Mitogenomics of five Olidiana leafhoppers (Hemiptera: Cicadellidae: Coelidiinae) and their phylogenetic implications

Similar morphological characteristics and limited molecular data of Olidiana resulted in their unknown phylogenetic statuses and equivocal relationships. To further understand the genus Olidiana, we sequenced and annotated five Olidiana complete mitochondrial genomes (mitogenomes). Our results show that Olidiana mitogenomes range from 15,205 bp to 15,993 bp in length and include 37 typical genes (13 protein-coding genes, 22 tRNAs, and 2 rRNAs) and a control region. Their nucleotide composition, codon usage, features of control region, and tRNA secondary structures are similar to other members of Cicadellidae. We constructed the phylogenetic tree of Cicadellidae using the maximum likelihood (ML) and Bayesian inference (BI) methods based on all valid mitogenome sequences. The most topological structure of the obtained phylogenetic tree is consistent. Our results support the monophyletic relationships among 10 subfamilies within Cicadellidae and confirm Iassinae and Coelidiinae to be sister groups with high approval ratings. Interestingly, Olidiana was inferred as a paraphyletic group with strong support via both ML and BI analyses. These complete mitogenomes of five Olidiana species could be useful in further studies for species diagnosis, evolution, and phylogeny research within Cicadellidae.

Within Coelidiinae, Olidiana McKamey (2006) is a relatively large genus, with 99 reported species from the Oriental and Palearctic realms (Li & Fan, 2017;Nielson, 1982;Nielson, 2015;Viraktamath & Meshram, 2019). Some species of Olidiana are relevant agricultural and forest pests and cause harm by directly feeding on plant sap or by indirectly spreading viral diseases (Frazier, 1975;Li & Fan, 2017;Nielson, 1982). Olidiana exhibits morphological characteristics extremely similar to those of other Coelidiinae genera, making species distinction a challenging task. Furthermore, some species of Olidiana have been incorrectly identified, resulting in more than one synonym for the same species (Cai & Shen, 1998;Li & Fan, 2017;McKamey, 2006;Nielson, 1982;Nielson, 2015;Walker, 1851;Xu, 2000;Zhang, 1990). In addition, the taxonomic statuses of some species of Olidiana are constantly changing, and several new genera (Singillatus, Tumidorus, and Zhangolidia) have been established by revising this genus (Li & Fan, 2017;Nielson, 2015). Collectively, generic classification remains unsatisfactory, making it challenging to determine phylogenetic relationships. Therefore, it is necessary to utilize the mitogenomes of Olidiana species to classify and determine the genetic relationships among Coelidiinae species.

Sample collection and DNA extraction
Details of sample collection are presented in Table S1. All specimens were preserved in absolute ethanol and stored at −20 • C until analysis. Genomic DNA was extracted from muscle tissues of adult males using the DNeasy R Tissue Kit (Qiagen, Germany). Total genomic DNA was eluted in 70 µL of double-distilled water. The remaining extraction steps were performed according to the manufacturer's protocol. The obtained genomic DNA was stored at −20 • C until further analysis.

Mitogenome annotation and sequence analysis
The locations of 13 protein-coding genes (PCGs) were identified using the ORF Finder tool of the National Center for Biotechnology Information and the invertebrate mitochondrial genetic code. Uncommon start and stop codons were identified by comparing our sequences with those of other Cicadellidae species. The locations and secondary structures of 22 transfer RNA (tRNA) genes were determined using tRNAscan-SE (Schattner, Brooks & Lowe, 2005) and ARWEN version 1.2 (Laslett & Canbäck, 2008). Ribosomal RNA (rRNA) genes were identified based on the loci of adjacent tRNA genes and compared with those of other Cicadellidae species (Wang, Li & Dai, 2017;Wang et al., 2019c). Repeat sequences within the control region were determined using the Tandem Repeats Finder tool (http://tandem.bu.edu/trf/trf.submit.%20options.html) (Benson, 1999). The annotated mitogenome sequences of the five Olidiana species have been deposited in GenBank with the accession numbers MN780581-MN780585.

