Cells of Ostreopsis were observed from the coasts of São Paulo State, Brazil, in the South Atlantic Ocean. Isolation was carried out on 18 December 2013 during the low tide around the pier of the Marine Station of the University of São Paulo at Ubatuba (23°30’3.16’’ S - 45°7’6.78’’ W). Macroalgae were collected from rocky surfaces during the low tide (< 1 m depth), placed into a bottle with ambient water and stirred vigorously. The gross particles were removed through a 200 µm mesh filter.

In the laboratory, the bottle sample was stirred, and the suspension was let to settle in a composite settling chamber. Epiphytic cells were examined with an inverted microscope Nikon TS-100 (Nikon, Tokyo, Japan) and photographed with a digital camera mounted on the microscope’s eyepiece (Cyber-shot DSCW300, Sony, Tokyo, Japan).

A single cell of Ostreopsis was isolated using a micropipette and placed in 24-well tissue culture plate with 0.2 µm-filtered seawater collected that day from the same locality, supplemented with f/2 medium without silicates. Two days later, the cells were placed into a 6-well tissue culture plate with f/2 medium made with filtered and sterilized seawater. Seawater was collected two kilometers offshore and in the laboratory, it was prefiltered through a Whatman GF/F glass filter (~0.7 µm), and subsequently passed through a Nuclepore polycarbonate filter (0.2 µm). Borosilicate glass bottles partially filled with filtered seawater were covered with aluminum foil and autoclaved during 15 minutes at 1-2 bar pressure, and a temperature of 120 °C. After one day, bottles were placed in the incubator for at least one day before use. The culture was incubated at 23 ºC, with 100 µmol photons m-2.s-1 from cool-white tubes; the photoperiod was 12:12 h L:D. The aliquots of the clonal culture (2 mL) were transferred to 50 mL polystyrene tissue culture flasks with 20 mL of culture medium and incubated under the same conditions. A volumen of 20 mL of fresh culture medium was added after two weeks. Aliquots of the culture were harvested during the first weeks for further observations with epifluorescence and confocal microscopes. After one month, cells with distorted shapes began to appear. The cells were fixed with 5 % glutaraldehyde and kept in the refrigerator. Morphological studies used epifluorescence and confocal microscopes, fixed cells were transported to the University campus (~250 km far away from the coastal laboratory). The fixed cells were stained with Fluorescent Brightener 28 (Sigma-Aldrich, St. Louis, MO, USA) (blue emission) and observed at × 1 000 magnification under an Olympus BX51 epifluorescence microscope equipped with an Olympus DP72 camera. Cells fixed with glutaraldehyde were also observed at ×630 magnification with an inverted confocal microscope TCS SP8 AOBS (Leica, Wetzlar, Germany), after staining with Fluorescent Brightener 28. The nomenclature for the plate tabulation followed Gómez, Qiu, Lopes, and Lin (2015).

By the use of the inverted microscope, cultured cells of Ostreopsis were micropipetted individually with a fine capillary into a clean chamber and washed several times in a series of drops of 0.2 µm-filtered and sterilized seawater. Finally, a total of 50 cells were placed in a 0.2 mL Eppendorf tube filled with several drops of absolute ethanol. The sample was kept at room temperature in darkness during one year, until the molecular analysis could be performed. Prior to DNA extraction, the tubes were centrifuged for 10 min at 10 000 rpm to settle the Ostreopsis cells, and the ethanol was aspirated. The cell pellet was resuspended in 100 μL of DNA lysis buffer (0.1 M EDTA pH 8.0, 1 % SDS, 200 μg. mL−1 proteinase K) and transferred into a 1.5 mL tube, and the original tube was washed with 100 μL of DNA lysis buffer for four more times. Then, the resultant 0.5 mL was incubated for 48 hours at 55 °C. DNA extraction and purification followed a previously reported protocol (Qiu, Huang, Liu, & Lin, 2011). At the end of the extraction process, Ostreopsis DNA was eluted in 50 μL of Tris-HCl solution. Next, 1 μL of the extracted DNA was used in PCR with primers Dino1662F and 28SR2 to amplify a ~1 900 base pair region of the rRNA gene cistron covering 3’ end region of the SSU, the entire ITS, and partial 5’ region of the LSU (3’end-SSU-ITS1-5.8S-ITS2-LSU) (Qiu, Huang, Liu, Zhang, & Lin, 2013). The PCR amplifications were carried out in 25 μL reaction volumes under thermocycling conditions, including a denaturing step of 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 45 s, and a final extension step of 72 °C for 10 min. PCR products were resolved by agarose gel electrophoresis with the DL2000 DNA Ladder (TaKaRa Bio, Dalian, China), and the bands with expected sizes were excised in order to remove primer dimers. DNA was purified and directly sequenced as previously reported (Qiu et al., 2011).

The DNA sequence was analyzed using Basic Local Search Tool (BLAST) against databases in GenBank. A total of 58 of LSU rDNA and 62 of ITS-5.8S-ITS2 hit sequences showing significant similarity to the sequences obtained in this study were retrieved from the databases. Combined sequences were aligned with ClustalW, using default parameters (Larkin et al., 2007), and obvious misalignments adjusted manually. The trimmed ITS-5.8S-ITS2 and D1-D2 LSU alignments were analyzed using ModelTest to select the most appropriate evolutionary model (Posada & Crandall, 1998). The selected General Time Reversible model with gamma distribution was employed for Maximum Like lihood analysis using PhyML3.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). Categories of substitution rates were set at four, and other parameters were estimated based on the datasets. The proportion of invariable sites and the gamma shape parameter were 0.00 and 1.1064 for the LSU dataset, and 0.2546 and 4.3634 for ITS, respectively. The sequence (LSU rDNA and ITS) obtained in this study was deposited in GenBank under accession number KX781270.

