A Study On Genetic Geography And Conservation Genetics Of The Rare And Endangered Plant Dendrobium Tenuifolia In East Asia

Dendrobium moniliforme(Linnaeus)Swartz which belongs to genus Dendrobium of Orchidaceae,is an endangered species and is distributed fragmentally in East Asia.It has highly ornamental and medicinal value.Its natural populations are facing the risk of extinction due to human overexploitation and habitat deterioration,it is imperative to launch an investigation into phylogeography and conservation genetics for proposing conservation strategies and sustained development of D.moniliforme.In the current study,18 wild D.moniliforme populations were collected from Japan(one population),Korea(one population),and the mainland(15 populations gathered from Yunnan,Sichuan,Guizhou,Guangxi,Hunan,Fujian,Jiangxi,Hubei,Anhui,and Henan provinces)as well as Taiwan(one population)of China,which represented its entire natural habitat range.We investigated evolution history,divergence patterns,genetic diversity,and genetic structure of populations based on chloroplast DNA(cpDNA),mitochondrial DNA(mtDNA),and fluorescent amplified fragment length polymorphism(AFLP)data.The major foundings are showed below:1.Phylogeography of D.moniliforme based on cpDNA and mtDNA datacpDNA(trnC-petN and trnE-trnT)and mtDNA(nadl/b-c and nad7/2-3)molecular markers were used to investigate its phylogeography from 18 natural populations.A total of 35 cpDNA haplotypes and 30 mtDNA haplotypes were identified.High level of haplotype diversity(cpDNA:Hd = 0.8862;mtDNA:Hd-0.8733),total genetic diversity(cpDNA:HT= 0.9011;mtDNA:HT= 0.8886),and significant population phylogeographic structure(cpDNA:NST=0.586>GST= 0.328;mtDNA:NST 0.722>GST=0.451)were detected within D.moniliforme populations.Mismatch distributions and neutral test indicated that all sampled populations and all populations sampled in mainland China expanded their range around 0.56 Ma and 0.54 Ma,respectively,based on cpDNA analysis,whereas mismatch distributions and neutral test of mtDNA data refused population expansion model.The results based on cpDNA and mtDNA was not congruent,different events happened in evolutionary history.Network based on the cpDNA and mtDNA haplotypes showed all haplotypes could be divided into two clades(Clade A and Clade B).There was no shared haplotypes between mainland China and Japan,implying D.moniliforme populations might have evolved allopatrically.Based on the obtained cpDNA and mtDNA results,we inferred that multiple refuges for D.moniliforme existed.2,Conservation genetics of wild populations of D.moniliforme based on fluorescent AFLP markersElucidation of the genetic diversity of an endangered species is a prerequisite for germplasm conservation.In the current study,160 accessions representing 15 wild populations from different regions were collected to investigate the genetic diversity of the endangered orchid herb,D.moniliforme,using fluorescent AFLP markers.It was found that this species was characterized by high levels of genetic polymorphism(100%polymorphic fragments),low levels of total genetic diversity(Ht = 0.1520),and moderate degrees of genetic differentiation among populations(FST=0.1026).STRUCTURE analysis showed that the 15 D.moniliforme populations formed three clusters corresponding to their geographical distribution.Analysis of molecular variance(AMOVA)revealed that only 19.20%of the genetic variation was existed among populations,while 80.80%of the genetic variation was occurred within populations.The Mantel test revealed that isolation by distance(IBD)did exist in D.moniliforme,but not among adjacent populations,which was possibly due to the large geographic distance and highly fragmented distribution of the species.Based on the obtained results,some useful conservation strategies were proposed for the effective and sustainable exploitation of D.moniliforme.3.Genetic diversity of cultivated populations of D.moniliforme based on fluorescent AFLP markersIn order to investigate the effectiveness and scientificity of ex situ conservation,it is necessary to regularly evaluate the level of genetic diversity and genetic structure of the ex situ conserved plants.To evaluate the effectiveness of ex situ cultivation of D.moniliforme for its genetic diversity conservation,we employed fluorescent AFLP markers to compare the genetic diversity and structure in four cultivated populations of D.moniliforme comprising 80 individuals from its germplasm cultivation base and their respective wild populations consisting of 47 individuals from four provinces in China.The four cultivated populations of D.moniliforme(Ht = 0.1868)showed reductions in gene diversity in comparison to their wild populations(Ht = 0.1955),while the differences of gene diversity between the cultivated and wild populations of D.moniliforme was not statistically significant.Cluster analysis revealed that individuals of wild and cultivated populations in the same regions were clustered together,implying ex situ cultivation of D.moniliforme effectively conserving the genetic resources of this medicinal species.Based on the results of evolution history and genetic structure of the studied populations,we proposed some corresponding conservation strategies:1)In order to conserve the genentic resources of D.moniliforme,populations in southwestern,central-southeastern of China,and the Islands should be taken into consideration as"Evolution Significant Units" in program for conserving the genetic resource of this endangered species.We proposed the populations with high level of genetic diversity and haplotype diversity should be protected preferentially.To enhance the stability and adjustability of D.moniliforme,seed gene flow and size of population should be increased.2)To protect as far as possible the genetic diversity of D.moniliforme,samples and seeds in all populations should be gathered in ex situ conservation;and in the same time,we should prevent outbreeding depression.