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Article

Mitogenomics of Three Ziczacella Leafhoppers (Hemiptera: Cicadellidae: Typhlocybinae) from Karst Area, Southwest China, and Their Phylogenetic Implications

1
School of Karst Science, Guizhou Normal University, Guiyang 550001, China
2
State Engineering Technology Institute for Karst Desertification Control, Guiyang 550001, China
3
Guizhou Provincial Key Laboratory for Rare Animal and Economic Insect of the Mountainous Region, Guiyang University, Guiyang 550005, China
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(9), 1002; https://doi.org/10.3390/d15091002
Submission received: 27 June 2023 / Revised: 7 August 2023 / Accepted: 5 September 2023 / Published: 8 September 2023

Abstract

:
Leafhoppers (Hemiptera, Auchenorrhyncha, Cicadellidae) are distributed worldwide and include around 2550 genera, more than 21,000 species, including almost 2000 species in China. Typhlocybinae is the second largest subfamily in Cicadellidae after Deltocephalinae. Previously, morphological characteristics were the diagnostic basis of taxonomy, but they were not combined with molecular biology. The genus Ziczacella Anufryev, 1970 has only six known species worldwide. The mitogenomes of Ziczacella steggerdai Ross, 1965, Ziczacella dworakowskae Anufriev, 1969 and Ziczacella heptapotamica Kusnezov, 1928 were sequenced and identified here for the first time. They all contained 13 PCGs, 22 tRNA genes, 2 rRNA genes, and a control region, and the complete mitochondrial genomes were 15,231 bp, 15,137 bp, and 15,334 bp, respectively. The results show heavy AT nucleotide bias. Phylogenetic analysis yielded the following topology: (Empoascini + Alebrini) + ((Erythroneurini + Dikraneurini) + (Zyginellini + Typhlocybini)). In this study, three newly sequenced species were closely related to Mitjaevia dworakowskae and M. shibingensis. We confirmed the monophyly of the four tribes within Typhlocybinae again, and Zyginellini should be combined with Typhlocybini, which supports Chris’s points.

1. Introduction

Erythroneurini, belonging to Typhlocybinae of Cicadelladae, is the largest tribe of Typhlocybinae, with about 2000 species worldwide [1]. The leafhoppers feed on the sap of host plants and are abundant in forests and grasslands [2]. They are not only important agricultural and forestry pests, but also carriers of plant pathogens [3,4]. Ziczacella Anufryev, 1970, as one genus of this tribe, is widely distributed in the Palaearctic and Oriental regions, and only six known species have been reported until now [5,6].
The traditional classification of leafhoppers has attracted great attention, including the classification of Typhlocybinae. At present, Typhlocybinae mainly contains six tribes, but this taxonomy remains controversial [7,8,9]. Previously, people have tried to use morphological data or sequence data of a few genes to estimate the phylogenetic relationships between leafhopper groups, but little research has been performed on Typhlocybinae [10,11,12]. Nowadays, the emergence of a new generation of sequencing technology has brought a breakthrough to solve this problem so that the mitochondrial genome data can verify the existing classification of Typhlocybinae [13,14,15].
The insect mitochondrial genomic DNA has a molecular weight of 14–20 KB and is a closed double-stranded DNA molecule. Usually, it contains 37 genes, including 22 transfer RNA (tRNAs) genes,13 protein-coding genes (PCGs), including cytochrome c oxidase subunits 1–3 (cox13), NADH dehydrogenase 1–6 and 4L (nad16 and nad4L), ATPase subunit 6 and 8 (atp6 and atp8), cytochrome b (cytb), two ribosomal RNAs genes (16S and 12S), and a control region, which is the region rich in A + T. The mitochondrial genome is very suitable for genomics research [16,17]. Compared with nuclear genes, the mitochondrial genome has many advantages, such as easy detection, low molecular weight, simple structure, conservative arrangement, and so on. Therefore, the mitochondrial genome is widely used to identify phylogenetic relationships and population genetic structure at different taxonomic levels [18,19].
Leafhoppers are important agricultural and forestry pests, feeding on the sap of various economic crops, and they are also vectors of plant pathogens [2,3,4]. These characteristics make it very important to study the genetic information and biological evolution of leafhoppers. In order to further enrich the mitochondrial genome data of leafhoppers and provide comparative data for related species, the complete mitochondrial genome sequences of Ziczacella steggerdai Ross, 1965, Ziczacella dworakowskae Anufriev, 1969, and Z. heptapotamica Kusnezov, 1928 from Karst areas in southwest China were sequenced and analyzed [5,20,21], and their phylogenetic relationship with other leafhoppers was analyzed (Figure 1). At present, there are no gene data about the erythroneurine genus Ziczacella in the GenBank, and this study is the first time the complete mitochondrial gene and phylogenetic relationship of Ziczacella has been analyzed. These new molecular data will contribute to the identification of leafhopper species, the comparison of genetic relationships, and the study of population genetics and evolution in the future. Thirty mitogenomic sequences were downloaded from the National Center for Biotechnology Information (NCBI) to estimate phylogenetic relationships (Table 1).

