Genetic Diversity And Phylogeography Of Gynostemma BL.

Gynostemma BL.belongs to the genus Cucurbitaceae,consisting of 16 species and 2 varieties,which fall into two subgenera and two sections,among them,9 species and 2 varieties only distribute in China.The distribution pattern of the genus indicates a natrue of Tropical Asia Type,the modem distribution centre of the genus ranges from drainage areas of the Yangtze River to Yunnan,southwest China.The natural populations of G pentaphyllum(Thunb.)Maldon are distributed as the genus,the special species have narrow distributed.The distribution of Gynostemma BL.gives a useful model for the study of phylogeography.The distribution pattern of Gynostemma BL.genetic variation and its hitorical causes has been studied in this paper,which will play an essential role in the study of endemic plant distribution pattern and geological changes in China.The patterns of genetic diversity of Gynostemma BL.was analysed using ISSR marker,and the haplotype of cpDNA rpl20-rps12 regions were analysed to infer phylogeography and the history of speciation,and the possible refugia was suggested.The results of cpDNA PCR-RFLP and rpl20-rps12 sequence gave a gloom result of the relationship in Gynostemma BL..This study focuses on four subjects:1.Genetic diversity Gynostemma BL.Studies were performed to investigate the genetic variation of fourteen natural populations of Gynostemma pentaphyllum(Thunb.)Makino,an outcrossing clonal plant species in China, using inter-simple sequence repeat(ISSR)markers.Fourteen selected primers were used to amplify DNA samples from 140 individuals,and totally 194 loci were detected.The percentage of polymorphic bands(PPB)showed that the genetic diversity was high at the species level(PPB=96.39%)but quite low at the population level(PPB=1.03-25.26%). Shannon's information index(I)and Nei's gene diversity(H_e)displayed a similar trend to PPB. According to the hierarchical analysis of molecular variances(AMOVA),the analysis of Shannon's diversity and Nei's analysis of gene diversity,the percentages of genetic variation among populations were 88.66%,90.05%and 88.94%,respectively,indicating a high level of inter-population genetic differentiation.The low levels of genetic diversity within populations and high genetic differentiation among populations were assumed to result from the limited gene flow and genetic drift.Mantel test revealed no significant correlation between genetic and geographic distance matrices(r=0.1628,P＞0.05).Alao,the genetic diversity of 6 species of Gynostemma BL.had been studied using ISSR marker,a significate difference of genetic diversity had been examined.G.pentaphyllum showed the highest diversity(PPB=34.17%) and G longipes had the lowest leave(PPB=1.67%).2.Phylogeographic study on Gynostemma BL.The relationships and distribution of cpDNA haplotypes suggested that Gynostemma BL. had been widely distributed in China during the Tretiary.Yunnan Province was considered to be the origin center.Two routes had been suggeted for Gynostemma BL.dispersed from origin center to East China.North route might be from Yunnan Province to Sichuan basin and Qinling Mountain,finally reached Jiangsu and Anhui.The south route from the origin center to Wuling Mountain.Several Pleistocene refugia were suggested.The refugia of Gynostemma BL.in China concentrated on Wuling Mountain and Sichuan basin.After the Quaternary, Wuling Mountain became the new origin center,which play an important roal in present distribuion pattern.3.Phylogeny study on Gynostemma BL.Based on cpDNA PCR-RFLP marker,the phylogeny of MP tree and NJ tree had been provided.Subgen.Trirostellum formed a monophyletic clade and separated from Subgen.Gynostemma.The cpDNA rpl20-rps12 analysis indicated Subgen.Gynostemma was presumed as the precursor of this genus.4.Strategies for conservation of Gynostemma BL.Based on the genetic data by ISSR,ZJS and FJS population were suggested to give the priority conservation,as they had the most genetic diverity in G.pentaphyllum.Th models baesd on haplotype richness had been used in Gynostemma BL.conservation study.We evaluated the genetic contributions of 18 populations based on cpDNA haplotypes.The special species G.laxiflomm,G.longipes and G.cardiospermum should be conserved with priority.Also,effective conservation strategies were proposed for conserving this traditional Chinese medicinal herb.Concerning the management of G.pentaphyllum,we suggested that in situ conservation be an important and practical measure for maintaining the genetic diversity and that a possibly maximum number of populations be conserved.In the long run, the most suitable strategy for in situ conservation of Gynostemma BL.is the protection of its habitats.At the same time the gene flow between plant species and natural species should been avoided.