The first assessment of cyanobacterial and diazotrophic diversities in the Japan Sea

The diversity of cyanobacteria and diazotrophs in the Japan Sea was investigated by analyzing sequences of cyanobacterial 16S rRNA genes and nitrogen fixation genes (nifH) from seawater sampled at depths ranging from the surface to 100?m at two stations. Of the 107 cyanobacterial 16S rRNA gene sequences obtained, 97 and three sequences were assigned to Synechococcus sub-cluster 5.1 and Prochlorococcus HL (II), respectively. Unlike other oceanic regions, at our two sampling stations the composition ratio of the sequences assignable to Synechococcus sub-cluster 5.3 was relatively high (8?%). No sequences of diazotrophic cyanobacteria were found in the cyanobacterial 16S rRNA genes. In the nifH clone library (36 sequences), ten sequences were identified as a UCYN-A group of diazotrophic cyanobacteria; the other 26 sequences (72?%) were assigned to proteobacteria. These results suggest that heterotrophic bacteria, including UCYN-A, dominate the diazotrophic community in the Japan Sea. Our study reveals the dominance of Synechococcus in cyanobacterial community and (photo)heterotrophic diazotrophs in the diazotrophic community in the Japan Sea, suggesting its unique characteristics.     

Controlling the growth of a cyanobacterial contaminant, Synechoccus sp., in a culture of Tetraselmis chui (PLY429) by varying pH: Implications for outdoor aquaculture production

In aquaculture, large volumes of phytoplankton are often grown outdoors to reduce costs. However, growing microalgae in an environment not as well-controlled as in the laboratory can lead to unwanted phytoplankton, including cyanobacteria, contaminating a culture. A cyanobacterial contaminant was isolated from an outdoor culture of Tetraselmis chui (PLY429) at the Milford Laboratory. This study investigated the growth of PLY429 and the cyanobacterium in pure cultures and a mixed culture in a pH range of 6.5-9.5. The division of PLY429 was greater at a pH range of 7.0-8.0; whereas, for the cyanobacterium, higher growth was obtained at pH 8.0-9.0. Results from combined cultures of PLY429 and the cyanobacterium grown at various pHs indicated that maintaining pH near 7.1 yields higher growth of PLY429 than those of the cyanobacterium. These findings suggest that controlling pH may reduce the population of a cyanobacterial contaminant in an aquaculture feed culture.     

Effect of temperature and phytoplankton concentration on Nile tilapia Oreochromis niloticus (L.) filtration rate

Nile tilapia Oreochromis niloticus (L.) held in timed‐pulse feeding chambers, were provided with algal‐rich water dominated by either green algae (Scenedesmus, Ankistrodesmus, Chlorella and Tetraedron) or cyanobacteria (Microcystis) to determine the effect of temperature and phytoplankton concentration on filtration rates. Green algae and cyanobacteria filtration rates were measured as suspended particulate organic carbon (POC) kg^?1 wet fish weight h^?1. Ivlev's filter‐feeding model described the relationships between filtration rates and suspended POC concentration of green algae and cyanobacteria. Filtration rates of both green algae and cyanobacteria increased linearly as water temperature increased from 17 °C to 32 °C and were significantly higher in the warm‐water regime (26–32 °C) than in the cool‐water regime (17–23 °C). Filtration rates at 95% saturation POC (FR₉₅) in green algal and cyanobacterial waters were 700 mg C kg^?1 h^?1 and 851 mg C kg^?1 h^?1 in the warm‐water regime and 369 mg C kg^?1 h^?1 and 439 mg C kg^?1 h^?1 in the cool‐water regime respectively. The FR₉₅ in warm water were achieved at lower POC concentrations than in cool water.     

