Studies On The Conservation Biology And Chloroplast Genomes Of The Endangered Resource Plant Yunnanopilia Longistaminea And Related Species

Yunnanopilia longistaminea(W.Z.Li)C.Y.Wu et D.Z.Li belongs to the monotypic genus of Opeliaceae family.The species is mainly distributed in the valley regions of Southwest China.Due to its delicious taste and unique flavor,the wild resource is undergoing dramatically declining due to excessive utilization.Based on the previous investigation about the wild distribution pattern and endangered status,the seeds ecophysiological characteristics,phylogeographic structure,genetic diversity and phylogenetic of the rare and endangered plant Y.longistaminea were studied in this thesis using the methods from botany,conservation biology,molecular biology and chloroplast genomics.Furtheremore,the species'endangered mechanisms were analyzed and the conservation strategies were also put forward.The main results and conclusions of the thesis are:1.Seed ecophysiological characteristics of Yunnanopilia longistamineaSeeds morphological characteristics,germination requires and dormancy type of Y.longistaminea were studied.The results indicated that freshly-matured seeds of Y.longistaminea belong to big seed.Seeds are sensitive to dehydration indicates the seeds belongs to recalcitrant.The seed germination of this species involves two distinct stages:radicle emergence and epicotyl(shoot)emergence.The optimum stratification temperature was 28? or 28?/20? to radicle emergence.The optimum GA3 solution for the seeds undergoing shoot emergence was 100 mg·L-1.The percentages of shoot emergence after 7 days cold stratification at 5? were slightly higher than those in other groups.The duration of cold stratification also significantly affects the epicotyl dormancy release of Y.longistaminea seeds.Under natural conditions,seeds of Y.longistaminea are subjected to a autumn?winter?spring temperature cycle.Warm moist conditions and cold stratification can improve radicle emergence and alleviate epicotyl dormancy,respectively.Thua,considering seeds have a fully developed embryo and mature and are dispersed in summer,and radicles and epicotyls emerge the following autumn and next spring,respectively,we concluded that Y.longistaminea seeds belong to physiological epicotyl dormancy.This study is also the first to describe physiological epicotyl dormancy in China.Optimal dormancy breakage methods are warm(28?/20?)?cold(5?)?GA3(100 mg·L-1)?warm(28?/20?).2.Phylogeographic and conservation genetics of Y.longistaminea based on cpDNA and ITS4-ITS5 analysisThe cpDNA(psbAF-trnHR atpB-rbcL,trnL-trnF,trnG-trnS)combination and nrDNA(ITS4-ITS5)were employed to analyze the 295 individuals of Y longistaminea of 16 populations in the main distribution area.The cpDNA combined fragment(length of 2595 bp)detected 11 haplotypes,and nrDNA fragment(length of 638 bp)detected 16 haplotypes.Both cpDNA(Hd=0.605,HT=0.953,HS=0.330,Pi=0.00241)and ITS4-ITS5(Hd=0.526,HT=0.883,HS=0.203,Pi=0.00151)had a high levels of genetic diversity and low levels of nucleotide diversity at the species level.However,both cpDNA(GST=0.949,NST=0.983)and ITS4-ITS5(GST=0.651,NST=0.834)showed a relatively low level of genentic diversity at the population level and and high genetic differentiation occurred among populations.According to the AMOVA analysis,the cpDNA analysis results showed that the genetic variation among populations was 98.5%,and the genetic variation within population was 1.5%;the ITS4-ITS5 analysis results indicated that the genetic variation among populations was 87.6%,and the genetic variation within population was 12.4%.The genetic variation analysis on Y.longistaminea showed that the genetic variation mainly occurred among populations.According to cpDNA(Hd=0.605,Pi=0.00241,FST=0.985)and ITS4-ITS5(Hd=0.526,Pi=0.00151,FST=0.875),the genetic differentiation among populations of Y.longistaminea was high.The results of ITS4-ITS5 neutral test and mismatch analysis show that there appeared expansion in the populations of Y.longistaminea.The divergence time was about cpDNA(2.72 Mya)and ITS4-ITS(2.99 Mya).We speculated that there might be two ice-age shelters of Y.longistaminea.3.ESUs division of Y.longistaminea based on cpDNA and ITS4-ITS5 analysis5 ESUs(ESU A-E)could be determined according to the haplotype geographical distribution,genetic distance and AMOVA analysis of the Y.longistaminea in the 16 populations.The priority preservation should be respectively ESU A,ESU E,ESU B,ESU C and ESU D in sequence.The populations in ESU A are not in the preservation area,and especially,there are rare adult individuals in GS population;and the human disturbance is the most serious in this area,which is in urgent need for targeted preservation.ESU B is on the isolated branches,and there is less gene exchanges among populations;there may appear degradation in the future,so the priority should first given to the PY population.ESU D has good preservation situation,and the populations are included in the natural reservation area,so the GXB population should be mainly preserved.4.Chloroplast genomics analysis Y.longistaminea and related speciesAccording to the results analysis on the chloroplast genomes sequencing and assembling of Y.longistaminea and related species,the chloroplast genomes of Y.longistaminea and related species consist of four parts:large single copy area,small single copy area and reverse repeat region.The total length of chloroplasts was between 144306 and 147461 bp.GC content was basically stable,around 37%.There were altogether 101 chloroplast genomes in the related species of Y.longistaminea(67 protein coding genes,30 trnA genes and 4 rRNA genes).The genealogical tree constructed according to the protein coding gene of the chloroplast genome showed that Y.longistaminea,Champereia manillana and Cansjera rheedii clustered into three independent monophyly.5.Endangered mechanism and conservation strategiesThe comprehensive analysis based on the results of this study indicated that the reasons of the endangering of Y.longistaminea include:fragmented distribution of populations,the genetic diversity mainly occurred among populations,and small gene exchanges;the special habitat requirements of seeds germination and the high frequency of local human disturbance inhibited the species natural regenerataion.Furthermore,the strategies for protection are proposed according to the endangered mechanism such as:in situ preservation for existing populations which are give priority preservation with the guidance of ESU should be conducted.In addition,ex situ conservation,ratification propagation and reintroduction were also suggested in the present study.