339 J. Phycol. 35, 339–355 (1999) GENETIC, MORPHOLOGICAL, AND TOXICOLOGICAL VARIATION AMONG GLOBALLY DISTRIBUTED STRAINS OF NODULARIA (CYANOBACTERIA) 1 Christopher J. S. Bolch 2,3 School of Plant Science, University of Tasmania, GPO Box 252-55, Hobart 7001, Australia Philip T. Orr, Gary J. Jones CSIRO Land and Water, Resource Sciences Centre, Block B, 80 Meiers Road, Indooroopilly, Queensland, 4068, Australia and Susan I. Blackburn CSIRO Marine Research, GPO Box 1538, Hobart, Tasmania, 7001, Australia Morphological, toxicological, and genetic varia- tion was examined among 19 strains of Nodularia. The strains examined could be morphologically dis- criminated into four groups corresponding to N. spumigena Mertens, N. sphaerocarpa Bornet et Fla- hault, and two strains that did not clearly corre- spond to currently accepted Nodularia species. Ge- netic variation was examined using nucleotide se- quencing of the phycocyanin intergenic spacer re- gion (cpcBA-IGS) and RAPD-PCR. The PCR-RFLP of the cpcBA-IGS differentiated four genotypes cor- responding to the four morphological groups. How- ever, nucleotide sequencing of 598 bp of the 690- bp fragment showed that one of the three strains corresponding to N. sphaerocarpa (PCC 7804) was genetically divergent from the other two, suggesting that it constitutes a distinct species. Nucleotide var- iation within the morphospecies groups was limited (1%), and all 14 Australian strains of N. spumigena possessed identical cpcBA-IGS sequences. The RAPD-PCR differentiated the same groups as the cpcBA sequencing and discriminated each of the sev- en different Australian populations of N. spumigena. Strains from within a bloom appeared genetically identical; however, strains isolated from different blooms could be separated into either a western or a southeastern Australian cluster, with one strain from western Australia showing considerable genetic divergence. The pattern of variation suggests that individual blooms of N. spumigena are clonal but also that Australian N. spumigena populations are ge- netically distinct from each other. Examination of genetic distance within and between blooms and within and between morphological groups showed clear genetic dicontinuities that, in combination with the cpcBA-IGS data, suggest that Nodularia contains genetically distinct morphospecies rather than a con- tinuous cline of genetic variation. Furthermore, these morphospecies are genetically variable, exhib- iting hierarchical patterns of genetic variation on re- gional and global scales. Production of the hepato- toxin nodularin was not restricted to one genetic lin- eage but was distributed across three of the five ge- notypic groups. A strain of N. spumigena from a nontoxic Australian population was found to fall within the range of genetic variation for other toxic Australian strains and appears to be a unique non- toxic strain that might have arisen by loss of toxin production capacity. Key index words: Australia; cyanobacteria; DNA; ge- netic diversity; hepatotoxins; intergenic spacer; No- dularia; Nodularia spumigena; Nodularin; PCR; phy- cocyanin operon; population genetics; RAPD; RFLP; systematics; taxonomy Abbreviations: cpcBA-IGS, phycocyanin intergenic spacer region; NODLN, nodularin Filamentous cyanobacteria of the genus Nodularia Mertens ex Bornet et Flahault are mainly aquatic, although they often can be isolated from the surface of moist soils. In aquatic environments, they can be found in both planktonic and benthic habitats, mainly in brackish, alkaline inland lakes and in coastal lagoons and estuaries (Nordin and Stein 1980, Baker 1992). The distinctive, metameric tri- chomes are composed of tightly packed discoid cells with regularly spaced heterocysts and intercalary ak- inetes (Baker 1992). The genus is now relatively well defined, but in the past the separation of species within the genus has fluctuated greatly. More than 20 taxa had been established within Nodularia until the revision by Nordin and Stein (1980) that reduced the number of species to only two, N. spumigena Mertens and N. harveyana Thuret ex Bornet et Flahault, separated mainly by trichome diameter. This situation was clearly overly simplistic, and more recent revisions now recognize seven species (three benthic, four planktic) on the basis of habitat, morphological lim- 1 Received 17 June 1998. Accepted 30 December 1998. 2 Present address and author for reprint requests: School of Plant Science, University of Tasmania, GPO Box 252-55, Hobart 7001, Australia; e-mail chris.bolch@utas.edu.au.