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Abstract Background The second Internal Transcriber Spacer (ITS2) is a fast evolving part of the nuclear-encoded rRNA operon located between the 5.8S and 28S rRNA genes. Based on crossing experiments it has been proposed that even a single Compensatory Base Change (CBC) in helices 2 and 3 of the ITS2 indicates sexual incompatibility and thus separates biological species. Taxa without any CBC in these ITS2 regions were designated as a 'CBC clade'. However, in depth comparative analyses of ITS2 secondary structures, ITS2 phylogeny, the origin of CBCs, and their relationship to biological species have rarely been performed. To gain 'close-up' insights into ITS2 evolution, (1) 86 sequences of ITS2 including secondary structures have been investigated in the green algal order Ulvales (Chlorophyta, Viridiplantae), (2) after recording all existing substitutions, CBCs and hemi-CBCs (hCBCs) were mapped upon the ITS2 phylogeny, rather than merely comparing ITS2 characters among pairs of taxa, and (3) the relation between CBCs, hCBCs, CBC clades, and the taxonomic level of organisms was investigated in detail. Results High sequence and length conservation allowed the generation of an ITS2 consensus secondary structure, and introduction of a novel numbering system of ITS2 nucleotides and base pairs. Alignments and analyses were based on this structural information, leading to the following results: (1) in the Ulvales, the presence of a CBC is not linked to any particular taxonomic level, (2) most CBC 'clades' sensu Coleman are paraphyletic, and should rather be termed CBC grades. (3) the phenetic approach of pairwise comparison of sequences can be misleading, and thus, CBCs/hCBCs must be investigated in their evolutionary context, including homoplasy events (4) CBCs and hCBCs in ITS2 helices evolved independently, and we found no evidence for a CBC that originated via a two-fold hCBC substitution. Conclusions Our case study revealed several discrepancies between ITS2 evolution in the Ulvales and generally accepted assumptions underlying ITS2 evolution as e.g. the CBC clade concept. Therefore, we developed a suite of methods providing a critical 'close-up' view into ITS2 evolution by directly tracing the evolutionary history of individual positions, and we caution against a non-critical use of the ITS2 CBC clade concept for species delimitation.

Abstract Background Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor. Results Phylogenetic analyses of the almost complete rDNA operon with an improved taxon sampling containing most known cyanobacterial lineages recovered the Paulinella chromatophore as sister to the complete PS-clade. The sequence of the complete carboxysomal operon of Paulinella was determined. Analysis of RubisCO large subunit (rbcL) sequences revealed that Paulinella shares the proteobacterial form 1A RubisCO with the PS-clade. The γ-proteobacterium Nitrococcus mobilis was identified as sister of the Paulinella chromatophore and the PS-clade in the RubisCO phylogeny. Gene content and order in the carboxysomal operon correlates well with the RubisCO phylogeny demonstrating that the complete carboxysomal operon was acquired by the common ancestor of the Paulinella chromatophore and the PS-clade through HGT. The carboxysomal operon shows a significantly elevated AT content in Paulinella, which in the rbcL gene is confined to third codon positions. Combined phylogenies using rbcL and the rDNA-operon resulted in a nearly fully resolved tree of the PS-clade. Conclusion The HGT of the carboxysomal operon predated the divergence of the chromatophore ancestor from the PS-clade. Following HGT and divergence of the chromatophore ancestor, diversification of the PS-clade into at least three subclades occurred. The γ-proteobacterium Nitrococcus mobilis represents the closest known relative to the donor of the carboxysomal operon. The isolated position of the Paulinella chromatophore in molecular phylogenies as well as its elevated AT content suggests that the Paulinella chromatophore has already undergone typical steps in the reductive evolution of an endosymbiont.

Background: Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor., Results: Phylogenetic analyses of the almost complete rDNA operon with an improved taxon sampling containing most known cyanobacterial lineages recovered the Paulinella chromatophore as sister to the complete PS-clade. The sequence of the complete carboxysomal operon of Paulinella was determined. Analysis of RubisCO large subunit (rbcL) sequences revealed that Paulinella shares the proteobacterial form 1A RubisCO with the PS-clade. The gamma-proteobacterium Nitrococcus mobilis was identified as sister of the Paulinella chromatophore and the PS-clade in the RubisCO phylogeny. Gene content and order in the carboxysomal operon correlates well with the RubisCO phylogeny demonstrating that the complete carboxysomal operon was acquired by the common ancestor of the Paulinella chromatophore and the PS-clade through HGT. The carboxysomal operon shows a significantly elevated AT content in Paulinella, which in the rbcL gene is confined to third codon positions. Combined phylogenies using rbcL and the rDNA-operon resulted in a nearly fully resolved tree of the PS-clade., Conclusion: The HGT of the carboxysomal operon predated the divergence of the chromatophore ancestor from the PS-clade. Following HGT and divergence of the chromatophore ancestor, diversification of the PS-clade into at least three subclades occurred. The gamma-proteobacterium Nitrococcus mobilis represents the closest known relative to the donor of the carboxysomal operon. The isolated position of the Paulinella chromatophore in molecular phylogenies as well as its elevated AT content suggests that the Paulinella chromatophore has already undergone typical steps in the reductive evolution of an endosymbiont.