2. Materials and Methods

2.1. Sampling and DNA Extraction

Samples of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica were collected from roadside weeds in Jiulongpo District, Chongqing City, China (29°5′51″ N, 107°19′57″ E), Shizhong District, Leshan City, Sichuan Province, China (30°01′39″ N, 103°46′37″ E), and Qixingguan District, Bijie City, Guizhou Province, China (27°09′42″ N, 105°05′47″ E), respectively. Samples were placed in a freezer at −20 °C and then stored in the Cicadellidae specimen room, School of Karst Science, Guizhou Normal University, under the voucher numbers GZNU-ELS-20220321, GZNU-ELS-20220322, and GZNU-ELS-20190716. Total genomic DNAs were extracted from three leafhopper species with abdomens and wings removed using a DNeasy Blood & Tissue kit (QIAGEN, Beijing, China) in turn, according to the manufacturer’s instructions. Due to the small size of the leafhopper body, in order to successfully extract DNA, each species used six leafhoppers.

2.2. DNA Sequencing and Assembly

Sequencing libraries were generated using the Truseq Nano DNA HT Sample Preparation Kit (Illumina, Alameda, CA, USA). Whole mitochondrial genome sequencing (150 bp paired-end reads) was performed using the Illumina Novaseq 6000 platform (Illumina, Alameda, CA, USA) at Berry Genomics, Beijing, China. The raw data were filtered using SOAPnuke v.1.3.0, and approximately 6.2 GB (Z. heptapotamica), 5.7 GB (Z. steggerdai), and 5.4 GB (Z. dworakowskae) of clean data were obtained and saved in fastq format. The average depth was 703.0 X, 529.8 X, and 424.1 X, respectively. After quality-proofing of the obtained fragments, the complete mt genome sequence was assembled manually using Geneious Prime V 2021.1.1 and then manually proofread based on sequencing peak figures. A homology search was performed by the Blast function in NCBI to verify the amplified sequence as the correct sequence [32,33].

2.3. Sequence Annotation and Analyses

The assembled mitogenome sequence was subsequently annotated using Geneious Prime V 2021 and the mitogenome of Mitjaevia protuberanta (GeneBank accession number: NC_047465.1) as the reference. All tRNA genes were identified with MITOS Web Server (http://mitos.bioinf.uni-leipzig.de/index.py, accessed on 19 December 2021) [34]. The nucleotide base composition, codon usage, A + T content values, and relative synonymous codon usage (RSCU) were calculated using MEGA 7.0 [35]. The typical second structures for tRNAs were manually drawn with Adobe Illustrator 2021 in accordance with the MITOS predictions. Circular mitogenome maps were drawn using the CG View server [36] (http://cgview.ca/, accessed on 6 January 2023) and Photoshop CS 6. The skewing of the nucleotide composition was calculated with the formulas AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [37]. The complete mitochondrial genome sequences of Z. steggerdai (Genbank: OQ657302), Z. dworakowskae (Genbank: OQ657303), and Z. heptapotamica (NC064506.1) were submitted to NCBI.