Ecology of Blue-Green Algae in Aquaculture Ponds

Cyanobacteria (blue-green algae) in the genera Anabaena, Aphanizomenon, Microcystis, and Oscillatoria often form extensive and persistent blooms in freshwater aquaculture ponds. Bloom-forming cyanobacteria are undesirable in aquaculture ponds because: 1) they are a relatively poor base for aquatic food chains; 2) they are poor oxygenators of the water and have undesirable growth habits; 3) some species produce odorous metabolites that impart undesirable flavors to the cultured animal; and 4) some species may produce compounds that are toxic to aquatic animals. Development of cyanobacterial blooms is favored under conditions of high nutrient loading rates (particularly if the availability of nitrogen is limited relative to phosphorus), low rates of vertical mixing, and warm water temperatures. Under those conditions, dominance of phytoplankton communities by cyanobacteria is the result of certain unique physiological attributes (in particular, N₂ fixation and buoyancy regulation) that allow cyanobacteria to compete effectively with other phytoplankton. The ability to fix N₂ provides a competitive advantage under severe nitrogen limitation because it allows certain cyanobacterial species to make use of a source of nitrogen unavailable to other phytoplankton. The ability to regulate cell buoyancy through environmentally-controlled collapse ad reformation of intracellular gas vacuoles is perhaps the primary reason for the frequent dominance of aquaculture pond phytoplankton communities by cyanobacteria. Cyanobacteria that can regulate their position in the water column gain a distinct advantage over other phototrophs in poorly mixed bodies of water. In addition to the physicochemical interactions that influence phytoplankton community dynamics, cyanobacterial-microbial associations may play an important regulatory role in determining community structure. Cyanobacteria are always found in close association with a diverse array of microorganisms, including eubacteria, fungi, and protozoans. These associations, which in the past have often been viewed as antagonistic, are increasingly seen as mutualistic and may function in a positive manner during bloom development.     

Comparative Nile tilapia and silver carp filtration rates of Partitioned Aquaculture System phytoplankton

Nile tilapia (Oreochromis niloticus) and silver carp (Hypophthalmichthys molitrix) held in timed-pulse feeding chambers were provided water dominated either by green algae (Scenedesmus, Ankistrodesmus and Tetraedron) or by cyanobacteria (Microcystis) to compare filtration rates (FRs). FRs were expressed as suspended particulate organic carbon (POC)/kg wet fish weight/h and as phytoplankton units filtered based on counts. Nile tilapia and silver carp filtration significantly reduced phytoplankton number of both taxonomic groups with the larger phytoplankton being filtered proportionally more than the smaller phytoplankton. Nile tilapia FR of green algae was significantly higher than silver carp; however, silver carp FR of cyanobacteria was higher than Nile tilapia. Ivlev's 90% saturation FRs (FR₉₀) in green-algal and cyanobacterial water sources were 702 and 812 mgC/kg/h for Nile tilapia and 414 and 1028 mgC/kg/h for silver carp, respectively. Silver carp were observed to reach these FR₉₀ values at lower POC concentrations than Nile tilapia with both green algae and cyanobacteria.     

Low-density silver carp Hypophthalmichthys molitrix (Valenciennes) polyculture does not prevent cyanobacterial off-flavours in channel catfish Ictalurus punctatus (Rafinesque)

Silver carp Hypophthalmichthys molitrix (Valenciennes) were co‐cultured with channel catfish Ictalurus punctatus (Rafinesque) in 0.4 ha earthen ponds to determine the impacts of carp grazing on pond phytoplankton communities and cyanobacterial off‐flavours in catfish. Carp were stocked at densities of 0, 75, or 250 fish ha^?1 in seven replicate ponds per treatment. The mean chlorophyll a concentrations (a measure of phytoplankton standing crop) steadily increased in all treatments from about 100 μg L^?1 in April to more than 400 μg L^?1 by mid‐October. Silver carp had no affect (P>0.1) on chlorophyll a concentrations across all sampling dates (April though October) or for sampling dates late in the growing season (August–October) when the prevalence of cyanobacterial off‐flavours among catfish populations is usually greatest. Silver carp did not eliminate odour‐producing cyanobacteria from pond phytoplankton communities: on sampling dates in September and October, three to six ponds in all treatments contained populations of the odour‐producing cyanobacteria Oscillatoria perornata, Anabaena spp., or both. Failure of silver carp to eliminate odour‐producing cyanobacteria resulted in a relatively high incidence in all treatments of ponds with off‐flavoured catfish. On sampling dates in September and October, catfish in three to five ponds in each treatment were tainted with either musty (2‐methylisoborneol) or earthy (geosmin) off‐flavours. The presence of silver carp had no obvious effect on off‐flavour intensity: on each sampling date, at least three ponds in each treatment contained catfish described as distinctly to extremely off‐flavored. Apparently, hypertrophic conditions in catfish ponds overwhelm the effect of silver carp grazing at the low carp densities used in this study.     

