Molecular Phylogeographic Study Of A "Living Fossil" Plant: Ginkgo Biloba L. (Ginkgoaceae)

Ginkgo biloba L.,often referred to as a living fossil,is a famous and widely cultivated plant.However the possible wild populations of Ginkgo are only restricted in China.Accordingly,determination of genetic diversity,population structure and glacial refugia are of crucial importance in order to understand the historical processes which Ginkgo has undergone.Our present work investigated the phylogeography of G biloba including 13 populations from China and samples of cultivated trees from Japan,North America and Europe.The analysis was carried out based on cpDNA Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP),cpDNA trnK gene and trnS-trnG intergenic spacer,Amplified Fragment Length Polymorphism (AFLP)and mtDNA nad2 intron.Genetic diversity and population structure were detected and the possible glacial refugia of G biloba in China were inferred in combination with the ecological investigations,geological history,fossil record and the pollen-based vegetation simulation during the last glacial maximum(LGM).Consequently,conservation strategies are proposed.There are 5 main conclusions of our research:1.cpDNA PCR-RFLP analysisTwelve populations from China and samples from Japan,Europe and North America,altogether 220 individuals,were analyzed using cpDNA trnK1-trnK2 intergenic spacer digested by Hinf I and MspI.In total nineteen cpDNA haplotypes were detected and formed two haplotypes diversity centres in SW China(JF,WC,PX SP and ES)and Eastern China(TM).Haplotype diversity(h)in SW China ranged from 0.7714～0.8875 and high haplotype diversity(0.8485)is also found in TM in Eastern China.Molecular variation analysis showed that 94.17%variation was detected within populations with Gst=0.2.Hence the inference was made that the possible glacial refuge of G.biloba is located in SW China.2.cpDNA trnK gene and trnS-trnG intergenic sequencesThirteen populations from China and samples from Japan,Europe and North America(145 individuals)were analyzed using cpDNA trnK gene and trnS-trnG intergenic spacer.A total of 1587 bp and 11 variation sites(5 are parsimony informative)were detected,which gave rise to a total of 9 cpDNA haplotypes. Nucleotide diversity and haplotype diversity are p=0.62×10^-3and h=0.289 respectively.In SW China,5 haplotypes were found with p ranging from 0.00 to 1.72×10^-3and h from 0.00 to 0.56.Four haplotypes(A,B,C and D)are unique to SW China.In Eastern China,3 haplotypes were found,among which 2 are unique,p= 0.53×10^-3and h=0.47.AMOVA revealed 61.94%variation exist within populations. High genetic differentiation was detected(Fst=0.38065,Gst=0.25038)among populations.Haplotype phylogenetic analysis and nested clade analysis revealed that two potential refugia existed during the last glaciation:the first was located in SW China(JF,WC,PX,SP and ES)and the second in Eastern China,especially Mt.TM was the first time to be identified as a refuge based on molecular analysis.Neutrality test and mismatch distribution neither support any significant postglacial population expansion,but there is some short distance migration from the margine of the refugia area in Southwestern China.The occurrence of haplotype E,which was derived from the only locally distributed haplotype I in TM,might be the result of a rapid range expansion during the last interglacial periods from Eastern China.Human transplantation has definitely played an important role in the spread of Ginkgo.3.AFLP analysisA total of 106 individuals from 13 populations in China and some countries in Europe were investigated.Six primer combinations produced 109 fragments,among which 50%are polymorphic.High genetic diversity was detected in ES(0.103),DH (0.100)and TM(0.102).Low genetic diversity was detected in the cultivated populations of TC(0.027),TX(0.043)and the samples from Europe(0.068).High genetic differentiation(28.73%)among populations was detected with Fst=0.29. Neighbor-Joining,principal coordinate analysis and misture analysis revealed three major phylogeographical groups:SW China,other populations in China and European samples.This pattern probably reflected the isolation of SW China during the last ice age and the SW populations and Eastern populations have independent evolution history.Four unique fragments were identified in SW populations and Eastern populations,respectively.Thus,two refugia areas were revealed.European samples are genetically divergent from Chinese populations,indicating their indirect origin from China,but it is more closely related to the Eastern populations in China.After excluding European samples,further grouping analysis showed that cultivated populations(TX,TC and SX)were separated into three different groups indicating they have different origins.AFLP patterns also indicated human transplantation was of great help for the spreading of Ginkgo.4.mtDNA nad2 intronCloning results of nad2 intron showed high variations within individuals.We inferred that PCR mismatch and probably the second structure of nad2 intron will prohibit the accrurate process of PCR.The random distribution of these variable sites can not be used as informative sites,so nad2 intron is not the suitable marker for the phylogeoraphica study of G biloba.5.Evolutionary history and conservation strategiesCpDNA haplotype distribution showed us two glacial refugia of G biloba during the last ice age.High genetic diversity was found in Southwestern China and Eastern TM population.The genetic structure detected in this study is critical for the establishment of the conservation strategies.In order to conserve the genetic resources of G biloba,we propose that the most desirable populations for the in situ conservation are those in SW(JF,WC,PX,SP and ES)and Eastern China(TM).The highest importance should be devoted to the conservation of the old Ginkgo trees in those populations.Because of the high genetic diversity these populations are desirable for an ex situ core collection.In addition the collection of germplasm from different populations is necessary during ex situ conservation in order to preserve the high genetic diversity of G biloba.