The Study On Preliminary Investigation And Molecular Phylogeny Evolution Of Chrysosplenium In Wulingshan Areas

The Chrysosplenium L.comprises 65 species in the world,35 species and 8 varieties in China.As core area of global Chrysosplenium distribution,Few research about Chrysosplenium field investigation and systematics had been reported since 1982 in China.Chrysosplenium has significant medicinal effect for flavonoids content.There are a lot of research in Chrysosplenium chemical compositions in three decades,but lacks on investigation updating and system classification.Wulingshan Areas is a mountain chain which spins Hubei,Hunan,Chongqing and Guizhou provinces about 170,000 square kilometers and contains 24 national nature reserve areas.Between 1987 and 1989,Academy of Sciences conducted a field study in Wulingshan Areas and found only 3 Chrysosplenium species.(C.macrophyllum ? C.lanuginosum ?C.sinicum)Now,more than twenty years on,there are greater changes in Area? population? administrative region and vegetation conditions.This project relies on the ministry of national science and technology works special-------Comprehensive Investigation on Biodiversity of Wulingshan Areas.Between 2014 and 2016,we collected about 300 samples of Chrysosplenium,consist of 13 sepcies?7 varieties and 1 new species.Investigation area include 7 county(city)?13 towns?7 national nature reserve areas.This study analyzed internal transcribed spacer(ITS)sequences characters of 21 species(including varieties)of Chrysosplenium,then we conducted Secondary Structure Prediction and ITS sequence-structure alignment.The authenticity of sequences and predicted secondary structures are verified by conservative domain and motif which both exist in eukaryotes.At last we analyzed Compensatory base changes(CBCs)in internal transcribed spacer 2(ITS2)secondary structures.The results are as follows:1.GC content in ITS1 and ITS2 is similar,5.8S GC content is above them which average 50.15%.The numbers of parsim-information sites and variable sites in ITS1 are higher than in ITS2.Among 12 species of Oppositifolia,average GC content in ITS1(48%)is higher than in ITS2(42.7%)and,have a very significant difference(P<0.000).On the contrary,Among 9 species of Alternifolia,average GC content in ITS1(43.3%)is lower than in ITS2(46.3%)and,have a very significant difference(P<0.000).Furthermore,among all 21 species,ITS1 GC content in Oppositifolia is higher than in Alternifolia and,have a very significant difference(P<0.000).ITS2 GC content in Oppositifolia is lower than in Alternifolia,and have a very significant difference(P=0.002).2.Except C.sinicum and C.nepalense,the genetic distances between different subgenus(Oppositifolia and Alternifolia)are significantly higher than within the same subgenus.Genetic distance between C.sinicum and C.nepalense is minimum(p=0.013),but any one of them with other species is greater than 0.1.3.By using five methods(MP?NJ?ML?UMPGA?Bayes),we construct five phylogeny trees and compare each other.We believe that the result of Bayes phylogeny trees is closest to morphological classification.In addition,resolution and confidence in bayes tree are higher than other trees.4.By comparing the predicted consensus secondary structures of ITS1 and of ITS2 in 21 species,we found that variation of secondary structures in ITS1 is higher than in ITS2.And base pairs in IT2 secondary structures have better stability.ITS1 secondary structures change among species of Alternifolia are milder than of Oppositifolia.By contrast,ITS2 secondary structures are conservative in both subgenus.In conclusion,ITS2 secondary structures are more conservative than ITS1 in Chrysosplenium;Secondary structures of Alternifolia are more conservative than of Oppositifolia in both ITS1 and ITS2.5.Chrysosplenium and its relative genus(Peltoboykinia)contain a contiguous conservative sequence,this sequence form several stable secondary structure(H3?H12?H13?T4?L8?L7)in ITS1.We suspect that those sturcture have strong connection with function of ITS 5.8S secondary structures in all species only differ in an internal loop,and structure of Alternifolia at the position of this internal loop show up as L1,but Oppositifolia show up as L2+L3.This distinction may be a reason for differences on the morphology between Oppositifolia and Alternifolia.Two types of terminal loop(T3a and T3b)are found in helix III of ITS2,and none of them exist in relative genus(Peltoboykinia).Moreover,we have not found any CBCs among species of Alternifolia,but CBCs among species of Oppositifolia are widespread.According to Coleman and Vacquier(2002),in all eukaryote groups,when sufficient evolutionary distance has accumulated to generate even one CBC in the relatively conserved pairing positions of the ITS2 secondary structure,taxa differing by the CBC are observed experimentally to be totally incapable of intercrossing,thus,we suppose that the possibility of intercrossing among species of Oppositifolia is much more less than among species of Alternifolia.Species belong to Oppositifolia tend to evolve independently,and Alternifolia are more easier to produce new variants.6.We found that C.sinicum and C.nepalensel have several unusual features in Chrysosplenium.(a)Their GC contents are higher than other species.(b)Although they belong to Oppositifolia,genetic distances between them and Alternifolia are smaller.(c)Instead of classed them in evolutionary branch of Oppositifolia in phylogenetic tree,they were classed in same evolutionary branch which parallel with branch of Oppositifolia and of Alternifolia.Furthermore,lots of CBCs existed between them and Oppositifolia,but hardly existed between them and Alternifolia.Therefore,we believe that the possibility of intercrossing between them and Alternifolia is higher than between them and other species of Oppositifolia.C.sinicum and C.nepalense would probably have been evolved independently a long time ago,and they Have been separated from other species of Oppositifolia.