Structure And Phylogeny Of The Complete Mitochondrial Genomes Of Eurymelinae(Hemiptera: Cicadellidae)

Leafhopper subfamily Eurymelinae,belonged to the supperfamily Membracoidea of Auchenorrhyncha in Hemiptera,is an important phytophagous insect group including more than 22,000 species reported worldwide.The taxonomic status of Eurymelinae has always been controversial,and the level of family classification is unstable.Until 2018,Dietrich combined the previous subfamilies of Idiocerinae and Macropsinae into the leafhopper subfamily Eurymelinae,this system was followed by many authors.Mitochondrial genome is characterized by small molecular weight,maternal inheritance,rapid evolutionary rate and genetic conservation.In recent years,with the development of sequencing technology,mitochondrial genome(mitogenome)has been widely used as a molecular marker in the discussion and analysis of insect taxonomic category.As of December 2022,NCBI has included 183 complete mitochondrial genome sequences of leafhoppers,with data concentrated in a large number of widely distributed subfamilies.More than 120 sequences are available of Cicadellinae,Typhlocybinae and Deltocephalinae,while the subfamily leafhopper Eurymelinae has been relatively poorly studied,with only 11 complete mitochondrial sequences being measured.In this study,the mitogenomes of representative species of nine genera of Eurymelinae were sequenced,which obtained 15 complete mitochondrial genomes(including one new record species).All 26 species of Eurymelinae complete mitogenome sequences(including 11 species registered in NCBI)were analyzed for genome structure.At the same time,177 Cicadellidae species were selected as ingroups and three Cercopidae species were selected as outgroups to construct four datasets of PCGs,PCGRNA,PCG12 and PCG12 RNA for phylogenetic reconstruction using the maximum likelihood and Bayesian inference method to discuss the phylogenetic relationship of Cicadellidae.The main results are as follows:1.Mitochondrial genomic structure of Eurymelinae(1)The mitochondrial genomic of all Eurymelinae ranges from 15,287 bp-16,842 bp in total sequence length,with 37 genes ranging from 10,934 bp-10,991 bp in length,6 bp-42 bp in spacer length in non-coding regions,and 991 bp-2,503 bp in control region length;the total sequence length difference is 1,555 bp.and the control region length difference is 1,512 bp.The total mitochondrial gene length difference is mainly caused by the difference in the control region,which is consistent with the published genome of Cicadellidae insects.(2)The complete mitochondrial gene sequences of all Eurymelinae species are reverse complementary double-stranded circular molecules,including 37 typical mitochondrial genes and one non-coding region.All species are arranged in the same way,with no gene rearrangement occurring.(3)The mitogenome base of Eurymelinae show a significant A+T bias,with the AT content of the whole sequence ranging from 76.4% to 79.6%.The highest AT content of Rhytidodus viridiflavus was rrn S-12 S,and the highest AT content of other wide-headed leafhoppers was in the control area.All the regions with the lowest AT content are proteincoding regions.(4)The start codons of the protein-coding genes of Eurymelinae are ATN(ATT,ATG,ATA,ATC)and TTG,and the termination codons are TAA,TAG and T.A maximum of four start codons of a single species are used.ATP6,ND2 and COX2 are relatively stable,so only one termination codons was used.(5)In the mitogenomes of Eurymelinae,except for the deletion of the dihydrouridine arm(DHU)deletion in the secondary structure of t RNA-Ser1,the remaining 21 t RNAs are typical clover structures.All species has two groups of homologous receptors for leucine(Leu)and serine(Ser),with the codons are standard invertebrate conventional anticodons.(6)Among the 13 protein-coding genes of mitogenomes of Eurymelinae,ATP8(Pi =0.5180)has the fastest evolution rate,while COX1(Pi = 0.0828)has the slowest evolution rate.2.Phylogenetic analysis of CicadellidaeIn this study,the phylogenetic tree is constructed using the Bayesian inference and maximum likelihood method with the following four datasets: PCG,PCGRNA,PCG12 and PCG12 RNA.The results are as follows:(1)Except for the subfamily Eurymelinae,the remaining 12 subfamilies of Cicadelidae are all monophyletic groups,supporting the classic subfamily system proposed by Evans.The subfamily relationships of the Cicadellidae insects involved in this study are different in phylogenetic relationships under different data sets or tree construction models,but in general they can be divided into three branches: the Idiocerini,Megophthalminae,Signoretiinae,Evacanthinae and Ledrinae are often clustered into one branch;the Cicadellinae,Typhlocybinae and Mileewinae are often clustered into one branch;the Coelidiinae,Iassinae,Macropsini,Ulopinae,Hylicinae and Deltocephalinae are often clustered into one branch.(2)In this study,the two tribes of Eurymelinae,the Idiocerini and the Macropsini,are distantly related,but the Idiocerini and Macropsini are monophyletic groups.In Idiocerini,except Idiocerus is not a monophyletic group,the other seven genera are monophyletic groups.Populicerus and Rhytidodus cluster into one branch and forms a sister group relationship,Koreocerus and Chinaocerus formed a sister group relationship,and their phylogenetic relationship are as follows:(((((Idiocerus + Metidiocerus)+(Populicerus +Idiocerus)+ Rhytidodus)+(Koreocerus + Chinaocerus)))+ Idioscopus + Busonia).In Macropsini,all genera in the group are monophyletic groups,Pedionis emerge as the sister group to Oncopsis,and their internal genetic relationship are as follows:(Macropsis +((Pedionis + Oncopsis)+ Pediopsoides)).