Accumulation and biochemical effects of microcystin-LR on the Patagonian pejerrey (Odontesthes hatcheri) fed with the toxic cyanobacteria Microcystis aeruginosa

We studied accumulation and biochemical effects of microcystin-LR (MCLR) in Odontesthes hatcheri after dietary administration of the cyanobacteria Microcystis aeruginosa (1.3 μg MCLR/g body mass, incorporated in standard fish food). After 12 h, MCLR content in liver did not differ between fish fed with crushed or intact cells, demonstrating O. hatcheri’s capacity to digest cyanobacteria and absorb MCLR. In the second experiment, fish received toxic cells, non-toxic cells, or control food; MCLR accumulation was monitored for 48 h. Protein phosphatase 1 (PP1), catalase (CAT), glutathione-S-transferase (GST) activities, and lipid peroxidation (as MDA) were measured in liver and intestine. Methanol-extractable MCLR was determined by PP1 inhibition assay (PPIA); extractable and protein-bound MCLR were measured by Lemieux oxidation-gas chromatography/mass spectrometry (GC/MS). MCLR accumulated rapidly up to 22.9 and 9.4 μg MCLR/g in intestine and liver, respectively, followed by a decreasing tendency. Protein-bound MCLR represented 66 to ca. 100 % of total MCLR in both tissues. PP1 activity remained unchanged in intestine but was increased in liver of MCLR treated fish.CAT and GST activities and MDA content were significantly increased by MCLR only in liver. We conclude that O. hatcheri is able to digest cyanobacteria, accumulating MCLR mostly bound to proteins. Our data suggest that this freshwater fish can be adversely affected by cyanobacterial blooms. However, the rapid decrease of the detectable MCLR in both tissues could imply that sublethal toxin accumulation is rapidly reversed.     

Changes in the nutritional parameters of muscles of the common carp (Cyprinus carpio) and the silver carp (Hypophthalmichthys molitrix) following environmental exposure to cyanobacterial water bloom

The present study evaluated the effect of naturally developing cyanobacteria on the composition of muscles of two commercially important freshwater fish species. Fish were exposed to cyanobacterial biomass including Microcystis aeruginosa and Microcystis ichthyoblabe for 4 weeks. Then, they were transferred to dechlorinated potable water without any cyanobacteria for another 4-week period, thus modelling their preparation for consumers. Samples of muscles were collected every week during exposure and subsequent stay in dechlorinated potable water. The cyanobacterial water bloom of 3.9–6 × 10⁵ cells mL⁻¹ (133–383 μg g⁻¹ of total MC DW) induced statistically significant effects only in the content of fatty acids ( P <0.05; P <0.01) in the common carp ( Cyprinus carpio ), while all studied parameters including the content of dry matter and fat ( P <0.01), proteins ( P <0.05), fatty acid composition ( P <0.05; P <0.01) and some amino acids ( P <0.05) were affected in the silver carp ( Hypophthalmichthys molitrix ). This study has shown that cyanobacteria in the environment of commercially produced fish may decrease the dietetic value of fish muscles.     

Filtration of Green Algae and Cyanobacteria by Freshwater Mussels in the Partitioned Aquaculture System

Abstract.— The freshwater mussel Elliptio complanata was provided water containing green algae and cyanobacteria delivered from the Partitioned Aquaculture System (PAS) at eight flow rates to determine algal filtration rates as mg of particulate organic carbon (POC)/kg wet tissue weight per h. The dominant taxon in cyanobacterial waters was Microcystis while the dominant taxa in green algal waters were Scenedesmus and Ankistrodesmus . The cell counts of Scenedesmus and Ankistrodesmus were the only algal taxa that were significantly different between the incoming water and water filtered by mussels. Filtration rates of POC obtained from green algal water were significantly greater than from cyanobacteria-dominated waters at all flow rates. A significant increase in mean filtration rate was observed as flow rates increased. The filtration rate of green algae increased as POC concentration increased, peaking at 28 mg C/L. A maximum filtration rate was not observed with cyanobacterial waters.     

