Construction of genomic marker sets based on the chloroplast genome of a green alga, Ulva pertusa (syn. Ulva australis), leads to simple detection of Ulva species.

In closed sea areas such as Tokyo Bay, a phenomenon known as a green tide often occurs, in which large amounts of Ulva seaweed grow abnormally and form mats along the coast. This is currently a serious environmental problem. Green tides are generated by the explosive growth of multiple types of Ulva algae. However, many Ulva species show similar characteristics to each other and are indistinguishable by appearance, making it difficult to identify the Ulva algae in green tides. In this study, we determined the entire nucleotide sequence of the chloroplast genome of Ulva pertusa (syn. Ulva australis) and identified two large inversions of gene order, suggesting the occurrence of genome inversions. We also detected structural polymorphisms among Ulva chloroplast genomes. Ulva pertusa was classified in a different clade from that containing U. lactuca and U. ohnoi, suggesting that U. pertusa is evolutionarily divergent from these species. Based on this knowledge, we constructed a genetic diagnosis system for Ulva algae. Using this approach, we established a simple method that can determine the species of Ulva algae by PCR using specific molecular markers, through which representative Ulva species such as U. lactuca, U. ohnoi and U. pertusa were easily distinguished.

Species composition, diversity, and distribution of the genus Ulva along the coast of Jeju Island, Korea based on molecular phylogenetic analysis.

Species diversity in the genus Ulva remains understudied worldwide. Using molecular analyses we investigated the species composition, diversity, distribution, and relative frequencies of the genus Ulva along the entire coast of Jeju Island, off the southern tip of Korea. Species identification was performed for 215 samples collected from 23 sites, based on comprehensive phylogenetic and model-based species delimitation analyses using the sequences of two molecular markers, chloroplast elongation factor Tu (tufA) and nuclear rDNA internal transcribed spacer (ITS). We identified 193 specimens as nine Ulva species, 14 specimens as Blidingia spp., and eight samples undetermined, based on the combined analysis of tufA and ITS phylogenies. Two model-based approaches generally supported nine groups of Ulva species. Previously documented species complex, such as U. ohnoi-U. spinulosa and U. procera-U. linza showed discordant relationships between the two phylogenies. The occurrence of U. torta on Jeju Island was first observed, despite its existence on the mainland previously reported. Ulva australis [16 of 23 sites; 34.4% (relative frequency)], U. ohnoi (16; 21.9%), and U. procera (11; 14%) were found to be the predominant species. Our study highlights that molecular analysis is critical for species delimitation in the genus Ulva and provides fundamental information for an understanding of green-tide assemblages on the "biological hotspot" coastal ecosystem, Jeju Island in Korea. This study will also help to monitor and manage local green tides at the areas that are currently encountering rapid climate changes.

Differential gene expression for carotenoid biosynthesis in a green alga Ulva prolifera based on transcriptome analysis.

BACKGROUND: Carotenoids are widely distributed in plants and algae, and their biosynthesis has attracted widespread interest. Carotenoid-related research has mostly focused on model species, and there is a lack of data on the carotenoid biosynthetic pathway in U. prolifera that is the main species leading to green tide, a harmful plague of floating green algae. RESULTS: The carotenoid content of U. prolifera samples, that is the main species leading to green tide, a harmful plague of floating green algae at different temperatures revealed that its terpenoid was highest in the samples subjected to high temperature at 28 °C (H), followed by the samples subjected to low temperature at 12 °C (L). Its terpenoid was lowest in the samples subjected to medium temperature at 20 °C (M). We conducted transcriptome sequencing (148.5 million raw reads and 49,676 unigenes in total) of samples that were subjected to different temperatures to study the carotenoid biosynthesis of U. prolifera. There were 1125-3164 significant differentially expressed genes between L, M and H incubation temperatures, of which 11-672 genes were upregulated and 453-3102 genes were downregulated. A total of 3164 genes were significantly differentially expressed between H and M, of which 62 genes were upregulated and 3102 genes were downregulated. A total of 2669 significant differentially expressed genes were observed between L and H, of which 11 genes were upregulated and 2658 genes were downregulated. A total of 13 genes were identified to be involved in carotenoid biosynthesis in U. prolifera, and the expression levels of the majority were highest at H and lowest at M of incubation temperature. Both the carotenoid concentrations and the expression of the analysed genes were lowest in the normal temperature group, while low temperature and high temperature seemed to activate the biosynthesis of carotenoids in U. prolifera. CONCLUSIONS: In this study, transcriptome sequencing provided critical information for understanding the accumulation of carotenoids and will serve as an important reference for the study of other metabolic pathways in U. prolifera.

Comparative study on mitogenomes of green tide algae.

