Phylogenetic And Phylogeographical Analysis Of The Main Groups Of Pierid Butterflies (Lepidoptera:Ditrysia:Pieridae)

The Pieridae is one of the lepidopteran groups,which is categorized into the suborder Ditrysia,covering about 1,240 species all around the world,mostly in zoogeographic regions of the Palearctic and Oriental.Morphological and Molecular biological evidences showed that the Pieridae contains four subfamilies?Coliadinae,Dismorphiinae,Pseudopontiinae and Pierinae?.However,up to date,the phylogenetic relationships among the four subfamilies and the major taxa within each subfamily have not reached a consensus result.In addition,studies on the phylochronology and phylogeography of their major taxa of the family are still scarce.In this paper,we firstly determined the complete mitgenome of the four pierid species,named the Baltia butleri,Talbootia naganum,Pontia callidice and Pontia daplidice,and compared them with the Prioneris clemanthe formerly determined by our laboratory and other 17 pierid species mitogenome sequence data with detail.Additionally,according to different mitogenomic datasets,including the concatenated13 PCGs,13 PCGs+2 rRNAs,2 rRNAs,2 rRNAs+22 tRNAs and 22 tRNAs sequences respectively,we reconstructed the phylogeny of the main Pieridae lineages with BI and ML methods to further discuss the phylogenetic relationship between their various groups.Meanwhile,we sequenced the mitochondrial COI,nuclear wingless and EF-1?of 40 pierid species,combined with other corresponding gene sequence data of pierids that known in GenBank to reconstruct the pieridae phylogeny on a larger sampling scalewith BI and ML methods.Based on BEAST relaxed Molecular clock algorithm,we estimated the origin and divergence time of major groups of Pieridae using relevant fossil records as calibration pionts.Additionally,we reconstructed their ancestral geographical distributions based on the modern biogeographic distributions,in order to clarify their historical biogeography.According to results of comparative analysis,the complete mitochondrial genomes'of Baltia butleri,Talbootia naganum,Pontia callidice and Pontia daplidice,are 15,124,15,155,15,109,and 15,124 bp in size respectively.The mitochondrial genomes of the four species all contain 13 PCGs,22 putative tRNA genes,2 rRNA genes,and a non-coding control area called A+T-rich region.In addition,like other butterfly mitogenomes,the three tRNAs between the A+T-rich region and ND2,are arranged in the pattern of tRNA^Met/tRNA^Ile/tRNA^Gln which is different from the pattern that found in the ancestral insects(tRNA^Ile/tRNA^Gln/tRNA^Met).The nucleotide compositions of the four mitogenomes are shown to be significantly A+T biased,with the values of 79.7%,79.8%,79.8%and 79.9%,respectively.The mitgenomes of four pierid species are closely arranged in structure,harboring some intergenic spacers and overlapping regions as other lepidopterans,for example,the four mitgenomes harbor10,9,13 and 11 intergenic spacers,with a total length of 85,85,113 and 103 bp,and several 1 to 8 bp overlapping regions respectively.Twelve of the 13 PCGs start with the typical ATN?ATG,ATA and ATT?codons,whereas the COI begin with CGA;nine PCGs?ATP6,ATP8,COIII,Cytb,ND1,ND2,ND4L,ND5 and ND6?take ATT as the stop codon,except Pontia daplidice ND5 takes TAG as the stop codon;ND3 uses TAG as stop codon,COI and COII use single T as the stop codon,ND4 uses incomplete TA as the stop codon.Except tRNA^ser?AGN?which lacks the dihydrouridine loop,all tRNAs have a typical clover secondary structure,with typical base pair mismatches as found in other insects reported;in addition,the predicted lrRNA and srRNA topologies are also generally in congruence with those previously reported from other lepidopterans.The A+T-rich regions the four mitogenomes are361,387,362,and 374 bp,respectively,with their A+T contents 91.4%,95.0%,90.9%,and 92.7%,respectively.Additionally,these regions also contain some typical structures characteristics of lepidopterans,such as a poly-T stretch upstream of the motif