Characterization and phylogenetic implications of newly sequenced mitogenomes of Five Mileewa and Processina species from China (Hemiptera: Cicadellidae: Mileewinae)

To accumulate evidence of the phylogeny of Mileewinae and the relationships among Mileewa, Processina, and Ujna genera, we sequenced the complete mitochondrial genomes of four Mileewa spp., namely, Mileewa mira, Mileewa lamellata, Mileewa sharpa, and Mileewa amplimacula. The first complete mitogenome of the genus Processina (P. sexmaculata), established by Yang, Deitz & Li from China and comprising five species, was also sequenced in this study. Annotation showed that the five mitogenomes were 14787 –15436 bp in length, and all harbored 37 typical genes. The AT content of the five mitogenomes ranged from 78.3% to 80.2%, which was similar to that of other sequenced Mileewinae species. For protein-coding genes (PCGs), ATN was the start codon, while atp8 and nad5 genes were initiated with TTG, and a great majority of them used TAA or TAG as stop codons, whereas cox2 and nad1 ended with an incomplete codon T–. All tRNAs had a typical cloverleaf secondary structure, except for trnS1, which had a reduced dihydrouridine arm. We further used 59 Membracoidea species and two outgroups to reconstruct phylogenetic trees based on 13 PCGs under an independent partition model with Bayesian inference and Maximum-likelihood methods. Among these two trees, each of the subfamilies Cicadellinae, Typhlocybinae, and Mileewinae were recovered as a monophyletic group with high support values, suggesting that Typhlocybinae was more ancient than Mileewinae and Cicadellinae. Within the Mileewinae subfamily, all species maintained the same relationships and topologies according to both the BI and ML analyses (PP > 0.8, BS > 83) as follows: (M. sharpa + (U. puerana + ((M. ponta + (M. mira + M. lamellata)) + ((M. albovittata + (M. margheritae + M. amplimacula)) + (M. rufivena + (P. sexmaculata + M. alara)))))), and the monophyly of the genera Processina, Mileewa and Ujna were not supported. This study further enriches the Mileewinae mitogenome database and will contribute to future research on the systematics, evolution, and classification of this group.

anal tube segment) with single caudal process. A great majority of Chinese Mileewini species belong to the genera Mileewa, which has 56 species, Ujna, which has eight species, and Processina, which has five species that are only recorded in China 11 .
Mileewa is a widespread and large genus of the tribe Mileewini established with Mileewa margheritae by Distant (1908). The genus has more than 80 valid species globally that have a dark appearance dorsally and forewing that is usually truncated or concave apically and sometimes distinctly expanded from base to apex. Moreover, in our recent study, seven species were newly recorded in this genus from China [11][12][13] . The genus Processina was completely established in China by Yang, Deitz & Li in 2005 and contains five species. Processina dashahensis (Yang et Li, 2005) is one of these species that was recorded in the Dashahe Natural Reserve of Guizhou Province. The other species originated from several places, such as Taiwan, Yunnan, Guizhou, and Sichuan. The traditional classification difference between the genera Mileewa and Ujna is that the head width is narrower than the pronotum or subequal to the pronotum width; the forewing apex is truncated or emarginated in Mileewa yet rounded in Ujna; and male style have long setae near their middle section in Mileewa versus an absent long setae in Ujna. In contrast, the genus Processina is distinguished from the other two genera mainly by its male style with a straight apical portion, rounded apex, and dense setae at the apex 14 . However, some ambiguous features make it difficult to distinguish between species in traditional classification studies. Therefore, there is an urgent need to increase our knowledge on the mitochondrial genomes of Mileewinae species and to use these molecular data to determine the phylogenetic relationships among other subfamilies and within this small group.
The insect mitochondrial genomes are mostly 14.5-17 kb double-stranded circular molecules comprising 37 typical genes: 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs: s-rRNA; l-rRNA), 22 transfer RNA genes (tRNAs), and a control region (CR) [15][16][17] . To date, six Mileewinae species from China have been sequenced, and these sequences have been deposited in the NCBI database: Mileewa rufivena (MZ326689) 18 , Mileewa ponta (MT497465) 19 , Mileewa margheritae (MT483998) 20 , Mileewa albovittata (MK138358) 21 , Mileewa alara (MW533151) 22 and Ujna puerana (MZ326688) 18 . They are all from the genera Ujna and Mileewa, while sequences of Processina species have not been deposited in NCBI. In this study, we sequenced and annotated the complete mitogenomes of five Mileewinae species: one from the genus Processina, and others from Mileewa. Furthermore, we analyzed the characteristics of these mitogenomes, including nucleotide composition, tRNA secondary structure, codon usage, gene overlaps, intergenic spacer, and CRs. The mitochondrial genome data in this study can help us understand the phylogeny and evolution of Mileewinae. The growing number of studies on the complete mitogenomes of Mileewinae will yield more evidence, which can render a better understanding of the taxonomic status of the subfamily Mileewinae among other Cicadellidae subfamilies.

