Pteridophytes have a longer evolutionary history than any other vascular land plant and, therefore, have endured greater loss of phylogenetically informative information. This factor has resulted in substantial disagreements in evaluating characters and, thus, controversy in establishing a stable classification. To compare competing casicatis, we obtained DNA sequences of a chloroplast gene. The sequence of 1206 nt of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL) was determined from 58 species, representing almost all families of leptosporangiate ferns. Phylogenetic trees were inferred by the neighbor-joining and the parsimony methods. The two methods produced almost identical phylogenetic trees that provided insights concerning major general evolutionary trends in the leptosporangiate ferns. Interesting findings were as follows: (i) two morphologically distinct heterosporous water ferns, Marula and Salvinia, are sister genera; (iu) the tree ferns (Cyatheaceae, Dicksoniaceae, and Metaxyaceae) are monophyletic; and (ui) polypodiolds are distantly related to the gleicheniolds in spite of the imilarit of their exindusiate soral morphology and are close to the higher indusiate ferns In addition, the affiities of several "problematic genera" were assessed.The extant ferns include -10,000 species and 250 genera in the world (1). They are the most conspicuous spore-bearing land plants and the principal members of land flora after the flowering plants. Ferns Based on morphology, the leptosporangiate ferns are usually classified into three major groups, Marsileaceae, Salviniaceae including Azollaceae, and the rest, which are often treated as different orders (5-7). Although both the Marsileaceae and the Salviniaceae have distinctive morphologies, both live in aquatic habitats and are characterized by heterospory, the latter being extremely rare in the leptosporangiate ferns. The phylogenetic relationship ofthese aquatic ferns to the homosporus ferns remains unsolved (8).Classification and phylogenetic relationships of the leptosporangiate ferns above the family level are controversial (1, 5,6,[8][9][10]. The reasons for discrepancy among classification schemes include disagreements in evaluation of morphological characters used. It is often difficult to identify homologous characters because similar characters are found in apparently different phylogenetic lineages; convergent or parallel evolution probably often occurred during the long evolutionary history of ferns (11). Furthermore, frequent extinctions produced missing links, which have resulted in difficulties elucidating phylogenetic interrelationships of major groups (1, 11). Micromolecular information (12) also is not useful to infer familial relationships for the same reasons.Recently molecular systematics in plants has progressed rapidly with in vitro DNA amplification (polymerase chain reaction, PCR) mediated by thermostable DNA polymerase and the direct sequencing methods. In angiosperm systematics, this molecular approach...