Study On Genetic Diversity And Molecular Phylogenetic Relationships Of Partial Species In Nymphalidae In China

Nymphalidae(Lepidoptera:Papilionoidea) is a big family in Papilionodea.Butterflies in this family are beautiful and distribute in all continents of the world except Antarctica.Larvae of most species in this family represents important pest to foodcrops,fruit tree,vegetable,fibre crop, forest,bamboo,and so on.The systematics of Nymphalidae butterflies based on morphological characteristics has been studied by many authors for more than one century.In recent years,some researchers restructure the phylogenetic relationships among part of species from Nymphalidae through using methods and techniques of molecular biology.However,as the diverse shapes and life history of Nymphalidae butterflies,the phylogenetic relationships of the species in this family are very unclear all the time,which have been argued by taxonomist.Furthermore,it can not be illustrated that the evolution significance of many research results because the unclear phylogenetic relationships of subfamilies,tribes and genera in Nymphalidae. Therefore,research on the genetic diversity and phylogenetic relationship of this family not only has theory significance,but also possesses important practical value for protection of biology diversity as well as pest management.RAPD(Random Amplification Polymorphic DNA) method using for the identification of polymorphism and analyzing phylogenetic relationships among and within closely related species was first described by Willianms and Welsh in 1990.RAPD markers are based on the amplification of unknown DNA sequences using single,short,random oligonucleotide primers.The RAPD method is advantageous over RFLPs in technical ease, quickness,low cost and fewer amounts of samples required.It has been widely applies to the organisms including bacteria,plants and vertebrates for taxonomy and species identification,as well as construction of genetic mapping,genetic relationships among species and population,and locating pathological important genes.The RAPD method has also been soon adopted by entomologists and widely used to study taxonomy and phylogenetic relationships of insects.The sequence analyses of mtDNA and nuclear genes are effective molecule markers for reconstructing the phylogenetic relationships and evolution research,which has been widely used to investigate population structure,deletion of gene,hybridization, biogeography and phylogenetic relationships.In this study,we investigated the population-level genetic diversity of Sasakia charonda or Pieris rapae in China based on RAPD markers; preliminarily examined phylogenetic relationships of Apaturinae, Nymphalinae,Limenitidinae and Nymphalidae by analyzing sequences of the mtDNA Cyt b,COI and nuclear EF-1αgenes;reconstructed the phylogenetic trees of Nymphalidae based on the mtDNA COI and nuclear EF-1αgenes (Data I) by using NJ,ML as well as Bayesian methods,and then mapped 3 morphological characters on the ML tree for investigating the evolutionary patterns of these morphological characters.On the basis of the results of the phylogenetic relationships of Nymphalidae reconstructed based on the sequences determined from the 13 species in our laboratory and additional sequences of other 43 species found in GenBank(DataⅡ) and fossil information of Satyrinae,Nymphalinae and Dynamine alexaen,the average divergence times among the subfamilies are then estimated for the first time. The main contents and conclusions are as follows:1.Genome DNA of butterflies from the breast muscle,the abdomen muscle, and the midleg and hindleg muscle of dry samples was extracted using phenol-chloroform protocol and saturated NaCl methods.Genome DNA extracted by two methods from the midleg and hindleg muscle was quite good for RAPD analyses.However,the genome DNA extracted by phenol-chloroform protocol methods from the midleg and hindleg muscle suits to analyses of sequences of mtDNA and nuclear genes.2.RAPD markers were applied to analyze genetic divergence among three populations of Sasakia charonda,Sasakia funebris and Hestina assimilis. Genomic DNA of fifty-eight individuals was extracted from dissected leg muscle using saturated NaCl method and then amplified by 10 random primers(10 bp) which were previously selected,a total of 200 clear and reproducible bands were generated,the RAPD patterns showed polymorphic variations among and within different genera and species. The amplified fragments were analyzed by the Phyltools 6.0,the molecular phylogenetic tree based on Nei's genetic distance of RAPD markers was constructed using UPGMA and Neighbor-Joining.The dendrogram indicated strong similarities within populations.Among three populations of Sasakia charonda,the individuals of Hubei population and Shanxi population closely clustered together;and then they were clustered with Liaoning population.These data suggested that several implications. Firstly,RAPD is very useful as an effective molecular marker to distinguish the divergence in Sasakia and its closed related genera. Secondly,it seems to be a tendency of correlations between the genetic and geographic distance among the 3 different populations of Sasakia charonda from the south to the north.Thirdly,the RAPD similarity levels follows the order of individuals > populations > species > genera.The results of dendrogram based on RAPD markers are consistent with the previous conclusions of morphologic classification.3.The