Arthrospira platensis
and
Arthrospira maxima
are species of cyanobacteria used in health foods, animal feed, food additives, and fine chemicals. This study conducted a comparison of the 16S rRNA gene and
cpcBA
-intergenic spacer (
cpcBA
-IGS) sequences in
Arthrospira
strains from culture collections around the world. A cluster analysis divided the 10
Arthrospira
strains into two main genotypic clusters, designated I and II, where Group I contained
A. platensis
SAG 86.79, UTEX 2340,
A. maxima
KCTC AG30054, and SAG 49.88, while Group II contained
A. platensis
PCC 9108, NIES 39, NIES 46, and SAG 257.80. However, although
A. platensis
PCC 9223 belonged to Group II-2 based on its
cpcBA
-IGS sequence, this strain also belonged to Group I based on its 16S rRNA gene sequence. Phylogenetic analyses based on the 16S rRNA gene and
cpcBA
-IGS sequences showed no division between
A. platensis
and
A. maxima
, plus the 16S rRNA gene and
cpcBA
-IGS sequence clusters did not indicate any well-defined geographical distribution, instead overlapping in a rather interesting way. Therefore, the current study supports some previous conclusions based on 16S rRNA gene and
cpcBA
-IGS sequences, which found that
Arthrospira
taxa are monophyletic. However, when compared with 16S rRNA sequences,
cpcBA
-IGS sequences may be better suited to resolve close relationships and intraspecies variability.
INTRODUCTION
Arthrospira
is a commercially important filamentous cyanobacterium with an annual production estimated at over 3,000 tons per year, the largest among microalgae (Pulz and Gross 2004). Found in tropical and subtropical regions in warm lakes with a high carbonate and bicarbonate content, and high pH and salinity (Tomaselli 1997),
Arthrospira
is a rich source of proteins, minerals, vitamin B
12
, β-carotene, and essential fatty acids, such as
γ
-linolenic acid. Owing to its high protein content of up to approximately 60-70% on a dry weight basis, the amino acids present in
Arthrospira
match the proportions recommended by the Food and Agriculture Organization (FAO) (Dillon et al. 1995). Plus, it does not require any chemical or physical processing in order to become digestible due to the absence of a cellulose cell wall (Vonshak 1997
b
). Indeed, it can be used as feed for fish, poultry, and farm animals (Belay 1997, Vonshak 1997
a
). Originating from the taxonomic studies of cyanobacteria by Geitler (1932), some confusion has arisen between the genera
Spirulina
and
Arthrospira
(Desikachary and Jeeji-Bai 1996). While the 16S rRNA gene sequences of these two genera indicate that they are only distantly related (Nelissen et al. 1994, Scheldeman et al. 1999), putative species of
Arthrospira
have been recognized in the so-called ‘
Spirulina
’ commercial industrial field.
Microscopy identification of cyanobacteria is rapid and sensitive. However, even with skilled and experienced operators, identification is sometimes uncertain. Thus, molecular approaches are particularly useful in the detection and identification of specific strains, especially those that are morphologically identical at the species level. Plus, genetic identification has been used to discriminate nuisance species in cyanobacterial genera, including
Microcystis, Anabaena, Nodularia
, and
Cylindrospermopsis
(Wilmotte and Golubic 1991, Neilan et al. 1995).
Phylogenetic analyses based on the 16S rRNA gene and
cpcBA
-intergenic spacer (
cpcBA
-IGS) take considerable time and effort, yet they provide certainty in the identification of genera using both the length and the sequence of the
cpcBA
-IGS. In addition to 16S rRNA gene sequence analyses (Otsuka et al. 1998, Honda et al. 1999, Turner et al. 1999), the genes encoding the major light-harvesting accessory pigment proteins, particularly the phycocyanin operon (
cpc
), including the IGS between
cpcB
and
cpcA
and the corresponding flanking regions (
cpcBA
-IGS), have also been targeted for phylogenetic studies of cyanobacteria (Neilan et al. 1995, Barker et al. 1999). Furthermore, to determine the phyletic relationships between
Arthrospira
strains, a region of the phycocyanin locus, namely
cpcBA
-IGS, has been sequenced, as its nucleotide substitution rate is potentially higher than
Origins and accession numbers for Arthrospira and Spirulina strainscpcBA-IGS,cpcBA-intergenic spacer.aNIES, National Institute for Environmental Studies Collection, Tsukuba, Ibaraki, Japan; UTEX, Culture Collection of Algae at the University of Texas at Austin, Austin, TX, USA; SAG, Sammlung von Algenkulturen der Universit?t G?ttingen, Germany; PCC, Pasteur Culture Collection of Cyanobacterial Strains, Paris, France; KCTC, Korean Collection for Type Cultures, Daejeon, Korea.b-: not amplified.