2.4. Phylogenetic Analyses

The phylogenetic analysis used the mitochondrial genomes of three newly sequenced species and other Typhlocybinae species downloaded from GenBank, containing four species from Empoascini, eight species from Erthroneurini, two species from Alebrini, five species from Typhlocybini, five species from Dikraneurini, and four species from Zyginellini. Atkinsoniella thalia and Scaphoideus maculatus were regarded as outgroups (Table 1). For phylogenetic analyses, 13 PCG and 2 rRNA genes were selected to construct phylogenetic trees. Thirty mitogenomic sequences were aligned and corrected using MAFFT v7; gaps and ambiguous sites in the alignments were then removed using Gblocks 0.91b [38,39]. The trimmed datasets were used to estimate the phylogeny by Bayesian inference (BI) using MrBayes 3.2.7 and maximum likelihood (ML) using IQ-TREE [40,41,42]. The best model was inferred by PartitionFinder (v2.1.1) [43]. BI selected GTR + I + G as the optimal model, running 10 million generations twice, sampling once every 1000 generations after the average standard deviation of the segmentation frequency drops below 0.01, with the first 25% of the samples being discarded burn-in, and the posterior probability (PP) of each branch was calculated. ML constructed with the IQ-TREE used an ultrafast bootstrap approximation approach with 10,000 replicates and calculated bootstrap scores for each node (BP).

3. Results and Discussion

3.1. Organization and Composition of the Genome

The genome organization and nucleotide composition of three new mitogenomes sequenced in this study are similar to other Typhlocybine species previously reported [26,29,44,45]. The complete mitogenomes of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica are double-stranded plasmids with 15,231 bp, 15,137 bp and 15,334 bp, respectively (Table 2). Usually, it contains 13 PCGs, 22 tRNA genes, 2 rRNA genes, and a control region. Twenty-four genes encode in the majority strand (L-strand), while the other fourteen genes encode in the minority strand (H-strand) (Figure 2). The mitogenome of Z. steggerdai has a total of 36 bp space in thirteen gene overlaps, ranging in length from 1 to 8 bp; the longest overlap region fell between tRNA-Trp and tRNA-Cys genes. In addition, there were ten 1–6 bp coding gene spacer regions, with a total length of 29 bp; the longest 6 bp intergenic spacer sequences were located between cox3 and tRNA-Gly. In the Z. dworakowskae mitogenome, gene overlaps were found at 13 gene junctions and involved a total of 51 bp, the longest 16 bp overlapping located between nad4 and nad4L. Intergenic spacer sequences were found at 12 gene junctions and involved a total of 29 bp; the longest 6 bp intergenic spacer sequences were located between cox3 and tRNA-Gly. The mitogenome of Z. heptapotamica harbors a total of 32 bp in 12 overlapping genes (1–8 bp-long), and 11 coding gene spacer regions (1–6 bp-long) are present. The longest 8 bp overlap region fell between tRNA-Cys and tRNA-Tyr genes, and the longest 6 bp intergenic spacer sequences were located between cox3 and tRNA-Gly.
The nucleotide composition of the whole mitogenome of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica was as follows: (A) 42.2%, 42.4%, and 42.4%; (T) 37.2%, 36.9%, and 36.9%; (G) 8.9%, 8.9%, and 9.0%; and (C) 11.7%, 11.8%, and 11.8%. The mitochondrial genomes of the three mitogenomes show heavy AT nucleotide bias, with an A + T% content for the whole sequence of 79.4%, 79.3%, and 79.2%, respectively (Figure 3). The PCGs show the lowest A + T% among whole genes, while the control region (CR) has the strongest A + T% bias. Analysis of 37 individual genes of the three mitogenomes shows that AT skews are mostly positive, while for GC skews, all the GC skews are negative. Positive AT skews indicate that the content of base A is higher than that of base T, while a negative value indicates the opposite. Only the AT skews of CR are negative (−0.255, −0.196, and −0.148). In conclusion, the genetic composition of the three species is mostly biased towards A and C (Table 3, Figure 3).

