title: Phylogenetic analysis of cultivation-resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum, Nostocales, Cyanobacteria) using single-cell and filament sequencing of environmental samples.
abstract: Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass-capillary isolation technique and a semi-nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni- to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni- to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.
authors.affiliation: Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 1760, České Budějovice, CZ-37005, Czech Republic.|Centre for Phycology, Institute of Botany ASCR, v.v.i., Dukelská 135, Třeboň, CZ-379 82, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Mareš
authors.affiliation: Center for Protein Engineering, University of Liège, Liège, 4000, Belgium.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Lara
authors.affiliation: Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, Brno, CZ-61137, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Dadáková
authors.affiliation: Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 1760, České Budějovice, CZ-37005, Czech Republic.|Centre for Phycology, Institute of Botany ASCR, v.v.i., Dukelská 135, Třeboň, CZ-379 82, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Hauer
authors.affiliation: Department of Limnology and Bio-Oceanography, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, Vienna, A-1090, Austria.
authors.forename: Bohuslav
authors.initials: B
authors.lastname: Uher
authors.affiliation: Center for Protein Engineering, University of Liège, Liège, 4000, Belgium.
authors.forename: Annick
authors.initials: A
authors.lastname: Wilmotte
authors.affiliation: Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 1760, České Budějovice, CZ-37005, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Kaštovský
keywords: 16S rRNA gene; Petalonema; Stigonema; cyanobacteria; morphology; phylogeny; single cell; single filament; taxonomy