Effect of inoculation of the cyanobacteria Oscillatoria sp. on tilapia biofloc culture

Cyanophytes are the most ancient photosynthetic organisms. During its evolution, they have developed various ecophysiological adaptation strategies to survive in extreme conditions. The environment prevailing under biofloc cultures provides various conditions appropriate for cyanobacterial proliferation. An outdoor experiment (7 weeks) was performed with a simple random design consisting of four inoculation levels (by triplicate) of Oscillatoria sp. (0.0, 0.1, 0.5 and 1 mg L^?1) in saltwater biofloc. The objective was to evaluate the effect of the cyanobacteria inoculation on water quality and tilapia production parameters. The results indicated that the amount of Oscillatoria sp. inoculated significantly affected water quality (pH, chlorophyll a, TSS and NO₃‐N) and tilapia parameters (final weight, feed conversion ratio, specific growth rate and survival). No significant effects on dissolved oxygen, total ammonia nitrogen (TAN) or NO₂‐N were observed. We recommend identifying the cyanobacteria species that are able to grow in a biofloc system and their possible adverse effects on the system.     

Development of phytoplankton communities and common off-flavors in a biofloc technology system used for the culture of channel catfish (Ictalurus punctatus)

The use of biofloc technology production systems continues to increase in the aquaculture industry worldwide. Recent research demonstrated that outdoor biofloc systems can be used to produce high yields of channel catfish (Ictalurus punctatus). However, studies have not yet been performed to determine the development and composition of phytoplankton communities and related off-flavor problems in these biofloc production systems. In this study, water samples were collected biweekly from May to November and channel catfish samples were collected during harvest in November from nine 18.6 m² biofloc culture tanks. Water and fillet samples were analyzed for levels of the common off-flavor compounds geosmin and 2-methylisoborneol (MIB). The development and composition of phytoplankton communities in each culture tank was also monitored. In addition, water and biofloc samples were evaluated to assess the microbial sources of geosmin and MIB within the culture tanks. Phytoplankton (including algae and cyanobacteria attached to bioflocs) biomass, as determined by concentrations of chlorophyll a in the water, gradually increased in all tanks over time. Phytoplankton communities that developed in the culture tanks were dominated by fast-growing, unicellular and small colonial types of green algae (chlorophytes) and diatoms (bacillariophytes) and slower growing, small colonial types of cyanobacteria (cyanophytes). A positive correlation (p < 0.05) between cumulative feed addition and chlorophyll a concentration was found. Although geosmin and MIB were present in the culture water of each tank during most of the study, levels were typically low and only one tank yielded catfish with geosmin and MIB in their flesh at levels high enough to be designated as off-flavor. A positive correlation (p < 0.05) between cumulative feed addition and MIB concentrations in the water of culture tanks indicates a greater potential for MIB-related off-flavor problems at high feed application rates. The microbial sources responsible for production of geosmin and MIB in the culture tanks remain unknown.     

An integrated recirculating aquaculture system (RAS) for land-based fish farming: The effects on water quality and fish production

To mitigate the serious water pollution caused by the rapid expansion of the aquaculture industry in recent years, the development of improved aquaculture systems with more efficient water usage and less environmental impact has become essential. In this study, a land-based recirculating aquaculture system (RAS) was established that consisted of purification units (i.e., a primary biological pond, two parallel horizontal subsurface flow constructed wetlands [CWs], and a long ecological ditch) and 4-5 series-connected recirculating ponds. This system was mainly designed to stock channel catfish (Ictalurus punctatus), fifteen spine stickleback (Spinibarbus sinensis) and yellow catfish (Pelteobagrus fulvidraco), and the culture efficacy was evaluated based on a 2-year field experiment covering two growing seasons. According to the results, the primary biological pond played a role in sedimentation or nutrient retention, although this was not as evident when the CWs were functioning. The water flowing through the wetland system at a hydraulic loading rate (HLR) of 600 mm/day displayed lower values for the temperature, pH, dissolved oxygen (DO), suspended solids, organic matter and nutrients, whereas the electrical conductivity (EC) was higher, suggesting the accumulation of dissolved solids in the system. Due to the recirculation treatment, the trophic status of the recirculating ponds increased gradually along the direction of the flow and was notably lower in comparison to the control. As a result, the fish production responded to the variation of the water quality, which was reflected in the measurements of culture efficacy (final weight, survival rate, SGR and yield). The three main rearing species showed a decreasing trend along the direction of the flow, which was higher compared to the control, whereas an opposite trend was observed for filter-feeding fish. A Pearson correlation analysis revealed that the main culture species were inclined to live in meso- or oligotrophic conditions, and the silver carp adapted to more eutrophic conditions. Because RAS can provide better environmental conditions year-round, the present culture method could be more suitable for species that are sensitive to water quality in typical subtropical areas.     

