Phylogenetic Position And Phylogeography Of Glyptostrobus Pensilis Based On Chloroplast Markers

Glyptostrobus pensilis(Staunt.)Koch was originally a monotypic genus plant in Taxodiaceae,but it has now been classified in Cupressaceae in APG IVclassification system.Glyptostrobus pensilis is a first-grade protected plant in China,and is also evaluated as Critically Endangered(CR)on the IUCN Red List of Threatened Species.In this study,we developed the polymorphic microsatellite(cpSSR)locus using the complete genome sequence of the G.pensilis chloroplast downloaded from the NCBI database.The phylogenetic position of G.pensilis in Taxodiaceae family was explored using the nuclear genome and chloroplast genome microsatellite(nSSR,cpSSR)markers.Using the developed polymorphic cpSSR locus to study the phylogeography of the representative populations of the existing G.pensilis global distrnbution(China,Vietnam,and Laos),to analyze its the level of genetic diversity and genetic differentiation,to define the phylogeography structure of G.pensilis,to estimate the refuge location during the ice period and the migration and expansion route during interglacial period,and to understand the population dynamics history.The research work can provide theoretical and practical basis for the conservation of genetic resources and population recovery of G.pensilis,and can also provide reference for the study of plant vicissitudes in China during the quaternary glacial period.The main research results are as follows:(1)There are 170 perfect SSR loci in the chloroplast genome of the G.pensils,and the SSR loci with a repeat number greater than or equal to 8 were screened.A total of 70 pairs of cpSSR primers were designed.All 70 pairs of primers can be successfully amplified,and produced single clear bands.The polymorphism of primers were detected using 16 samples of G.pensilis from different populations,and 10 pairs of polymorphic cpSSR primers were obtained.The genetic diversity of polymorphic loci were used to detect using 16 samples.The results showed that the genetic diversity of polymorphic loci was high.Therefore,the polymorphic loci which screened can be used in the following research on the phylogenetic position and phylogeography structure of G.pensilis and understand its evolutionary history.(2)Phylogenetic analysis was performed on 10 species of 8 genus of the Taxodiaceae using chloroplast genome(cpSSR)and nuclear genome(EST-SSR).CpSSR loci could be amplified in most Taxodiaceae samples,with an average amplification rate of 98%.Among 23 EST-SSR loci,18 pairs had amplification success rate of more than 50%.The results of phylogenetic tree(NJ)and PCoA constructed based on cpSSR markers showed that the phylogenetic tree was roughly divided into two major clusters,and G.pensilis and Taxodium are a cluster group.Other species of the Taxodiaceae are a large cluster of clusters.The results of phylogenetic tree(NJ)and PCoA constructed based on EST-SSR markers were approximately the same as those of based on chloroplast gene genome markers.The two marker methods were mutually validated.The G.pensilis and Taxodium are closely related and have relatively late phylogenetic position in Taxodiaceae.(3)The phylogeography of 14 populations(China,Laos and Vietnam)was studied by using 10 polymorphic chloroplast microsatellites.The results of genetic diversity showed that 153 haplotypes were detected in 343 plants of 14 populations,with an average number of haplotypes of 12.79 and an average number of effective haplotypes of 8.40.Private haplotypes existed in each population,and the average frequency of private haplotypes(Pr)was 0.379.The genetic diversity(He)of each population was high,ranging from 0.62 to 0.995.The geographical distribution map of haplotype reveals that there are not existence of share haplotypes between countries,and the number of private haplotypes in each population is significantly higher than that of shared haplotypes.The degree of genetic differentiation of G.pensilis was very high,the total genetic diversity(HT)was 0.991,and the population genetic mean diversity(Hs)was 0.856.The genetic differentiation coefficient RST(0.469)was significantly larger than GST(0.136),which indicated that the population of G.pensilis had an obvious phylogeography structure.The results of molecular variance(AMOVA)analysis showed that genetic variation of G.pensilis populations mainly came from among groups(47.70%).The value of genetic differentiation coefficient(FST)was 0.5972,which was greater than 0.25,indicating that there was a high degree of genetic differentiation of among the populations.The results of STRUCTURE analysis showed that the optimal K value was 3,indicating 14 populations was mainly divided into three clusters according to the country,which was consistent with the results of DAPC analysis.The phylogenetic tree results also showed three clusters,one cluster in Laos,one cluster in Vietnam,and one cluster in south and north China.(4)The DIY ABC software was used to speculate the evolutionary history of the G.pensilis population,and the results showed that the posterior probability of scenario 4 was the highest.We hypothesize that the G.pensilis evolved from China to Vietnam to Laos,and the glacial refugias were located in northern China near Jiangxi,Hunan and Fujian provinces,and in southern China Guangdong province and parts of Laos and Vietnam.(5)Through the above research,we have proposed a protection strategy for the protection of G.pensilis populations.To strengthen the protection of the existing population and to protect the natural population of Laos at the highest level;To establish seed orchards or nursery in areas similar to G.pensilis habitats.At the same time,seed breeding,cuttings and other vegetative propagation methods are adopted to find the best planting method of G.pensilis and expand the population.