Abstract The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was inves- tigated. The strains were analyzed using molecular meth- ods with different taxonomic resolutions, including re- striction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S in- ternal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, as- signed to heterocyst differentiation, was used for denatur- ing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera. Keywords Cyanobacteria · Diversity · Phylogeny · Denaturing gradient gel electrophoresis (DGGE) · Internal transcribed spacer (ITS) · 16S rRNA · RFLP Introduction Cyanobacteria constitute one of the largest group of pho- tosynthetic prokaryotes. They are found world-wide in highly diverse ecosystems from marine to terrestrial sys- tems. The genus Nostoc dominates in terrestrial systems and includes cyanobacteria with the capacity to form sym- bioses with an exceptionally broad range of plants such as bryophytes (liverworts and hornworts), fungi, Azolla (a water fern), cycads (gymnosperms) and Gunnera (an an- giosperm) (Bergman et al. 1996; Rai et al. 2000). Tradi- tionally, the taxonomy of Nostoc species has been based on morphological and physiological observations (Vag- noli et al. 1992). In the classification system of cyanobac- teria proposed by Rippka et al. (1979), the genus Nostoc is classified to section IV, which is defined as filamentous cyanobacteria able to differentiate specialized N 2 -fixing cells (heterocysts), and are in addition characterized by di- vision in one plane only. However, the extreme morpho- logical flexibility of Nostoc species, which is influenced by the life cycle stage and environmental conditions (Vagnoli et al. 1992; Mollenhauer et al. 1994; Dodds et al. 1995), makes identification and taxonomy based on mor- phology alone problematic. Therefore, supplementary ge- netic information should be incorporated to give a poly- phasic classification system for the genus Nostoc. Many symbiotic Nostoc isolates are referred to as Nos- toc punctiforme (Zimmerman et al. 1991, 1992; Mollen- hauer et al. 1996). The classification of N. punctiforme, type strain PCC 73102, is exclusively based on morpho- logical criteria, cell size and the formation of a puncti- forme stage (sheath-bound filaments with terminal hetero- cysts) in the life cycle of these cyanobacteria when grown on artificial medium. So far, no genetic analysis has been used to classify symbiotic Nostoc isolates above the strain level. Recently, studies on the genetic diversity of Nostoc isolates from different hosts have been reported. In cyanobacteria isolated from bryophytes, cycads and Gun- nera there is a high genetic diversity among symbiotic cyanobacteria, and the specificity of the cyanobacteria- plant symbioses is relatively low (West and Adams 1997; Rasmussen and Svenning 1998; Costa et al. 1999; Nilsson et al. 2000). However, by using PCR fingerprinting and sequence analyses of the tRNA Leu intron, a high level of genetic and taxonomic resolution can be established, and consequently provide information about diversity at the strain level. Knowledge about the genetic diversity of the genus Nostoc above the strain level is therefore important to ap- proach the taxonomy of symbiotic isolates. Most studies addressing taxonomy and phylogeny have been done on free-living cyanobacteria. The conserved 16S rRNA gene Ulla Rasmussen · Mette M. Svenning Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera Received: 27 December 2000 / Revised: 25 April 2001 / Accepted: 14 May 2001 / Published online: 29 June 2001 ORIGINAL PAPER U. Rasmussen (✉) Department of Botany, Stockholm University, 10691 Stockholm, Sweden e-mail: ulla.rasmussen@botan.su.se, Tel.: +46-8-163779, Fax: +46-8-165525 M.M. Svenning Department of Biology, Faculty of Science, University of Tromsö, 9037 Tromsö, Norway Arch Microbiol (2001) 176 : 204–210 DOI 10.1007/s002030100313 © Springer-Verlag 2001