[en] We present the results of a phylogenetic study, based on ARDRA of the rDNA operon, of 38 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 54 tested cultures. Three living samples from Earthrise Farms ponds (September 1997), four freeze-dried samples from EF ponds (August 1996, February and March 1997) and a powder of ‘Spirulina pacifica’ were also included in the study. The strain Spirulina laxissima SAG 256.80 was used as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by ARDRA, and thus the Internal Transcribed Spacer (ITS) was selected as a molecular taxonomic marker. The ITS sequences situated between the 16S and the 23S rRNA genes were amplified by PCR and yielded amplicons of about 540 bp. The amplicons were digested with four restriction enzymes (EcoR V, Hha I, Hinf I, Mse I) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters (Clusters I and II), of which Cluster I was divided into Subclusters I.A and I.B. Four freeze-dried samples from EF cultivation ponds (Summer 1996 and Winter 1997), as well as a sample of powder sent as ‘Spirulina pacifica’ appeared to contain a mixture of genotypes from Clusters I and II. 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.
Direct use of cells for PCR seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of BSA (Bovine Serum Albumin) in the PCR mix.
In order to study in more depth the genotypic relationships of Arthrospira, we have obtained the ITS sequence of 19 cultures and 7 samples (living or freeze-dried samples from EF ponds, dried natural samples and one commercial pill). The data confirmed the existence of Clusters I and II, but also subdivided each of them into two Subclusters (A and B). In three cultures, simultaneous presence of types II.A and II.B was detected. It is likely that sequences of both types are contained in different copies of the ITS and that the three cultures represent cryptic duplicates of one unique genotype.
The strains cultivated in the EF ponds belong to types I.A, II.A and II.B, while the winter ponds samples were a mixture of types I and II.
Though there was surprisingly little sequence variability in the ITS sequences, we designed PCR primers which are specific for the two clusters (44 different positions) and for the four subclusters (2 to 4 different positions).
