Phylogeny And Population Genetics Of Gynostemma BL. (Cucurbitaceae)

Gynostemma BL.,which belongs to the family Cucurbitaceae,is a genus containing 16creeping herbaceous species,which can be divided into 2 subgenera,among them,14 species and 2 varieties are distributed in China.The species of this genus mainly grow in moist mountains,forests,thickets,and streamside of East and Southeast Asia.The main distribution range in China is in the Yangtze river basin and its south areas.In recent years,many scientists pay much attention to genus Gynostemma because of its great medicinal value.However,field investigation found that the geographical environment of the wild population of genus Gynostemma is complex.The species in this genus have formed a series of ecological variation types during the process of differentiation,and multiple morphological characteristics cross and transition are serious among species.It brings great challenges in classification and phylogeny of the genus Gynostemma based on the morphology.At present,the researches related to genus Gynostemma mainly focus on chemical composition and pharmacological properties,morphological and cytological studies,and phylogenetic studies based on a few molecular markers,while the chloroplast genome of this genus has not been reported.Therefore,it is necessary to construct the complete chloroplast genome?CPG?of Gynostemma species.Herein,this study analyzed the phylogenetic relationship and population genetics of the genus Gynostemma by using CPGs,nuclear SNP constructed by Genotyping-by-Sequencing and the quantitative data of morphology,so as to reveal the evolution and population dynamic history of the wild population of the genus Gynostemma in subtropical China.The main conclusions are as following:?1?The CPG sequence and map of Gynostemma pentaphyllum?Thunb.?Makino was obtained by extracting the chloroplast DNA and Illumina sequencing.The results indicate that the length of the CPG of G.pentaphyllum was 157,576 bp with a CG content of 37.00%.A total of 139 functional genes were predicted,including 91 protein-coding genes,40 tRNA genes,and 8 rRNA genes.This study was the first obtaining of the CPG sequence of Gynostemma,which would provide a good reference for the phylogenetic studies of Gynostemma and Cucurbitaceae.?2?In the comparative study of the 8 CPGs of genus Gynostemma conducted that all of the CPGs displayed a typical quadripartite structure,2 copies of inverted repeats?IRs,26,174bp average?segregated by two SC regions,namely a large single copy region?LSC,86,834bp average?and small single copy region?SSC,18,649 bp average?.Each of them encoded133 genes,including 87 protein-coding genes,37 tRNA genes,8 rRNA genes,and 1pseudogene infA.The CPG was conserved in structure,gene composition and gene order.Through the search of three kinds of long repeated sequences and SSR in CPGs,it was found that there were a large number of repeat sequences in CPG of Gynostemma,and most of them were distributed in the gene intergenic region and intron.In addition,studies on sequence variation and mVISTA showed that protein-coding region?CDS?of CPG was more conservative than non-coding region?CNS?,and reverse repeat?IR?region was more conservative than single-copy?SC?region.The phylogenetic relationship among the species within the genus showed that it could be divided into 2 subgenera,which was consistent with the morphological classification.?3?The comparative analysis of the 10 CPGs from the representative genera of Cucurbitaceae family showed that the chloroplast genomes of Cucurbitaceae plants were relatively conservative,which had stable structure and no gene rearrangement event.Selective pressure analysis showed that three protein-coding genes?accD,clpP,and matK?were under selection and their coding proteins often have functions in chloroplast protein synthesis,gene transcription,energy transformation,and plant development.It might play a key role in the species response during the process of the development of terrestrial ecosystems.This study also indicated that the CPG of Cucurbitaceae was GC deleted type and had strong A/T preference at the third codon position.An unconventional translation initiation codon of psbL gene was found and provided evidence for RNA editing,which can modify the mutation and regulate the chloroplast gene expression.It is a kind of correction mechanism in plant chloroplasts.Applying BI and ML methods,p hylogenetic analysis strongly supported the position of Gomphogyne,Hemsleya,and Gynostemma as the relatively original lineage in Cucurbitaceae.This study suggested that the CPG sequences were useful for phylogenetic studies.It would also determine potential molecular markers and candidate DNA barcodes for coming studies and enrich the valuable CPG resources of Cucurbitaceae.?4?In this study,the phylogenetic relationships of 13 species and 2 varieties from Gynostemma were further explored through quantitative morphological data and the analyses of CPG and GBS at the molecular genome level.The results showed that there were 2subgenera of Gynostemma,namely,Subgen.Gynostemma and Subgen.Triostellum.However,we suggested that G.pubescens,G.pentaphyllum var.dasycarpum,G.burmanicum,and G.burmanicum var.molle should be incorporate into"G.pubescens",and it could be combined into a generalized species named"G.pentaphyllum"with G.longipes,G.laxum and G.pentaphyllum.The differentiation time calculated by BEAST shows that the differentiation of Gynostemma began in the late Paleogene?23.02ma?,and the differentiation of the two subgenus was in the Miocene?12.75ma?,while the differentiation of most species was mainly concentrated in the late tertiary Pliocene and the early quaternary Pleistocene?3Ma?.At the last interglacial period?0.13 Ma?,Gynostemma was mainly distributed in the southeast of East Asia.When the last glacial period?0.02 Ma?came,the distribution range of subgenus Gynostemma gradually moved to the west inland and reached the Hengduan mountain area and the Qinling-Daba mountains,while the distribution range of the subgenus Triostellum shrinks locally and forms their own refuge.And there may be hybridization and ge netic drift in the diffusion process after the ice age,thus forming the phylogenetic relationship and distribution pattern of the present species.In addition,the genetic structure of Gynostemma based on the SNP data was concerned in this study,and it was found that the species of Gynostemma can be divided into east and west regions.We speculated that the formation and diffusion of species in genus Gynostemma were affected by strong geographical environment and climatic conditions.?5?Finally,this is the first study to investigate the genetic diversity level and genetic structure of G.pentaphyllum populations in the genomic level.The results showed that the genetic diversity of 30 G.pentaphyllum populations was low?He=0.175,Ho=0.183?,the degree of differentiation was high(F_ST=0.984),and the genetic variation mainly existed among populations.The populations could be divided into north and south subgroups,and the populations in the southeastern edge of Qinghai-Tibet plateau,Yunnan-Guizhou plateau and Qinling-Daba area have certain gene flow in the long-term evolutionary history.In conclusion,this study constructed the CPGs of the genus Gynostemma and carried out the comparative analysis within the genus and among its sister genera?Cucurbitaceae?,combining with the nuclear GBS and quantitative morphological data to discussed systematics classification and phylogenetic evolutionary relationships of the genus Gynostemma.Moreover,the population genetic evolution and dynamic history of this genus were studied using the simplified genomic data analysis,so as to deepen people's understanding of the molecular genetic background of Gynostemma and provide effective information for the plant protection in east Asia.