Since 2007, the annual green tide disaster in the Yellow Sea has brought serious economic losses to China. There is no research on the genetic similarities of four constituent species of green tide algae at the genomic level. We previously determined the mitochondrial genomes of Ulva prolifera, Ulva linza and Ulva flexuosa. In the present work, the mitochondrial genome of another green tide (Ulva compressa) was sequenced and analyzed. With the length of 62,311 bp, it contained 29 encoding genes, 26 tRNAs and 10 open reading frames. By comparing these four mitochondrial genomes, we found that U. compressa was quite different from the other three types of Ulva species. However, there were similarities between U. prolifera and U. linza in the number, distribution and homology of open reading frames, evolutionary and codon variation of tRNA, evolutionary relationship and selection pressure of coding genes. Repetitive sequence analysis of simple sequence repeats, tandem repeat and forward repeats further supposed that they have evolved from the same origin. In addition, we directly analyzed gene homologies and translocation of four green tide algae by Mauve alignment. There were gene order rearrangements among them. With fast-evolving genomes, these four green algal mitochondria have both conservatism and variation, thus opening another window for the understanding of origin and evolution of Ulva.

Genetic evidence in tracking the origin of Ulva prolifera blooms in the Yellow Sea, China.

Recurrent green tides have been recorded in the Yellow Sea for 11 consecutive years. The origin of floating green algae in the Yellow Sea, however, remains a subject of debate. Previous studies suggest that the major bloom-forming green alga Ulva prolifera represent a unique ecotype different from other attached populations of U. prolifera in China. In this study, 97 green algal samples collected during the 2012 green-tide event and from other locations along the coastline of China were analyzed. Based on the sequences of nuclear ribosomal RNA gene (rDNA) internal transcribed spacer (ITS) region and 5S rDNA spacer region, the green alga U. prolifera in the samples were identified. The intraspecific genetic diversity within U. prolifera was then examined using sequences of 5S rDNA spacer and a marker of sequence characterized amplified region (SCAR) highly specific for bloom-forming U. prolifera in the Yellow Sea. The screening results for SCAR marker demonstrated that U. prolifera attached to aquaculture rafts in Subei Shoal belong to the same ecotype of the bloom-forming U. prolifera in the Yellow Sea. These findings offer genetic evidence that aquaculture rafts in Subei Shoal are a major source of floating green algae in the Yellow Sea.

Living apart-together: Microhabitat differentiation of cryptic nematode species in a saltmarsh habitat.

Coexistence of highly similar species is at odds with ecological theory of competition; coexistence, then, requires stabilizing mechanisms such as differences in ecological niche. In the bacterivore nematode Litoditis marina species complex, which occurs associated with macro-algae, four cryptic lineages (Pm I-IV) co-occur in the field along the south-western coast and estuaries of The Netherlands. Here we investigate the temporal and/or spatial niche differentiation in their natural environment using a qPCR-based detection and relative quantification method. We collected different algal species (i.e. two Fucus species and Ulva sp.) and separated algal structures (i.e. receptacula, thalli, non-fertile tips and bladders) at different sampling months and times (i.e. twice per sampling month), to examine differences in microhabitat use between coexisting L. marina species. Results demonstrate that the cryptic species composition varied among different algal species and algal structures, which was also subject to temporal shifts. Pm I dominated on Fucus spp., Pm II showed dominance on Ulva sp., while Pm III overall had the lowest frequencies. Microhabitat partitioning was most pronounced between the two cryptic species which had similar microbiomes (Pm I and Pm II), and less so between the two species which had significantly different microbiomes (Pm I and Pm III), suggesting that species which share the same microhabitats may avoid competition through resource partitioning. The interplay of microhabitat differentiation and temporal dynamics among the cryptic species of L. marina implies that there is a complex interaction between biotic components and abiotic factors which contributes to their coexistence in the field.

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae, Chlorophyta): evidence for ongoing speciation.

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm-temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata-ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS-based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.

Isolation and characterization of Ulva prolifera actin1 gene and function verification of the 5' flanking region as a strong promoter.

Ulva prolifera is a green macroalgae with an extremely high growth rate that can accumulate biomass with considerable protein content. To set up an available seaweed expression system, a prior step is to isolate endogenous and strong constitutive promoters. For this reason, the full-length genomic actin1 gene from U. prolifera (Upactin1) was cloned and its 5' flanking sequence was obtained by genome walking. The Upactin1 open reading frame consisted of 1134 nucleotides encoding 377 amino acid residues. Besides 4 exons and 3 introns in the coding region, an extra leader intron was identified in the 5' untranslated region. According to quantitative GUS assays based on transient expression, the promoter activity of the Upactin1 5' flanking region was found to be several times higher than that of the widely-used cauliflower mosaic virus 35S (CaMV35S) in all tested species of Ulva. In addition, precise deletion of the leader intron led to a significant decrease of promoter strength in U. prolifera, and almost entire loss of strength in U. linza and U. pertusa. To our knowledge, this is the first report to prove function of a leader intron in algae. The 5' flanking region of Upactin1 was shown to be a much stronger promoter than the foreign CaMV35S, and its activity was highly dependent on the presence of the leader intron. We propose that the Upactin1 promoter could serve as an endogenous and strong constitutive element for genetic engineering of U. prolifera.

