Characterization And Evolution Of Hymenopteran Mitochondrial Genomes And Their Phylogenetic Utility

Metazoan mitochondrial genome is usually used for determining population structure. phylogenetic relationships and general evolutionary events because of its small genome size, stable gene content,uniparental inheritance,lack of extensive recombination and the accelerated rate of nucleotide substitution.Previous studies indicated that there are a lot of special characters in hymenopteran mitochondrial genomes,such as high A+T content and frequent gene rearrangement.However,limited mitochondrial genomes,representing only a few families in this species-rich insect order,have been sequenced.Therefore,characterization of more hymenopteran mitochondrial genomes is needed to address evolutionary questions and traits,and to revole phylogenetic relationships of this group.In this study,we conducted a large scale sequencing and annotation of hymenopteran mitochondrial genomes,and comparied them with other sequenced mitochondrial genomes in Hexapoda.especially in Hymenoptera.to explore the evolutionary traits and mechanisms in mitochondrial genomes.Additionally,we assessed the utility of complete mitochondrial genome sequences as markers for phylogenetic analyses of Hymenoptera and Holometabola. Consequently we concluded eight main points as follows:(1) Eleven mitochondrial genomes representing five hymenopteran families and one mitochondrial genome representing Mecoptera(Hexapoda:Holometabola) were sequenced and annotated for the first time.Gene content,intergenic region,gene arrangement,codon usage,tRNA and rRNA secondary structures as well as characters of A+T-rich region were analyzed in each sequenced mitochondrial genome.A large intergenic region was found between atp8 and atp6 in Evania appendigasrer,which could form an mRNA secondary structure and therefore was supposed to facilitate cleavage between these abutting proteins transcript.An extremely A+T-riched(99.1%) 1515 bp tandem repeat region with three types of repeat elements was located between cox1 and cox2 in Diadegma semiclausum,and the most likely ancestral element originated from the 3' end of cox1.The formation of this region was proposed to be independent tandem duplications followed by mutation/insertion/deletion.The inversion of A+T-rich region in Hexapoda was evidenced structurally for the first time due to its exsistence in Diachasmimorpha longicaudata,Spathius agrili and Cotesia vestalis mitochondrial genomes.The most extensive rearrangement events in Hymenoptera were found in Cotesia vestalis mitochondrial genome,including seven protein-coding and 11 tRNA genes.(2) Overlapping genes were rarely rearranged,while those rearranged genes usually have intergenetic regions inbetween.A conserved motif was found between trns1 and nad1.which was supposed to be associated with the termination of genome transcription(mtTERM).trnS2 and trnK usually use abnormal anticodon TCT and TTT respectively,which were probably related to gene rearrangement.(3) The values of uncorrected nucleotide diversity(Pi),ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions(Ka/Ks) and Jukes-Cantor corrected Ka/Ks indicated that mitochondrial genomes evolved faster in Hymenoptera than in other holometabolous orders.For individual genes in holometabolous mitochondrial genomes,nad6,atp8 and nad4l evolved the fastest while cox1,cox2,cob and nad5 evolved much slowly.In Hymenoptera,Apis mellifera,Bombus ingitus,Melipona bicolor,Cephalonomia gallicola,Radoszkowskius oculata and Macrocentrus camphoraphilus had a high Ka/Ks value,while D.longicaudata,Nasonia longicornis and Perga condei had a low Ka/Ks value.The values of Ka/Ks are variable among species of different subfamilies in the famliy Braconidae.Most species had more transitions than transversions in Hymenoptera. Evania appendigaster and R.oculata had more substitutions than other species.(4) In Hymenopteran mitochondrial genomes,gene arrangement is conserved in the basal suborder,Symphyta,while frequent gene rearrangements are observed in the other suborder, Apocrita.Gene rearrangements mainly occurred in four hot spots,i.e.A+T-rich region-nad2, nad2-cox1,cox2-atp8 and nad3-nad5.Most rearranged genes are tRNAs,but only a few protein-coding or rRNA genes were rearranged.Translocation,inversion,shuffling and remote inversion events were found to be present in nearly equal frequency.The remote inversion was supposed to be caused by two independent recombination events.(5) The hymenopteran mitochondrial genomes had the highest A+T content in Holometabola,followed by Lepidoptera,while the Thysanoptera and Isoptera had the lowest one.A+T content is usually higher in Apocrita than in Symphyta except for E.appendigaster, which even had a lower A+T content than P.condei,a species of Symphyta.(6) In all braconid species,strand asymmetry was reversed,which supported the hypothesis that reversal of strand asymmetry is caused by inversion of replication/transcriotion -related A+T-rich region.(7) A broad survey of strand asymmetry among 133 sequenced hexapod mitochondrial genomes showed that reversal of strand asymmetry evolved triple in Hexapoda,as here in Braconidae and other two families,Philopteridae(Phthiraptera) and Aleyrodidae(Hemiptera). The sign of AT skew on individual gene is associated with gene direction while GC skew is associated with replication orientation.Accordingly,we proposed a new hypothesis of "Asymmetric deamination" for strand asymmetry,that is.more C deaminations during transcription than replication whereas more A deamination during replication than transcription.(8) Phylogenetic analyses show that complete mitochondrial genome sequences could recover the documented basal position of Hymenoptera among holometabolous orders.The sister-group relationship between Neuropterida(Neuroptera+Megaloptera in this study) and "Mecoptera+Diptera" was resolved.The clade Proctotrupomorpha (Proctotrupoidea+Chalcidoidea in this study) and the sister-group relationship between Ichneumonoidea and Aculeata were supported within Hymenoptera.When the families of Evaniidae,Bethylidae,Mutillidae are included in analyses,the supported relationship in hymenopteran lineage is "Ichneumonoidea+(Evaniidae+((Bethylidae+Bethylidae)+(Apoidea+ (Eumenidae+Vespoidea))))",indicating that Evaniidae is a sister group of Aculeata. lchneumonoidea a sister of"Evaniidae+Aculeata",and Vespoidea a paraphyletic group.In Braconidae.Aphidiinae is sister to Cyclostomes,Microgastroids and Helconoids form the clade Noncyclostomes.and a relationship of"(Aphidiinae+Cyclostomes)+Noncyclostomes" was supported.