18s Ribosomal Dna Sequences Indicate a Monophyletic Origin of Charophyceae1

Nuclear‐encoded small‐subunit (18S) ribosomal RNA genes ofChara australisR. Brown(C. corallinavar.nobilisf.nobilisR.D.W.) andNitella flexilis (L.)Ag. were amplified by polymerase chain reaction, cloned, and completely sequenced. Using structural criteria, the sequences were aligned with 18S ribosomal DNAs (rDNAs) from 11 other chlorophyllb‐containing algae and six higher plants (embryophytes). Phylogenetic trees were inferred by distance, neighbor‐joining, parsimony, and maximum‐likelihood approaches; confidence intervals were estimated by bootstrapping, and nonrandomness of tree structure was confirmed by permutation tests. 18S rDNAs ofC. australisand twoNitellaspecies were found to be specifically related and, together with 18S rDNAs ofChlorokybus atmophyticusGeitler,Klebsormidium flaccidum(A. Br.) Silva, Mattox, et Blackwell, and twoColeochaetespecies, support a robust monophyletic group (Charophyceae). Although most trees favored a specific sister‐group relationship between Charophyceae and embryophytes, statistical tests revealed that a sister‐group relationship between Charophyceae and Chlorophyceae could not be ruled out. Additional complete sequences from 18S rDNAs of lower land plants may be required to resolve phylogenetic relationships among these organisms.