Study On The Conservation Biology Of The Rare And Endangered Sinojackia Microcarpa (Styracaceae)

Conservation biology is a discipline that mainly studies the protection of animals and plants,as well as technology and methods for protecting their living environment(What effective measures should be taken to protect biodiversity).Sinojackia microcarpa is a rare and endangered plant endemic to China,and it was listed as the first of Extremely Small Population of Wild Plant by the Department of Forestry of China.At present,very few studies have focused on conservation biology of S.microcarpa.The present survival situation of the wild S.microcarpa,its population characteristics,morphological diversity of leaf and population genetic diversity were investigated by using methods of field investigation and laboratory test,sampling and investigation,morphology observation,molecular marker respectively.This study also analyzed the main obstacle factors affecting the survival and development of S.microcarpa.Finally,a theoretical basis for population regenerated,reconstruction and artificial propagation and utility of S.microcarpa were provided.The main results are listed as follows:(1)Identification of S.microcarpa populationsReference to the model specimens of Li'an and Jiande' populations,according to the comprehensive comparative analysis of phenotypic diversity and genetic diversity of population leaves,the results showed that four populations distributed in ZJFY,ZJTL,ZJJH and ZJYW also belonged to S.microcarpa species,which extends the distribution range of S.microcarpa populations in Zhejiang Province.To characterize the suspected population and species,the leaf surface and genetic diversity of AHJX population were similar with the populations of ZJLA and ZJJD,which was also classified to the S.microcarpa populations.The results expanded the distribution range of S.microcarpa populations outside the Zhejiang Province.However,the suspicious distribution populations of JXPZ might be a transition type among the populations of S.microcarpa,S.rehderiana and S.huangmeiensis.(2)Leaf-type diversity of eight populationsThe obsevartion results of leaf length(LML),leaf veins(VN),petiole length(LSL),leaf base angle(LBA),leaf width(LW),leaf front width(LFW),leaf basal width(LBW)indicated that the optimum leaf sample of S.microcarpa was related to the optimum environment for growth and development.There were significant differences in each character between inter population and intra population.There was a decreased trend of LML,LW and LSL with the increase of latitude.The results of cluster analysis showed that the 8 populations gathered into 2 clusters based on the dividing line 1 of genetic distance.The phenotypic characteristics were basically similar among the 4 populations of ZJTL,ZJYW,ZJLA and ZJJD.They could be grouped into 1 class.Furthermore,the populations in ZJFY,AHJX and JXPZ were similarly grouped into 1 class,but the population in ZJJH was divided into another class.Therefore,the phenotypic characteristics of leaf traits of six populations,on the one hand,were a high similarity,and on the other hand,were basically grouped into 1 class according to geographical distance.(3)Genetic differentiation analysis of eight populationsTo investigate the effects of different factors on the experiment of ISSR genetic diversity for the rare and endangered S.micoocarpa,potential factors(magnesium2+,dNTP,primers,Taq polymerase and DNA template)were compared and analyzed at different levels.Annealing temperature and the number of cycle,which also had a significant influence on the result of the experiment,were optimized based on the optimal ISSR-PCR amplification system.The results showed that a suitable ISSR-PCR amplification system for S.microcarpa was established.A total volume of 20.0?L system contained about 1 ?DNA templates,10×Buffer 2 M1,1.0 U Taq polymerase(takara),tendency for 2.5 mmol·L-1 magnesium2+,dNTP tendency for 0.25 mmol·L-1,0.5 ?mol·L-1 primers.Amplification conditions were:94? modified 5 min,and then enter the following cycle,94? to 55?,30 s summarize stretching up to 72 s,30 s to the 72? for a total of 40 cycles,finally extends 72 ? for 5 min,cooled to 4 ?.This study established the rare and endangered plant S.microcarpa best PCR-behind the ISSR reaction system for the study of the genetic diversity of the plant,and layed a foundation for it's landscape applications.Leaf samples were collected from 185 individuals.156 bands were generated from 14 pairs of informative SSRs primers with an average polymorphism percentage bands(PPB)of 45.67%.The average index of Nei's genetic diversity(He)and Shannon's information(I)were 0.10 and 0.17 respectively.The genetic consistency(GI)was 0.28?0.87.The range of genetic distance(GD)were 0.14-1.28.With 10 informative ISSR primers,95 bands were amplified with an average PPB of 41.45%.The average of He and I were 0.13 and 0.20 respectively.The genetic consistency(GI)was 0.60 to 0.98.The range of genetic distance(GD)was 0.02 to 0.51.Cluster analysis showed that 8 wild populations could be classified into three groups,based on SSRs and ISSR genotypes using the UPGMA analysis when the threshold value was 0.40 and 0.14.There was some significant association between the phylogenetic relationship and the geographic locations in the S.microcarpa populationsThe results also indicated that the wild populations of S.microcarpa from the Zhejiang,Anhui province and Jiangxi province valley had a poor genetic diversity in SSRs and ISSR genotype,thus it is of great importance to explore genetic evolution and gene resources and to preserve the local populations in this area and expanded area.