Population Genetic Structure And Phylogeography Of Ginkgo Biloba L. (Ginkgoaceae): Evidence From CpDNA Haplotypes And Nuclear Microsatellites

Ginkgo biloba L., one of the Tertiary relict plants, was referred to as the living fossil and a widely cultivated plant. The questions about the possible wild populations and evolutionary history of Ginkgo are crucial to understand the historical processes which Ginkgo had undergone. Now we studied on the population genetic structure and phylogeography of G. biloba including 33 populations from China and samples of cultivated populations from Korea, Japan, Europe and North America. This work was carried out based on cpDNA trnK gene and trnS-trnG and atpH-atpl intergenic spacer,14 pairs of SSR loci to investigate the genetic diversity and the glacial refugia and patterns of population genetic structure at the scale of China. There are some main conclusions of our research:1. cpDNA trnK gene, trnS-trnG and atpH-atpl intergenic spacer sequencesThirty-three populations (430 individuals) were analyzed using cpDNA trnK, trnS-trnG and atpH-atpl intergenic spacer. A total of 15 cpDNA haplotypes were detected. Nucleotide diversity and haplotype diversity areπ=0.580×10-3 and h= 0.352 respectively. SW China and Eastern China had higher genetic diversity. AMOVA revealed 55.50% variation exist within populations. High genetic differentiation was detected (Fst=0.44506) among populations.Phylogenetic analysis and nested clade analysis of cpDNA haplotypes revealed that two refugia existed during the glaciation:the first was located in SW China and the second in Mt. TM in Eastern China. The present populations derive their genetic make-up from two inferred clusters.2. Development of SSR markersWe isolated and characterized 11 polymorphic microsatellite loci from G. biloba using a modified biotin-capture method. These loci showed high allelic diversity with PIC ranging from 0.432 to 0.909 (average= 0.752). The observed and expected heterozygosities ranged from 0.208 to 0.708 (average= 0.484) and from 0.501 to 0.934 (average= 0.795), respectively. These microsatellite markers can be used as genetic markers for further studies on genetic diversity and the gene flow among populations of this species.3. SSR analysisA total of 980 individuals from 33 populations in China, Korea, Japan, Europe and North America were investigated using 14 pairs of SSR loci. High genetic diversity was detected in WC (0.741), XA (0.810) and TM (0.788). Low genetic differentiation among populations was detected with Fst= 0.119. Structure analysis revealed two major phylogeographical groups in China:SW China, Eastern China and the mixture of other populations. This pattern probably reflected the isolation of the SW populations and Eastern populations have independent evolution history.Thus, SSR analysis combined cpDNA data revealed two refugia areas. 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.4. Evolutionary and cultivation historycpDN A haplotypes and SSR analysis had certained two glacial refugia of G. biloba during the interglacial period. The phylogeny tree based on Bayesian method showed there were two lineages in G. biloba. The posterior probability of these two lineages was 1 and indicated that two significant evolutionary unit had been formed. BEAST showed the most recent common ancestor of the two lineages was 1.9 million years ago. Mismatch distribution supported significant postglacial population expansion in Eastern China in 701 kya BP, the southwest region did not experience significant expansion, but there are some outside groups in the refugia undergone a short-range diffusion. The rare allelic richness based on SSR analysis showed that the farther away from the glacial refugia, the rare allelic richness decreased, indicating the postglacial recolonization from the nearest refugia. Central China was the secondary contact zone of both glacial refugia.Ginkgo has a very long cultivation history in Eastern Asian. SSR analysis revealed that some cultivation population had different sources. Korean population showed the similarity with central and southwest China, while the Japanese populations were similar to Central and Eastern China, the higher genetic diversity indicated that they might be introduced from China for many times and from various locations. Genetic diversity in Europe and North America are very low. As the wind pollination of woody plants, the seeds of G. biloba hadn't the structure of long-distance dispersal, but the ratio of pollen flow/seed flow was 4 and lower than other wind pollination plants. This may indicate human activities promoted gene flow among Ginkgo populations.5. Conservation strategiesThe genetic structure detected in this study suggested some ideas for the management of G. biloba populations, especially for Southwest populations, a natural reserve must be established to protect the ancestral refugia in this area. In contrast, the population in Mt. Tianmu should have the highest conservation priority in ex situ conservation. In some cultivated populations and marginal populations, the low genetic diversity requires careful consideration.