We are doing it wrong: Putting homology before phylogeny in cyanobacterial taxonomy.

The rapid expansion of whole genome sequencing in bacterial taxonomy has revealed deep evolutionary relationships and speciation signals, but assembly methods often miss true nucleotide diversity in the ribosomal operons. Though it lacks sufficient phylogenetic signal at the species level, the 16S ribosomal RNA gene is still much used in bacterial taxonomy. In cyanobacterial taxonomy, comparisons of 16S-23S Internal Transcribed Spacer (ITS) regions are used to bridge this information gap. Although ITS rRNA region analyses are routinely being used to identify species, researchers often do not identify orthologous operons, which leads to improper comparisons. No method for delineating orthologous operon copies from paralogous ones has been established. A new method for recognizing orthologous ribosomal operons by quantifying the conserved paired nucleotides in a helical domain of the ITS, has been developed. The D1' Index quantifies differences in the ratio of pyrimidines to purines in paired nucleotide sequences of this helix. Comparing 111 operon sequences from 89 strains of Brasilonema, four orthologous operon types were identified. Plotting D1' Index values against the length of helices produced clear separation of orthologs. Most orthologous operons in this study were observed both with and without tRNA genes present. We hypothesize that genomic rearrangement, not gene duplication, is responsible for the variation among orthologs. This new method will allow cyanobacterial taxonomists to utilize ITS rRNA region data more correctly, preventing erroneous taxonomic hypotheses. Moreover, this work could assist genomicists in identifying and preserving evident sequence variability in ribosomal operons, which is an important proxy for evolution in prokaryotes.

Reptodigitus Chapmanii (Nostocales, Hapalosiphonaceae) Gen. Nov.: A Unique Nostocalean (Cyanobacteria) Genus Based on a Polyphasic Approach1.

The Nostocales is a monophyletic, heterocytous lineage of cyanobacteria capable of akinete production and division in multiple planes, depending upon family-level clade. While present in a variety of ecosystems, the diversity of the Nostocales has been poorly elucidated. Due to environmentally -induced phenotypic plasticity, morphology alone is often insufficient to determine the true phylogenetic placement of these taxa. In order to bridge this gap, taxonomists now employ the polyphasic approach, combining methods such as morphological analysis, phylogenetic analysis based on DNA sequence and genetic identity based on ribosomal genes, and secondary structure of the 16S-23S ITS and 16S rRNA gene sequences, as well as ecological characterization. Using this combined approach, a new genus and species (Reptodigitus chapmanii gen. et sp. nov.) isolated from the St. Johns River (Jacksonville, Florida, USA) within the Nostocales is herein described. Phylogenetic analyses place this taxon within the Hapalosiphonaceae, sister to the clade containing Fischerella, Hapalosiphon, and Westiellopsis. The 16S-23S ITS secondary folding structure analysis also supports the erection of this new genus.

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones.

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen-fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well-supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.

Nostoc thermotolerans sp. nov., a soil-dwelling species of Nostoc (Cyanobacteria).

A filamentous, soil-dwelling cyanobacterial strain (9C-PST) was isolated from Mandsaur, Madhya Pradesh, India, and is described as a new species of the genus Nostoc. Extensive morphological and molecular characterization along with a thorough assessment of ecology was performed. The style of filament orientation, type and nature of the sheath (e.g. distribution and visibility across the trichome), and vegetative and heterocyte cell dimensions and shape were assessed for over one year using both the laboratory grown culture and the naturally occurring samples. Sequencing of the 16S rRNA gene showed 94 % similarity with Nostocpiscinale CENA21 while analyses of the secondary structures of the 16S-23S ITS region showed unique folding patterns that differentiated this strain from other species of Nostoc. The level of rbcl and rpoC1 gene sequence similarity was 91 and 94 % to Nostocsp. PCC 7524 and Nostocpiscinale CENA21, respectively, while the nifD gene sequence similarity was found to be 99 % with Nostocpiscinale CENA21. The phenotypic, ecological, genetic and phylogenetic observations indicate that the strain 9C-PST represents a novel species of the genus Nostoc with the name proposed being Nostoc thermotolerans sp. nov. according to the International Code of Nomenclature for Algae, Fungi, and Plants.