We amplified, TA-cloned, and sequenced the 16S–23S internal transcribed spacer (ITS) regions from single isolates of several cyanobacterial species, Calothrix parietina, Scytonema hyalinum, Coelodesmium wrangelii, Tolypothrix distorta, and a putative new genus (isolates SRS6 and SRS70), to investigate the potential of this DNA sequence for phylogenetic and population genetic studies. All isolates carried ITS regions containing the sequences coding for two tRNA molecules (tRNA and tRNA). We retrieved additional sequences without tRNA features from both C. parietina and S. hyalinum. Furthermore, in S. hyalinum, we found two of these non-tRNA-encoding regions to be identical in length but different in sequence. This is the first report of ITS regions from a single cyanobacterial isolate not only different in configuration, but also, within one configuration, different in sequence. The potential of the ITS region as a tool for studying molecular systematics and population genetics is significant, but the presence of multiple nonidentical rRNA operons poses problems. Multiple nonidentical rRNA operons may impact both studies that depend on comparisons of phylogenetically homologous sequences and those that employ restriction enzyme digests of PCR products. We review current knowledge of the numbers and kinds of 16S–23S ITS regions present across bacterial groups and plastids, and we discuss broad patterns congruent with higher-level systematics of prokaryotes.

A total of 27 strains of Oculatella were isolated, characterized and sequenced, and analysed phylogenetically with an additional environmental clone from the Atacama Desert and 10 strains isolated and sequenced by others. The strains were clearly separated based upon phylogenetic analyses conducted with a concatenated alignment of the 16S rRNA and 16S-23S ITS region of the ribosomal operons in the genus Oculatella. Differences in secondary structure of the conserved domains of the ITS region, as well as comparative analysis of P-distance among ITS regions, served to separate the strains into distinct taxonomic units. Seven new species of Oculatella were described, including four from arid to semi-arid soils (O. atacamensis, O. mojaviensis, O. coburnii, O. neakameniensis) and three from more mesic habitats, including a temperate lake (O. hafneriensis), a desert waterfall (O. cataractarum) and a Hawaiian sea cave (O. kauaiensis). The soil forms show statistically significant morphological differences, but the ranges overlap to a degree that they are not diagnosable by morphology, and these four cryptic species are characterized here using molecular characters. The more mesic species, including the type species from Mediterranean hypogea, O. subterranea, are all morphologically distinct from each other and from all four soil taxa. This report is the first to use solely molecular criteria to distinguish cryptic species of cyanobacteria.