Establishment of a propagation technique and ecological surveys for the protection of Rebun lady's slippers

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About 100 flowers of Cypripedium macranthos var. rebunense were artificially polhnated annually. Pollination rate was 90%. From among the formed capsules 10 were sampled for immature seed culture, and the remainings were left for natural seed propagation. Immature seeds were cultured at ICU and NAIST, and seedlings were obtained. However, acclimation of some of these seedlings tried in 2000 was unsuccessful. Those kept in laboratories are still growing. In 2001 some of them were transplanted to the experiment field after cold-treatment New shoots are expected to emerge in the spring of 2002. The increase in the number of flowered plants among those propagated by natural seeding reached 18% within the preservation area of 70mm^2 in Rebun Island during the observation period. No new seedlings from seeds artificially sown under or outside of the periphery of mature plants have been identified yet. Seedling formation is usually observed under mature plants. For seed germination under natural environment seeding time and condition of water supply seem to have great influence. Seeds just prior to full maturation seem to have more germination potential. Comparative nucleotide sequence analysis of 10 Cypripedium species which grow in the northern hemisphere was made using chloroplast DNA to understand their phylogenetic relationship. C.calceolus found in Rebun Island and Kitami district of Hokkaido was demonstrated to be distinctly different from those in western Europe and China. It is least possible that C.calceolus in Rebun Island is a denizen from these regions as has been claimed. We consider that C.calceolus in Rebun Island should be treated as an endangered species