Isolation of the non-fastidious microalga with astaxanthin-accumulating property and its potential for application to aquaculture

Astaxanthin has recently attracted considerable attention for its biological properties such as the antioxidant activity as well as a coloring agent used for farmed fish. However, its biological production, mainly by a green microalgae Haematococcus pluvialis, is costly because of its fastidious growth characters. Therefore, for future applications of biological astaxanthin production in aquaculture, non-fastidious microalgal strains were isolated from environmental samples, examined for their astaxanthin-accumulating activity, and characterized phylogenetically. While β-carotene was found in all tested isolates, one isolate, GK12, from activated sludge of a sewage treatment plant accumulated 2.5 ± 0.36 mg/g dry cell of free astaxanthin de novo under photoautotrophic culture condition, which was comparable to photoautotrophic cultures of other known astaxanthin-accumulating microorganisms. Mixotrophic culture conditions increased GK12 biomass, but astaxanthin content was decreased, suggesting that the simple photoautotrophic cultivation is more efficient way for GK12 to produce astaxanthin than mixotrophic cultivation. A phylogenetic study of SSU rDNA strongly suggested that GK12 is a novel species in the genus Monoraphidium, Chlorophyta. Besides making efforts to increase astaxanthin-productivity of known astaxanthin accumulators, it is also beneficial to study GK12 from the view point of applications to aquaculture.     

Large-scale microalgae production for nursery rearing of marine bivalves

The feasibility of large-scale bloom induction of nutritionally suited natural phytoplankton species, to feed a semi-industrial nursery of edible shellfish, built on the Belgian coast, was tested.The outdoor microalgal production unit consisted of four tanks of 100 m² surface each (two of 1 m depth and two of 0·5 m depth), equipped with different mixing devices.The cultures were run as chemostats in which seawater was enriched with commercial inorganic N, P, and Si fertilizers. Depending on the season, between 5–10 and 80% of the culture volume could be harvested daily, with algal densities ranging from 50 000 to 500 000 cells per ml.By manipulation of operational parameters such as detention time, nutrient levels and nutrient ratios (N:Si:P), unsuited or less suited species of algae (e.g. Chlorella and Phaeodactylum) could be replaced by more desirable species (e.g. Skeletonema, Nitzschia, Chaetoceros).Various biological and technological problems encountered during year round operation, including collapsing of the culture, seawater enrichment, water quality, fouling and water treatment, are commented.     

水射流空化强化光催化氧化技术在养鱼污水处理中的应用初探

研究了水射流空化强化TiO2光催化氧化技术处理养鱼污水的效果,同时考察了单独的水射流空化处理、光解处理、光催化氧化处理的效果。试验结果表明:水射流空化强化TiO2光催化技术处理养鱼污水0.5 h后,杀菌率达97%;处理4 h后,氨氮和COD的去除率分别达25%和29%。在降解氨氮和COD方面,水射流空化技术和TiO2光催化氧化技术结合表现出明显的协同效应。水射流空化强化光催化氧化是一种绿色无污染的工艺,而且高速射流产生的冲击波对水体可以起到增氧的作用,有利于养殖鱼类的生长。     

茜坑水库浮游植物时空分布及水质风险评价

浮游植物是判断水体富营养化程度的关键指标之一,藻类水华中有害蓝藻释放的蓝藻毒素会危害人类健康,对饮用水的安全构成威胁。2010年4月至2011年3月,按月对广东省深圳市茜坑水库的浮游植物群落进行了调查。结果表明,茜坑水库共采集到浮游植物7门、66属、127种,种类组成以绿藻(54%)和硅藻(25%)为主,水库属绿藻-硅藻型水体;浮游植物细胞密度年均值为1.10×107个/L,绿藻门、硅藻门、隐藻门和蓝藻门细胞密度所占比例较高;浮游植物多样性在茜坑水库各样点间不存在显著性差异(P>0.05),表明水体交换能力较强;结合3种水质评价方法,初步判断茜坑水库为轻污-中污型水体,并通过对潜在产毒蓝藻现存量的分析来评价水质安全存在的风险。7月的水质风险最大,应进行藻毒素测定。对理化因子的测定显示,水库总氮和总磷浓度均超过《GB3838-2002地表水环境质量标准》中Ⅱ类标准值。     

Multiplex species-specific PCR identification of native and non-native oysters (Crassostrea) in Brazil: a useful tool for application in oyster culture and stock management

