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

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (<it>Raphanus sativus</it> L.)

<p>Abstract</p> <p>Background</p> <p>Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in <it>Brassicaceae</it>. Although the gene <it>orf138</it> has been isolated as a determinant of Ogura-type CMS, no homologous sequence to <it>orf138</it> has been found in public databases. Therefore, how <it>orf138</it> sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene <it>orf138</it>.</p> <p>Results</p> <p>Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional <it>atp9</it>. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional <it>trnfM</it>. The gene <it>orf138</it> was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene <it>orf138</it> was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported <it>Brassicaceae</it> species, although no homology was found for the remaining 20% of sequences.</p> <p>Conclusions</p> <p>Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known <it>Brassicaceae</it> mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of <it>Brassicaceae</it>, and novel genes such as <it>orf138</it> could have been created by the shuffling process of mitochondrial genome.</p

Genetic Diversity and Distribution of the Ciguatera-Causing Dinoflagellate <i>Gambierdiscus</i> spp. (Dinophyceae) in Coastal Areas of Japan

<div><p>Background</p><p>The marine epiphytic dinoflagellate genus <i>Gambierdiscus</i> produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese <i>Gambierdiscus</i> revealed the existence of two distinct phylotypes: <i>Gambierdiscus</i> sp. type 1 from subtropical and <i>Gambierdiscus</i> sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese <i>Gambierdiscus</i> are still unclear, because a comprehensive investigation has not been conducted yet.</p><p>Methods/Principal Finding</p><p>A total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese <i>Gambierdiscus</i> was high since five species/phylotypes were detected: including two reported phylotypes (<i>Gambierdiscus</i> sp. type 1 and <i>Gambierdiscus</i> sp. type 2), two species of <i>Gambierdiscus</i> (<i>G. australes</i> and <i>G</i>. cf. <i>yasumotoi</i>) and a hitherto unreported phylotype <i>Gambierdiscus</i> sp. type 3. The distributions of type 3 and <i>G</i>. cf. <i>yasumotoi</i> were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and <i>G. australes</i> occurred from the subtropical to the temperate area, with a tendency that type 1 and <i>G. australes</i> were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and <i>G. australes</i> exhibited mouse toxicities.</p><p>Conclusions/Significance</p><p>This study revealed a surprising diversity of Japanese <i>Gambierdiscus</i> and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.</p></div

Bayesian inference (BI) phylogeny of the D8–D10 region of the LSU rDNA of <i>Gambierdiscus</i> species/phylotypes.

<p>Nodal supports are of Bayesian posterior probability (pp) and Bootstrap (bt) values obtained by BI analysis and Maximum likelihood (ML) analysis, respectively. Nodes with strong supports (pp/bt  = 1.00/100) are shown as thick lines. For sequences obtained via cloning, a variant ID, starting with C, is shown followed by strain ID (i.e. T080908_1_C1). Sequences obtained in the present study are indicated in color.</p

Map of research area.

<p>Location of each sampling station and its ID as well as presence (yellow circle) or absence (gray circle) of <i>Gambierdiscus</i> cells in the sample is shown. Each island is marked as A: Hokkaido, B: Honshu (main Isl.), C: Shikoku, D: Kyushu, E: Okinawa Island, F: Miyako Island, Ikema Island and Irabu Island (from right to left), G: Ishigaki Island and Iriomote Island (from right to left). A: the boreal area, B, C and D: the temperate area, E, F and G: the subtropical area.</p

Oligonucleotide primers used for PCR amplification and DNA sequencing.

*<p>: Annealing site in the ITS1-5.8S-ITS2 rDNA sequence of <i>Gambierdiscus yasumotoi</i> GYASU, GU968498 (Vandersea et al., 2010 Direct Submission).</p>**<p>: Annealing site in the LSU rDNA sequence of <i>Prorocentrum micans</i>, X16108 (Lenaers et al., 1989 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060882#pone.0060882-Lenaers1" target="_blank">[51]</a>).</p>***<p>: Annealing site in the SSU rDNA sequence of <i>Pfiesteria piscicida</i>, AY245693 (Litaker et al., 2003 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060882#pone.0060882-Litaker4" target="_blank">[44]</a>).</p

Summary of genetic distances of the SSU rDNA over the p distance calculated by uncorrected genetic distance (UGD) model among and within 66 consensus sequences ( = 66 strains; each consensus sequence was constructed with cloned sequences and/or directly sequence of each strain) of <i>Gambierdiscus</i> species/phylotypes, and other protists.

<p>bp*:Nucleotide bases of the SSU rDNA used for calculations.</p><p>n**: Numbers of species (species/phylotypes) used for calculations.</p><p>Reference***: Each ref. calculated molecular similarity (we converted values to uncorrected <i>p</i> distance) of individual SSU rDNA.</p><p>NC****: Not calculated.</p><p>2 orders*****: The values include between-species data set for 2 orders; Thalassiosirales and Naviculales.</p