Phylogeny Of The Leafhopper Subfamily Deltocephalinae (Hemiptera:Cicadellidae) Based On The Mitochondrial Genomes

Deltocephalinae is the largest subfamily of Cicadellidae,many of these species are important agricultural pests.The members of Deltocephalinae can not only direct sucking plant juice predatory host nutrition,but also transmit plant virus disease.Because of its huge number of species and significant morphological differences,the range and classification of high-level in Deltocephalinae is constantly changing.At present,most phylogenetic studies on Deltocephalinae are mainly based on the data of partial gene sequences and morphological characters.With the development of sequencing technology,insect mitochondrial genome(mitogenome)become an important molecular marker in the study of insect systematics.Mitogenome has been widely used in phylogenetic studies of different taxa level within various insects due to its maternal inheritance,compositional stability and genetic conservation.Now the number of mitogenomes in Deltocephalinae is limited,and with no comparative genomics and phylogenetic studies based on mitogenome data.Therefore,supplementation of the mitogenome data and subsequent comparative analysis are of great significance for studying the phylogeny of Deltocephalinae.At this study,we chose 30 leafhoppers species from 22 tribes of Deltocephalinae.After High-throughput sequencing,assembly,annotation and gap closing,the complete or nearly complete mitogenome sequences were obtained.With the combination of nine species from Genbank,we conducted the comparative mitochondrial genomics studies for 39 Deltocephalinae leafhoppers.Additionally,all mitogenomes of leafhoppers and treehoppers so far in Genbank were selected as ingroups,and three species of Cicadoidea and four species of Cercopoidea were selected as outgroups,and phylogenetic analysis were implemented based on both site-homogeneous and site-heterogeneous models.Main conclusions were drawn as below:(1)Mitogenomes of Deltocephalinae leafhoppers are closed double-stranded circular molecules,with the length ranging from 14,805 to 16,626 bp.Both whole mitogenome and various parts showed significant AT nucleotide bias.The third codon position of protein-coding genes(PCGs)had higher A + T content than that of the frst and second positions,all mitogenomes showed positive AT-skews and negative GC-skews.(2)Gene order of the most Deltocephalinae leafhopper mitogenomes were identical to that of the putative ancestral insect Drosophila yakuba.Only five species had gene rearrangement in the t RNA genes,Japananus hyalinus and Eupelix cuspidata changed the t RNA cluster trn W-trn C-trn Y to trn Y-trn W-trn C,Macrosteles quadrilineatus and Macrosteles quadrimaculatus changed the t RNA cluster trn W-trn C-trn Y to trn C-trn W-trn Y,Hodoedocus sp.changed the t RNA cluster trn A-trn R-trn N-trn S1-trn E-trn F to trn R-trn E-trn F-trn A-trn Ntrn S1.(3)Most of the PCGs started with the standard ATN codons,and also some started with TTG.Except for a few terminated with the incomplete T or TA,most of the PCGs using TAA or TAG as the stop codons.Evolutionary rate were different for each PCG,the average ratio of all Ka/Ks were less than 1 which indicated that these genes were evolving under purifying selection.The results showed that cox1 had the lowest evolutionary rate,indicating that the highly conservative of this region.(4)There were some gene overlaps in each Deltocephalinae leafhopper mitogenomes,and two 7 bp overlaps(atp8-atp6、nad4-nad4l)were stable to be found.All mitogenomes had some non-coding regions between genes,and the longest intergenic spacer was the control region.The difference in length of control region among various species were large,and tandem repeat units were found in some species.(5)There were no exactly the same topologies in the results of phylogenetic trees yielded by homogeneous and heterogeneous models,and the difference mainly exist in the inner groups of Deltocephalinae.Monophyly of Membracoidea was strongly supported in all phylogenetic trees,and the relationship(Membracoidea +(Cicadoidea + Cercopoidea))were well supported by all results.All treehoppers got together to one clade and paraphyletic to leafhoppers.(6)All Deltocephalinae leafhoppers got together to one clade and separated from other subfamilies leafhoppers,indicating the monophyly of this group.Half of the homogeneous models results supported all other but Deltocephalinae leafhoppers got to one clade,and Deltocephalinae was sister to these groups.The phylogenetic relationships within Deltocephalinae were different under various combinations.Some well supported nodes which had high support values were the species from the same tribe or a few tribes,and these relative stable topologies were consistently recovered by all analyses.And also,the poorly supported nodes with low support values were the clades which always shift between various combinations.(7)Within Deltocephalinae,except for Athysanini and Scaphoideini,all the representative species from the same tribe could get together to one clade which made these tribes monophyletic.A few relative stable relationships within some groups were inferred from most analyses,Pendarini and Cicadulini;Acinopterini,Acostemmini,Goniagnathini and Luheriini;Eupelicini and Stenometopiini;Cochlorhinini and Athysanini(Norvellina sp.);Bonaspeiini,Athysanini(Tambocerus sp.)and Selenocephalini;Deltocephalini,Tetartostylini and Paralimnini,and all the above mentioned tribes could got together and had close relationships with each other separately.Acinopterini,Acostemmini,Goniagnathini,Luheriini,Penthimiini and Hecalini were the early derived groups in Deltocephalinae.