Studies On Conservation Genetics Of Camellia Huana

Camellia huana T.L.Ming et W.J.Zhang belongs to Camellia（Theaceae）,which is a small shrub with yellow flowers.It is one of the species of yellow-flowered Camellia.The distribution of C.huana is very narrow,being restricted to evergreen,broad-leaved forests in the limestone hills of Tian’e County in northern Guangxi and Luodian County,Ceheng County and Xingren County in southern Guizhou,China.C.huana is the most northerly species of sect.Chrysantha and shows a discontinuous distribution with other sect.Chrysantha species.It is of great significance in the introduction of yellow-flowered Camellia to the north.Owing to the ornamental and medicinal value,more and more wild plants of C.huana were transplanted by nearby residents,even though many natural populations have disappeared.C.huana has listed as an Endangered（EN）species on the Threatened Species List of China’s Higher Plants.A total of 358 individual C.huana samples were collected from five natural populations in the Tian’e County of Guangxi,and seven other populations directly transplanted from wild habitats:three in the Luodian County of Guizhou province,and the remaining four in the Tian’e County of Guangxi.A chloroplast small single copy region（SSC）（including ndhF,rpl32,trnL-UAG,CCSA）,a single copy nuclear gene PAL（phenylalanine ammonia-lyase,PAL）and 12 pairs of microsatellite primers were employed to analyze the genetic diversity and structure of C.huana,and develop effective conservation strategies and management measures based on the results.The main findings are as follows:1)The entire distribution area of C.huana was surveyed in 2018 and 2019,and only five natural populations were found,in Tian’e County,Guangxi.There was no natural populations in Guizhou.Field studies also have found there are a large number of plants transplanted directly from wild in the local resident near the wild habitat.2)C.huana possessed a low level of genetic diversity based on sequencing data.Chloroplast DNA fragments were amplified and sequenced to obtain a total of 120sequences,and cpDNA consensus sequences were 5,200 bp in length containing nine polymorphic sites with no indels.Genetic diversity indices of total nucleotide（Pi）and haplotype（Hd）diversity in all populations were 0.00042（individually ranging from 0 to0.00021）and 0.759（individually ranging from 0 to 0.533）,respectively,as inferred from cpDNA.A single copy nuclear gene PAL were amplified and sequenced to obtain a total of240 sequences,consensus sequences were 660 bp in length containing seven polymorphic sites with no indels.Genetic diversity indices of total nucleotide（Pi）and haplotype（Hd）diversity in all populations were 0.00131（individually ranging from 0.00067 to 0.00197）and 0.631（individually ranging from 0.442 to 0.711）,respectively,as inferred from PAL.We identified eight chloroplast haplotypes,haplotype C5 was the most abundant haplotype with wide distribution in five populations（MGS,ECC,LJPT,DLT and NRT）.Haplotypes C5 and C8 took central positions while the other haplotypes were distributed on outside nodes in the network.This distribution pattern suggested that haplotypes C5 and C8 likely represent the ancient haplotypes for this distribution area.Haplotype C5 was separated from haplotype C3 by four mutational steps.In addition,there are only one or two mutational steps between any two adjacent haplotypes.Among a total of eight nuclear haplotypes were yielded in the species,haplotype H1 and H2,as the most dominant haplotypes in the genetic composition of the species,were detected in the almost all populations,except population HLT and TBC,respectively.When referred to the PAL subsets,haplotype H2 and H1 took central position in the network with a higher proportion of individuals than remaining haplotypes.This distribution pattern predicted that haplotype H1 and H2 likely represent the ancestral haplotypes for this geographic area.The remaining haplotypes were distributed at each node,and there was only one mutational step between any pair of haplotypes.Microsatellite markers in C.huana revealed a low level of genetic diversity.A total of99 alleles at 12 SSR loci were revealed across 358 individuals from 12 C.huana populations,with a mean of 8.25 alleles per locus.The number of mean alleles（Na）per population varied from 2.833（populations XDZ and LBT）to 5.417（population MGS）,with an average number of 3.931 across all populations.The average number of effective alleles（Ne）for all populations was 2.272,varying from 1.722（population XDZ）to 2.908（population MGS）.The average values of Ho and He for all populations were 0.457 and0.466,respectively;Ho values varied from 0.353（population JSP）to 0.589（population TBC）and He values from 0.339（population JSP）to 0.605（population MGS）.3)High level of genetic differentiation was detected in the C.huana species based on microsatellite markers.The genetic differentiation coefficient(F_ST)between populations was between 0.027 and 0.419.Among them,F_STT value was less than 0.15 in twenty groups,F_STT value was between 0.15 and 0.25 in seventeen groups,and the F_STT value of remaining twenty-nine groups was more than 0.25.The genetic differentiation coefficient derived from cpDNA and PAL were 0.9318 and 0.0853,respectively.Thus,this indicated that we found high and moderate genetic differentiation among populations of C.huana for cpDNA and PAL,respectively.The AMOVA results showed that only 6.82%genetic variation was partioned within populations for cpDNA,whereas 91.47%and 78.41%were partioned within populations for PAL and SSR markers,respectively.In addition,no significant correlation was detected between genetic and geographic distance both at microsatellite markers and DNA sequencing data.4)According to the results of our STRUCTURE analysis,the 12 C.huana populations should be split into two genetic groups consistent with geographical distribution using SSR markers.Populations XDZ,JSP and LBT were assigned to Group I;the remaining populations（MGS,ECC,TBC,LJPT,WGT,NJT,DLT,NRT and HLT）composed Group II.The existence of two groups was also supported by principal co-ordinate analysis（PCoA）.5)Both in situ and ex situ conservation and management strategies should be put into effort in purpose of maintaining the maximum amount of the genetic diversity.According to the results of our STRUCTURE analysis,the five natural C.huana populations should be split into two management units,and we should place more emphasis on in situ conservation.High levels of genetic differentiation among populations suggested that the transplanted populations originated from the neighboring limestone mountains,respectively.Therefore,some ex situ conservation measures are necessary for transplanted populations,with as many sample individuals as possible covering all populations.