwent the strongest purifying selection, which is consistent with previous studies of psyllids [6].
pressure.The DNA barcoding gene COX1 underwent the strongest purifying selection, which is consistent with previous studies of psyllids [6].2).

Nucleotide Diversity and Codon Usage
The sliding window analysis showed that the nucleotide diversity (Pi) of the 13 PCGs in Egeirotrioza is highly variable, with the highest Pi obtained for ATP8 (0.390) followed by ND1 (0.367) and ND4 (0.280), and the lowest Pi for COX1 (0.100) (Figure 5).The relative synonymous codon usage (RSCU) patterns exhibited by the three mitochondrial genomes are largely similar, as depicted in Figure 6, which includes the RSCU values for all possible synonymous codons corresponding to the 22 amino acids and 62 available codons used in the 13 PCGs of Egeirotrioza species.Among the Egeirotrioza species, UUA is the preferred codon, while Leu2, Ile, Phe, and Mte are the most frequently utilized amino acids.When considering the three species, E. gracilis and E. xingi favor the codons AUU, UUA, UUU, and AUA, whereas E. rufa prefers the codons UUA, AUU, UUU, and AUA.Prior to this study, the genus Egeirotrioza never had any molecular markers published in GenBank and BOLD systemv4.Our results on nucleotide diversity show that the Egeirotrioza COX1 gene can be used as an effective molecular marker for classification.This study has generated barcode reference data for Egeirotrioza species in order to use DNA barcoding as a rapid tool for accurate identification of the psyllid to aid phytosanitary measures.Meanwhile, the published mitochondrial genome of Egeirotrioza also facilitates the application of multi-marker DNA meta-barcoding technology for rapid pest monitoring.Codons ending in A/T are used more frequently, and the AT content at the third codon position is higher than that at the first and second codons, indicating that the third codon is more vulnerable to AT alterations [38].2).The rRNA lengths are as follows: 1892 (E.rufa), 1891 (E.gracilis), and 1898 (E.xingi).The AT contents (%) are 79.08 (E.rufa), 76.95 (E.gracilis), and 78.60 (E.xingi).The AT-skew values of all mitochondrial genomes are negative (−0.035 to −0.016), while the GC-skew values are positive (0.0314 to 0.0357) (see Table 2).

Nucleotide Diversity and Codon Usage
The sliding window analysis showed that the nucleotide diversity (Pi) of the 13 PCGs in Egeirotrioza is highly variable, with the highest Pi obtained for ATP8 (0.390) followed by ND1 (0.367) and ND4 (0.280), and the lowest Pi for COX1 (0.100) (Figure 5).The relative synonymous codon usage (RSCU) patterns exhibited by the three mitochondrial genomes are largely similar, as depicted in Figure 6, which includes the RSCU values for all possible synonymous codons corresponding to the 22 amino acids and 62 available codons used in the 13 PCGs of Egeirotrioza species.Among the Egeirotrioza species, UUA is the preferred codon, while Leu2, Ile, Phe, and Mte are the most frequently utilized amino acids.When considering the three species, E. gracilis and E. xingi favor the codons AUU, UUA, UUU, and AUA, whereas E. rufa prefers the codons UUA, AUU, UUU, and AUA.Prior to this study, the genus Egeirotrioza never had any molecular markers published in GenBank and BOLD systemv4.Our results on nucleotide diversity show that the Egeirotrioza COX1 gene can be used as an effective molecular marker for classification.This study has generated barcode reference data for Egeirotrioza species in order to use DNA barcoding as a rapid tool for accurate identification of the psyllid to aid phytosanitary measures.Meanwhile, the published mitochondrial genome of Egeirotrioza also facilitates the application of multimarker DNA meta-barcoding technology for rapid pest monitoring.Codons ending in A/T are used more frequently, and the AT content at the third codon position is higher than that at the first and second codons, indicating that the third codon is more vulnerable to AT alterations [38].

Phylogenetic Relationships
The heterogeneous divergence analysis using AliGROOVE found that the cds_faa matrix exhibited lower heterogeneity than the cds_fna, cds12_fna, cds12_rrna, and cds_rrna matrices (Figure 7).We used a supermatrix including cds_faa (5375 sites), cds_fna (7148 sites), cds_rrna (12,580 sites), cds12_fna (7148 sites), and cds12_rrna (9006 sites) to reconstruct the phylogenetic relationship of Triozidae based on BI and ML methods.The two approaches using these five matrices generated five BI and six ML trees, respectively (Figure 8 and Figures S1-S10).