3.2. Protein-Coding Genes and Codon Usage

The three Ziczacella mitogenomes contained 13 PCGs; the total length is 10,955 bp, 10,949 bp, and 10,970 bp, respectively. Among the 13 protein-coding genes of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica, nine PCGs (nad2, nad3, nad6, cox1, cox2, cox3, atp8, atp6, and cytb) encode on the majority strand (H-strand), while the other four PCGs (nad1, nad4, nad5, and nad4L) encode on the minority strand (L-strand) (Table 2). The longest gene was the nad5 gene (1657, 1636, and 1675), and the shortest was the atp8 gene (153, 153, and 153) in Z. steggerdai, Z. dworakowskae, and Z. heptapotamica, respectively. The proportion of A, T, G, and C is 41.7%, 35.6%, 10.0%, and 12.7% in Z. steggerdai mitogenome; 41.6%, 35.6%, 10.0%, and 12.8% in Z. dworakowskae mitogenome; and 41.6%, 35.5%, 10.1%, and 12.8% in Z. heptapotamica. The A + T content of the PCGs in Z. steggerdai mitogenome (77.3%) is higher than in Z. dworakowskae mitogenome (77.2%) and Z. heptapotamica mitogenome (77.1%) (Table 3).
Among the 13 protein-coding genes, the atp8 genes started with TTG, while all other PCGs contained the usual ATN (ATA, ATT, and ATG) start codon. In Z. steggerdai mitogenome and Z. heptapotamica mitogenome, three PCGs utilize ATG (cox3, nad4L, and cytb), four PCGs utilize ATT (cox2, nad3, nad5, and nad6), one PCG utilizes TTG (atp8), and five PCGs utilize ATA (nad1, nad2, nad4, atp6, and cox1) as the start codon. Eleven PCGs (nad1, nad2, nad3, nad4, nad4L, nad6, cox1, atp6, atp8, cox3, and cytb) have TAA as a stop codon, whereas the cox2 and nad5 genes use a single T. However, in the Z. dworakowskae mitogenome, unlike the other two mitochondrial genomes, nad4 is started by ATT.
The 13 PCGs in the three mitogenomes comprised 5076 codons, 5045 codons, and 5111 codons, respectively. Statistics on the available codon count and the relative synonymous codon usage (RSCU) of Z. steggerdai found that the most abundant codons were AAA (Lys), AAU (Asn), AUU (Ile), and AUA (Met). Z. dworakowskae mitogenome, where the five most frequently used codons are AAU (Asn), AAA (Lys), UAU (Tyr), UUA (Leu2), and AUA (Met). The five most frequently found codons in the mitochondrial genome of Z. heptapotamica were AAU (Asn), AAA (Lys), UAU (Tyr), UUA (Leu2), and AUU (Ile). Relative synonymous codon usage (RSCU) showed that the most frequently utilized amino acids are Asn, Lys, Phe, Ile, Tyr, and Met (Table 4). The majority of codons all end with A or U, which leads to the high A + T bias of the entire mitogenome (Figure 4 and Figure 5).

3.3. Transfer and Ribosomal RNA Genes

The mitogenomes of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica included 22 transfer RNA genes, as in most invertebrates [46,47], of which 14 are encoded in the major strand (H-strand), while the other eight are encoded in the minor strand (L-strand) (Table 2). Their nucleotide lengths ranged from 61 (Ala, Arg) to 70 bp (Lys, Val), and the total lengths of tRNA genes were 1429 bp, 1428 bp, and 1422 bp, respectively. The tRNA of the three species has a positive AT and negative GC skew; the AT skew of 22 tRNA is positive, and the GC skew is positive. Compared to the conventional insect mitochondrial gene order, no tRNA gene rearrangements are found. All of the tRNA genes can be folded into typical cloverleaf secondary structures except for tRNA-Ser1 in three newly sequenced mitochondrial genomes, which lack the dihydrouridine (DHU) stem and form a simple loop [48,49]. It can be clearly seen from the secondary structure of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica that, in addition to the typical Watson–Crick pairings (A-U and G-C), there are also some typical pairings such as U-G, and a total of 14, 16, and 16 G-U weak base pairs are found, respectively (Figure 6, Figure 7 and Figure 8).
The two ribosomal RNAs (12S and 16S ribosomal RNA) are separated by tRNA-Val. The three Ziczacella mitogenomes and the 16s rRNA gene (Z. steggerdai: 1176 bp, Z. dworakowskae: 1176 bp, and Z. heptapotamica: 1179 bp) are located between tRNA-Leu1 and tRNA-Val; the 12S rRNA gene (Z. steggerdai: 725 bp, Z. dworakowskae: 723 bp, Z. heptapotamica: 725 bp) is between tRNA-Val and the A + T rich region. Both rRNA genes are encoded on the L-strand. The rRNA genes displayed a positive AT skew and a negative GC skew, with an A + T content of 82.6% in Z. steggerdai, 82.6% in Z. dworakowskae, and 82.7% in Z. heptapotamica.

