Research On Conservation Ecology Of Primulina Rare Plants

Plants of Gesneriaceae are the optimum research objects to study plant speciation and evolution by their rich morphology variations within genera.Primulina Hance was originally described as a monospecific genus and was once considered with single species,currently Primulina was enlarged greatly so that it becomes one of the largest genus of Gesneriaceae in China,at least 174+ species were confirmed and reported which distributed from southern China,i.e.Guangxi,Guizhou,Northern Guangdong,Southern Hunan,Hainan,South-eastern Yunnan and Chongqin,especially on the karst topography.Primulina displays rapid variation specific adaptability from a narrow genetic basis,having high scientific value in understanding plant sympatric speciation.We combined means and methods of multi-disciplines,such as:Palynology;Taxonomy;Transcriptome analysis;Molecular systematics and 3S technology to investigate the evolutionary significance of adaptation mechanism and interspecific differentiation at both the population and species levels of this genus.In addition,the content of differentiation on environmental adaptation and molecular systematics to testify each other,combined with population genetic analysis.At the same time,the population genetic structure was analyzed by SSR molecular marker technique.Furthermore,successful implementation of this research shed more light on the mechanism of sympatric speciation of Primulina based on the encological differentiation of environmental gradients,laid the foundation of revealing the origins and generation of biodiversity about this genus,and also had very important realistic significance on protecting the biodiversity and sustainable utilization of resources.The results were summarized as followings:1.Pollen grains of 43 species of the genus Primulina from South China were observed under a scanning electron microscope.The morphological variability of pollen and the taxonomic implications of pollen morphology were observed,in addition,the result of cluster analysis(UPGMA)was performed on the palynology data and reported here,showing:(1)Pollen grains of Primulina were all tri-colpate,sexine ornamentation all were reticulate,while the distinction exists in size,shape and exine sculpture among different species.(2)Cluster analysis resulted in the delimitation of six main sub-clusters with seven groups,the result showed that the great morphological hetero-geneityis in the genus is polyphyletic;and through combine,comparative analysis and species distributions,indicated that the developing and forming of karsts physiognomy in South China played an important role in the taxa differentiation of Primulina.2.Molecular of systematics:Despite the comprehensive revisions of Chirita by Wood and Wang W.T.in 20th Century,while Primulina was recently revised according to molecular studies by Michael Moller,Aton Weber and Wang Y.Z.,the genus is still very problematic with regard to subdivision.Based on sequence data from the internal transcribed spacer region of nuclear DNA(ITS)and the trnL-F regions of chloroplast DNA from 104 species we got the data in this study and 21 species which downloaded from NCBI of Primulina,as well 11 species of other genera,ie.Petrocodon,Ornithoboea and Loxostigma as the outgroup.Molecular data were analyzed using Bayesian and Maxiimum Likelihood(ML)methods with separate nuclear and chloroplast data sets,as well as a combined data set.Morphological investigation on specimens and field observation were investigated simultaneously.The results showed that phylogenetic trees resulting from separate analyses proved highly congruent and the combined analysis of the two data sets produced a well-supported topology of the species examined,and supported 4 major clades within the genus,while two clades was far relationship to another two,one of which was distributed in the Hainan island and another had the zigzag monochasial,paraelades Cyme,P.anisocymosa as the representative.In addition,ten new species were described here.3.We sequenced transcriptomes for 6 represent species of Primulina across the phylogeny of the genus using the Illumina HiSeq2000 platform,while Petrocodon jingxiensis as outgroup to compare.A total of 66979379700nt datas including more than one million clean reads per every species were processed into 344108 contig with a mean length of 1188 bp,while an N50 value of 1197 bp after pooling.By NR annotation,we got a total of 207937 unigenes with significant BLAST matches(E-value cutoff of 10-5).By COG annotation,possible functions of 85257 unigenes were classified and subdivided into 25 COG