Materials and methods
Sample collection and taxonomic identification. Specimens of adult male M. mira, M. lamellata, M.
sharpa, M. amplimacula, and P. sexmaculata were collected from China (Table S1). All specimens were collected, immediately preserved in 100% ethanol, and stored at −20 °C in a laboratory freezer prepared for DNA extraction. The species used in this study (males) were identified by Maofa-Yang based on morphological characteristics, especially male genitalia, according to the taxonomy system described by   6 .
DNA extraction and sequencing. After identifying the species, total genomic DNA was extracted from the head and thorax tissues of five single adult species, using the Tissue and Blood Genome DNA Extraction Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocols. Voucher DNA was stored at −20 °C, while the external genitalia were kept in glycerol. Both were deposited at the Institute of Entomology, Guizhou University, Guiyang, China (GUGC). The five new mitogenomes were subjected to next-generation sequencing on the Illumina NovaSeq6000 platform (Berry Genomics, Beijing, China) with a paired-end 150 sequencing strategy.
Mitogenomes assembly, annotation, and analysis. The clean sequence reads were assembled using Getorganelle 1.7.5 23 based on the Mileewa rufivena mitochondrial genome sequence from GenBank (Accession number: MZ326689.1) 18 . The five mitogenomes were initially annotated using MitoZ 2.4-alpha with invertebrate mitochondrial genetic codes 24 . The annotated results from MitoZ were then imported into Geneious Prime 2021.1 software for further editing. We used the MITOS (http:// mitos. bioinf. uni-leipz ig. de/ index. py) web server 25 with invertebrate genetic codes to further revise the tRNA and PCG locations and generate the secondary structures of tRNAs and the ARWEN program 26 to check the MITOS results for tRNA secondary structures and locations. ORF finders in Geneious Prime were also used to annotate PCGs using the invertebrate genetic code. l-rRNA genes were defined according to adjacent tRNA genes (trnL1 and trnV), whereas s-rRNA genes were located by comparison with other homologous Cicadellidae species s-rRNA genes. Mitogenomic circular maps were generated using OGDRAW website (https:// chlor obox. mpimp-golm. mpg. de/ OGDraw. html) 27 29 . Tandem repeats in the CR were recognized using the Tandem Repeats Finder program (https:// tandem. bu. edu/ trf/ trf. basic. submit. html) 30 . Finally, the five newly sequenced mitogenomes of Mileewinae were submitted to GenBank with a tbl file using GB2sequin (https:// chlor obox. mpimp-golm. mpg. de/ GenBa nk2Se quin. html) 31 32 and Tettigades auropilosa (KM000129)-were used as outgroups. The detailed information and accession numbers for these mitogenomes are listed in Table S2.
For the five newly sequenced species, each PCG in the mitogenome was manually extracted using the Geneious Prime 2021.1 program. Thirteen PCG sequences were aligned in batches with MAFFT 33 using the'-auto' strategy and codon alignment mode. The alignments were refined using the codon-aware program MACSE v. 2.03 34 , which preserves the reading frame and allows the incorporation of sequencing errors or sequences with frameshifts. Ambiguously aligned fragments of 13 alignments were removed in batches using Gblocks 35 . The alignments of each individual gene were concatenated using the Phylosuite 28 program. The best partitioning scheme and evolutionary models for 39 predefined partitions were selected using PartitionFinder2 36 with a greedy algorithm 37 and Akaike Information Criterion (AIC) in Bayesian inference (BI) analyses and auto-generated in IQ-TREE 38 with Bayesian Information Criterion (BIC) in maximum-likelihood (ML) analyses. Detailed model information is shown in Table S3. BI analysis was inferred using MrBayes 3.2.6 39 with four chains under an independent partition model (two parallel runs, 10,466,900 generations) with sampling every 100 generations, in which the initial 25% of sampled data were discarded as burn-in, and the remaining trees were used to generate a consensus tree and calculate Bayesian posterior probability (PP) values after the average standard deviation of split frequencies was < 0.01. ML analysis was performed using the IQ-TREE program 38 under an edge-linked partition model for 1000 replicates of ultrafast 40 bootstraps and generated bootstrap support (BS). BI and ML trees were viewed and edited using the iTOL online tool (https:// itol. embl. de/) 41 .