3.4. Control Region

Like the typical insect mitochondrial genome, Z. steggerdai, Z. dworakowskae, and Z. heptapotamica have a control region, which is located between 12S rRNA and tRNA-Ile, which is the longest in the mitogenomes. The nucleotide composition of the A + T rich region of the three mitogenomes is as follows: Z. steggerdai 98.6%; Z. dworakowskae: 99.2%; and Z. heptapotamica: 96.3%, respectively (Table 3). The AT content in this region was the highest in the whole mitochondrial genome.

3.5. Phylogenetic Relationships

In this study, the phylogenetic tree was constructed based on 13 PCGs and 2 rRNAs from 28 species of Typhlocybinae (Alebrini, Dikraneurini, Empoascini, Erythroneurini, Typhlocybini, and Zyginellini) and two outgroups. Phylogenetic analysis showed that Z. steggerdai, Z. dworakowskae, and Z. heptapotamica are sister groups with Mitjaevia dworakowskae and Mitjaevia shibingensis. The result yielded the following topology: (Empoascini + Alebrini) + ((Erythroneurini + Dikraneurini) + (Zyginellini + Typhlocybini)) (Figure 9). Empoascini, Dikraneurini, Alebrini, and Erythroneurini are identified as monophyletic, with the sister group of Alebrini and Empoascini as the base branch, followed by the sister group of Erythroneurini and Dikraneurini, and finally, the species of Zyginellini and Typhlocybini belonging to the same branch.
In previous studies, Typhlocybinae was often divided into six monophyletic tribes: Alebrini, Dikraneurini, Empoascini, Erythroneurini, Typhlocybini, and Zyginellini [50]. Zyginellini was proposed by Dworakowska in 1979, and its morphological difference from Typhlocybini is the hind wing vein CuA connected to vein MP. Maximum likelihood (ML) and Bayesian inference (BI) are highly supported and have consistent topologies in most phylogenetic analyses. Both methods support that Typhlocybini and Zyginellini merge into a single branch, and the two tribes should be treated as synonyms, because their species are so intertwined that they are hard to separate from each other. The results are slightly different from the traditional classification system, but similar to the results of most phylogenetic relationships of Typhlocybinae based on molecular data. We support the view proposed by Dietrich (2013); that is, Zyginellini may not be a monophyletic group and should be merged with Typhlocybini [9]. The Typhlocybine species are very rich, so like most phylogenetic studies based on molecular data, this study is only based on a small number of species to explore the phylogenetic status of newly sequenced species. However, more sequencing data of leafhoppers are needed to construct a more complete phylogenetic tree in order to clarify the relationship between the tribes of Typhlocybinae.

4. Conclusions

In summary, the complete mitochondrial genome sequences of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica were sequenced for the first time. We analyzed the basic composition, location, and other characteristics of PCGs, tRNA genes, rRNA genes, and control regions and further elucidated the relationship between them and other species in Typhlocybinae. They are close to most other sequenced leafhoppers in structures and compositions. In addition, based on the mitochondrial gene sequences of 30 leafhopper species, a phylogenetic tree was established by the maximum likelihood method and Bayesian method. The result showed that this collection of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica is a sister group to the collection of Mitjaevia dworakowskae and Mitjaevia shibingensis. Meanwhile, Alebrini, Dikraneurini, Empoascini, and Erythroneurini are proven again as monophyletic, while Zyginellini and Typhlocybini should be gathered into a single branch. The results of this study confirm that Zyginellini is a junior synonym of Typhlocybini; that is, the two tribes should be combined and placed into the same taxon as a monophyletic group.

