Monophyly of the genus Closterium and the order Desmidiales (Charophyceae, Chlorophyta) inferred from nuclear small subunit rDNA data

We newly sequenced the nuclear-encoded small subunit (SSU) rDNA coding region for 21 taxa of the genus Closterium. The new sequences were integrated into an alignment with 13 known sequences of conjugating green algae representing six traditional families (i.e. Zygnemataceae, Mesotaeniaceae, Gonatozygaceae, Peniaceae, Closteriaceae, and Desmidiaceae) and five known charophycean sequences as outgroups. Both maximum likelihood and maximum parsimony analyses supported with high bootstrap values one large clade containing all placoderm desmids (Desmidiales). All the Closterium taxa. formed one clade with 100% bootstrap support, indicating their monophyly, but not paraphyly, as suggested earlier. As to the taxa within the genus Closterium, we found two clades of morphologically closely related taxa in both maximum likelihood and maximum parsimony trees. They corresponded to the C. calosproum species complex and the C moniliferum-ehrenbergii species complex. It is of particular interest that the homothallic entity of C. moniliferum v. moniliferum was distinguished from and ancestral to all other entities of the C. moniliferum-ehrenbergii species complex. Superimposing all 50 charophycean sequences on the higher order SSU rRNA structure model of Closterium, we investigated degrees of nucleotide conservation at a given position in the nucleotide sequence. A characteristic signature structure to the genus Closterium was found as an additional helix at the tip of VI region. In addition, eight base deletions at the tip of helix 10 were found to be characteristic of the C calosporum species complex, C gracile, C incurvum, C. pleurodermatum, and C pusillum v. maius. These taxa formed one clade with an 82% bootstrap value in maximum parsimony analysis.