| Parnassia is one of the genera in Celastraceae,occurring mainly in temperate regions.All species of Parnassia are perennial herbs with white or(green)flowers solitary on unbranched scapes.It has high ecological and economic value.In terms of morphological systematics,the predecessors proposed a classification which contains six groups within Parnassia based on four morphological traits.However,in term of molecular phylogenetic studies,the predecessors constructed the phylogenetic relationship among Parnassia based on only a few common fragments,which contributed to problem such as low statistical values.Furthermore,the identification of samples may be wrong,so that the results are not representative and persuaded.Parnassia brevistyla,P.delavayi and P.leptophylla belong to Subsect.Xiphosandra.Following extensive field investigations and examination of specimens,we found that the reliability of their taxonomic characteristics remained unknown and all three species have been retained.In addition,angiosperm seed is a kind reproductive organ with a large number of important genetic information,have relatively stable traits,and the trait of seeds is very stable.But there are no reports on the morphological taxonomy of phenotypic traits related to the seeds of Parnassia.In order to solve the above problem,this study used high-throughput sequencing technology to obtain the chloroplast genome sequences of six Parnassia species(P.palustris,P.longipetala,P.trinervis,P.crassifolia,P.delavayi and P.wightiana),and conducted molecular phylogenetic studies for 64 samples of 27 species.Meanwhile,the micro-morphological data of 22 Parnassia seeds were obtained by scanning electron microscopy,rational taxonomic treatment of Subsect.Xiphosandra and the systematic study of Parnassia was carried out.The main results and conclusions for this study are as follows:1.Therefore,this study obtain a total of 72 GB of raw data,using Illumina HiSeq 4000 genome analyzer platform via Illumina PE150 strategy.The BLAST results of six respectively species were found to be considerably consistent,2,225,059 reads for P.palustris,1,555,160 reads for P.longipetala,4,263,164 reads for P.trinervis,1,307,560 reads for P.crassifolia,2,052,332 reads for P.delavayi and 3,645,249 reads for P.wightiana.The genome size of them via de novo assembly were:P.palustris 148,700 bp,P.longipetala 152,748 bp,P.trinervis 153,788 bp,P.crassifolia 151,655 bp,P.delavayi 151,552 bp and P.wightiana 151,671 bp.The structural results based on Geneious analysis showed that the GC content of genus Parnassia is 36.9%-37.2%.The plastome encodes 115 genes for each species of Parnassia(105 P.palustris),in which 81 genes are protein coding(P.palustris 71),29 are tRNA genes,and 4 are rRNA genes.All the plastomes have 18 genes duplicated in IRs and 20 genes contain introns,except P.palustris lost an ndhA.Our chloroplast phylogenomic analyses based on cpDNA homologous sequences,strongly supported the monophyly of each branch,and further supported the Parnassia's system location.2.Based on the aligned plastomes we found 37 hypervariable regions,and 19 fragments showing high levels of polymorphism(ccsA-psaC,psaC-rps1 5,ndhG-ndhA,ndhA,ycfl,rps3-rps19,rps8-rps3,psbB-petB,clpP-psbB,rpl20-clpP,psbE-petL,petA-psbF,rbcL-accD,atpB-rbcL,trnL-UAA-ndhJ,ycf3-rps4,psaA-ycf3,petN-trnE-UUC,atpF-atpl)were screened by a large number of PCR.Our chloroplast phylogenomic analyses of 64 samples of 27 Parnassia species,combining these hypervariable region fragments and ITS,strongly supported that there are five clades of this genus:P.faberi and P.longipetala formed the basal clade ?(staminodes unlobed or labiated);P.bifolia(staminodes 2-lobed,petal margin entire),formed a monophyletic group ? as a transitional group;P.cabulica,P.nubicola,P.oreophila,P.laxmannii and P.trinervis formed clade ?(staminodes 3-lobed,petal margin erose);P.palustris(staminodes 7-23-branched,petal margin entire),formed a monophyletic group IV;clade V consisting of 15 remaining Parnassia plants,with three kinds staminodes(unlobed,3-lobed and 5-lobed)and with three kinds petal margins(entire,erose and fimbriate).In addition,the combined analysis of molecular phylogenetic tree and morphological traits indicate that the morphological characteristics of the Parnassia(such as petal margin and staminodes)could be evolved many times,and some species had hybridization phenomena(such as P.wightiana)Phylogenetic analyses genusted a fully resovled tree with 100%statistical values at all the clades,indicated that the hypervariable regions fragments of chloroplast genomes may potentially provide the opportunity to better resolve the phylogeny of Parnassia.3.The results of seed electron microscopy of 22 species of Parnassia(covering six groups)showed that seed coats can be divided into three main categories:1)The seed coat is capsular-shaped,with a pore-shaped reticular:P.palustris,P.nubicola,P.lutea,P.oreophila;2)Transitional group:P.bifolia;3)Normal development of seed coat,scalariform:P.faberi,P.noemiae,P.obovata,P.amoena,P.longipelata,P.mysorensis,P.chinensis,P.foliosa,P.crassifolia,P.perciliata,P.cooperi,P.wightiana,P.gansuensis,P.delavayi,P.brevistyla,P.tenella,P.trinervis.According to the micromorphological characteristics of seed coat,the identification key to species of Parnassia was estab ished.4.The results of classical taxonomy of P.brevistyla,P.delavayi and P.leptophylla based on more than 1,300 specimens,indicated that the shape of basal leaves and the depth of staminode lobes,are vary continuously within populations and should not be used to distinguish separate species.In summary,the molecular evolution and phylogenetic analysis of the chloroplast genome sequence of Parnassia supports the six classifications system using morphological traits of this genus;molecular evidence and data analysis supported P.brevistyla,P.delavayi and P.leptophylla were the same species,so that P.brevistyla and P.leptophylla should be treated as synonyms of p.delavayi;in addition,the micromorphological study of seed coat of most Parnassia species,indicate seed coat has a high taxonomic value.The results of the study will help to understand the systematic and phylogenetic relationship among the species of the genus,and laid the foundation for a more in-depth evolutionary position in the angiosperm group and characteristics evolution trend study of Parnassia in the future. |
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