Author Contributions

Conceptualization, J.W. and N.Z.; methodology, J.W. and T.P.; visualization, J.W.; software, J.W. and T.P.; validation, Y.S., T.P. and C.L.; investigation, J.W.; writing—original draft preparation, J.W. and N.Z.; draft review, Y.S. and C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32260120), the World Top Discipline Program of Guizhou Province: Karst Ecoenvironment Sciences (No. 125 2019 Qianjiao Keyan Fa), the Science and Technology Project of Guiyang City ([2020]7-18), the Innovation Group Project of the Education Department of Guizhou Province ([2021]013), and the Natural Science Foundation of Guizhou Province (Qiankehejichu-ZK [2023] General 257).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. External morphology of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
Figure 1. External morphology of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
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Figure 2. Circular maps of the mitochondrial genome of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica.
Figure 2. Circular maps of the mitochondrial genome of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica.
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Figure 3. AT and GC skews were calculated for 37 mitochondrial genes of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica.
Figure 3. AT and GC skews were calculated for 37 mitochondrial genes of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica.
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Figure 4. Count in the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
Figure 4. Count in the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
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Figure 5. Relative synonymous codon usage (RSCU) in the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
Figure 5. Relative synonymous codon usage (RSCU) in the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
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Figure 6. Predicted secondary cloverleaf structure for the tRNAs of Z. steggerdai.
Figure 6. Predicted secondary cloverleaf structure for the tRNAs of Z. steggerdai.
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Figure 7. Predicted secondary cloverleaf structure for the tRNAs of Z. dworakowskae.
Figure 7. Predicted secondary cloverleaf structure for the tRNAs of Z. dworakowskae.
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Figure 8. Predicted secondary cloverleaf structure for the tRNAs of Z. heptapotamica.
Figure 8. Predicted secondary cloverleaf structure for the tRNAs of Z. heptapotamica.
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Figure 9. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic tree for three newly sequenced species based on 13 PCGs and 2 rRNAs of Typhlocybinae. The first number is a bootstrap proportion (BP) of maximum likelihood (ML) analyses, and the second number at each node is Bayesian posterior probabilities (PP).
Figure 9. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic tree for three newly sequenced species based on 13 PCGs and 2 rRNAs of Typhlocybinae. The first number is a bootstrap proportion (BP) of maximum likelihood (ML) analyses, and the second number at each node is Bayesian posterior probabilities (PP).
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Table 1. List of the mitochondrial genomes analyzed in the present study.
Table 1. List of the mitochondrial genomes analyzed in the present study.
TribeSpeciesLength (bp)GenBank No.Reference
AlebriniShaddai sp.17,575 bpMZ014457.1[22]
Sobrala sp.16,732 bpMZ014458.1[22]
DikraneuriniDikraneura zlata15,330 bp MZ014450.1[22]
Dikraneurini sp.15,306 bpMZ014451.1[22]