Sequence alignment and phylogenetic analysis
To determine the phylogenetic relationships among members of Cicadellidae, 74 species from 12 subfamilies of Cicadellidae as well as 6 treehopper species were included, with two Cercopoidea species (Tettigades auropilosa and Cosmoscarta bispecularis) used as outgroups (Table S2). Phylogenetic analysis was performed by independently aligning the sequences of 13 PCGs and 2 rRNA genes. For each PCG sequence, terminal codons were removed before alignment using MAFFT version 7.0 in the Translator X online server (http://translatorx.co.uk/) with the L-INS-i strategy (Abascal, Zardoya & Telford, 2010;Castresana, 2000). Each rRNA gene was individually aligned using MAFFT with the G-INS-I strategy, and poorly aligned sites were removed using Gblocks 0.91b (Katoh, Rozewicki & Yamada, 2019). The resulting 15 alignments were concatenated using MEGA version 6.
Five datasets were concatenated for phylogenetic analysis: (1) PCGs, all codon positions of the 13 PCGs with 10,044 nucleotides; (2) PCG12, first and second codon positions of the 13 PCGs with 6,696 nucleotides; (3) AA, amino acid sequences of the 13 PCGs with 3,348 amino acids; (4) PCG12R, first and second codon positions of the 13 PCGs and 2 rRNA genes with 8,448 nucleotides; and (5) PCGR, all codon positions of the 13 PCGs and 2 rRNA genes with 11,796 nucleotides. The substitution saturation of four datasets (PCG, PCG12, PCG12R, and PCGR) was tested by plotting the number of transitions and transversions against genetic divergence using DAMBE (Xia, 2013).
A phylogenetic tree was constructed using the ML method with IQ-TREE (Nguyen et al., 2015;Zhang et al., 2019). The best-fit model was selected for each partition under corrected AIC using Partition Finder 2 (Table S3) (Lanfear et al., 2017) and evaluated using the ultrafast bootstrap approximation approach for 10,000 replicates. BI was performed using MrBayes version 3.2.6 (Suchard & Huelsenbeck, 2012;Zhang et al., 2019). Following the partition schemes suggested by Partition Finder, all model parameters were set as unlinked across partitions. Two independent runs with four simultaneous Markov chains (one cold and three incrementally heated at T = 0.2) were performed for 100 million generations, with sampling every 1,000 generations.  (Table S2). Their lengths were within the ranges of complete mitogenomes reported for other Cicadellidae species (14,805 bp for Nephotettix cincticeps and 16,811 bp for Idioscopus laurifoliae) (Song, Cai & Li, 2017;Wang et al., 2018). The mitogenomic architecture closely matched that of the inferred insect ancestral mitogenome (Crease, 1999): the newly sequenced mitogenomes had closed, circular DNA, typically comprising 37 genes (13 PCGs, 22 tRNAs, and 2 rRNAs) and a noncoding control region ( Fig. 1). Of the 37 genes, most were encoded by the majority strand (J-strand) (9 PCGs and 14 tRNAs), whereas the minority strand (N-strand) encoded 14 genes (4 PCGs, 2 rRNAs, and 8 tRNAs) (Fig. 1, Table S4). However, the lengths of the 37 genes did not significantly differ between the five Olidiana species and other Cicadellidae species. The AT content of the five mitogenomes ranged from 78.0% ( (Table 1). Additionally, the five Olidiana mitogenomes comprised 1-4-bp-long intergenic spacers at eight different loci, except for the trnY -COI intergenic spacer, which had 2-10-bp-long intergenic spacers. A total of 12 gene pairs were directly adjacent to each other, whereas the other gene pairs overlapped with each other, with overlap lengths of 1-4 bp, except for trnW -trnC and trnS2-ND1, which had large overlap lengths of 7-15 bp (Table S4). The gaps among the 37 genes in the mitogenomes were relatively smaller than those among genes in most reported Cicadellidae mitogenomes (Wang et al., 2018;Wang et al., 2019a;Wang et al., 2019b;Wang et al., 2019c;Wang et al., 2020a;Wang et al., 2020b).