New Ulvaceae (Ulvophyceae, Chlorophyta) from mesophotic ecosystems across the Hawaiian Archipelago.

Ulvalean algae (Chlorophyta) are most commonly described from intertidal and shallow subtidal marine environments worldwide, but are less well known from mesophotic environments. Their morphological simplicity and phenotypic plasticity make accurate species determinations difficult, even at the generic level. Here, we describe the mesophotic Ulvales species composition from 13 locations across 2,300 km of the Hawaiian Archipelago. Twenty-eight representative Ulvales specimens from 64 to 125 m depths were collected using technical diving, submersibles, and remotely operated vehicles. Morphological and molecular characters suggest that mesophotic Ulvales in Hawaiian waters form unique communities comprising four species within the genera Ulva and Umbraulva, each with discrete geographic and/or depth-related distributional patterns. Three genetically distinct taxa are supported by both plastid (rbcL and tufA) and nuclear (ITS1) markers, and are presented here as new species: Umbraulva kaloakulau, Ulva ohiohilulu, and Ulva iliohaha. We also propose a new Umbraulva species (Umbraulva kuaweuweu), which is closely related to subtidal records from New Zealand and Australia, but not formally described. To our knowledge, these are the first marine species descriptions from Hawai'i resulting from the collaboration of traditional Hawaiian nomenclature specialists, cultural practitioners and scientists. The difficulty of finding reliable diagnostic morphological characters for these species reflects a common problem worldwide of achieving accurate identification of ulvalean taxa using solely morphological criteria. Mesophotic Ulvales appear to be distinct from shallow-water populations in Hawai'i, but their degree of similarity to mesophotic floras in other locations in the Pacific remains unknown.

The mitochondrial genome of the bloom-forming green alga Ulva prolifera.

From 2007 to 2015, massive green tides formed by Ulva prolifera O.F. Müller have broken out for nine consecutive years in the Yellow Sea of China. Here, we determined the complete mitochondrial genome sequence of U. prolifera. The circular U. prolifera mitogenome is 63 845 bp in size, with the overall A + T content of 66.04% which is similar to the other three ulvophyceans (60.66%-67.83%). The mitogenome encodes 74 genes and the coding sequences account for 77.3% of the genome. Nine introns are identified in five genes and each of these introns contains an ORF. Two long tandem repeats identified have a period of 11 bp with a copy number of 62.5 and 19.5, respectively. The genome organization of U. prolifera is similar to that in Ulva sp., and its reduced spacer region makes it more compact than the latter. The present data will provide important information on phylogenetics and molecular evolution of Ulva species.

Changes to the biomass and species composition of Ulva sp. on Porphyra aquaculture rafts, along the coastal radial sandbank of the Southern Yellow Sea.

Compositions, changes and biomass of attached Ulva species on Porphyra rafts along the radial sandbank in the Yellow Sea were investigated, and potential contributions to green tides was analyzed. Ulva prolifera, Ulva flexuosa and Ulva linza were all appeared throughout the investigated period. U. prolifera and U. flexuosa dominated attached Ulva population on Porphyra rafts. Attached Ulva species biomass showed obviously spatial and temporal variations. Temperature, Ulva microscopic propagules and human activities were main factors to influence attached Ulva species biomass. The total attached Ulva species biomass was more than 20,000 fresh weight tons in April, and the green tide causative species U. prolifera accounted 51.03% in April 2013 before green tides occurred. The high biomass of attached Ulva species would contribute most to green tides in the Yellow Sea. But how attached Ulva species on Porphyra rafts contributing to green tides in the Yellow Sea should be further studied.

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa.

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/ß1-6Galß1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galß1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.

Understanding the recurrent large-scale green tide in the Yellow Sea: temporal and spatial correlations between multiple geographical, aquacultural and biological factors.

The coast of Jiangsu Province in China - where Ulva prolifera has always been firstly spotted before developing into green tides - is uniquely characterized by a huge intertidal radial mudflat. Results showed that: (1) propagules of U. prolifera have been consistently present in seawater and sediments of this mudflat and varied with locations and seasons; (2) over 50,000 tons of fermented chicken manure have been applied annually from March to May in coastal animal aquaculture ponds and thereafter the waste water has been discharged into the radial mudflat intensifying eutrophication; and (3) free-floating U. prolifera could be stranded in any floating infrastructures in coastal waters including large scale Porphyra farming rafts. For a truly integrated management of the coastal zone, reduction in nutrient inputs, and control of the effluents of the coastal pond systems, are needed to control eutrophication and prevent green tides in the future.

