The dinoflagellate Alexandrium tamarense was collected and kindly provided by Prof. Hung-Non Chou in Institute of Fisheries Science, National Taiwan University. An axenic culture of Alexandrium tamarense was cultured in f/2 medium. Cultures were kept at 28˚C and a 12 h light:12 h dark cycle with the light intensity of ca. 200 umol photons m⁻²∙S⁻¹.

Cultivable bacteria were isolated and maintained on a Zobell 2216E prepared with aged filtered natural seawater, supplemented with GSe trace elements and vitamins were used as necessary [15]. Bacterial isolation was performed by harvesting 1 ml of a late-logarithmic phase A. tamarense culture by brief centrifugation (12 000 g for 20 s). The spent medium was removed and the algal cell pellet resuspended in 100 μL of sterile seawater and vortexed. The cell suspension was diluted 10-fold and 100 μL of each dilution spread onto 2216E agar plates and incubated in the dark at 25˚C for 3 - 6 days. Bacterial colonies with distinct colony morphology were picked. Bacterial isolates were grown in 2216 broth, glycerol was added (25% v/v), and the cells were stored at −80˚C.

Genomic DNA of the sample was extracted using Wizard^® DNA Kit (Promega, Madison, USA) following the manufacturer’s instruction. The quality of extracted DNA was checked by 0.8% agarose gel electrophoresis and spectrophotometry (optical density at 260 nm/280 nm ratio). The V3-V4 region of bacterial 16S rRNA were amplified by PCR for high-throughput pyrosequencing. The 16S rRNA gene V3-V4 region of bacteria was amplified using the universal primers of the forward 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and the reverse 806R (5’-GGACTACHVGGGTWTCTAAT-3’). PCR amplifications of the 16S rRNA V3-V4 region were performed. PCR program was as follows: 95˚C for 3 min; 27 cycles of 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 45 s with a final extension of 72˚C for 10 min. The amplicon mixture was applied to the HiSeq 2500 MiSeq Genome Sequencer (Illumina, San Diego, CA, USA).

Extraction of high-quality DNA was performed and the raw sequences were selected and the low-quality sequences were removed. Alignment of the extracted high-quality sequences were performed using PyNAST and UCLUST. The unique sequence set was classified into operational taxonomic units (OTUs) under the threshold of 97% identity using UCLUST. Chimera Slayer was applied to remove the potential chimeric sequences in the representative set of OTUs. Alpha-diversity is used to analyze the microbial diversity. The index for the community richness includes Chao―the Chao1 estimator (http://www.mothur.org/wiki/Chao) and Ace―the ACE estimator (http://www.mothur.org/wiki/Ace). The index for the community diversity includes the Shannon index (http://www.mothur.org/wiki/Shannon) and the Simpson index (http://www.mothur.org/wiki/Simpson). Sequencing depth index is Coverage―the Good’s coverage (http://www.mothur.org/wiki/Coverage). MOTHUR (version v.1.30.1, http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity) is used for data analysis.

As shown in Table 1, a total of 102,131 of bacterial V3-V4 16S rRNA raw sequence reads were generated from the samples. The number of unique and classifiable representative OTU sequences for bacteria was 98. Based on homologous sequence alignment method and clustering with information extracted from the RDP and BLAST databases, the lowest level of taxonomy of the identified OTUs was determined. The rarefaction and Shannon diversity curves for the samples reached the saturation phase (Figure 1), indicating the majority of bacterial phylotypes of the sample had already been covered. As shown in Table 1, the Shannon index, Simpson diversity index, Chao1 and observed species of each sample were used to evaluate the species richness and diversity indicates large distinction of species richness and diversity of the bacterial community within the sample.

Eight bacterial phyla were identified in the Alexander tamarense culture sample in this study (Figure 2), namely

Proteobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Actinobacteria, Verrucomicrobia, Gemmatimonadetes and Cyanobacteria. The predominant phylum was Proteobacteria, accounting for 59.18% of the total bacterial sequences. Bacteroidetes, Planctomycetes, Actinobacteria, and Firmicutes were the subdominant phyla, which contributed to 14.28%, 13.26%, 6.12% and 4.08% of the total bacterial sequences, respectively. Minor phyla, including Verrucomicrobia, Gemmatimonadetes and Cyanobacteria, together contributed to 3.06% of the total bacterial sequences.

The major bacterial genera belonged to Proteobacteria including Rhodobacteraceae, Hyphomonadaceae, Phycisphaera, Dinoroseobacter, of 25.79%, 13.05%, 12.43% and 11.1%, respectively. The most abundant genus, Rhodobactgeraceae, was comprised of 10 different types of OTUs (Figure 2). Based on the phylogenetic tree, there is still 5.29% unknown data could be further assigned to the potential new species.

In order to develop the furthering interaction study of symbiotic bacteria with the host dinoflagellate, the culture-independent isolation of the cultivable symbiotic bacteria strains from toxic Alexander tamarense and the bacterial diversity based on phylogenetic analysis of the 16S rDNA sequences of the isolated bacteria strains were performed. Total 12 bacterial strains were isolated from the Zobell 2216 marine agar. Based on the result as shown in Figure 3 and Figure 4, the obtained bacterial diversity is in well consistent with the phylogenetic

analysis using pyrosequencing anslysis.