ATAGA,the ATTTA motif is followed by a section of?TA?n?n=6-10?similar to the microsatellite structure.BI and ML phylogenetic trees generated upon the datasets of 13 PCGs,13 PCGs+2 rRNAs,2 rRNAs and 2 rRNAs+22 tRNAs showed that?1?the Pieridae of this study is composed of three well-supported clades?Dismorphiinae,Coliadinae,and Pierinae?,with the relationship of?Dismorphiinae+?Coliadinae+Pierinae??;?2?the subfamily Dismorphiinae?Leptidea morsei herein?is sister to the grouping of other two subfamilies?Coliadinae and Pierinae?;?3?the Coliadinae and Pierinae are both monophyletic and sister to each other;?4?the Pierinae subfamily contains two sister tribes,the Anthocharidini and Pierini;?5?the Anthocharidini is made up of two sister genera Anthocharis and Hebomoia;?6?the Pierini is constituted by two clades:the first one is the?Pieris+?Baltia+?Pontia+Talbotia???,the second one is the?Prioneris+?Delias+Aporia??;?7?the Coliadinae subfamily contains three genera,with their relationship of??Catopsilia+Colias?+Gonepteryx?.Furthermore,this study indicates that tRNA sequence data alone was not sufficient enough to reconstruct the deep phylogeny robustly.In addition,BI and ML phylogenetic trees based on COI,wingless and EF-1?gene were reconstructed,and the results showed that:?1?the two resultant trees harbored the same topologies,the Pieridae was made up of four clades?subfamilies Coliadinae,Dismorphiinae,Pseudopontiinae and Pierinae?with relatively strong supports,with their relationship of?Coliadinae+??Dismorphiinae+Pseudopontiinae?+Pierinae??.?2?the subfamily Pseudopontiinae of this study only represented by Pseudopontia paradoxa was sister to the Dismorphiinae.?3?the subfamily Coliadinae totally contained 10 genera,and was made up of two clades:the first one is the Eurema,within which the Pyrisitia proterpiah and Teriocolias zelia are suggested to be included;the second is the?Kricogonia+Nathalis?+?Gandaca+?Gonepteryx+Dercas+?Catopsilia+?Anteos+?Zerene+Colias?????.It is proposed that the Coliadinae should be divided into two sister tribes,namely the Euremaini and Coliasini.?4?the subfamily Dismorphiinae contained 6 genera,and was made up of two clades:the first one is the Leptidea,the second one is the?Pseudopieris+?Moschoneura+?Dismorphia+?Patia+Enantia????;it is suggested that the Dismorphiinae should be divided into two sister tribes of the Leptideaini and Dismorphiaini.?5?the subfamily Pierinae contained 45 genera,and was made up of six tribes?Anthocharidini,Pierini,Elodinini,Leptosiaini,Nepheroniini and Colotini?.It is suggested that Colotis group should be lifted up to the tribal taxa of Colotini;and Hebomoia should be included in the Nepheroniini,rather than in the Anthocharidini.The divergence times of the major pierid lineages were estimated based on the obtained phylogeny of Pieridae in conjunction with their fossil records and their host plants in this study using the relaxed Molecular clock methods.At the same time,according to the recent geographical distributions,their ancestral geographical distributions was reconstructed,combined with the corresponding earth's environmental events in the earth's history,and their spatio-temporal diversification patterns were discussed.The results showed that the origin time of the Pieridae was approximately 94.68 million years agao?Ma??95%confidence interval:101.24-89.13Ma?in the Cenomanian Periods of late Cretaceous,with the Nearctic as the originating center;the subfamily Coliadinae was probably originated at about 57.87Ma?95%confidence interval:69.13-46.44 Ma?in the Paleocene,at the Nearctic areas;the subfamily Dismorphiinae and Pseudopontiinae was originated at about 76.78 Ma?95%confidence interval:86.66-66.52 Ma?in the late Cretaceous,with the subfamilies Pseudopontiinae and Dismorphiinae originating at the Afrotropical and Neotropical areas,respectively.The subfamily Pierinae was probably originated at about 85.34 Ma?95%confidence interval:87.13-84.16 Ma?in the Periods of Santonian of the late Cretaceous,with the Oriental as the differentiation centre.