The wild cells from recently collected samples were oval to tear-shaped fully covered of brown pigments, and some cells showed a large red body (Fig. 1A, Fig. 1B, Fig. 1C and Fig. 1D). In apical-antapical view, the dorso-ventral diameter of wild cells ranged from 35 to 65 µm with an average value of 55.1 µm, and the transdiameter (width) ranged from 20 to 40 µm with an average value 32.6 µm (n = 15). The average ratio between the dorsoventral axis and the transdiameter was 1.69. The cell dimensions of our cells and those from other ocean regions were compared in Table 1.

Table 1
Morphometric gathering of cultured Ostreopsis ovata/O. cf. ovata data

In the cultured cells, the apical pore complex (APC) plate was located on the dorsal side. It was about 8 µm long and slightly curved, enclosed by the plate 2’ and also touched apical plates 1’, and 3’. There were three apical plates (Fig. 1E, Fig. 1F, Fig. 1G, Fig. 1H, Fig 1I). The 1’ plate occupied the center of the epitheca. It was hexagonal and touched the plates 2’, 3’, 1’’, 2’’, 6’’ and 7’’ (Fig. 1G, Fig. 1H, Fig. 1I). The plate 2’ was elongated and the 3’ plate was irregularly pentagonal in the dorso-central epitheca (Fig. 1G, Fig. 1H and Fig. 1I). There were seven precingular plates. Plates 1’’, 2’’, 3’’, 4’’, 5’’ and 7’’ were irregularly quadrangular. The 1’’, 4’’, and 5’’ plates were the smallest. The 2’’ and 3’’ plates were dorso-ventrally elongated. The 6’’ plate was pentagonal and the largest of the precingular series (Fig. 1F, Fig. 1G, Fig. 1H and Fig. 1I).

Fig. 1
Light micrographs of Ostreopsis cf. ovata from Ubatuba, São Paulo, Brazil; A-B. Recently collected wild cells; C-M. Cultured strain UBA-BR; A. Note the prominent red accumulation body; B. Note the size differences; C. Apical-antapical view; D. Lateral view; E. Empty epitheca; F-G. View of the epitheca of calcofluor stained cells under epifluorescence microscopy; H-I View of the epitheca with overlay images of confocal microscopy; J-M. View of the hypotheca of calcofluor stained cells under epifluorescence microscopy. Scale bar = 10 µm.

In the hypotheca, there were five post cingular plates. Most of the hypotheca was occupied by the plates 2’’’, 3’’’, 4’’’, and a posterior intercalary plate (1p) (Fig. 1J, Fig. 1K, Fig. 1L, Fig. 1M). The 2’’’ plate was quadrangular and dorso-ventrally elongated. The 3’’’ and 4’’’ plates were quadrangular and extended for most of the dorsal half of the hypotheca. The 1’’’ plate much smaller than the other postcingular plates was difficult to observe. The 5’’’ plate was oblong and irregularly triangular (Fig. 1K and Fig. 1L). The 1p plate (or alternatively interpreted as the second antapical plate, 2’’’’) was showed an elongated pentagonal shape. The first antapical plate was small, triangular and located between the plates 2’’’ and 1p (Fig. 1J). The plate formula was Po, 3’, 7’’, 5’’’, 1p, and 1’’’’ or alternatively Po, 4’, 6’’, 5’’’, 2’’’’. The cell surface was smooth. The number of large pores per 25 µm2 of the thecal surface was 3-4 (Fig. 1J).

In the LSU rDNA phylogenetic tree, the sequences under the names Ostreopsis ovata and O. cf. ovata branched into three clades. The most basal clade contained two sequences from strains isolated at Madeira Island in the NE subtropical Atlantic Ocean. Other clade contained a sequence from Malaysia. Our new LSU rDNA sequence branched in the main clade together with sequences of strains from the Mediterranean Sea, European Atlantic coasts, subtropical NE Atlantic (Madeira and Canary Islands), and other sequences from Rio de Janeiro in Brazil and Japan (Fig. 2).

Fig. 2
Maximum likelihood tree of Ostreopsis spp. Basedon the LSU rDNA (D1-D2) sequences. The sequence obtained in this study is bold-typed. Support of nodes is base don bootstrap values of Maximum Likelihood (ML)/Neighbor Joining (NJ) methods with 1000 resamplings. Only values greater than 60 are shown. Coolia monotiswas used as an outgroup.

Our sequencing effort also yielded a new ITS sequence of Ostreopsis cf. ovata from a Brazilian strain. The ITS marker seems to have a greater resolving power because the sequences of Ostreopsis ovata split into five clades. Two sequences from Japan (AB674909) and Korea (HE793379) branched in the most basal clade. The rest of sequences branched into two sister clades and each further subdividing into two subclades. Our new ITS sequence also branched in a clade with sequences of strains from the Mediterranean Sea, European Atlantic, subtropical NE Atlantic, other sequences from Brazil at Rio de Janeiro, and a few sequences from Japan (Fig. 3). Its sister subclade contained two sequences from the NE subtropical Atlantic at Madeira Island (FM244642) and the Mediterranean Sea at Greece (FM244736). The other clade was also divided into two subclades with sequences of strains isolated from Malaysia, Indonesia and Oceania at Cook Islands (Fig. 3).

Fig. 3
Maximum likelihood tree of Ostreopsis spp. based on the ITS1-5.8S-ITS2 sequences. The sequence obtained in this study is bold-typed. Support of nodes is based on bootstrap values of Maximum Likelihood (ML)/Neighbor Joining (NJ) methods with 1 000 resamplings. Only values greater than 60 are shown.