The Study On The Distribution At Small-scale,Clonal Structure And Genetic Diversity Of Kingdonia Uniflora On Taibai Mountain

As one of the most endangered plants species in China for its extremely narrow distribution and stringent habitation requirements,Kingdonia uniflora is a clonal subalpine perennial herb belonging to the monotypic genus of Circaeasteraceae,which is the key species in biodiversity research for ancient origin and isolated taxonomic position.Researchers have shown that K.uniflora was only unevenly distributed in the coniferous forests,coniferous and broad-leaved mixed forest and subalpine shrub understory environments on the north and northeast slope in Taibai Mountain.Previous studies focused on the studies of biological characteristic,geographical distribution,anatomy,pollination and morphological of K.uniflora,but the distribution at smallscale understory,the clonal structure and genetic diversity along the altitude are still unknown,therefore,the protection of this species exist some blindness.In this study,we attempted to utilize methods of physiology to solve the issues of distribution at small-scale habitats of ecology.Moreover,the inter-simple simple sequence repeats(ISSR)and simple sequence repeat(SSR)techniques were used to investigate the genetic diversity and genetic variation in K.uniflora among four natural populations at different altitudeson north slope of Taibai Mountain.On the other hand,we used SSR makers to describe the clonal diversity and clonal structure of K.uniflora populations along the altitudes through different sampling strategies.The results are as follows:(1)We studied the photosynthesis of K.uniflora in the field to test whether understory sunflecks result in photoinhibition and thereby potentially influencing its patchy distribution.K.uniflora exhibited the low dark respiration rates(Rds),low light compensation points(LCPs)and low light saturation points(LSPs)characteristic of shade-tolerant plants,allowing it to survival during the long periods of low understory light.Moreover,K.uniflora was able to regulate light energy utilization by nonphotochemical quenching(NPQ)in low light.And the higher apparent quantum yield(?)of K.uniflora in SL regions indicates a greater capacity to utilize weak light.The net photosynthetic rate(Pn)of K.uniflora could be reduced by the induced nonstomatal factors subjected to continuous saturated light.The continuous light radiation and enriched sunfleck could caused photoinhibition in K.uniflora.The UV-B in sunflecks could be an underlying factor in the Pn decrease and photoinhibition of K.uniflora.In addition,application of simulated sunflecks indicated that K.uniflora leaves do not need continuous light.The photosynthetic responses of K.uniflora to sunflecks indicated that sunflecks are a limiting factor in the small-scale distribution of K.uniflora.(2)The genetic diversity and the pattern of genetic structure in four natural populations of K.uniflora along the altitudinal gradient were evaluated by 12 intersimple sequence repeats(ISSR)markers and 17 simple sequence repeat(SSR)markers.A total of 87 unambiguous and reproducible bands were generated,of which 41(PPL=47.13%)were polymorphic by 12 ISSR primers.The effective number of alleles per locus ranged from 1.151 to 1.246 with an average being 1.191.The PIC values for the evaluated 12 ISSR primers were 0.606~0.796,indicating that the ISSR primers are suitable for investigated genetic characters of K.uniflora.A total of 60 alleles at 17 SSR loci were revealed across 108 individuals of K.uniflora.The number of alleles per locus for 17 microsatellite loci ranged from 2 to 6,with an average of 3.53,and the expected heterozygosities(He)and observed heterozygosities(Ho)ranged from 0.231 to 0.350 and from 0.093 to 0.154,respectively.No linkage disequilibria were detected at these loci at P<0.05 significance levels.None of locus with null alleles in all populations though null alleles might occur at few locus in some populations suggested by Micro-Checker software,indicating that the SSR loci are suitable for investigated genetic characters of K.uniflora.There was a relatively low level of genetic diversity in natural populations of K.uniflora for both markers(I=0.170 and 0.187,for ISSR and SSR respectively;PPL=47.13% for ISSR;He=0.332 for SSR).And the population of K.uniflora at 2889 m in XBS had greater genetic diversity than populations at lower and higher altitudes(for ISSR and SSR),corresponded to the pattern of ‘L<M>H'.Furthermore,Nei's analysis of gene diversity and AMOVA analysis revealed that the high level of differentiation occurred among the populations based on ISSR and SSR markers(for ISSR: ?ST=0.74,GST=0.737;for SSR: ?ST=0.67,GST=0.792).It is therefore likely that K.uniflora maintained low levels of genetic diversity due to clonality,self-compatible breeding system and its pollinators,which restrict the exchange of genes.The results of STRUCTURE revealed that the four natural populations of K.uniflora grouped into three clusters.The DBL population grouped into one cluster,the population of HHP grouped into another cluster and distinctly separated from XBS population and SBS population.The UPGMA clustering analysis suggested that four populations were classified two filiations,the one was HHP population,the other one included the rest populations.(3)The K.uniflora showed patchy distribution independently along the altitudinal gradient in Taibai Mountain.The the clonal diversity of K.uniflora in five populations by mechanical sampling along the altitudinal gradient using microsatellite(simple sequence repeat;SSR)analysis demonstrated that the proportion of distinguishable genets(G/N)and Simpson's diversity index were 0.15 and 0.74,respectively,confirmed the lower clonal diversity in clonal species.The correlation analysis showed that clonal diversity increased and then decreased along altitudes,the value of PD in XBS population at 2889 was significantly higher than that in other populations.The clonal structure showed none of the five populaitons were composed of a single genet,and there was no identical genotype among five populations.K.uniflora populations exhibits significant clonality and the two maximum clones were 7m and 7.1m,appeared in HHP and SBS populations.The HHP and SBS plots were composed by monoclonal in seven 1m×1m plots.Majority genets in seven 1m×1m plots inclined to clumped distribution and minority genets presented descrete distribution,indicating clear mosaic within populations of K.uniflora and was suggestive of an un-strict guerrilla strategy.The number of ramets in 1m×1m SL plot was more than in 1m×1m SR plot at XBS.The genets in 1m×1m SR plot were inclined to descrete distribution,indicating the genets generally might escape from the sunfleck-enriched habitat.It reflectd sunflecks play an important role in understory small-scale distribution of K.uniflora.Both mantel tests and spatial autocorrelation analysis revealed significant positive clonal structures at small scales.