Characterization of Arthrospira / Spirulina strains: Molecular Aspects

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)

Remarkable conservation of internally transcribed spacer sequences of Arthrospira ("Spirulina") (Cyanophyceae, Cyanobacteria) strains from four continents and of recent and 30-year-old dried samples from Africa

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and H, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters LA and I.B were separated by two substitutions, whereas subclusters II.A, and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly LA and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample ("dihe' 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two :Forms of the four diagnostic positions that distinguish subclusters genotype H.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.Molecular diversity of the Arthrospira genu