Unusually expanded SSU ribosomal DNA of primary osmotrophic euglenids: molecular evolution and phylogenetic inference

Abstract

Expansion segments within eukaryotic nuclear SSU ribosomal RNA have been characterized in many diverse organisms. So far, only a few studies have examined the evolutionary history of SSU rDNA variable regions for monophyletic groups. A euglenozoan SSU rDNA data set was analyzed combining phylogenetic inference and examination of expansion segment evolution. Although SSU rDNA length expansion could be ascribed to all Euglenozoa, most unusual length variation occurs within primary osmotrophic euglenids, particularly within the genus Distigma. The longest SSU rRNA gene reported to date can be found in D. sennii, comprising more than 4500 bases. RT-PCR analyses revealed that the complete gene is transcribed into RNA without posttranscriptional modifications. Further investigations uncovered that most of the length extension is due to elongated variable regions V2 and V4, but virtually all variable regions except for V3 are extended within primary osmotrophic euglenids. Analyses of secondary structure revealed several insertion points within variable regions, some of which are of phylogenetic importance. Varying GC content has been detected among species and between expansion segments and core regions. Nevertheless, individual expansion segments of one species as well as variable sequence positions within core regions tend to evolve in parallel concerning nucleotide frequencies. The presence of a large internal repeat within V2 of Distigma sennii hints at a possible mechanism for large-scale sequence length expansion.