Phylogeography of Ostreopsis along West Pacific Coast, with Special Reference to a Novel Clade from Japan

BACKGROUND: A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. METHODS/PRINCIPAL FINDING: In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. CONCLUSIONS/SIGNIFICANCE: This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate <em>Ostreopsis</em> spp. in Coastal Waters of Japan

<div><p>Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate <i>Ostreopsis</i> species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of <i>Ostreopsis</i> spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of <i>Ostreopsis</i>. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.</p> </div

Cell enumeration of <i>Ostreopsis</i> species in environmental samples.

<p>Number of cells of four species of <i>Ostreopsis</i> were determined by qPCR considering normalization with RE in environmental samples collected from various locals of Japanese coastal waters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057627#pone-0057627-t005" target="_blank">Table 5</a>. Cell number of <i>O</i>. cf. <i>ovata</i> (red), <i>Ostreopsis</i> sp. 1 (blue), <i>Ostreopsis</i> sp. 5 (green), <i>Ostreopsis</i> sp. 6 (purple) as determined by qPCR with RE. Total cell number of <i>Ostreopsis</i> species counted by microscopy (white). Numbers on y-axis represented Log₁₀ cells g⁻¹ fresh weight algae. Values are mean ± SD.</p

Standard curves of linear plasmid spiked with or without an environmental sample.

<p>Standard curves of linear target plasmid containing the 28S rDNA spiked with DNA from the environmental sample (Maizuru100904) using various <i>Ostreopsis</i> species-specific primer and probe sets (A: <i>O</i>. cf. <i>ovata</i>; B: <i>Ostreopsis</i> sp. 1; C: <i>Ostreopsis</i> sp. 5; D: <i>Ostreopsis</i> sp. 6) were constructed with a 10-fold dilution series of the plasmid. Error bars represent standard deviation of triplicate PCR reactions. Black circle: plasmid containing the 28S rDNA spiked with DNA from the environmental sample. Open circle: plasmid containing the 28S rDNA without DNA from the environmental sample.</p

Scheme of the cell quantification method by qPCR used in this study.

<p>Normalization with DNA recovery efficiency (RE) by addition of exogenous plasmid DNA was considered in the qPCR method.</p

Standard curves of circular and linear plasmid with or without the addition of an environmental sample.

<p>Standard curves of pGEM-3z assay generated by circular and linearized pGEM-3Z plasmid with or without the addition of an environmental sample were generated. Error bars represent standard deviation of triplicate PCR reactions. Black circle: circular pGEM-3Z with an environmental sample. Open circle: circular pGEM-3Z. Black triangle: linear pGEM-3Z with the environmental sample. Open triangle: linearized pGEM-3Z.</p

Cell quantification of each species in mixed samples of cultured cells of various species.

<p>Cell quantification of each <i>Ostreopsis</i> species in three mixed culture samples (A–C) spiked with the environmental sample (Maizuru100904) by qPCR considering normalization with RE. A: qPCR quantification result of four species in mixed culture sample (Sample A) containing 900±200 cells of cultured <i>O.</i> cf. <i>ovata,</i> 1,500±350 cells of <i>Ostreopsis</i> sp. 1, 1,500±230 cells of <i>Ostreopsis</i> sp. 5 and 1,260±110 cells of <i>Ostreopsis</i> sp. 6. B: qPCR result in Sample B containing 90,000±5,000 cells of cultured <i>O.</i> cf. <i>ovata,</i> 100±10 cells of <i>Ostreopsis</i> sp. 1, 80±10 cells of <i>Ostreopsis</i> sp. 5 and 50±5 cells of <i>Ostreopsis</i> sp. 6. C: qPCR result in Sample C containing <i>O.</i> cf. <i>ovata,</i> 80,000±4,000 cells of <i>Ostreopsis</i> sp. 1, 70±10 cells of <i>Ostreopsis</i> sp. 5 and 50±10 cells of <i>Ostreopsis</i> sp. 6. White columns: cell number determined by qPCR method considering normalization with RE, Black columns: actual number of cells spiked in environmental sample. Numbers on x-axis represented Log₁₀ cell number. Values are mean ± SD.</p

List of environmental samples used in experiments to enumerate <i>Ostreopsis</i> spp. using qPCR.

<p>List of environmental samples used in experiments to enumerate <i>Ostreopsis</i> spp. using qPCR.</p

Culture sample list.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057627#pone-0057627-g001" target="_blank">Fig. 1</a> of Sato et al. (2011) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057627#pone.0057627-Sato1" target="_blank">[44]</a>.</p

Primer and probe specificity.

<p>Primer and probe specificity.</p