The relative synonymous codon usage (RSCU) values of the five mitogenomes were shown in Fig. 2 and Table S4. The results indicated that the RSCU values and codon numbers of each mitogenome were similar. The four most frequently used codons were UUU (Phe), UUA (Leu2), AUU (Ile), and AUA (Met), wherein all of which are formed by A and U. This suggests that the most frequently used codons could be found with a preference toward more A and T than G and C.
Transfer and ribosomal RNA genes. All five new species contained 22 typical tRNA genes; of them, 14 were located on the H-strand, and the other eight on the L-strand (Table 1, Fig. 1). The length of tRNAs ranged 61-72 bp, and the concatenated total length ranged from 1422 bp (M. sharpa) to 1440 bp (M. amplimacula). The tRNA genes also presented a high AT bias with AT content ranging from 79.9% to 81.4%, and all exhibited a positive AT skew (0.014-0.02, except for −0.003 in M. amplimacula) and GC skew (0.16-0.197) ( Table 2). The secondary structures of the 22 tRNAs in the five newly sequenced mitogenomes were generated and are presented in Figure S1-S5. All tRNAs could be folded into the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm and was replaced by a simple loop, which has been observed in other Cicadellidae mitogenomes 18  Overlapping and intergenic spacers. All overlaps and intergenic spacers were identified and were displayed in Table 1. We identified 10 (P. sexmaculata) to 16 (M. sharpa) overlaps distributed in five mitogenomes ranged from 1 to 12 bp. The longest overlap was 12 bp in M. mira and located between nad6 and cytb. The five mitogenomes had 5-14 intergenic spacers, with sizes ranging from 1 to 24 bp. The longest (24 bp) intergenic spacer was identified between the trnS2 and cytb genes in P. sexmaculata. Two and three locations had same intergenic spacers and overlaps between genes across the five mitogenomes, respectively: trnP-nad6 and nad4L-trnT (2-bp long intergenic spacers), trnN-trnS1 (1-bp long overlaps), atp6-atp8 (7-bp long overlaps),   www.nature.com/scientificreports/ and trnW-trnC (8-bp long overlaps). Additionally, the differences in overlaps and intergenic spacers between the four Mileewa mitogenomes and P. sexmaculata mitogenome were found to be located at nad4-nad4L (7-bp long overlaps in four Mileewa species; 1-bp long in P. sexmaculata) and nad5-trnF (1-bp long overlaps in four Mileewa species; 3-bp long intergenic spacers in P. sexmaculata). Furthermore, 23-bp (nad4-trnH), 13-bp (cox1-trnL2), and 14-bp long (trnY-cox1) intergenic spacers were identified in the mitogenome of P. sexmaculata but not in any of the other Mileewinae species.  Table 1). The CR was the longest non-coding region, with the highest AT content, ranging from 83.3% (P. sexmaculata) to 86.6% (M. amplimacula). Except for −0.059 in P. sexmaculata, AT skew was positive (0.004-0.057). In addition, M. lamellata (0.096) and P. sexmaculata (0.042) had a positive GC skew in the CR, whereas the remaining three species had a negative GC skew (−0.06 to −0.03) ( Table 2). The detailed structural information of the CR among the five mitogenomes is shown in Figure S6-S10. The number of repeat units in the CR across the five   www.nature.com/scientificreports/ Phylogenetic relationships. Phylogenetic relationships were analyzed using 13 PCGs of 59 Membracoidea species and two outgroups mitogenome datasets. Two phylogenetic trees were reconstructed using the BI and ML methods (BI-PCGS and ML-PCGS, respectively) (Figs. 3,4). According to these two trees, 11 Mileewinae species formed a monophyletic group with high support values (BS = 100, PP = 1) and were located at the apex of the trees, and each subfamily was completely recovered as a monophyletic group in both ML and BI analyses. Both analyses demonstrated that 11 Mileewinae species were clustered together with a sister relationship with the subfamilies Idiocerinae, Evacanthinae, and Ledrinae, and these four subfamilies (Mileewinae, Idiocerinae, Evacanthinae, and Ledrinae) clustered together to form a sister group to Cicadellinae, with high support values (BS > 86, PP = 1). Moreover, the subfamilies Mileewinae, Idiocerinae, Evacanthinae, Ledrinae, and Cicadellinae clustered in one clade to form a sister group to Typhlocybinae. Our study indicated that Typhlocybinae is more ancient than Mileewinae and Cicadellinae and that Mileewinae is not a sister group of Typhlocybinae, which differs from a previous study 18,42,45,50 .

