Sequencing And Analysis Of The Complete Mitochondrial Genomes Of Four Species From Blondeliini(Diptera:Tachinidae)

Objective:The complete mitochondrial genomes of four species from the tribe Blondeliini were sequenced.The homology and difference of the mitochondrial genomes of four species were compared and analyzed by comparative genomics.Based on the mitochondrial protein coding genes(PCGs),the phylogenetic tree of the family Tachinidae was reconstructed to study the phylogeny of Tachinidae and the evolutionary relationship among higher taxa of Tachinidae.Methods:The mitochondrial genomes of four species of Blondeliini were sequenced by long PCR plus primer walking method combined with High-throughput sequencing.The DNA fragments were assembled and annotated by bioinformatics principles and techniques.Based on 13 protein coding genes(PCGs),Sarcophaga ruficornis Fabricius and Fucellia costalis Stein as outer group,4 species of Blondeliini and 15Tachinids as inner group,the phylogenetic tree was reconstructed by Maximum likelihood method to study the phylogeny of family Tachinidae and tribe Blondeliini.Results:(1)The nearly full length of the mitochondrial genomes of the four species are15,841 bp(Compsilura concinnata),15,250 bp(Vibrissina turrita),15,041 bp(Trigonospila ludio)and 15,483 bp(Eophyllophila elegans),respectively.The mitochondrial DNA molecules of the Blondeliini are typical circular closed double-stranded structure,composed of 38 basic structural elements(13 PCGs,2r RNA genes,22 t RNA genes and a non-coding control region),of which 2 r RNA genes(16S r RNA,12S r RNA),8 t RNA genes(t RNA^Val,t RNA^Leu(CUN),t RNA^Pro,t RNA^His,Trna^Phe,t RNA^Tyr,t RNA^Cys,t RNA^Gln)and 4 PCGs(ND1,ND4L,ND4,ND5)are coded by the N strand,and the remaining 23 genes are coded by the J strand.The sequence and direction of the genes are relatively conservative,and no gene rearrangements have been found.(2)The content composition of four nucleotides in the mitochondrial genomes of the four species are not balanced,it shows obvious AT bias.(3)The mitochondrial genomes of the four species from tribe Blondeliini all have gene overlapping and spacing between genes,the overlap length 1?50 bp.The maximum overlap length is located between the 16S r RNA and t RNA^Val in E.elegans.Most of the gene gaps are 1?16 bp in length.There is the largest gap between the 16S r RNA and t RNA^Val in C.concinnata,which is 60 bp in length.(4)16S r RNA is located between t RNA^Leu(CUN)and t RNA^Val,and 12S r RNA is located between t RNA^Val and Control Region.The length of 16S r RNA are between 1,259 bp(T.ludio)and 1,342 bp(V.turrita),and 12S r RNA is between 783 bp(V.turrita)and 821 bp(T.ludio).(5)Except for COI which uses TCG as the start codon,the other 12 protein coding genes use ATN as the start codon.Most protein coding genes use TAA and TAG as stop codons,while TA or T as incomplete stop codons only in a few genes,TTA is the most frequently used codon.In C.concinnata,there are no CCG,CGC or CGG codons,and the frequency of use is 0.(6)The 22 t RNA genes are discontinuously distributed in the mitochondrial genome,ranging in size from 63 bp(t RNA^Arg in C.concinnata)to73 bp(t RNA^Val in V.turrita and E.elegans),the total length ranges from 1,466 bp(C.concinnata)to 1,492 bp(T.ludio),which accounts for 9.25%to 9.91%of the complete mitochondrial genome length.Except for the secondary structure of t RNA^Ser(AGN),which lacks the DHU-arm to form a circular structure,the other 21 tRNAs assemble into clover-shaped typically.In addition to the typical A-U and C-G pairing,there are U-U or A-C mismatched base pairs in different stems in Blondeliini occasionally.U-G mismatch occurs in T.ludio.(7)In phylogenetic studies,the species of subfamily Exoristinae are assembled into a branch,indicating that Exoristinae is a monophyletic clade;therein,most of Blondeliini and Eryciini are sister groups,Winthemiini and(Exoristini+(Goniini+(Blondeliini+Eryciini)))are sister groups;In Blondeliini,Eophyllophila elegan is located at the base of the branch of(Goniini+(Blondeliini+Eryciin)),meaningwhile Trigonospila ludio and Winthemiini are assembled into a branch,it is speculated that Blondeliini is a monophyletic clade.(8)Dexiinae is located at the base of the branch of Tachinidae,and is the most primitive subfamily,subfamily Phasiinae and Tachininae are sister groups,Exoristinae is the most evolved subfamily in Tachinidae.Conclusions:The phylogeny of the family Tachinidae was studied by sequencing and comparative analysing the complete mitochondrial genomes of four species from tribe Blondeliini,the results showed that the lengths of the mitochondrial genomes which are 15,841 bp(Compsilura concinnata),15,250 bp(Vibrissina turrita),15,041 bp(Trigonospila ludio)and 15,483 bp(Eophyllophila elegans).It is suggested that subfamily Exoristinae is a monophyletic clade;the tribe Blondliini is a paraphyletic clade,genera Trigonospila and Eophyllophila should be removed from the tribe Blondeliini,and their taxonomic status need to be further studied;Dexiinae is the most primitive subfamily in Tachinidae,subfamily Phasiinae and Tachininae are sister groups,Exoristinae is the most evolved subfamily in Tachinidae.