ANALYSIS OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING SEQUENCES OF THE 5.8S RIBOSOMAL DNA AND INTERNAL TRANSCRIBED SPACER REGIONS¹

Abstract

The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS‐1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS‐1 (WKS‐I type) or A. tamarense CU‐15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS‐1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS‐1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150‐177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28‐bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.

Azadinium poporum from the Argentine Continental Shelf, Southwestern Atlantic, produces azaspiracid-2 and azaspiracid-2 phosphate

“…Lysis was made using warm lysis buffer (65 8C), cells were transferred to a suspension of glass beads and disrupted and homegenised by reciprocal shaking at maximum speed (6.5 m s À1 ) in a FastPrep instrument (Thermo Savant, Illkirch, France), 4 mL of RNAse was added, and elution was made in 50 mL of elution buffer. Polymerase chain reaction (PCR) for large subunit ribosomal DNA (LSU rDNA) was performed using primers D1 C (5 0 -ACCCGCTGAATTTAAGCATA-3 0 ) and D2R (5 0 -CCTTGGTCCGTGTTTCAAGA-3 0 ) (Scholin et al, 1994), and PCR for internal transcribed spacer ribosomal DNA (ITS rDNA) was performed using primers ITSa (5 0 -CCAAGCTTCTAGATCGTAACAAG-G(ACT)TCCGTAGGT-3 0 ) and ITSb (5 0 -CCTGCAGTCGACA(GT)ATGCT-TAA(AG)TTCAGC(AG)GG-3 0 ) (Adachi et al, 1996). Genomic DNA was amplified in a 20 mL PCR reaction containing 16.3 mL of milliQ water, 2.0 mL of 10Â HotMaster Taq Buffer (Eppendorf), which included MgCl 2 , 0.2 mL of each primer (10 mM), 0.2 mL of dNTP (10 mM), 0.1 ml HotMaster Taq polymerase (Eppendorf), and 10 ng of DNA mL À1 .…”

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

“…The LSU primers were DIR-F (5′-ACCCGCTGAA TTTAAGCATA-3′) and Dir-2C (5′-CCTTGGTCCGTGTTT CAAGA-3′), according to Scholin et al (1994). The PCR steps were a hold at 94°C for 9 min, followed by 20 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 9 m. For ITS, we used ITS A (5′-CCAAGCTTCTAGATCGTAACAAGGHT CCGTAGGT-3′) and ITS B (5′-CCTGCAGTCGACAKA TGCTTAARTTCAGCRGG-3′) (Adachi et al, 1996), with PCR steps comprising a hold at 94°C for 5 min, followed by 35 cycles of 94°C for 20 s, 57°C for 10 s and 70°C for 5 min. The primers for rbcL were 130F (5′-AACWACWACTTGG ATTTGGAA-3′) and 1600R (5′-GCATGAATATGMTG WACCAT-3′) (Yoon et al, 2002), with PCR steps comprising a hold at 94°C for 2 min, followed by 39 cycles of 94°C for 20 s, 46°C for 30 s and 70°C for 5 min.…”

The first report ofVulcanodinium rugosum(Dinophyceae) from the South China Sea with a focus on the life cycle

“…A partial LSU rDNA sequence, including the D1 and D2 domains, was amplified by polymerase chain reaction (PCR) using primers D1R and D2C (Scholin et al 1994). The total ITS1-5.8S-ITS2 was amplified using ITSA and ITSB primers (Adachi et al 1996). The PCR protocol was as follows: 94 8C for 3.5 min; followed by 35 cycles of 94 8C for 50 s, 45 8C for 50 s, 72 8C for 80 s; plus a final extension of 72 8C for 10 min.…”