categories.129 pathways and 124373 unigenes were subjected to the KEGG Pathway analysis to reconstruct the metabolic pathway network;among which,metabolism related pathways and biosynthesis of secondary metabolites were represented the largest groups.Of these unigenes,19295 were successfully annotated and 203528 were identified as coding sequences(CDSs).We identified a total of 32741 simple sequence repeats(SSRs),as trimmer motif style is the most abundant one in silico EST-SSR distribution.We screened 309 putative single-copy nuclear genes by OrthoMCl(v1.4)of 18 species(include 11 species datas form NCBI)and used the supergene from single-copy nuclear genes for phylogeny construction analysis by Bayes(v3.1).In addition,we identified a total of 85 candidate orthologs under positive selection which could be used for further ecological genomics studies in Primulina and its relatives.Furthermore,the assembled unigenes with annotations and the massive gene-associated molecular markers would help guide the following chapter population genetic research.4.Based on molecular,morphological and cytological studies,and combined the transcriptome analysis,the previously monotypic genus Deinostigma which formerly endemic to Vietnam now as a new distributed genus in China.Deinostigma now has been expanded 3 species in China,including one cultivated species,i.e.Deinostigma cicatricose,D.cyrtocarpa and D.tamiana,which all previously included in Primulina.Now Deinostigma distributed form Vietnam to S China.5.Through sequenced transcriptome from 7 populations of Primulina xiuningensis complex,we got 436949258 raw reads,420906808 clean reads,which were average assembled into 235626 scaffolds and N50 average of 450 nt.The GC content of the transcriptome averaged 52.19%.We found that trimmer motif style was the most abundant one in silico EST-SSR distribution.By NR arnotation,we got a total of 153,231 unigenes with significant BLAST matches(E-value cutoff of 10-5).By COG annotation,possible functions of 43.83%all-unigenes were classified and subdivided into 25 COG categories.129 pathways and 96081 unigenes were subjected to the KEGG Pathway analysis to reconstruct the metabolic pathway network;among which,21579 unigenes were assigned to metabolism related pathways,and 849 unigenes were divided into the Biosynthesis of secondary metabolites,were represented the largest groups.In addition,we screened a total of 184 putative orthologous genes,used them in phylogeny construction by Maximum likelihood method,and identified the candidate orthologs which could be related with adaptive evolution.The transcriptome of P.xiuningensis complex not only showed great potential in gene content,but also provided an efficient application of RNA-seq in development of genomic resources.6.We used 10 polymorphic microsatellite loci from transcriptome analyses data to assess the levels of genetic diversity and structure for 24 individuals from 3 populations in the type location.The result of genetic diversity showed that 63 alleles were identified across the ten microsatellites analysed while 3-10 alleles,as the mean value of 6 alleles,per locus.The mean number of alleles per locus(MNA)in all individuals was 6.3±2.16;the values of observed heterozygosity(HO)and expected heterozygosity(HE)were 0.702 and 0.634 respectively,and FIS<0 showed few inbreeding.Fst result indicated a wide genetic variation between the No.1 and No.3 populations,however,no significant genetic differentiation were showed on No.2 population,which were in the geographic middle position to other two populations;Fst analysis(0.0413)and AMOVA result supported small significant result as 76%of the total variance to the within-individuals component based on the microsatellite data,and the PCoA and structure analysis indicated that there just one clusters in total.Bottleneck analysis revealed that there was no historic bottleneck in these populations under TPM model,while Mode-shift test showed all the alleles form all populations are the L-shape.7.In this study,we selected 13 eco-environmental factors according to previous studies and the growth habitat of Primulina,calculated the weight of various ecological factors which affected habitat suitability through the application of maximum entropy model;then used the multiply models and geographic information system(GIS)to obtain the suitable range of each factor which affected its growth,and also assess the spatial distribution of habitat suitability.Based on this,forecast the change of spatial distribution in future,and the measures and proposals of sustainable utilization and protection for the plant species were put forward.