Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral.

BACKGROUND: Endozoicomonas bacteria symbiosis with various marine organisms is hypothesized as a potential indicator of health in corals. Although many amplicon analyses using 16S rRNA gene have suggested the diversity of Endozoicomonas species, genome analysis has been limited due to contamination of host-derived sequences and difficulties in culture and metagenomic analysis. Therefore, the evolutionary and functional potential of individual Endozoicomonas species symbiotic with the same coral species remains unresolved. RESULTS: In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genomes (SAGs) for uncultured coral-associated Endozoicomonas spp. We obtained seven novel Endozoicomonas genomes and quantitative bacterial composition from Acropora tenuis corals at four sites in Japan. Our quantitative 16S rRNA gene and comparative genomic analysis revealed that these Endozoicomonas spp. belong to different lineages (Clade A and Clade B), with widely varying abundance among individual corals. Furthermore, each Endozoicomonas species possessed various eukaryotic-like genes in clade-specific genes. It was suggested that these eukaryotic-like genes might have a potential ability of different functions in each clade, such as infection of the host coral or suppression of host immune pathways. These Endozoicomonas species may have adopted different host adaptation strategies despite living symbiotically on the same coral. CONCLUSIONS: This study suggests that coral-associated Endozoicomonas spp. on the same species of coral have different evolutional strategies and functional potentials in each species and emphasizes the need to analyze the genome of each uncultured strain in future coral-Endozoicomonas relationships studies. Video Abstract.

Draft Genome Sequence of Okeania sp. Strain KiyG1, Assembled from Single-Amplified Genomes Collected from Cape Kiyan, Okinawa, Japan.

The genus Okeania is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of Okeania sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.

Characterization of Macroscopic Colony-Forming Filamentous Cyanobacteria from Okinawan Coasts as Potential Sources of Bioactive Compounds.

Marine macroscopic colony-forming filamentous (MMCFF) cyanobacteria are considered as prolific producers of bioactive compounds. Thus, knowledge of the diversity of MMCFF cyanobacteria as related to bioactive compound production has become very important. However, basic taxonomic studies of MMCFF cyanobacteria are lacking. Many cyanobacterial taxa are still misidentified or undescribed. In this study, a total of 32 cyanobacterial colonies from nine coastal regions of Okinawa Prefecture were investigated for a diversity assessment. A polyphasic approach including morphological and molecular studies based on 16S rRNA gene sequences was performed to characterize Okinawan MMCFF cyanobacteria. Both morphological and molecular phylogenetic results showed that MMCFF cyanobacteria from Okinawan coasts are very diverse. We found morphotypes of Lyngbya-like, Phormidium-like, and Leptolyngbya-like groups among Okinawan cyanobacterial samples. Genetically, samples were distributed in various clades in the phylogenetic tree, including within Moorena, Okeania, Caldora, Neolyngbya, Dapis, as well as several unknown clades. In addition, cytotoxic activities of three samples from Kiyan coast were tested against HeLa cells. All three crude extracts of these samples showed strong cytotoxic activity with IC50 < 1 µg/ml.

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales, Rhodophyta) from East Asia.

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.

Kabirimine, a New Cyclic Imine from an Okinawan Dinoflagellate.

On our quest for new bioactive molecules from marine sources, two cyclic imines (1, 2) were isolated from a dinoflagellate extract, inhibiting the growth of the respiratory syncytial virus (RSV). Compound 1 was identified as a known molecule portimine, while 2 was elucidated to be a new cyclic imine, named kabirimine. The absolute stereochemistry of 1 was determined by crystallographic work and chiral derivatization, whereas the structure of 2 was elucidated by means of spectroscopic analysis and computational study on all the possible isomers. Compound 1 showed potent cytotoxicity (CC50 < 0.097 µM) against HEp2 cells, while 2 exhibited moderate antiviral activity against RSV with IC50 = 4.20 µM (95% CI 3.31-5.33).

Minnamide A, a Linear Lipopeptide from the Marine Cyanobacterium Okeania hirsuta.

Minnamide A is a lipopeptide with a unique repeating structure consisting of hydroxy and proposed ß-branched methyl groups. The absolute configuration of minnamide A was determined by a combination of chemical degradation, chiral HPLC analyses, and synthetic methods. Minnamide A showed growth-inhibitory activity toward HeLa cells with an IC50 value of 0.17 µM and rapidly induced cell death at a concentration of 2 µM. Minnamide A induced the copper-mediated accumulation of reactive oxygen species.

Unexpected High Diversity of Terrestrial Cyanobacteria from the Campus of the University of the Ryukyus, Okinawa, Japan.