Phylogenetic Relationships
The heterogeneous divergence analysis using AliGROOVE found that the cds_faa matrix exhibited lower heterogeneity than the cds_fna, cds12_fna, cds12_rrna, and cds_rrna matrices (Figure 7).We used a supermatrix including cds_faa (5375 sites), cds_fna (7148 sites), cds_rrna (12,580 sites), cds12_fna (7148 sites), and cds12_rrna (9006 sites) to reconstruct the phylogenetic relationship of Triozidae based on BI and ML methods.The two approaches using these five matrices generated five BI and six ML trees, respectively (Figures 8 and S1-S10).The phylogenetic analyses using all the matrices produced highly consistent topologies, thus supporting the monophyly of Egeirotrioza.The phylogenetic relationships show that Egeirotrioza is closely related to Leptynoptera (Figure 8).The genus Evegeirotrioza was established by Li [10].Subsequently, Burckhardt and Ouvrard [11] proposed the lecular correlation.In our phylogenetic studies, we formed the structure (Egeirotrioza xingi + (Evegeirotrioza rufa + Evegeirotrioza gracilis)) (Figures 8 and S1-S10).Evegeirotrioza has been well restored to the taxa of Egeirotrioza, so we reject Evegeirotrioza at both morphological and molecular levels and restore the monophyly of Egeirotrioza [39].As Egeirotrioza has never been reported as a molecular marker, the genus was not included in previous phylogenetic studies based on molecular morphology.Trioza is considered by most authors as an artificial receptacle for species not showing any particular morphological modifications.In our results, the position of Trioza (five species) was located in four groups, and the relationships of Leptynoptera and Bactericera are highly consistent with previous studies [6].In Triozidae, a species-rich, probably monophyletic family, most of the genera are ill-defined and artificial, and the phylogenetic relationships between genera remain largely unknown.Here, we have provided the mitochondrial DNA of Egeirotrioza, the first molecular analysis of its phylogenetic position, and also new fundamental data on the phylogenetic relationships of Psyllidae.
Due to the limited samples, the position of the genus Trioza was unclear and it also did not support the monophyly of Trioza, which was located in four groups (Figure 8).Additional taxon sampling, data, and analyses are necessary to resolve this ambiguity in future studies.For instance, low-coverage sequences are often utilized to extract more molecular markers to reconstruct stable phylogenetic relationships, as successfully demonstrated in other groups [40][41][42].

Conclusions
Three mitochondrial genomes of Egeirotrioza were obtained, including two complete mitochondrial genomes and one linear mitochondrial genome.The newly sequenced mitochondrial genomes exhibit similar structural features and nucleotide compositions to the previously published data of Triozidae.The nucleotide diversity (Pi) analysis showed that the diversity of ATP8 is the highest, and that of COX1 is the lowest.The three mitochondrial genomes display similar relative synonymous codon usage (RSCU) patterns.In adding published data, we could also reconstruct the phylogenetic relationships among Triozidae.In our results, the monophyly of Egeirotrioza was well supported, while the position of the genus Trioza was unclear due to the limited samples.The phylogenetic analyses using all the matrices produced highly consistent topologies, thus supporting the monophyly of Egeirotrioza.The phylogenetic relationships show that Egeirotrioza is closely related to Leptynoptera (Figure 8).The genus Evegeirotrioza was established by Li [10].Subsequently, Burckhardt and Ouvrard [11] proposed the synonymy of Evegeirotrioza with Egeirotrioza based on the morphological character of the genital plate, which is conical when viewed from the side.Evegeirotrioza was morphologically considered to be synonymous with Egeirotrioza, but there was no evidence of molecular correlation.In our phylogenetic studies, we formed the structure (Egeirotrioza xingi + (Evegeirotrioza rufa + Evegeirotrioza gracilis)) (Figures 8 and S1-S10).Evegeirotrioza has been well restored to the taxa of Egeirotrioza, so we reject Evegeirotrioza at both morphological and molecular levels and restore the monophyly of Egeirotrioza [39].As Egeirotrioza has never been reported as a molecular marker, the genus was not included in previous phylogenetic studies based on molecular morphology.
Trioza is considered by most authors as an artificial receptacle for species not showing any particular morphological modifications.In our results, the position of Trioza (five species) was located in four groups, and the relationships of Leptynoptera and Bactericera are highly consistent with previous studies [6].In Triozidae, a species-rich, probably monophyletic family, most of the genera are ill-defined and artificial, and the phylogenetic relationships between genera remain largely unknown.Here, we have provided the mitochondrial DNA of Egeirotrioza, the first molecular analysis of its phylogenetic position, and also new fundamental data on the phylogenetic relationships of Psyllidae.
Due to the limited samples, the position of the genus Trioza was unclear and it also did not support the monophyly of Trioza, which was located in four groups (Figure 8).Additional taxon sampling, data, and analyses are necessary to resolve this ambiguity in future studies.For instance, low-coverage sequences are often utilized to extract more molecular markers to reconstruct stable phylogenetic relationships, as successfully demonstrated in other groups [40][41][42].

