The internal transcribed spacer 2 (ITS2) is required in ribosome biogenesis [57
] and its gradual removal from mature rRNA is driven by its specific secondary structure [60
Using three newly obtained ITS2 sequences from Ankyra judayi, Atractomorpha porcata and Sphaeroplea annulina (Sphaeropleaceae) in this study we aimed to pursue two consecutive questions concerning the phylogenetic relationships within Chlorophyceae. (1) What is the phylogenetic position of the newly sequenced algae relative to the 'core Sphaeropleales' and could the biflagellate DO-group be regarded as monophyletic? (2) How does the secondary structure of the new ITS2 sequences look like and is an autapomorphic feature of the secondary structure associated with the monophyletic DO-group?
Considering the question (1) Buchheim et al. [6
] and Wolf et al. [23
] approached the problem with 18S + 26S rDNA and 18S rDNA data, but the relationship between the 'core Sphaeropleales' and the Sphaeropleaceae remained unclear. However, in their studies, Ankyra
clustered in a monophyletic clade named Sphaeropleaceae. We confirm this 'Sphaeroplea
' clade with all three genera being strongly separated from other clades. As a result of a Bayesian analysis on a combined 18S and 26S rDNA dataset Shoup and Lewis [61
] also found the Sphaeropleaceae as the most basal clade within the Sphaeropleales, but again the analysis lacked a strong backing. Beside these difficulties the 'core Sphaeropleales' were already shown to be monophyletic with high certainty [6
The DO-group (Sphaeropleales including the 'Sphaeroplea
' clade) as emended by Deason et al. [10
], for which the directly opposed basal body orientation and basal body connection features are verified [63
], is now strongly supported by molecular phylogenetic analyses. There was already evidence of an extended DO-group [6
], however, for some groups ultrastructural results are still lacking, and even though the collective basal body orientation and connection imply a monophyletic DO-group, until now no molecular phylogenetic analysis could show this with solid support [6
]. We demonstrate for the first time with robust support values for the equivocal nodes that the 'core Sphaeropleales', the 'Sphaeroplea
' clade, and the Sphaeropleales are monophyletic.
Regarding question (2), for all structures of the 'Hydrodictyon
' and the 'Scenedesmus
' clade, helix I shows the typical branching (Y-structure). Initially, An et al. [68
] proposed a secondary structure model with an unbranched helix I for ITS2 sequences of 'Scenedesmus
' clade members. Thereafter, van Hannen et al. [34
] updated the model by folding the nucleotide sequences based upon minimum free energy and found a branched helix I as the most energetically stable option. The branching is result of an insertion of approximately 25 nucleotides capable of folding as an individual stem within the 5' end of the first helix. However, ITS2 sequence and secondary structure information of further 'core Sphaeropleales
' members, e.g. the 'Ankistrodesmus
' clade and the 'Bracteacoccus
' clade, lacks hitherto. In contrast, the Y-structure is absent within the 'Sphaeroplea
' clade and any other investigated group so far. Thus this feature is – contrary to our expectation – not an autapomorphic character for the biflagellate DO-group as a whole but for the 'core Sphaeropleales'.
Regarding future work, the resolution among the main clades of Chlorophyceae was statistically poorly supported in previous studies [68
]. Pröschold and Leliaert [24
] reviewed the systematics of green algae by applying a polyphasic approach, but did not yield a clear resolution regarding a sister taxon to the Sphaeropleales. Since they are not yet available, ITS2 sequences of chaetopeltidalean and chaetophoralean taxa could not be included in the present study and therefore the phylogenetic relationships between the main Chlorophyceae clades remain open. We recommend involving sequence and secondary structure information of chaetopeltidalean and chaetophoralean ITS2 sequences in future studies to find out if the monophyletic biflagellate DO-group could be further extended to a general monophyletic DO-group containing quadri- and biflagellate taxa. A genome-wide approach indicates that Sphaeropleales and Chlamydomonadales are sister taxa, however only a few organisms are included in this study [56
]. An additional uprising question is when the Y has evolved within the 'core Sphaeropleales'. This could be resolved by inclusion of other members (e.g. Bracteacoccus
) in further studies.
The two major reasons contributing to the robust results presented here are the change of the phylogenetic marker and the inclusion of secondary structure information. In contrast to previous phylogenetic work concerning Chlorophyceae, this study is based on the ITS2, which offers a resolution power for relationships from the level of subspecies up to the order level, because of their variable sequence but conserved secondary structure [26
]. Hitherto commonly used markers in contrast are a lot more restricted. Using 4SALE [40
] with implemented structure consideration, we could achieve for the first time a global simultaneously generated sequence-structure alignment (c.f. Fig. ) yielding specific sequence and structural features distinguishing different algae lineages (c.f. Fig. ).