Comparative Mitogenomics And Genetic Diversity Of Mirid Bugs(Hemiptera: Miridae)

Most of mirid bugs(Hemiptera: Miridae)are economically important as agricultural insect pests.In recent years,mirid bugs have been becoming important pests greatly destroying alfalfa,vegetables and many crops in China.Mirids show high species diversity with small body size,and generally many mirid species occur in the same field.To date,little is known about systematic evolution and genetic diversity of mirids.Information on systematic evolution and genetic diversity is important to understand mechanisms of ecological adaptation and disastrous rule,which will be helpful for effectively controlling these pests.Here,we sequenced and annotated mitogenomes of five mirid species,i.e.Apolygus lucorum,Adelphocoris suturalis,Ade.fasciaticollis,Ade.lineolatus and Lygus pratenszs,and then performed comparative mitogenomic analyses combined with previously sequenced mirid mitogenomes.Next,the potential of these mitochondrial genes as molecular markers were evaluated.Finally,population genetic diversity of these five mirids collected from different alfalfa planting region in China were analyzed by using the cox1 gene sequences.The main results as follows:1.We sequenced and annotated the mitogenomes of these five mirid bugs,Apo.lucorum,Ade.suturalis,Ade.fasciaticollis,Ade.lineolatus and L.pratenszs.The mitogenome sequences of five mirids have been deposited in Gen Bank of NCBI under accession numbers: KU234536 ~ KU234540.Ade.lineolatus and Apo.lucorum were completely sequenced,whereas Ade.suturalis,Ade.fasciaticollis and L.pratenszs were incomplete mitogenomes,which lack of trn I,trn Q,trn M and a large non-coding region.The completely sequenced mitogenomes contained 37 typical mitochondrial genes(i.e.13 PCGs,22 t RNA genes and two r RNAs)and a large non-coding region(putative control region).The order and orientation of the mitochondrial genes were identical to that of the putative ancestral insect mitogenome.Gene overlaps and spacers were presented in several conserved positions in the mirid mitogenomes,e.g.trn S2/nad1(7 bp),trn W/trn C(-8 bp),atp8/atp6(-7 bp)and nad4/nad4L(-7 bp).These five mirid mitogenomes showed similar nucleotide composition:high A+T content(75.74%~77.04%),positive AT-skews(0.11~0.17)and negative GCskews(-0.22~-0.12),as is usually observed in insect mitogenomes.The pattern of codon usage in these five mirid mitogenomes was consistent with previous findings in insects and the most frequently used codons were ended with A or T,namely that A+T-rich codons were preferably used.2.Combined with 15 sequenced mirid mitogenomes(including 5 mitogenomes in this study),we provided detailed comparative mitogenomic analyses at various taxonomic levels.The genome size of eight complete mirid bugs are between 14,768 bp in Apo.lucorum and 17,747 bp in L.hesperus.The gene content,gene order,base composition,codon usage were observed in Miridae.Among 13 PCGs,four genes(cox1,cox3,nad1 and nad3)had no length variability in the 15 examined mirid mitogenomes,nad2 genes have largest variability,the length of nad5 was most variable,but conserved within each genus.The t RNAs in the three Adelphocoris species could be folded into a classical clover-leaf secondary structure,However,trn S1(AGN)in Apo.lucorum and L.pratenszs species lacked the DHU stem-loop structures.All 22 t RNA in Apolygus and Lygus species used the standard anticodon,whereas two t RNA in the three Adelphocoris species were exceptions: trn S1 changed the anticodon GCU with UCU and trn K changed the anticodon CUU with UUU.The numbers of informative sites and the genetic distances of K2 P varied among 13 PCGs,the smallest gene atp8 showed the highest informative sites and K2 P value in Miridae,whereas the cox1 gene showed least informative sites and K2 P value in Miridae.The Ka/Ks values for all PCGs were far lower than one(< 0.59),suggesting that these genes were evolving under purifying selection,but the Ka/Ks values of cox1-barcode sequence were always larger than 1(1.34 ~15.20),indicating that different genes had different evolutionary rates,whereas the same genes in different taxonomic levels showed large differences.3.Phylogenetic relationships of 15 mirid mitogenome sequences were inferred using BI and ML methods based on two mitogenomic datasets(P123,P123RT).The results all consistently supported the relationship of Nesidiocoris +(Trigonotylus +(Adelphocoris +(Apolygus + Lygus))),as revealed by nad4,nad5,rrn L and 22 t RNAs,respectively.Taken sequence length,evolutionary rate and phylogenetic signal together,the individual nad4,nad5 and rrn L genes could been used as potential molecular markers for phylogenetics,population genetics and species identification in Miridae.4.The population genetic diversity of five mirid bugs among 16 different alfalfa planting region in China were investigated using the mitochondrial cox1 gene sequence,the results showedthat the total 34 haplotypes were found in five mirid bugs,there were one haplotype in 4 population,and the remaind populations were found 2 ~ 8 haplotypes.Most of these populations have significant population genetic differentiation(P < 0.05),whereas the two populations of Ade.lineolatus had the lower genetic differentiation(Fst = 0.04).In addition,the two populations of Apo.lucorum had some gene flow(Fst =-0.02).Most L.pratenszs among 10 different geographic populations have significant genetic differentiation(P < 0.05),but there was no significant genetic differentiation among a few populations.Phylogenetic analyses showed that each mirid bugs had the independent evolutionary branchs,in which the L.pratenszs haplotypes have divided into two branches,there was no significant geographical branch structure in these two branches.However,AMOVA analyses showed that there was significant genetic differentiation among these two branches.