Conclusions
Three mitochondrial genomes of Egeirotrioza were obtained, including two complete mitochondrial genomes and one linear mitochondrial genome.The newly sequenced mitochondrial genomes exhibit similar structural features and nucleotide compositions to the previously published data of Triozidae.The nucleotide diversity (Pi) analysis showed that the diversity of ATP8 is the highest, and that of COX1 is the lowest.The three mitochondrial genomes display similar relative synonymous codon usage (RSCU) patterns.In adding published data, we could also reconstruct the phylogenetic relationships among

Figure 2 .
Figure 2. Mitochondrial genome map showing the mitochondrial genome characteristics of representative species within the genus Egeirotrioza.The arrows indicate the direction of gene transcription.Normative abbreviations are used to represent PCGs and rRNAs, and single letter abbreviations are used to represent tRNAs.Red, green, blue, and orange represent PCGs, tRNA, rRNA, and CR, respectively.The GC content of the complete mitochondrial genome is shown in the second circle.The GC-skew of the complete mitochondrial genome is shown in the third circle.The innermost circle shows the length of the complete mitochondrial genome.

Figure 2 .
Figure 2. Mitochondrial genome map showing the mitochondrial genome characteristics of representative species within the genus Egeirotrioza.The arrows indicate the direction of gene transcription.Normative abbreviations are used to represent PCGs and rRNAs, and single letter abbreviations are used to represent tRNAs.Red, green, blue, and orange represent PCGs, tRNA, rRNA, and CR, respectively.The GC content of the complete mitochondrial genome is shown in the second circle.The GC-skew of the complete mitochondrial genome is shown in the third circle.The innermost circle shows the length of the complete mitochondrial genome.

Figure 3 .
Figure 3. Difference in AT contents of protein-coding genes of Egeirotrioza mitochondrial genomes.(A) First codon positions; (B) second codon positions; (C) third codon positions.

Figure 3 .
Figure 3. Difference in AT contents of protein-coding genes of Egeirotrioza mitochondrial genomes.(A) First codon positions; (B) second codon positions; (C) third codon positions.

Figure 5 .
Figure 5. Sliding window analysis of 13 PCGs and 2 rRNAs in the mitochondrial genomes of 3 species of Triozidae.The red line represents the nucleotide diversity (Pi) value (window size = 200 bp, step size = 20 bp).

Figure 6 .
Figure 6.Relative synonymous codon usage (RSCU) of mitochondrial protein-coding genes of 3 Triozidae species.The X-axis shows different amino acids, and the Y-axis shows the RSCU value (the number of times a certain synonymous codon is used/the average number of times that all codons encoding the amino acid are used).

Figure 5 .
Figure 5. Sliding window analysis of 13 PCGs and 2 rRNAs in the mitochondrial genomes of 3 species of Triozidae.The red line represents the nucleotide diversity (Pi) value (window size = 200 bp, step size = 20 bp).

Figure 5 .
Figure 5. Sliding window analysis of 13 PCGs and 2 rRNAs in the mitochondrial genomes of 3 species of Triozidae.The red line represents the nucleotide diversity (Pi) value (window size = 200 bp, step size = 20 bp).

Figure 6 .
Figure 6.Relative synonymous codon usage (RSCU) of mitochondrial protein-coding genes of 3 Triozidae species.The X-axis shows different amino acids, and the Y-axis shows the RSCU value (the number of times a certain synonymous codon is used/the average number of times that all codons encoding the amino acid are used).

Figure 6 .
Figure 6.Relative synonymous codon usage (RSCU) of mitochondrial protein-coding genes of 3 Triozidae species.The X-axis shows different amino acids, and the Y-axis shows the RSCU value (the number of times a certain synonymous codon is used/the average number of times that all codons encoding the amino acid are used).

Figure 8 .
Figure 8. Phylogenetic trees of Egeirotrioza based on the cds_faa matrix with the PMSF model in IQ-TREE.Support values on nodes indicate r SH-aLRT/UFBoot2, respectively.

Figure 8 .
Figure 8. Phylogenetic trees of Egeirotrioza based on the cds_faa matrix with the PMSF model in IQ-TREE.Support values on nodes indicate r SH-aLRT/UFBoot2, respectively.

Table 1 .
Collected information of newly sequenced species in this study.

Table 2 .
Nucleotide compositions of three newly sequenced species in this study.