genetic diversity and genetic relationship of five populations of the Pieris rapae in Shanxi province determined using random amplified polymorphic DNA(RAPD).Nei's genetic distances between populations were very low and minor different,showed that a high similarity among the five populations.The dandrogram based on Nei's genetic distance of RAPD markers was constructed using UPGMA and Neighbor-Joining. This result indicated that Daixian population was closer with Xiaxian population,then the two populations clustered with Datong population, and finally,aider the Taiyuan population grouped together with them,the four populations clustered with the CZ population.The genetic relationship was not consistent with the geographic distribution.4.The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1471 bp of cytochrome oxidase subunit I(COI).The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data,along with those of 4 species obtained from GenBank,to construct the MP and the NJ trees using Athyma jina,Penthema adelma,Polyura nepenthes and Charaxes bernardus as outgroups.Within the Apaturinae,the clustering results are approximately identical to the classical morphological classification.The mtDNA data suggests the genus Mimathyma as a monophyletic group.Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees.It also supports the taxonomic revision of removing several species from Apatura to other genera,namely Mimathyma schrenckii,M.chevana,M. nycteis,Chitoria subcaerulea,C.fasciola,C.pallas,and Helcyra subalba.5.The phylogenetic relationships of the subfamily Nymphalinae(sensu Chou,1994) was analyzed based on 1488 bp of mtDNA cytochrome oxidase subunit I(COI) gene sequence data obtained from 24 individuals, along with those of 8 species obtained from GenBank.The phylogenetic trees were reconstructed by NJ,ML and Bayesian methods through using Byblia anvatara as outgroup.Our data indicated that the tribes Nymphalini and Hypolimni(sensu Chou,1994) are not monophyletic groups,and the genus Junonia should be removed from Nymphalini to Hypolimni(= Junoniini).Based on our data,the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML and Bayesian methods,namely((((Nymphalis + Kaniska) + Polygonia) + Aglais) + Vanessa) +(Symbrenthia + Araschnia).This investigation provides a little novel information for Chinese researches of butterflies.6.To investigate the phylogenetic position of the subfamily Limenitidinae (sensu Harvey's,1991),we reconstructed the phylogenetic trees of Limenitidinae,Apaturinae,Nymphalinae,Satyrinae,Danainae, Heliconiinae,Charaxinae,Morphinae and Libytheinae(sensu Harvey's, 1991) based on COI and EF-1αsequences by using ML,NJ and Bayesian method.The results show that species classified into Biblidini,Cyrestini, Pseudergolini and Limenitidini by Harvey cluster together,respectively, and Limenitidini is farther with Biblidini,Cyrestini and Pseudergolini, supporting that Biblidini,Cyrestini,Pseudergolini and Limenitidini should be classified into subfamilies Biblidinae,Cyrestinae,Pseudergolinae and Limenitidinae,respectively.The phylogenetic analyses of the re-delimited Limenitidinae is presented based on 1471 bp of mtDNA cytochrome oxidase subunit I(COI) gene sequence data obtained from 21 individuals spanning 9 genera for the first time,along with those of 17 species obtained from GenBank.Our data support the monophyly of the subfamily Limenitidinae.The genus Limenitis can be split into two subclusters.Moreover,the results give one of the strongest hypotheses for the tribe relationships within Limenitidinae.7.The phylogenetic relationships of the Nymphalidae(sensu Chou,1994) was analyzed based on 432 bp of mtDNA Cyt b gene sequence data obtained from 51 individuals in 9 subfamilies.The phylogenetic trees were reconstructed by NJ method through using Papilio machaon as outgroup.The phylogenetic tree shows that each subfamily is not monophyletic and the phylogenetic relationships are disordered,resulting from short sequences of Cyt b and saturation on the 3^rd codon,which have led to reconstruct an untrue phylogenetic tree.Then,we reconstruct phylogenetic relationships of Nymphalidae based on the COI and EF-1αsequences(DataⅠand/or DataⅡ) to find the "true tree".The phylogenetic trees constructed based on DataⅠand DataⅡshow that phylogenetic relationships of partial subfamilies in Nymphalidae are as ((((Nymphalinae + Apaturinae) +(Cyrestinae + Biblidinae)) + Pseudergolinae) +(Heliconiinae + Limenitidinae)).The results indicate that it seems reasonable to divide the subfamily into two Vibes:Apaturini and Chitorini and replaced the Morphinae into Satyrinae.8.We mapped the states of the three characteristics(i.e.,spot of anal angle, eyespots,and process from outer margin of hind wing) on the internal nodes of the combined phylogenetic tree,which was based on the COI and EF-1αsequences(DataⅠ) by using maximum likelihood method.The results suggested the 3 characteristics might have repeatedly evolved in the family Nymphalidae.9.We constructed the phylogenetic trees of Nymphalidae based on a combined dataset of the COI and EF-1αsequences(DataⅡ) using ML and Bayesian methods.On the basis of the results of the phylogenetic relationships among subfamilies in Nymphalidae and the relative-rate tests as well as fossil information,the average divergence times among the subfamilies are estimated as 44.2 - 87.1 million years ago.The results will help us for understanding the origin and evolution of this family.