Michalowskiya breviprocessa15,591 bpMW264489.1Unpublished
Uniformus sp.14,440 bp MW272457.1Unpublished
Uzeldikra longiprocessa14,752 bp NC_069160.1Unpublished
EmpoasciniEmpoasca onukii15,167 bp NC_037210.1[23]
Ghauriana sinensis15,491 bp MN699874.1[24]
Empoasca serrata15,131 bp MZ014453.1[22]
Alebroides salicis15,890 bp MZ014449.1[22]
ErythroneuriniEmpoascanara dwalata15,271 bp MT350235.1[25]
Empoascanara sipra14,827 bpNC_048516.1[26]
Empoascanara wengangensis14,830 bpMT445764.1[25]
Mitjaevia dworakowskae16,399 bp MT981880.1[27]
Mitjaevia shibingensis15,788 bpMT981879.1[27]
Ziczacella steggerdai15,231 bpOQ657302This study
Ziczacella dworakowskae15,137 bpOQ657303This study
Ziczacella heptapotamica15,334 bpNC064506.1This study
TyphlocybiniEupteryx gracilirama17,173 bpMT594485.1[28]
Eupteryx minuscula16,944 bpMN910279.1[28]
Eurhadina acapitata15,419 bp MZ457331.1Unpublished
Eurhadina dongwolensis15,708 bpMZ457332.1Unpublished
Eurhadina jarrayi15,332 bpMZ014455.1[22]
ZyginelliniLimassolla emmrichi14,677 bpMW272458.1Unpublished
Limassolla lingchuanensis15,716 bpNC_046037.1[29]
Parazyginella tiani17,562 bpNC_053918.1[30]
Zyginella minuta15,544 bp NC_052876.1[31]
outgroupAtkinsoniella thalia15,034 bpNC_062847.1Unpublished
Scaphoideus maculatus15,486 bp NC_060770.1Unpublished
Table 2. Annotations of the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
Table 2. Annotations of the mitogenomes of Z. steggerdai (ZS), Z. dworakowskae (ZD), and Z. heptapotamica (ZH).
GenePositionSize (bp)IntergenicStart CodonStop CodonStrand
ZSZDZHZSZDZHZSZDZHZSZDZHZSZDZH
tRNA-Ile1–641–641–64646464000 H
tRNA-Gln62–13062–13063–129696967−3−3−2 L
tRNA-Met134–202134–202134–202696969334 H
nad2203–1174203–1174203–1174972972972000ATAATAATATAATAATAAH
tRNA-Trp1174–12371174–12371174–1237646464−1−1−1 H
tRNA-Cys1230–12921230–12921230–1292636363−8−8−8 L
tRNA-Tyr1296–13571296–13571296–1356626261333 L
cox11360–29011360–29011359–2900154215421542222ATAATAATATAATAATAAH
tRNA-Leu22907–29712907–29712906–2970656565555 H
cox22972–36502972–36502971–3649679679679000ATTATTATTTTTH
tRNA-Lys3651–37203651–37203650–3719707070000 H
tRNA-Asp3721–37853721–37853720–3784656565000 H
atp83784–39363784–39363783–3935153153153−2−2−2TTGTTGTTGTAATAATAAH
atp63933–45833933–45833932–4582651651651−4−4−4ATAATAATATAATAATAAH
cox34584–53634584–53634583–5362780780780000ATGATGATGTAATAATAAH
tRNA-Gly5370–54315370–54315369–5430626262666 H
nad35432–57855432–57855431–5784354354354000ATTATTATTTAATAATAAH
tRNA-Ala5787–58475787–58475786–5846616161111 H
tRNA-Arg5847–59075847–59075846–5906616161−1−1−1 H
tRNA-Asn5907–59725907–59725906–5971666666−1−1−1 H
tRNA-Ser15972–60385972–60385971–6037676767−1−1−1 H
tRNA-Glu6040–61036040–61026039–6102646364111 H
tRNA-Phe6105–61696104–61686104–6168656565111 L
nad56170–78266169–78046169–7843165716361675000ATTATTATTTTTL
tRNA-His7824–78877802–78657841–7904646464−3−3−3 L
nad47887–92097865–92027904–9226132313381323−1−1−1ATAATTATATAATAATAAL
nad4L9209–94879187–94659226–9504279279279−1−16−1ATGATGATGTAATAATAAL
tRNA-Thr9490–95529468–95309507–9569636363222 H
tRNA-Pro9553–96189531–95969570–9635666666000 L
nad69621–10,1069599–10,0849638–10,123486486486222ATTATTATTTAATAATAAH
cytb10,107–11,24310,085–11,22110,124–11,260113711371137000ATGATGATGTAATAATAAH
tRNA-Ser211,247–11,31011,225–11,28811,264–11,327646464333 H
Nad111,304–12,24511,282–12,22311,321–12,259942942939−7−7−7ATAATAATTTAATAATAAL
tRNA-Leu112,243–12,30712,221–12,28512,260–12,324656565−3−30 L
16S12,308–13,48312,286–13,46112,325–13,503117611761179000 L
tRNA-Val13,484–13,55313,462–13,53113,504–13,569707066000 L
12S13,554–14,27813,532–14,25413,570–14,294725723725000 L
D-loop14,279–15,23114,255–15,13714,295–15,3349538831040000 H