Origins and accession numbers for Arthrospira and Spirulina strains cpcBA-IGS, cpcBA-intergenic spacer. aNIES, National Institute for Environmental Studies Collection, Tsukuba, Ibaraki, Japan; UTEX, Culture Collection of Algae at the University of Texas at Austin, Austin, TX, USA; SAG, Sammlung von Algenkulturen der Universit?t G?ttingen, Germany; PCC, Pasteur Culture Collection of Cyanobacterial Strains, Paris, France; KCTC, Korean Collection for Type Cultures, Daejeon, Korea. b-: not amplified.
that of the widely used 16S rRNA gene sequence (Lyra et al. 2001).
Accordingly, the aim of this study is to clarify the diversity of cultivated strains of
Arthrospira
using sequence data from a highly variable DNA fragment, including a comparison of the phylogeny of
Arthrospira
strains based on the 16S rRNA gene and
cpcBA
-IGS. Plus, to define and delimit the genus
Arthrospira
, this study also includes other cyanobacteria. Finally, this study attempts to identify a specific molecular marker to fingerprint different strains of
Arthrospira
and develop tools for species and strain identification.
MATERIALS AND METHODS
- Strains and growth conditions
Ten
Arthrospira
cultures and one
Spirulina
culture were obtained from different culture collections across the world.
Table 1
shows a list of the 10
Arthrospira
strains, along with their origin, culture history, and the corresponding DNA database accession numbers for the 16S rRNA gene and
cpcBA
-IGS. The cultures were maintained in an SOT medium (Choi et al. 2003), containing per liter 16.8g of NaHCO
3
, 0.5g of K
2
HPO
4
, 2.5g of NaNO
3
, 1g of K
2
SO
4
, 1g of NaCl, 0.2g of MgSO
4
·7H
2
O, 0.04 g of CaCl
2
·2H
2
O, 0.01g of FeSO
4
·7H
2
O, 0.08g of Na
2
EDTA, 0.03 mg of H
3
BO
3
, 0.025 mg of MnSO
4
·7H
2
O, 0.002 mg of ZnSO
4
·7H
2
O, 0.0079 mg of CuSO
4
·5H
2
O, and 0.0021 mg of Na
2
MoO
4
·2H
2
O. Stock cultures of 10 mL were kept under low light (20 μmol photons m
-2
s
-1
) and at a constant temperature of 25 ± 1℃.
- DNA extraction, amplification, and sequencing
The cultured strains were harvested using centrifugation, and the genomic DNA extracted following the method of Neilan et al. (1995) using lysozyme and proteinase K. The 16S rRNA gene was then amplified using the universal primers 27F (5′-AGAGTTTGATCMTGCTGGCTCAG-3′) and 1512R (5′-GGYTACCTTGTTACGACTT-3′) (Cho and Giovannoni 2003). The PCR products were purified using a Qiagen QIAquick PCR purification column and cloned using a pDrive vector (Qiagen, Hilden, Germany). The
cpcBA
-IGS was amplified using primers located within the coding region of
cpcB
and
cpcA
, respectively, where the forward primer was CPC1F (5′-GGC KGC YTG YYT GCG YGA CAT GGA-3′) and the reverse primer was CPC1R (5.-AAR CGN CCT TGR GWA TCD GC-3′) (Kim et al. 2006). The amplified fragments (annealing temperature 55℃) were purified using a Qiagen QIAquick PCR purification column and cloned using a pDrive vector (Qiagen). The PCR products were then sequenced using the SP6 and T7 primers with the chain-termination method on an ABI377 automated sequencer (Solgent Ltd., Daejeon, Korea). The nucleotide sequences determined in this study have been deposited in the GenBank database under accession numbers DQ393278-DQ393296.