Terrestrial cyanobacterial strains were isolated from the Nishihara campus of the University of the Ryukyus, Okinawa, Japan. The 13 sampling sites were distributed in a 200 m radius and appeared as dry, blackened stains. From these small areas, 143 cyanobacterial strains were established. The strains were divided into five morphotypes, including unicells, unicells with baeocytes, non-branching filaments, false-branching filaments, and heterocystous strains. From the strains, 105 partial 16S rRNA gene sequences were obtained and could be classified into 30 generic types. Among them, 22 unique strains and over 1100 bps of data were selected for further phylogenetic analyses. These sequences were positioned into six main clades corresponding to cyanobacterial orders: Nostocales, Chroococidiopsidales, Chroococcales, Oscillatoriales, Pleurocapsales, and Synechococcales. Almost all sequences had no identical matching data in GenBank and many of them had no closely related data. These data suggest that the terrestrial cyanobacteria are very divese even within close sampling areas, such as within the campus of the University of the Ryukyus. The established strains are not only important for classification of terrestrial cyanobacteria but also for possible application studies in the future.

A metabarcoding framework for facilitated survey of endolithic phototrophs with tufA.

BACKGROUND: In spite of their ecological importance as primary producers and microbioeroders of marine calcium carbonate (CaCO3) substrata, endolithic phototrophs spanning both prokaryotic (the cyanobacteria) and eukaryotic algae lack established molecular resources for their facilitated survey with high throughput sequencing. Here, the development of a metabarcoding framework for the elongation factor EF-Ttu (tufA) was tested on four Illumina-sequenced marine CaCO3 microfloras for the characterization of their endolithic phototrophs, especially the abundant bioeroding Ostreobium spp. (Ulvophyceae). The framework consists of novel tufA degenerate primers and a comprehensive database enabling Operational Taxonomic Unit (OTU) identification at multiple taxonomic ranks with percent identity thresholds determined herein. RESULTS: The newly established tufA database comprises 4057 non-redundant sequences (from 1339 eukaryotic and prokaryotic phototrophs, and 2718 prokaryotic heterotrophs) including 27 classes in 10 phyla of phototrophic diversity summarized from data mining on GenBank(®), our barcoding of >150 clones produced from coral reef microfloras, and >300 eukaryotic phototrophs (>230 Ulvophyceae including >100 'Ostreobium' spp., and >70 Florideophyceae, Phaeophyceae and miscellaneous taxa). Illumina metabarcoding with the newly designed primers resulted in 802 robust OTUs including 618 phototrophs and 184 heterotrophs (77 and 23% of OTUs, respectively). Phototrophic OTUs belonged to 14 classes of phototrophs found in seven phyla, and represented ~98% of all reads. The phylogenetic profiles of coral reef microfloras showed few OTUs in large abundance (proportion of reads) for the Chlorophyta (Ulvophyceae, i.e. Ostreobium and Phaeophila), the Rhodophyta (Florideophyceae) and Haptophyta (Coccolithophyceae), and a large diversity (richness) of OTUs in lower abundance for the Cyanophyta (Cyanophyceae) and the Ochrophyta (the diatoms, 'Bacillariophyta'). The bioerosive 'Ostreobium' spp. represented four families in a large clade of subordinal divergence, i.e. the Ostreobidineae, and a fifth, phylogenetically remote family in the suborder Halimedineae (provisionally assigned as the 'Pseudostreobiaceae'). Together they harbor 85-95 delimited cryptic species of endolithic microsiphons. CONCLUSIONS: The novel degenerate primers allowed for amplification of endolithic phototrophs across a wide phylogenetic breadth as well as their recovery in very large proportions of reads (overall 98%) and diversity (overall 77% of OTUs). The established companion tufA database and determined identity thresholds allow for OTU identification at multiple taxonomic ranks to facilitate the monitoring of phototrophic assemblages via metabarcoding, especially endolithic communities rich in bioeroding Ulvophyceae, such as those harboring 'Ostreobium' spp., Phaeophila spp. and associated algal diversity.

Genetic diversity and distribution of the ciguatera-causing dinoflagellate Gambierdiscus spp. (Dinophyceae) in coastal areas of Japan.

BACKGROUND: The marine epiphytic dinoflagellate genus Gambierdiscus 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 Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet. METHODS/PRINCIPAL FINDING: 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 Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities. CONCLUSIONS/SIGNIFICANCE: This study revealed a surprising diversity of Japanese Gambierdiscus 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.

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.

Taxonomy of Nephroselmis viridis sp. nov. (Nephroselmidophyceae, Chlorophyta), a sister marine species to freshwater N. olivacea.

The genus Nephroselmis (Nephroselmidophyceae), which had been placed in the Prasinophyceae, is one of the primitive green flagellates that are important to our understanding of the early evolution of green plants. We studied a new species of Nephroselmis isolated from Japan, Fiji and South Africa. This species has been known for a long time as undescribed species 'N. viridis.' N. viridis possesses some ultrastructural characters shared with only the freshwater type species N. olivacea, including a disc-like structure beneath the pyrenoid and bipolar spiny body scales with 1-5-8-5-1 spines. Molecular phylogenetic analysis based on 18S rDNA also supports a sister relationship between N. viridis and N. olivacea. However, N. viridis is distinguishable from N. olivacea by the shape of its starch sheath, its scales, its pigment composition and its habitat. In this paper, we designate the formal description of N. viridis sp. nov. We also describe variability in the 18S rDNA introns of various N. viridis strains. This detailed study of N. viridis provides some insights into the evolution of Nephroselmis.

