Abstract
BackgroundThe 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.ResultsHigh 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.ConclusionsOur 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.
Highlights
Introduction of a numbering system forITS2 positions The ITS2 consensus structure diagram (Figure 1A) provided the opportunity to introduce a novel numbering system of ITS2 nucleotides for unambiguous positional descriptions of base pairs, Compensatory Base Change (CBC), hemi-Compensatory Base Change (hCBC), and indels
Comparison of these universal helices across taxa identified several base-paired positions that retained pairing by covariation, or by a change in only one position
The CBC clades sensu Coleman are not stable over time, since later evolving CBCs result in new CBC clades which are nested in their ‘parent CBC clades’ changing the status of the former towards paraphyletic grades, here germed CBC grades
Summary
Introduction of a numbering system forITS2 positions The ITS2 consensus structure diagram (Figure 1A) provided the opportunity to introduce a novel numbering system of ITS2 nucleotides for unambiguous positional descriptions of base pairs, CBCs, hCBCs, and indels. Helix 3 is usually much longer than the other helices, and its apical region shows high sequence conservation, often including a four nucleotide motif (YGGY) [6,7] This motif is close to the crucial cleavage site C2 where the degradation process of ITS2, i.e. the formation of the mature 5.8S and 28S rRNA, is initiated by a hitherto unidentified endonuclease [8,9,10,11]. The presence of a stable and functionally important RNA secondary structure can be revealed by comparing homologous positions among different organisms, and searching for non-conserved, but co-evolving nucleotides, which maintain base pairing in the RNA transcript, indicating the presence of intra-molecular RNA helices [4,14,15]. The existence of the non-canonical ‘wobble’ base pair (G-U), which is thermodynamically stable in RNA molecules, allows even single-sided changes that perfectly retain base pairing, and are named hemi-Compensatory Base Change (hCBC; e.g. G-U ® G-C; [15,16])
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