- Phylogenetic analysis
The 16S rRNA gene and
cpcBA
-IGS sequences obtained from the
Arthrospira
strains were initially compared with sequences available in the National Center for Biotechnology Information database using BLAST network services (
http://www.ncbi.nlm.nih.gov/BLAST
) to determine their approximate phylogenetic affiliations (Altschul et al. 1997). The sequences were aligned using PHYDIC 3.0, and unambiguously aligned nucleotide positions then used for phylogenetic analyses using PHYDIC 3.0 (Chun and Goodfellow 1995). The similarity values between the sequences were calculated from distance matrices by reversing the Jukes-Cantor distance formula (Jukes and Cantor 1969). Phylogenetic trees were then inferred by neighbour joining (NJ) (Saitou and Nei 1987) using the Kimura two-parameter model. The resulting NJ tree was evaluated by bootstrap analyses based on 1,000 resamplings. Due to the spacer variability, a phylogenetic analysis of the matrix was also performed using just the two coding regions. Finally, an overview of the phylogenetic position of
Arthrospira
in cyanobacteria was created by comparing the 16S rRNA gene and
cpcBA
-IGS sequences to corresponding cyanobacterial sequences available in databases and the sequences obtained in this study for
Spirulina laxissima
SAG 256.80 and
Oscillatoria sancta
NIER 10027.
RESULTS AND DISCUSSION
- Phylogenetic tree of 16S rRNA gene
Near-complete 16S rRNA gene sequences were determined for 7
Arthrospira
strains and 1
Spirulina
strain received from culture collections across the world. A phylogenetic tree was then reconstructed using a NJ analysis based on aligning the 8 sequences with
Escherichia coli
K-12 and
Bacillus subtilis
as the outgroup (
Fig. 1
). The corrected sequence alignment, providing the basis of the phylogenetic analyses, corresponded to positions 8-1512 according to the
E. coli
numbering system and was 1,383 nucleotides (nt) in length after removing all gaps and ambiguous positions.
The cluster analysis resolved the 7
Arthrospira
strains into two main genotypic clusters, designated Group I and II. Group I contained
A. platensis
SAG 21.99, PCC 9223, UTEX 2340, and
A. maxima
KCTC AG30054, while Group II contained
A. platensis
NIES 39, SAG 257.80, and PCC 9108. The bootstrap value between the Group I and Group II clusters was 78.4% in the phylogenetic tree, and the 16S rRNA gene similarity was 99.5%. Thus, the clusters were poorly supported by the bootstrap analysis. However, in the 16S rRNA gene sequences for the
Arthrospira
strains, the number of different nucleotides was less than 7 out of a total of 1,420 nt. In the geographical analysis, the strains in Group I originated from Chad, Namibia, and Kenya in Africa, while the strains in Group II originated from Chad in Africa and Peru in South America. In a previous report, a complete analysis of the dendrogram structure grouped the strains into two well-separated genotypic groups (Viti et al. 1997). The genotypic diversity of several strains attributed to these two species was also previously investigated on the basis of morphological criteria using a very sensitive total DNA restriction profile analysis (Scheldeman et al. 1999). In this case, the strains were also divided into two well-separated genotypic groups.
Neighbour-joining tree showing relationships among Arthrospira strains and other cyanobacteria, inferred from 16S rRNA gene sequence analyses. Bootstrap values >50% are shown. Bacillus subtilis (AL009126) and Escherichia coli (AE000452) were used as an outgroup to define the root of the tree. Bar, 0.1 substitutions per nucleotide position. SAG, Sammlung von Algenkulturen der Universität Göttingen, Germany; NIES, National Institute for Environmental Studies Collection, Tsukuba, Ibaraki, Japan; PCC, Pasteur Culture Collection of Cyanobacterial Strains, Paris, France; KCTC, Korean Collection for Type Cultures, Daejeon, Korea; UTEX, Culture Collection of Algae at the University of Texas at Austin, Austin, TX, USA.
The similarity of the 16S rRNA genes between the
Arthrospira
strains and
Lyngbya aestuarii
PCC 7419 was about 95%. However, the similarity of the 16S rRNA genes between the
Arthrospira
strains and
S. laxissima
SAG 256.80 was about 89%. Thus, as shown by the 16S rRNA gene sequences (Nelissen et al. 1996, Ishida et al. 1997), it would seem that
L. aestuarii
PCC 7419 is also closely related to
Arthrospira
and a sister to the clade
Planktothrix/Arthrospira
.