TAXONOMY AND PHYLOGENY OF NEPHROSELMIS CLAVISTELLA SP. NOV. (NEPHROSELMIDOPHYCEAE, CHLOROPHYTA)(1).

Nephroselmis clavistella D. G. Faria et S. Suda sp. nov. is collected from coastal sand samples from the eastern and western coasts of Okinawa-jima Island, Japan. The description of the cultured strains is based on light and electron microscopic observations. The cultured strains are phylogenetically analyzed based on 18S rDNA sequences. The cells are remarkably right-left flattened and appear round or ellipse when viewed from their right or left side, and are ∼5.0 µm in diameter. The posterior flagellum curved around the cell body at rest. A single, parietal, crescent chloroplast is yellowish green and contains one conspicuous eyespot in its anterior-ventral edge near the short flagellum base. A pyrenoid with one starch sheath is located dorsal of the chloroplast. The cells are divided by transverse binary cell division, as is common in other species of this genus. The cell body is covered with five types of scales, and among them four scale types are similar to Nephroselmis rotunda. The fifth scale type is a distinctive spiny and club-shaped stellate scale with 10 spines, four of the 10 spines extended ∼150 nm and each are slightly curved with a hook at the end, whereas six spines are club-shaped blunt ended. This scale morphology, an important taxonomic characteristic, has never been described before for the genus Nephroselmis. The cell's morphology is distinctive from previously described Nephroselmis species, and its unique scale characteristics led us to name this newly proposed species "clavistella," meaning club star.

REVISION OF THE MASTOPHOROIDEAE (CORALLINALES, RHODOPHYTA) AND POLYPHYLY IN NONGENICULATE SPECIES WIDELY DISTRIBUTED ON PACIFIC CORAL REEFS1.

The subfamily Mastophoroideae (Corallinaceae, Rhodophyta) is characterized by species possessing nongeniculate, uniporate tetrasporangial conceptacles without apical plugs, the presence of cell fusions, and the absence of secondary pit connections. However, molecular phylogenetic studies not including the type genus Mastophora indicated that the Mastophoroideae was polyphyletic. Our molecular phylogenetic analysis of the subfamily including the type genus using DNA sequences of SSU rDNA and plastid-encoded gene of PSII reaction center protein D1 (psbA) revealed that Mastophora formed a robust clade only with Metamastophora. The other mastophoroid genera were divided into six lineages within the family Corallinaceae. Five supported lineages-(i) Pneophyllum; (ii) Hydrolithon gardineri (Foslie) Verheij et Prud'homme, Hydrolithon onkodes (Heydr.) Penrose et Woelk., and Hydrolithon pachydermum (Foslie) J. C. Bailey, J. E. Gabel et Freshwater; (iii) Hydrolithon reinboldii (Weber Bosse et Foslie) Foslie; (iv) Spongites; and (v) Neogoniolithon-were clearly distinguished by the combination of characters including the presence or absence of palisade cells and trichocytes in large, tightly packed horizontal fields and features of tetrasporangial and spermatangial conceptacles. Therefore, we amend the Mastophoroideae to be limited to Mastophora and Metamastophora with a thin thallus with basal filaments comprised of palisade cells, tetrasporangial conceptacles formed by filaments peripheral to fertile areas, and spermatangia derived only from the floor of male conceptacles. This emendation supports Setchell's (1943) original definition of the Mastophoroideae as having thin thalli. We also propose the establishment of three new subfamilies, Hydrolithoideae subfam. nov. including Hydrolithon, Porolithoideae subfam. nov. including the resurrected genus Porolithon, and Neogoniolithoideae subfam. nov. including Neogoniolithon. Taxonomic revisions of Pneophyllum and Spongites were not made because we did not examine their type species.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria.

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 16S rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204T), Planktothrix rubescens (type strain CCAP 1459/22T), Planktothrix pseudagardhii sp. nov. (type strain T1-8-4T), Planktothrix mougeotii (type strain TR1-5T), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207T), Tychonema bourrellyi (type strain CCAP 1459/11BT) and Limnothrix redekei (type strain NIVA CYA 277/1T).

Morphological variability of colonies of Microcystis morphospecies in culture.

Serial observations were carried out on cultures of five morphospecies of the genus Microcystis Kützing ex Lemmermann 1907, Microcystis aeruginosa (Kützing) Kützing, Microcystis ichthyoblabe Kützing, Microcystis novacekii (Komárek) Compére, Microcystis viridis (A. Brown) Lemmermann, and Microcystis wesenbergii (Komárek) Komárek in Kondratieva. Many strains maintained colony forms characteristic of their morphospecies, and others showed morphological variations, some of which were characteristic of other morphospecies. M. novacekii displayed several morphotypes including some characteristics of M. aeruginosa and M. ichthyoblabe. M. wesenbergii also showed great morphological variability and showed morphotypes characteristic of M. aeruginosa. Distinction among these morphospecies, therefore, seemed to be obscure or impossible. We conclude that the current classification of the genus Microcystis, chiefly based on morphological characteristics, is no longer valid and must be reviewed in light of our observations that one strain may have various colony forms.