Molecular phylogenetic analyzes of pteridophytes based on rbcL sequence comparison

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In this study, we first developed widely-applicable method in amplifying and sequencing rbcL gene from pteridophytes. We tested many kinds of primers for PCR amplification of rbcL fragments and got universal primers for pteridophytes which is also applicable for seed plants. Performing this method with DNA sequencer which we bought by this grant, we became able to determine about 1200bp sequence of rbcL of one wild pteridophyte species in 2.5 days. We selected one species each from 32 family, 107 genera, that cover whole variation of pteridophytes, and determined their rbcL sequences. Based on them, we constructed molecular tree and global phylogenetic relationships in fern families were revealed. In this study, we also analyzed smaller groups of ferns (epecially Aspleniaceae) using the same molecular method. Aspleniaceae is one of the largest family of ferns, containing more than 700 species. Its Intrafamiliar phylogeny was poorly known. We determined rbcL sequences from 61 species of Aspleniaceae. We elucidated global phylogenetic relation in the family as well sd smaller groups in the family : genus Hymenasplenium (20 species) and H.obliquissimum species (17 samples) complex. Molecular phylogenetic analyzes based on rbcL sequencing was valid from inter-family to intra-species levels.