- Phylogenetic tree of cpcBA-IGS
The
Arthrospira
strain analyses were conducted using both the coding sequences and the spacer. Plus, the outgroup was the
cpcBA
-IGS from the chloroplast of
Cyanidium caldarium
. The NJ tree derived from the translated cpcBA-IGS sequences (
Fig. 2
) clustered the
Arthrospira
species into two groups, where Group I clustered with the Group II clusters in 100% of the bootstrap trees and the
cpcBA
-IGS similarity was more than 95.5%. Group I contained
A. platensis
SAG 86.79, SAG 21.99, UTEX 2340,
A. maxima
SAG 49.88, and KCTC AG30054, whereas Group II was subdivided into two lineages: one clade containing
A. platensis
PCC 9108, NIES 39, and NIES 46 supported by 96.6% of the bootstrap replications, and the other clade consisting of
A. platensis
PCC 9223 and SAG 257.80. The similarity of the strains belonging to Group I was 100%. However, while
A. platensis
PCC 9223 belonged to Group II-2 based on its
cpcBA
-IGS sequence, it also belonged to Group I based on its 16S rRNA gene.
With respect to the terminal branching patterns, the results suggested a high degree of congruence between the 16S rRNA genes and the
cpcBA
-IGS-based phylogenies of the nonmarine members of the picophytoplankton clade, yet with less well delineated subgroups in the more conserved 16S rRNA gene phylogeny (Robertson et al. 2001,
Neighbour-joining tree showing relationships among Arthrospira strains and other cyanobacteria, inferred from cpcBA-intergenic spacer sequence analyses. Bootstrap values >50% are shown. The chloroplast of Cyanidium caldarium was used as an outgroup to define the root of the tree. Bar, 0.1 substitutions per nucleotide position. PCC, Pasteur Culture Collection of Cyanobacterial Strains, Paris, France; NIES, National Institute for Environmental Studies Collection, Tsukuba, Ibaraki, Japan; SAG, Sammlung von Algenkulturen der Universität Göttingen, Germany; KCTC, Korean Collection for Type Cultures, Daejeon, Korea; UTEX, Culture Collection of Algae at the University of Texas at Austin, Austin, TX, USA.
Crosbie et al. 2003). In a previous report, 16S rRNA gene data and
cpcBA
-IGS data also indicated that several taxa were genetically heterogeneous and that the taxonomy of cyanobacteria needed to be reconsidered. Manen and Falquet (2002) suggested that 16S rRNA gene data and
cpcBA
-IGS data were often in agreement. Furthermore, another study investigated the use of the
cpcBA
-IGS region of the phycocyanin operon, flanked by
cpcB
and
cpcA
, as a marker for the subgenus characterization of cyanobacteria (Neilan et al. 1995, Bolch et al. 1996).
The phylogenetic analyses based on the 16S rRNA gene and
cpcBA
-IGS sequences showed no division between
A. platensis
and
A. maxima
, plus the 16S rRNA gene and
cpcBA
-IGS clusters did not exhibit any well-defined geographical distributions, and instead overlapped in a rather interesting way. Thus, it is now widely accepted that
S. laxissima
SAG 256.80 is distinct and relatively distant from
Arthrospira
strains.
- Structure and characterization of cpcBA-IGS
The sequences of the amplified fragments of the genus
Arthrospira
comprised the last 259 nt of
cpcB
, a 111 nt spacer, and the first 33 nt of
cpcA
. These regions were identified based on sequence comparisons using the BLASTN Sequence (
http://www.ncbi.nlm.nih.gov/BLAST
). For comparison, the
Spirulina
(Oscillatoriales) sequence comprised 259 nt of
cpcB
, a 100 nt spacer, and
Mosaic distribution of variable informative sites in cpcBA-intergenic spacer sequence of Arthrospira strains. For easier observation, the strains are classified according to the clusters in Fig. 2. The numbers above the alignment indicate the nucleotide position of the informative sites. Blocks of orthologous sequences are boxed. PCC, Pasteur Culture Collection of Cyanobacterial Strains, Paris, France; SAG, Sammlung von Algenkulturen der Universität Göttingen, Germany; NIES, National Institute for Environmental Studies Collection, Tsukuba, Ibaraki, Japan; KCTC, Korean Collection for Type Cultures, Daejeon, Korea; UTEX, Culture Collection of Algae at the University of Texas at Austin, Austin, TX, USA.
