The Complete Mitochondrial Genomes Of Four Pierid Butterfly Species And Their Phylogenetic And Phylochronological Implications

Pieridae is one of the major groups of butterflies(Lepidoptera,Ditrysia).Due to the diffculties in identifying morphological synapomorphies within the group and the scarcity of the fossil records,only a few studies on higher phylogeny of Pieridae have been reported to date.In this paper,we determined the complete mitogenome sequence of three Aporia species(Aporia bieti,Aporia hippia,and Aporia martineti)and Mesapia peloria,with detailed comparison with other available pierid butterfy species with respect to their genomic structure and composition,such as gene content,tRNA secondary structure,rRNA secondary structure,and gene order.Moreover,the phylogeny of the Pieridae comprising the three main subfamilies(Pierinae,Coliadinae and Dismorphiinae),was reconstructed for the first time based on the sequence data of mitochondrial thirteen protein-coding genes(PCGs).Additionally,the divergence time of these main Pieridae groups was estimated using a relaxed molecular clock method,aimed at presenting a timescale for the origin and diversifcation of the Pieridae.The four complete mitogenome sequences of A.bieti,A.hippia,A.martineti,and M.peloria,are 15,147,15,154,15,192,and 15,159 bp in size,respectively.Each genome contained 13 PCGs,22 putative tRNA genes,2 rRNA genes,and a non-coding control area called A+T-rich region.The mitogenomes of three pierid species,namely A.bieti,A.hippia and M.peloria all harbor 13 intergenic spacer sequences,with their sizes ranging frrom 1 to 43 bp(totalling 92 bp),1 to 47 bp(totalling 87 bp),1 to 57 bp(totalling 102 bp),respectively;whereas A.martineti mitogenome harbors 11 intergenic spacer sequences with their sizes ranging from 1 to 46 bp(totalling 87 bp).In addition,some overlapping sequences are found to be existed between tRNATrp and tRNACys,ATP8 and ATP6,ND4 and ND4 L of each mitogenome of the four pierid species in this study.Their PCGs are 11145 bp,11151 bp,11154 bp and 11151 bp,containing a total of 3715,3717,3718,and 3717 codons,respectively.For each species,twelve of their mitogenomic 13 PCGs start with the typical ATN codons,whereas the COI begins with CGA.For the stop codon of each species,the ND3 uses TAG,the COI,COII,ND4,and ND5 use single nucleotide T,whereas the remaining eight of the 13 PCGs(ATP6,ATP8,COIII,Cytb,ND1,ND2,ND4 L,ND6)use the standard TAA.All the mitogenomes of four pierid species contain 22 tRNA genes,and among which 14(trnM,trn I,trnW,trnL2(tRNALeu(UUR)),trnK,trnD,trnG,trnA,trnR,trnN,trnS1(tRNASer(AGN)),trnS2(tRNASer(UCN)),trnE,trnT)are encoded by the heavy chain and the other eight(trnQ,trnC,trnY,trnF,trnH,trnP,trn L1(tRNALeu(CUN)),trnV)are encoded by the light chain.All the tRNAs form the typical clover leaf secondary structure except the tRNAser(AGN),which lacked the dihydrouridine loop.In addition,all the tRNA genes harbor base pair mismatches,such as G·U,U·G,U·U,and A·A.The mitogenomic large subunit ribosomal RNA genes(16S rRNAs)are 1326 bp,1326 bp,1328 bp,and 1323 bp in size,with 84.2,84.6,84.4 and 85.0% A+T content respectively;while the small subunit ribosomal RNA genes(12S rRNAs)are 771 bp,774 bp,774 bp,and 772 bp in size,with 84.6,84.8,85.2 and 85.2% A+T content respectively.The mitogenomic A+T-rich regions are 382,418,362,and 378 bp in size,with 94.7,93.1,92.8,and 94.7% A+T content,respectively.Each A+T-rich region of the mitogenomes contains a poly-T stretch upstream of the characteristic motif ATAGA,and a few of microsatellite-like elements preceded by the ATTTA motif,such as(TA)n(N= 6-10).Phylogenetic analysis using mitogenomic 13 PCG sequence data with maximum likelihood(ML)and Bayesian inference(BI)methods showed that the Pieridae family contains three well supported clades(subfamilies Dismorphiinae,Coliadinae,and Pierinae),and their relationships were(Dismorphiinae +(Coliadinae + Pierinae)).Furthermore,the Pierinae contains three well supported subclades: the first is the Pieris,the second is the grouping of(Aporia + Delias + Prioneris),and the third is Hebomoia + Anthocharis,with the first and second constituting the Pierini,the third making up the Anthocharini.The phylogenetic analyses also indicated that the genera Hebomoia and Anthocharis are closely related and sister to each other,supporting the traditional classification of tribe Anthocharidini;the Mesapia peloria is shown to be clustered within the Aporia group,suggesting that the genus Mesapia should be recategorized into the genus Aporia and reduced to the taxonomic subgenus level.Lastly,phylogenetic analyses of this study verifies that the Coliadinae contains two well supported subclades Gonepteryx and(Colias + Catopsilia),as the traditional morphological view holds.Phylochronological analysis using fossil and hostplant calibrations with relaxed molecular dating method showed that the Pieridae began to diverge at about 92.45 million years ago(mya)in the middle or late Cretaceous Period,which is consistent with the flourishing of the angiosperms that replace the gymnosperms and provide the Pieridae with abundant host plants;the divergence between Coliadinae and Pierinae is dated back at about 86.34 mya,which is consisten with the shift of the host plant of Pierinae,from Fabales to Brassicales;the two subfamilies Pierinae and Coliadinae are dated to diverge at about 76.46 mya and 51.05 mya respectively;Pieris and Aporia are dated to diverge at about 35.4 mya and 31.74 mya respectively in the Eocene,and this species radiation of the two genera is correlated with the fastest,strongest and biggest global warming event happened at that time in the history of the Earth.