Phylogeography And Population Genetics Of The Critically Endangered Aquatic Plant Caldesia Grandis(Alismataceae)

Caldesia grandis Samuel.(Alismataceae)is a perennial,erect marsh herb.This species is confined to mountainous bogs and marshes in the temperate and tropical Old World,such as Malaysia,India,China,etc.Caldesia grandis in China has been reported from Hunan,Guangdong,Fujian,Zhejiang,and Yunnan Provinces in Mainland China as well as from the island of Taiwan.However,in recent years,due to human disturbances and the loss of natural habitats,wild populations of C.grandis have been significantly decreased in size and number.The species was listed as Critically Endangered(CR)by IUCN.In this study,in order to make appropriate conservation and management strategies for the conservation of this endangered species,we study the genetic diversity and phylogeography of C.grandis from nine natural populations based on RAD-seq data and chloroplast single nucleotide polymorphism data.The main results are presented here below:(1)The whole chloroplast(cp)genome of the three individuals of C.grandis(NX,LPH,BH)were sequenced using high throughput sequencing technology and assembled respectively.The whole cp genomes of C.grandis were 168,211 bp ? 168,304 bp with a typical quadripartite structure.All cp genomes had a similar structure,GC content,and gene organization;there are no IR border expansion and contraction.Fourteen polymorphic cpSSRs and 28 highly divergent hotspots were identified and applied to design primers for further study.Ten pairs of primers were verified by PCR amplification and five primers that could be valid amplified were chosen for phylogeographic and population genetic analyses of C.granids in the current study.(2)Phylogeography analyses were performed in nine populations of C.grandis using five newly developed cp primers.The highest haplotype polymorphism(h=0.579)was detected in the rps20-clpP region and the ndhF-rpl32 sequence displayed the highest nucleotide polymorphism(?=0.00338).In our study,five haplotypes were here identified and each population only contains one haplotype except YL population.Moreover,these haplotypes were divided into two clades,one is West(LW,LPH,GH,BH),and the other is East(GD,NX,QT,TR,YL),which are divided by the midline of the Nanling Mountains.The divergence time of the two clades was approximately 1.18 Mya during the Pleistocene period,and the divergence time of each haplotype was around 0.1 to 0.2 Mya.Additionally,The analysis of molecular variation indicated that the main components of genetic variation from among populations(99.32%),whereas 0.68% was from individuals within populations;When divided into two clades of east and west,the main components of genetic variation from among groups(94.94%),rest of the observed differentiation was explained from individuals within populations and among populations within groups,0.39% and 4.67% respectively.Also,the analysis of mismatched distribution inferred that the expansion event might have occurred in C.grandis in China.(3)In the RAD-seq analyses,a total of 8863 SNPs were filtered from 9 C.grandis populations based on De novo strategy and revealed a low genetic diversity of C.grandis(Ho = 0.102,He = 0.111).However,significant genetic differentiation(Fst = 0.333)between BH and GH was shown here,as revealed by the analysis of population genetic differentiation.Moreover,both the genetic structure and discriminant analysis of principal components inferred that historical gene flow might exist among populations of C.grandis,five genetic components were detected in all populations,five populations(GD,NX,LPH,TR and BH)were grouped together,and the remaining populations were isolated from each other.(4)Based on our results,we proposed the valuable protection strategies for the critically endangered species C.grandis in China.We suggested that each population should be as an independent evolutionary unit,at least according to the unique haplotype.Also,both in situ and ex situ should be implemented to protect this species.