Discussion
In the present study, we sequenced and comparatively analyzed the complete mitogenomes of five species belonging to two genera of Mileewinae.  ). We compared these five mitogenomes to those of established Mileewinae species [18][19][20][21][22] and found that the length of each PCG and tRNA gene is quite similar, while the difference is mainly observed in rRNAs and CRs. All the PCGs have relatively conserved characteristics. We found, for the first time, that a single A mismatch appeared in both the trnS1 (anticodon arms) and trnR (acceptor arms) genes in the five newly sequenced mitogenomes. Some common overlapping and intergenic spacer regions across the five mitogenomes were identified as well, such as a 7-bp long overlapping region between atp8 and atp6, an 8-bp long overlap between trnC and trnW, and a 2-bp long intergenic spacer region present in both trnP-nad6 and trnT-nad4L. Moreover, we found that P. sexmaculata, which is the first Processina species with a sequenced mitogenome, has a 23-bp long intergenic spacer located between trnH and nad4. A 24-bp intergenic spacer was found between cytb and trnS2 in P. sexmaculata mitogenome but not in the mitogenomes of other Mileewinae species. We further generated the BI and ML trees with concatenated alignment of PCGs. Our results indicated that each subfamily well separated, and their monophyly weresupported. The phylogenetic relationships were as follows: (Deltocephalinae + ((Hylicinae + ((Megophthalminae + (Smiliinae + (Aetalioninae + Centrotinae))) + (Macropsinae + (Coelidiinae + Iassinae)))) + (Typhlocybinae + (Cicadellinae + (Mileewinae + (Idiocerinae + (Evacanthinae + Ledrinae)))))) (Figs. 3,4). The BI-PCGS tree had more higher support values than the ML-PCGS tree; this topology is not completely consistent with the findings of recent studies 18,42,45,51 , possibly due to the difference of the data and phylogeny models used. Furthermore, 11 Mileewinae species clustered in a monophyletic clade (PP = 1, BS = 100), which is consistent with the studies by Yu et al., Dietrich et al., and our previous study [18][19][20][21][22]52 . We also found that Mileewinae was at the apex of the phylogenetic tree, while Deltocephalinae appeared at the basal branch of the trees, which was partly the same result as that reported by Yu et al. In addition, Cicadellinae, Typhlocybinae, and Mileewinae were each recovered as a monophyletic group. In contrast to the studies by Yu et al. and Dietrich et al. 18,50 , based on the phylogenetic trees from this study (Figs. 3, 4), we considered that Typhlocybinae is more ancient than Mileewinae and Cicadellinae and suggested that Typhlocybinae is not a sister group to Mileewinae. Within the Mileewinae subfamily, all species maintained the same relationships and topologies according to both the BI and ML analyses. The phylogenetic relationships with high support values (PP > 0.8, BS > 83) within the subfamily Mileewinae were as follows: (M. sharpa + (U. puerana + ((M. ponta + (M. mira + M. lamellata)) + ((M. albovittata + (M. margheritae + M. amplimacula)) + (M. rufivena + (P. sexmaculata + M. alara)))))) (Figs. 3,4). These results are also different from those presented in a recent study of Yu et al., who mentioned the topology as (U. puerana + (M. ponta + (M. rufivena + M. alara) + (M. albovittata + M. margheritae))). Among the studied topologies, the monophyly of the genera Processina, Mileewa and Ujna were not supported. These three genera species of Mileewinae were not clearly separated. Nevertheless, we only used one species from Processina that raises concerns about its representativeness. This study provides a referenceable framework to understand the relationships between Mileewinae and other subfamilies and further enriches the mitogenome database of the tribe Mileewini. Molecular data of more variable genera of Mileewinae are required to determine the phylogenetic relationships within this group and define the monophyly of Mileewinae.