Control region
The control region of Olidiana mitogenomes ranged from 1,075 bp (for O. alata) to 1,804 bp (for O. longsticka). The differences in length in the control region were mainly attributed to the length and number of tandem repeats (R). All variable repeats in Olidiana mitogenomes were identified. Only a short unit (R) with two copies, both 115 bp in length, was present in O. alata. In O. longsticka and O. olbliquea, the first repeat region (R1) was 448 and 226 bp in length, respectively, and both comprised two units. The other two repeat regions, i.e., R2 and R3, were located after R1, and they were 345 and 433 bp (O. longsticka) and 129 and 28 bp (O. olbliquea) in length, respectively; both comprised three copies. O. ritcheri and O. tongmaiensis comprised four types of units: R1, 2 × 135 bp; R2, 2 × 195 bp; R3, 3 × 116 bp; and R4, 4 × 78 bp and R1, 2 × 281 bp; R2, 3 × 191 bp; R3, 3 × 81 bp; and R4, 3 × 4 bp, respectively (Fig. 4). Similar to the long intergenic spacers in other insect species, the repeat regions in leafhoppers may be attributed to an alternative origin of mitogenome replication (Dotson & Beard, 2001). The AT content (84.1%-85.8%) of the control regions was generally higher than that of the other regions. This is in part due to damage or accumulation of mutations in the mitochondrial DNA (Martin, 1995).  (Table 1). Moreover, these control regions were compared with previously reported control region sequences; their differences were very large, and no obvious correlation or similarity was found with existing sequences.

DISCUSSION
The phylogenetic positions of the subfamilies Ledrinae and Deltocephalinae were different from those observed in previous studies, in which Deltocephalinae was located at the base of the Cicadellidae phylogenetic tree (Du et al., 2017b;Wang et al., 2019b;Wang et al., 2019c). However, in our study, the main topological structure showed that Ledrinae, instead of Deltocephalinae, was located at the base of the phylogenetic tree. This result confirms that Ledrinae is an ancient group of leafhoppers. This result is in agreement with that reported by Chen et al. (2019) (Li & Fan, 2017;Viraktamath & Meshram, 2019;Zhang, 1990). Interestingly, the seven Olidiana species could be divided into three groups based on significant differences in their morphological characteristics, which were characterized by body color, shape, and position of the processes on the aedeagus shaft. Therefore, based on complete mitogenome phylogenetic analysis and comparison of morphological characteristics, we propose Olidiana as a paraphyletic genus and suggest that it should be further examined based on the shape and position of the processes on the aedeagus shaft.

CONCLUSIONS
In this study, we sequenced and annotated the complete mitogenomes of five Olidiana species. The general genomic characteristics (gene content, gene size, gene order, base composition, PCG codon usage, and tRNA secondary structure) of the Olidiana mitogenomes were mostly consistent with those of reported Cicadellidae mitogenomes. In addition, we performed phylogenetic analyses to infer the probable relationships among the Cicadellidae subfamilies as well as to confirm the phylogenetic relationship among the Olidiana species. Our results support the presence of a monophyletic relationship among the 10 Cicadellidae subfamilies and confirm that Iassinae and Coelidiinae are sister groups with high approval ratings. Interestingly, phylogenetic analyses of the mitogenomes support our assertion that Olidiana is a paraphyletic genus, with the following topology: (O. tongmaiensis + (O. longsticka + (O. olbliquea + O. alata)) + (H. fascianus + (O. ritcheriina + (Olidiana sp. + O. ritcheri)))). Our findings will not only improve our understanding of the phylogenetic relationships of related insects but also contribute toward their taxonomic classification within Cicadellidae. Further studies of the combination of morphological and molecular characteristics of additional species are warranted to confirm the taxonomy of Cicadellidae.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the National Natural Science Foundation, China [Grant no. 31672342] and the Program of Excellent Innovation Talents, Guizhou Province, China [Grant no. 20164022]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.