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Background  Within Chlorophyceae the ITS2 secondary structure shows an unbranched helix I, except for the ' Hydrodictyon ' and the ' Scenedesmus ' clade having a ramified first helix. The latter two are classified within the Sphaeropleales, characterised by directly opposed basal bodies in their flagellar apparatuses (DO-group). Previous studies could not resolve the taxonomic position of the ' Sphaeroplea ' clade within the Chlorophyceae without ambiguity and two pivotal questions remain open: (1) Is the DO-group monophyletic and (2) is a branched helix I an apomorphic feature of the DO-group? In the present study we analysed the secondary structure of three newly obtained ITS2 sequences classified within the ' Sphaeroplea ' clade and resolved sphaeroplealean relationships by applying different phylogenetic approaches based on a combined sequence-structure alignment.    Results  The newly obtained ITS2 sequences of  Ankyra judayi, Atractomorpha porcata  and  Sphaeroplea annulina  of the ' Sphaeroplea ' clade do not show any branching in the secondary structure of their helix I. All applied phylogenetic methods highly support the ' Sphaeroplea ' clade as a sister group to the 'core Sphaeropleales'. Thus, the DO-group is monophyletic. Furthermore, based on characteristics in the sequence-structure alignment one is able to distinguish distinct lineages within the green algae.    Conclusion  In green algae, a branched helix I in the secondary structure of the ITS2 evolves past the ' Sphaeroplea ' clade. A branched helix I is an apomorph characteristic within the monophyletic DO-group. Our results corroborate the fundamental relevance of including the secondary structure in sequence analysis and phylogenetics.

ITS2 data corroborate a monophyletic chlorophycean DO-group (Sphaeropleales)