Arthrospira ('Spirulina') strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer

We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira ('Spirulina') cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 51 tested cultures. The strain Spirulina laxissima SAG 256.80 was included as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by amplified ribosomal DNA restriction analysis, and thus the internally transcribed spacer was selected as molecular taxonomic marker. The internally transcribed spacer sequences situated between the 16S and the 23S rRNA were amplified by polymerase chain reaction and yielded amplicons of about 540 bp. Direct use of cells for polymerase chain reaction seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of bovine serum albumin in the polymerase chain reaction mix. The amplicons were digested with four restriction enzymes (EcoRV, Hhal, Hinfl, Msel) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology.