Species Germplasm Conservation Of The Wild Plants With Extremely Small Populations-Cinnamomum Chago

As an endangered species endemic to Yunnan province,Cinnamomun chago B.S.Sun et H.L.Zhao possesses large economic and phylogenetic values.Although this species has been listed as one of the 321 proposed Plant Species with Extremely Small Populations(PSESP)in southwest China for the comprehensive surveys and germplasm conservation,the study of conservation biology for this species remains relatively unexplored.In the present study,a systematic and comprehensive study was conducted on the distribution status,eco-physiological characteristic of seed and conservation genetics to evaluate its endangered category,as well as to elucidate the endangered causes and mechanism for C.chago.Furthermore,some management and conservation strategies and suggestions to protect species germplasm of this species were proposed.The major results and conclusions are summarized as follows:1.Eveluation of endangered status and endangered category of C.chagoAccording to field investigation,the remnant populations of C.chago were fragmentarily occurred in Yunlong and Yangbi county of Yunnan Province.The number of populations and individuals were extremely few,and only 64 adults were found in 5 confirmed field populations.The wild populations were narrowly distributed with an extent of occurrence of 923.23 km2 and an area of occupancy of 60.19 km2.Two types of habitat for C.chago were categorized,containing evergreen broad-leaved forest for populations NMP and DSB and artificial economic forest for populations XC,LG and SBX.Of these 5 populations,none was found to distribute in protected area.The habitat of the populations has been seriously destroyed by human disturbance,such as unrestrained seeds collection and deforestation by the local villagers.C.chago had evolved as a species with a typical aging population structure with few seedlings or saplings were found.Therefore,based on IUCN Red List Categories and Criteria(Version 3.1),C.chago can be evaluated as EN(Endangered)species.It needs to be urgently rescued in China.2.Eco-physiological characteristics of seed for C.chagoSeed of C.chago belongs to large seeds,consisting of endocarp,cotyledon,and embryo.The hard and dense endocarp of seed is constituted by three different stone cells layers.Except the cavernous stone cells distributed in innermost layer,the remaining stone cells,closely spaced in outer layers of endocarp,possess strong mechanical strength as the thickened and highly lignifying cell wall and the high content of silicon.The endocarp has poor air permeability but good water permeability.The water should gradually seep into the seed through the short columnar stone cells with rounded simple pore and the vascular bundle in basilar endocarp of seed.The germination percentage was significantly increased to 33.18±19.42%after the basilar endocarp was broken.Gibberellin had no significant effect on germination percentage of seeds,indicating that there was no physiologically after-ripening process in embryo of C.chago.Temperature significantly affected germination percentage,and the optimal temperatures for seeds was 20-25?,under which the germination percentage was 53.33±22.74%.The germination percentage of Brassica pekinensis was inhibited significantly by the methanol extract of seeds,indicating that there are endogenous inhibitor to impede seed germination in C.chago seeds.Therefore,the dormancy of C.chago seeds belongs to combinational dormancy(PY+PD).Additionally,a total of 8 hormone signaling pathways,35 active enzymes and 132 candidate genes were detected by the enrichment analysis of differently expressed genes(DEGs)from transcriptome of initial seeds(CK),seeds that water absorbed for 24 h(As)and germinating seeds(Gr).Among the 132 candidate genes,the most were categorized as down-regulated genes.3.Conservation genetic analysis of C.chagoThe genetic diversity,genetic structure and demographic history of 5 populations in C.chago were investigated by 11 novel EST-SSRs and 4 DNA sequences(psbA-trnH,CHS,LFY and RPB2).As a result,a low level genetic diversity but a significant genetic differentiation(FST=0.2198,P<0.001)among populations was estimated at the species level(Na=2.509,NP=3.8,Ra=2.423,AE?1.635,Ho=0.177,He=0.340)based on EST-SSRs.The 5 populations presented obvious inbreeding depression as a higher value for inbreeding coefficient FIS(0.379<FjIS<0.661)and a lower value for outcrossing rate t(0.204<t<0.450),The AMOVA analysis revealed that 78.02%of the genetic variation was partitioned within populations.The population structure was cluster into two groups,of which group 1 was consisted of population XC,LG and SBX and the other group was constituted of population NMP and DSB.The Structure analysis was more detailed and distincted the 5 populations to 4 clusters,of which the population XC,LG and SBX was grouped,respectivly.By mantel tests,Genetic distance and geographic distance were not correlated(P>0.05).The historical and contemporary gene flow between populations were extremely low.The remnant populations have experienced recent historic bottleneck and the effective population sizes in all populations are smaller than 50.Consistent with the results of EST-SSRs,there was a significant genetic differentiation among populations based on the DNA sequences(HT>HS).The AMOVA analysis of 3 nDNA revealed that the most genetic variation was partitioned among populations,but the most genetic variation of cpDNA was partitioned wiyhin populations.Additionally,the neutrality tests and mismatch distribution analysis revealed that C.chago without a recent population expansion.3.Endangered mechanism and conservation strategies of C.chagoAccording to the above results,we proposed that the endangered causes of C.chago were mainly attributed to three aspects.Firstly,the species had extremely small populations and few individuals,and the distribution range was narrowly and fragmentarily.Secondly,population regeneration is difficult due to the serious human disturbance and combinational dormancy of seed.Finally,the genetic diversity and effective population sizes of remnant populations were extremely low and obvious inbreeding depression phenomenon presented within populations.Therefore,we suggest devise different conservation management for populations with different genetic characteristics and ecological differences.To begin with,we should take priority for in situ conservation for individuals and habit of every population,especially the population XC with higher genetic diversity.Furthermore,artificial assisted propagation should be carried out to expand wild population size of population with extremely low effective population size,such as SBX,NMP and DSB(Ne<15).Additionally,to improve and protect the genetic diversity of wild and artificial populations of C.chago,we should promote proper gene exchange among individuals to reduce the inbreeding frequency.It is noticeable that the loss of valuable and special haplotypes within population due to hybridization or introgression must be avoided in this process.