Table 3. Nucleotide compositions, AT skew, and GC skew in different regions of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica mitochondrial genomes.
Table 3. Nucleotide compositions, AT skew, and GC skew in different regions of Z. steggerdai, Z. dworakowskae, and Z. heptapotamica mitochondrial genomes.
RegionC%A%G%T%A + T%AT SkewGC Skew
ZSZDZHZSZDZHZSZDZHZSZDZHZSZDZHZSZDZHZSZDZH
whole11.711.811.842.242.442.48.98.99.037.236.936.879.479.379.20.0640.0700.071−0.134−0.136−0.135
PCGs12.712.812.841.741.641.610.010.010.135.635.635.577.377.277.10.0770.0780.079−0.119−0.123−0.118
tRNA12.012.012.040.540.440.39.69.79.837.937.937.978.478.378.20.0330.0320.031−0.110−0.110−0.101
rRNA11.011.111.049.349.449.66.46.36.333.333.233.182.682.682.70.1950.1970.199−0.269−0.273−0.272
CR0.80.52.136.839.941.00.50.31.661.959.355.398.699.296.3−0.255−0.196−0.148−0.231−0.143−0.135
Table 4. Codon and relative synonymous codon usage (RSCU) in the mitogenomes of three mitogenomes (* stands for stop codon).
Table 4. Codon and relative synonymous codon usage (RSCU) in the mitogenomes of three mitogenomes (* stands for stop codon).
Amino
Acid
CodonZ. steggerdaiZ. dworakowskaeZ. heptapotamicaAmino
Acid
CodonZ. steggerdaiZ. dworakowskaeZ. heptapotamica
CountRSCUCountRSCUCountRSCUCountRSCUCountRSCUCountRSCU
PheUUU3151.552891.432941.47TyrUAU2531.563211.633051.56
UUC910.451140.571060.53 UAC720.44720.37850.44
Leu2UUA3173.073123.083093.03HisCAU751.46721.37681.27
UUG490.47580.57810.79 CAC280.54330.63390.73
Leu1CUU1061.03830.82760.75GlnCAA1141.56851.43941.36
CUC190.18330.33330.32 CAG320.44340.57440.64
CUA1010.98900.89820.8AsnAAU3771.674201.514281.49
CUG270.26310.31310.3 AAC750.331350.491470.51
IleAUU3471.712761.583021.55LysAAA4361.753901.543921.55
AUC580.29740.42880.45 AAG610.251180.461140.45
MetAUA3341.73101.682861.68AspGAU641.44471.42581.73
AUG590.3600.32540.32 GAC250.56190.5890.27
ValGUU561.87501.68441.44GluGAA1301.68721.53791.55
GUC110.37130.44130.43 GAG250.32220.47230.45
GUA471.57491.65501.64CysUGU321.28331.27421.29
GUG60.270.24150.49 UGC180.72190.73230.71
Ser2UCU591.6571.43541.24TrpUGA631.59581.36501.23
UCC180.49190.48230.53 UGG160.41270.64310.77
UCA782.12721.81661.52ArgCGU111.1680.84111.29
UCG150.4170.18110.25 CGC20.2170.7460.71
ProCCU511.47271.08251.01 CGA222.32161.68141.65
CCC270.78351.4321.29 CGG30.3270.7430.35
CCA531.53321.28341.37Ser1AGU381.03411.03481.11
CCG80.2360.2480.32 AGC140.38270.68330.76
ThrACU781.54711.44731.42 AGA551.49651.63691.59
ACC400.79531.08551.07 AGG180.49310.78430.99
ACA731.45571.16611.19GlyGGU441.69311.57161.21
ACG110.22160.32160.31 GGC70.2780.41110.83
AlaGCU351.84201.33131.33 GGA291.12241.22100.75
GCC60.32100.6760.62 GGG240.92160.81161.21
GCA321.68271.8141.44*UAA3241.693511.653651.66
GCG30.1630.260.62 UAG590.31750.35740.34
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Wang, J.; Zhang, N.; Pu, T.; Li, C.; Song, Y. Mitogenomics of Three Ziczacella Leafhoppers (Hemiptera: Cicadellidae: Typhlocybinae) from Karst Area, Southwest China, and Their Phylogenetic Implications. Diversity 2023, 15, 1002. https://doi.org/10.3390/d15091002

AMA Style

Wang J, Zhang N, Pu T, Li C, Song Y. Mitogenomics of Three Ziczacella Leafhoppers (Hemiptera: Cicadellidae: Typhlocybinae) from Karst Area, Southwest China, and Their Phylogenetic Implications. Diversity. 2023; 15(9):1002. https://doi.org/10.3390/d15091002

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Wang, Jinqiu, Ni Zhang, Tianyi Pu, Can Li, and Yuehua Song. 2023. "Mitogenomics of Three Ziczacella Leafhoppers (Hemiptera: Cicadellidae: Typhlocybinae) from Karst Area, Southwest China, and Their Phylogenetic Implications" Diversity 15, no. 9: 1002. https://doi.org/10.3390/d15091002

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