Genetic and marine cyclonic eddy analyses on the largest macroalgal bloom in the world.

In 2008, a massive Ulva prolifera bloom, with a 3-million-ton biomass covering an area of 1.29 × 10(4) km(2) at its largest, suddenly appeared from May to July in South Yellow Sea. The mechanism behind the rapid growth of these seaweeds was investigated. Molecular phylogenetic analysis of free-floating algal samples from the Yellow Sea suggested that U. prolifera belong to one population, and that temporary cyclonic eddies in the Yellow Sea in late spring and early summer may help promote the proliferation of this bloom by providing seaweeds with sufficient growth time, abundant nutrition, and favorable habitats. The initial investigation on the relationship between marine cyclonic eddies and the route of free-floating algae extends our knowledge on how the emergence of free-floating macroalgal blooms in coastal areas could yield a large biomass.

Culture and hybridization experiments on an ulva clade including the Qingdao strain blooming in the yellow sea.

In the summer of 2008, immediately prior to the Beijing Olympics, a massive green tide of the genus Ulva covered the Qingdao coast of the Yellow Sea in China. Based on molecular analyses using the nuclear encoded rDNA internal transcribed spacer (ITS) region, the Qingdao strains dominating the green tide were reported to be included in a single phylogenetic clade, currently regarded as a single species. On the other hand, our detailed phylogenetic analyses of the clade, using a higher resolution DNA marker, suggested that two genetically separate entities could be included within the clade. However, speciation within the Ulva clade has not yet been examined. We examined the occurrence of an intricate speciation within the clade, including the Qingdao strains, via combined studies of culture, hybridization and phylogenetic analysis. The two entities separated by our phylogenetic analyses of the clade were simply distinguished as U. linza and U. prolifera morphologically by the absence or presence of branches in cultured thalli. The inclusion of sexual strains and several asexual strains were found in each taxon. Hybridizations among the sexual strains also supported the separation by a partial gamete incompatibility. The sexually reproducing Qingdao strains crossed with U. prolifera without any reproductive boundary, but a complete reproductive isolation to U. linza occurred by gamete incompatibility. The results demonstrate that the U. prolifera group includes two types of sexual strains distinguishable by crossing affinity to U. linza. Species identification within the Ulva clade requires high resolution DNA markers and/or hybridization experiments and is not possible by reliance on the ITS markers alone.

Recurrence of the world's largest green-tide in 2009 in Yellow Sea, China: Porphyra yezoensis aquaculture rafts confirmed as nursery for macroalgal blooms.

In the summer of 2008, the world's largest green-tide occurred in the Yellow Sea, China. The hypothesized cause was the expansion of Porphyra yezoensis aquaculture along the Jiangsu coastline and the re-occurrence of a green-tide in 2009 was predicted. In this study, satellite and field images showed the formation of the June 2009 green-tide which again originated from the Jiangsu coast. The responsible species, its source and biomass accumulation were studied to support the previous hypothesis. Morphological and phylogenetic analysis demonstrated the homology of Ulva prolifera in the 2008 green-tide with the U. prolifera from P. yezoensis aquaculture rafts. About 91-505kg/ha of U. prolifera was attached to the P. yezoensis aquaculture rafts and a total biomass of 4956 tonnes was estimated during the harvesting of P. yezoensis. This is sufficient to seed a bloom when they are dislodged from the rafts as a result of harvesting practices.

Tracking the algal origin of the Ulva bloom in the Yellow Sea by a combination of molecular, morphological and physiological analyses.

In 2008, Qingdao (36 degrees 06'N, 120 degrees 25'E, PR China) experienced the world largest drifting macroalgal bloom composed of the filamentous macroalga Ulva prolifera. No convincing biologic evidence regarding the algal source is available so far. A series of field collections of both Ulva sp. and waters in various sites along Jiangsu coasts were conducted in March to May of 2009. Density of microscopic Ulva germlings in the waters sampled from different sites ranged from 7 to 3140 individuals L(-1), indicating the wide-spreading and long-term existence of the algae in the investigated region. Morphological and the nuclear ribosomal internal transcribed spacer ITS nrDNA and the chloroplast-encoded rbcL gene comparisons of 26 algal samples revealed that the algae collected from land-based animal aquaculture ponds mostly resembled the dominating blooming alga in 2008. Mismatch of Porphyra farming period with the occurrence of the green tide bloom, as well as the negative identification results of the sampled green algae from the Porphyra rafts eliminated Porphyra rafts as the principal and original source of the dominating blooming alga.