Genetic Diversity Of Cypripedium Japonicum Thunb. With SCoT Analysis And The Phylogeography Study Of Sect. Flabellinervia

Section Flabellinervia (Pfitz) Hennessy ex Cribb. which belongs to Orchidaceae, genus Cypripedium L., including C. japonicum and C. formosanum. C. japonicum is a rare orchid distributes only in East Asia including just China, Korea and Japan. C. formosanum is an endemic species to Taiwan. In order to make the scientific and seasonable conservation strategies for this endangered slipper orchid, SCoT molecular marker was used to study the genetic diversity as well as its genetic structure of C. japonicum by detecting 16 populations which were from the whole distribution areas. Meanwhile,with the aim of understanding the cause of formation and conjecturing its origin,refuge and migratory routes,5 non-coding regions accD-psaI、petL-psbE、 psbK-trnS、trnK-rps16、trnQ-rps16 (accD-psaI、trnK-rps16、trnQ-rps16 of C. japonicum with our laboratory research results) was used to study the phylogeography of 20 populations in section Flabellinervia. The phylogeography research on the species with disjunctive distribution pattern is helpful to explore the genetic variation and to study the phylogeography structure of East Asia and the adjacent islands.The results indicated that Cypripedium japonicum had low genetic diversity at species level (PPB=61.04%, H=0.1449, SI=0.224) and population level (PPB=10.88%, H=0.0331, SI=0.0510). The populations had obvious genetic variation (Gst=77.88%), and low gene flow (Nm=0.1420). The analysis of UPGMA and PCoA both showed that the populations of C. japonicum clustering two main groups:China-Korea. Japan. The STRUCTURE analysis showed three groups:Japan, Eastern-Central China and Western China-Korea. Populations of Japan had obvious genetic differention. So, due to its low genetic diversity, in-situ protection of C. japonicum should be taken and FP、 SZ、JF、SN populations with high genetic diversity should be protected with priority.The phylogeography results of section Flabellinervia based on cpDNA showed that there were 22 haplotypes in 20 populations. The nucleotide diversity (71) at spexies level was 0.00299 and the haplotype diversity (h) was 0.874. The genetic div- ersity was in the medium range and the gene flow between populations was low (Nm=0.17). The network of haplotype had three groups:China-Korea, Japan, and Taiwan. Both of the MP、 BI results had two main branches:China-Korea, and Japan-Taiwan. The haplotype results also indicated that populations of Japan had obvious genetic differention. The divergence time showed that section Flabellinervia differentiated at Tertiary Miocene. The divergence time of Japan populations was similar with China-Korea populations, the divergence time of Taiwan populatiuons were the latest. We speculated that section Flabellinervia formed the distribution pattern at early time. The ancestors with continuous distribution were separated in glacial time. The separate areas had individual refuge and evolved independently until now.Combined with geological event, we speculated the ancestor of section Flabellinervia had been widely distributed in high latitude areas of the Northern Hemisphere and retreated southward under the influence of the cold climate in Oligocene. The species firstly arrived in areas with high latitude in Japan and Korea and then to China. They retreated southern to Taiwan through the Ryukyu Islands in the late Miocene. Cypripedium formosanum has relatively close genetic relationship to populations in Japan. In the quaternary glacial period, populations in China-Korea expanded from Central and Eastern China. Founder effect occured in populations of Korea that had only one haplotype. The populations in Japan expanded and had relatively high haplotype diversity and nucleotide diversity. The populations in Taiwan had no obvious expansion.