Phylogeography Of Camellia Flavida

Camellia flavida is an endangered species of yellow camellia growing in limestonemountains in southwest China.The current classificationofC.flavidais controversial.The size of natural populations of C.flavida,especially its variant C.flavida var.patens,has declined dramatically due to illegal transplanting,thus its distribution has become fragmented seriously.A total of 188 individual plants from 20 populations across theentire distribution range in southwest China were analyzed using two sequences:a chloroplast DNAsequence from the small single copy region and a single-copy nuclear gene called phenylalanine ammonia-lyase(PAL),By combining these two types of markers,we conducted a phylogeographic analysis of C.flavida to determine the population genetic structure and phylogeographic patterns of C.flavida,clarify the species classification and boundaries and propose recommendations for guiding future preservation actions.The main results were as follows:1)Alignment of cpDNA sequences from C.flavida produced a consensus sequence of 5178 base pairs.Seventeen haplotypes were defined with 38 polymorphic sites.The SAMOVA revealed that the cpDNA dataset of C.flavida can be partitioned into three groups.All var.flavida formed a group(ND,MQ,NF,DY,LLS,LR,SC,NX,LM,LL,MZ,LD,BZ,LX and LN).Var.patenswas separated into two groups:group(LHS,WM)and group(SG,NXS,NGL).The ML tree showed similar cpDNA haplotype phylogenetic relationship with the network analysis.The aligned PAL gene sequences from C.flavida were 653 base pairs long and contained 69 polymorphism sites,resulting in 87 distinct haplotypes.In contrast to the chloroplast DNA results,C.flavida populations can also optimally be placed into three groups.These three groups included var.flavida 1(ND,MQ,NF,DY,LLS,LR,SC,NX,LM,LL,MZ,LD),var.flavida 2(BZ,LX,LN),var.patens(SG,LHS,NXS,NGL,WM).In the PAL haplotype network,Haplotypes from var.patens were clustered together and situated in the middle of the network,separating haplotypes of var.flavida 1from that of var.flavida 2.Although with low bootstrap value,in the ML tree all the haplotypes of var.flavida 2 and most haplotypes of var.patens clustered together respectively.These three genetic groups are consistent with their morphological characteristics.Our nuclear DNA results demonstrate that there are three differentiated groups within C.flavida:var.flavida 1,var.flavida2,and var.patens.Samples from population BZ and the type herbarium specimens of C.flavida were collected from the same location;therefore,we suggest that var.flavida 2 is the genuine C.flavida.Species should then be better characterized using such nuclear markers.2)Both chloroplast and nuclear markers revealed high levels of genetic diversity(n =17,h = 0.94101,and ? = 0.00157 for cpDNA;n = 87,h = 0.97993,and ?= 0.00880 for the PAL gene).At the levels of variety,var.flavida(cpDNA:n=3,h=0.66086,?=0.00090;PAL:n=21,h=0.89852,?=0.00455)showed a higher genetic diversity than var.patens(cpDNA:n=14,h=0.92962,?-0.00092;PAL:n=67,h=0.97599,?=0.00902).According to the three SAMOVA groups of PAL,The var.flavida 1 group showed the highest level genetic diversity(cpDNA:n = 11,h = 0.91036,?= 0.00090;PAL:A = 58,h = 0.97242,? =0.00874).The var.flavida 2 group(cpDNA:n = 3,h = 0.67692,? = 0.00029;PAL:n = 9,h=0.82449,?= 0.00214)and var.patens(cpDNA:n = 3,h = 0.66086,? = 0.00090;PAL:n=21,h = 0.89852,?= 0.00455)have relatively lower level genetic diversity.3)The large genetic differentiation(GST= 0.976,NST-0.974)and low Nm value(0.01)derived from analysis ofcpDNA suggest anabsence of gene flow via seed dispersal.63.46%of the genetic variation in cpDNA was attributable to variation among varieties.The variation among populations(34.78%)was greater than the variation within populations(1.76%).The proportion of genetic variation between the three groups was also high(57.95%).Population differentiation determined with nuclear PAL gene data was also high(GST=0.253 and NST 0.414).The presence of significant phylogeographic structure was verified using the PAL data(GST<Nsr,P<0.05).The molecular difference between var.flavida and var.patens in the PAL data was low(17.81%).The gene flow(Nm)among populations was 0.35,and was primarily achieved by pollen transfer.The highly diverse and high levels of genetic differentiation can be attributed to the high habitat heterogeneity in limestone karst.4)Our nuclear DNA results demonstrate that there are three differentiated groups.The three groups should be recognized as three management units for conservation concerns.Control of illegal harvesting is critical for conservation of these species,and in situ conservation measures should be established first.There was high genetic diversity at var.flavida 1,and in situconservation measures should be preferentiallyconsidered.In var.flavida 2,there are only three populations,and var.flavida 2 is the genuine C.flavida.Thus,populations of var.flavida 2 should be candidates for ex situ conservation by using plants from cutting propagation.Within var.patens,no haplotype is shared among the three distribution points and high genetic divergence was detected.Because the number of populations is limited and there are few individuals in each population,these populations are reasonable candidates for ex situ conservation in germplasm banks.