(4)Research on the distribution pattern and characteristics of eight populationsThe populations of S.microcarpa were located at 29°04'27"?30°41'30"N and 116°54'57"?119°55'46"E of Zhejiang,Anhui and Jiangxi provinces.The tolerant maximum and lowest temperatures of S.microcarpa were 41.0? in summer(July)and-16.0? in winter(January),respectively.There were some different population structures of S.microcarpa among different populations.Overall,its distribution pattern was generally the aggregation type.The young and middle age population accounted for the highest proportion among the whole population.Its distribution showed an obvious Pyramid structure and the survival curve tends to the type of Deevy-III.There was a high mortality rate(qx)in the fifth age class and the qx value was 0.83.The number declined from the fourth age to the sixth grade.Only 0.30%plants could grow in the seventh age class.The average life expectancy(ex)showed a high peak at grade I and ?,qx and lethality rate(kx)at this time were relatively low,,suggesting that the growth vigor of S.microcarpa is the best in the most vigorous period in the middle and young stage.(5)Studies on flowering biology of S.microcarpaIn order to understand the reproductive biology of S.microcarpa and its possible effects on its status of being endangered,the floral syndrome and breeding system were investigated.A field investigation of the flowering course and functional floral morphology of S.microcarpa was conducted.The flower of S.microcarpa has a raceme and hermaphrodite flower structure,with 4.87±1.01 small flowers.There are some differences in morphology among flowers,and it flowers lasted 6.59±0.975 days.The stigma is always 4.63 ± 1.82 mm higher than anther during the bloom.Three cleft and gradually elongate the head of the stigma.The pollen powder is in the order of extraversion.During the flowering course,the stamen is closed to the center gradually and finally surrounds the pistil.The flowering course for one flower of this species can be divided into six periods based on the flower morphology and gender function expression:Bud stage,prophase of flowering,female stage,bisexual stage,male stage,and fade stage.The flowering period of a single plant is 20 d,and the flowering period of the whole population is about 25 d.Pollen lifespan after flowering is 2 to 5 d.The highest pollen vitality is in 1 to 2 d,and the highest receptivity of flowering is at the second to the fourth day.The flowering periods of the populations in different geographical environment are slightly different.The flowering phenology is delayed in those populations distributed in shade regions.The outcrossing index and pollen-ovule raio of S.microca,pa were 4 and 5342±1520 respectively.The results of pollination experiments showed that there was no apomixes.It was self-compatibility,and entomoplily played a larger role in the process of pollination.These indicated that the outcrossing is the main form in breeding system of S.microcarpa and it is also selfcompatibility.Thus,the value of gene flow(Nm)equal to 3.18 by the way of SSRs,and Nm of ISSR equal to 0.93,the propagation system of S.microcarpa belongs to a mixed-mating system.(6)Effects on Photosynthetic Physiology of S.microcarpaThe results from the Li-6400 portable photosynthesis system in S.microcarpa measured after treatment with shading(0.0,70.0%,90.0%)showed that with increasing of shady,the total of chlorophyll,light saturation point(LSP)and the net photosynthetic rate(Pn)in S.microcarpa were increased firstly and afterward decreased.It reached the peak at the 70.0%shade,and the value were 622.50 ± 10.87 ?mol·m-2·s-1,2.52 ±0.14?mol·m-2·s-1,respectly.Light compensation point(LCP),was decreased firstly andv subsequently increased,reached the bottom at the 70.0%shade with 208.84 ± 0.21?mol·m-2·s-1.The LCP?AQY?phototrophic and fluorescence factors clearly revealed that the S.microcarpa is a shade plant.Further evidence by chlorophyll fluorescence showed that the efficiency of energy conversion of open PS?(Fv/Fin)maintained stable,whereas its optoelectronic production(Yield),electron transport rate(ETR),photochemical quenching(qP),Non photochemical quenching of chlorophyll fluorescence(qN),rised firstly and then decreased.This was consistent with the change in Pn,like 70.0%shading treatment,reaching the peak at 0.730 ±0.022?16.800±4.34 ?mol·m-2·s-1?0.979±0.023?0.871± 0.019,respectively.These results suggested that the growth of S.microcarpa promotes under slight drought stress conditions,which might be probably associated with the dissipation of excess light energy.(7)Endangered mechanism and conservation strategiesThe presence of moderate to high genetic diversity in S.microcarpa indicated that the current endangered status of this species was not caused by genetic factors(e.g.genetic diversity decline,genetic drift and inbreeding)and biological characteristics.The main threat to this plant species may be the extremely limited distribution and rarity individuals.Additionally,site condition had led to a bottleneck in population natural regeneration,and colonization of new populations was lack of effective ways.We suggest that management policies should be improved to maintain the appropriate effective population size of S.microcarpa and to protect its natural habitats.Furthermore,adult S.microcarpa trees are critical resources,not only to maintain current genetic diversity but also to provide provenance for its future recovery.Thus,protecting adult trees should be the priority in conservation to ensure ongoing recruitment.Meanwhile,appropriate human disturbance can promote population natural regeneration due to site condition.In addition,we recommend that seeds should be collected for germplasm storage and artificial seedlings for ex situ conservation.Supporting the recovery of wild populations,the population recovery and reintroductions of S.microcarpa should also be carried out.