How slippage-derived sequences are incorporated into rRNA variable-region secondary structure: implications for phylogeny reconstruction

We analyzed the type and frequency of mutational changes in hypervariable rRNA regions, using the highly length-variable region V4 of the small subunit rRNA locus of tiger beetles (Cicindelidae) as an example. Phylogenetic analysis of indels in closely related species showed that (1) most indels are single nucleotides (usually A or T and sometimes G) or di-nucleotides of A and T. These occur at numerous foci, and they exhibit a strong bias for duplication of 5' single and di-nucleotide motifs but not 3' motifs. (2) Insertions/deletions in stem-forming regions affected paired and unpaired bases with about equal frequency but they did not disrupt the secondary structure. (3) Recurring mutations involving short repeats of the same bases caused parallel evolution of similar sequence motifs in the rRNA of different lineages. The observed types of change are consistent with the propostion that slippage is the main mutational mechanism. Slippage-derived sequences tend to be self-complementary, and therefore the stem-loop structure could be self-organizing as a consequence of the underlying mutational mechanism. Thus, the secondary structure in the cicindelid V4 region may be conserved due to the dynamics of the mutational mechanism rather than to functional constraints. These processes may also have a tendency to produce similar primary sequences irrespective of phylogenetic associations. The findings have implications for sequence alignment in phylogenetic analysis and should caution against the use of secondary structure to improve the determination of positional homology in hypervariable regions.