the first 33 nt of
cpcA
. For
Phormidium autumnale
(Oscillatoriales), the
cpcB
-
cpcA
locus comprised 224 nt of
cpcB
, a 89 to 106 nt spacer, and 281 nt of
cpcA
(Teneva et al., 2005). The sequence analyses of
Nodularia
isolates (Nostocales) resulted in 283 nt of
cpcB
, a spacer of 78-79 nt, and 306 nt of
cpcA
(Barker et al. 1999), whereas for the different
Aphanizomenon
colonies (Nostocales), this region comprised 227 nt of
cpcB
, a spacer of 77-103 nt, and 47 nt of
cpcA
(Barker et al. 2000). For
Synechococcus
(Chroococcales), the sequence comprised 274 nt of
cpcB
, a 43 to 60 nt spacer, and 195 nt of
cpcA
(Robertson et al. 2001). Interestingly, the length of the
cpcBA
-IGS spacer in Chroococcales was shorter than that in the other cyanobacteria (Nostocales and Oscillatoriales).
The information sites of the
cpcB
-
cpcA
DNA matrix for
Arthrospira
(with the taxa arranged according to the results in
Fig. 2
) are shown in
Fig. 3
. The mosaic distribution of the substitutions suggests recombination events at these phycocyanin loci. The first block comprises positions 1-259 (3′ end of
cpcB
translated sequences), while the second block comprises positions 260-403 (
cpcB
-
cpcA
spacer and 5′ end of
cpcA
translated sequences).
Fig. 3
shows the trees obtained using positions 1-259 and 260-403, respectively. While the first block clearly differentiates Groups I and II, as defined in
Fig. 3
, the substitutions in the second block do not separate Clade 1 and 2, but rather differentiate Group II-2 from all the other strains. In Group II-1 and II-2, the nucleotide substitution was T to C and A to T at 115 and 247, respectively. No amino acid changes were found in any of the groups. However, in Group I and II, the substitutions at 224 and 242 involved a change from Ser to Gly and Cys to Ala, respectively.
The LARD maximum-likelihood method (Holmes et al. 1999) was applied to find the breakpoint in the alignment that gave the highest likelihood under an evolutionary model incorporating recombination. This point was located after position 259, in agreement with
Fig. 3
. Thus, the
Arthrospira
tree topology based on the
cpcBA
-IGS fragment did not reflect the true cladogenesis due to horizontal transfers. Also, there was no correlation between the DNA matrix and the geographical origin. These results were the same as those reported by Manen and Falquet (2002), and also reminiscent of the mosaic distribution of informative sites previously observed in the
rbc
LX gene of
Nostoc
(Rudi et al. 1998). In the genus
Arthrospira
, the phycocyanin locus also indicates intragenic recombination and suggests exchanges of genetic material between strains. Genetic exchanges have also been postulated in
Nodularia
(Barker et al. 2000) and
Phormidium
(Teneva et al. 2005).
In summary, the current study supports some previous conclusions based on 16S rRNA gene and
cpcBA
-IGS sequences that
Arthrospira
taxa are monophyletic. Thus, the combination of morphological features with molecular markers, such as 16S rRNA gene sequences and
cpcBA
-IGS sequences, as used in this study, can be useful tools for identification and / or phylogenetic studies at the species level or higher taxonomic ranks in the order Oscillatoriales. When compared with 16S rRNA sequences,
cpcBA
-IGS sequences may be better suited to resolve close relationships and intraspecies variability. The probe based on
cpcBA
-IGS that showed a high specificity for cyanobacteria enabled the amplification and sequencing to be simple and fast.
Acknowledgements
This research was supported by grants from the Advanced Biomass R&D Centre in the Global Frontier Program from the Korean Ministry of Education, Science & Technology and the Centre for Aquatic Ecosystem Restoration in the Eco-STAR project from the Korean Ministry of Environment.
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