Molecular phylogenetic analyzes of pteridophytes based on rbcL sequence comparison

在本研究中，我们首次建立了一种广泛适用的从蕨类植物中扩增和测序rbcL基因的方法。对多种扩增rbcL片段的引物进行了筛选，得到了适用于蕨类植物的通用引物，也适用于种子植物。用我们用这笔钱购买的DNA测序仪进行这种方法，我们可以在2.5天内测定出一种野生蕨类植物的rbcL约1200bp的序列。我们从覆盖整个蕨类植物变异的32科107属中各选取1种，并测定了它们的rbcL序列。在此基础上，构建了分子系统树，揭示了蕨类植物的系统发育关系。在这项研究中，我们也使用相同的分子方法分析了较小的蕨类植物(特别是三叶蕨科)。山杨科是蕨类植物中最大的科之一，有700多种。它熟悉的系统发展史鲜为人知。我们测定了61种侧柏科植物的rbcL序列。我们阐明了该科的全球系统发育关系，以及科中较小的类群：膜壳属(20种)和斜纹夜蛾属(17个样本)复合体。基于rbcL测序的分子系统发育分析从科间到种内都是有效的。

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.