In an effort to develop molecular tools for oyster identification, this study reveals the usefulness of a multiplex polymerase chain reaction (PCR) for the reliable, rapid and low-cost identification of the four oyster species found along the Brazilian coast: Crassostrea gasar, Crassostrea rhizophorae, Crassostrea gigas and Crassostrea sp. Canela originally found at Pará, Brazil. In order to perform a simultaneous identification of these species, we used a set of five primers developed and adapted from the cytochrome oxidase subunit I (COI) and internal transcribed spacer 1 (ITS1) fragments, respectively. Amplification was successful in all four species and PCR products were visualized in agarose gel. A single reaction was capable of distinguishing between the species: C. gigas, with two fragments (236 bp for COI and 718 bp for ITS1); C. gasar, with one fragment (718 bp for ITS1); Crassostrea sp., with one fragment (621 bp for ITS1) and C. rhizophorae with two fragments (377 bp for COI and 718 bp for ITS1). This molecular approach provides a simple and rapid identification of the oyster species from the Brazilian coast, thus increasing the efficient and quality of oyster culture programs by reducing the risk of wrong species identification.     

Identification and characterization of a green-type mutant of Porphyra tenera Kjellman var. tamatsuensis Miura (Bangiales, Rhodophyta)

We identified two green-colored conchocelis strains of cultivated Porphyra, ‘Oba-green’ and HGT-6 (F₁ strain of ‘Oba-green’), based on PCR-RFLP and sequence analyses of the nuclear ITS region and the plastid RuBisCO spacer region. These molecular analyses confirmed that the two strains were green-type mutants of the endangered species Porphyra tenera, although their blade color was not green. Under the same culture conditions, blade length increase was significantly higher in HGT-6 than in a wild strain HGT-1 of P. tenera, and the blade shape of HGT-6 was extremely elongate compared with that of HGT-1. From the growth characteristics and ITS-1 sequence data, HGT-6 was confirmed to be a green-type mutant of P. tenera var. tamatsuensis, a vigorously growing cultivar. Although photosynthetic pigment contents were lower in HGT-6 than in HGT-1, total content of four major free amino acids was higher and the blade thickness was almost the same in HGT-6 and HGT-1. These results suggest that the green-type mutant HGT-6 has potential as breeding material for further development of Porphyra breeding.     

Effect of dietary cyanobacteria on growth and accumulation of microcystins in Nile tilapia (Oreochromis niloticus)

A 12-week growth trial was conducted in a flow-through system to investigate the chronic toxic effect of dietary intake of cyanobacteria on growth, feed utilization and microcystins accumulation in Nile tilapia (Oreochromis niloticus L.) (initial body weight: 5.6 g). Six isonitrogenous and isocaloric diets were formulated to include different contents of cyanobacteria with the dietary microcystins increasing from 0 to 5460.06 ng/g diet. The results showed that dietary intake of cyanobacteria could increase the growth of tilapia while there are no impacts on feed conversion efficiency or mortality. Feeding rate was higher for the diets containing highest cyanobacteria. Microcystins were mostly accumulated in fish liver. The relationship between microcystins contents in muscle, liver, spleen and dietary intake could be described by quadratic equations.Microcystins content in the muscle of Nile tilapia in present study exceeded the upper limit of the tolerable daily intake (TDI) of microcystins suggested by the WHO (0.04 μg/kg body weight/d). It is suggested that Nile tilapia fed on toxic cyanobacteria is not suitable for human food.     

Species identification of imported and Japanese commercial green algal products based on phylogenetic analyses using the nrITS2 and 5S rDNA spacer regions

The identification of imported green algal products represents a critical challenge to the Japanese Customs Service in terms of ensuring effective control of imported quota items (species of genera Monostroma and Enteromorpha and their products). To assist officials in this difficult task, we tested the applicability of phylogenetic analyses using the nuclear-encoded ribosomal DNA internal transcribed spacer 2 (nrITS2) and 5S ribosomal DNA (rDNA) spacer regions to identify green algal products at the species level. Our nrITS2-based phylogenetic analysis was able to roughly separate the Ulva-related samples into three clades: the Ulva linza–procera–prolifera (LPP) complex and two other clades, and identified the Monostroma-related samples as M. nitidum. Subsequent detailed analysis within the LPP complex using the 5S rDNA spacer region divided the samples into two groupings (the U. linza clade and U. prolifera clade), while it detected possible hybrid samples of U. linza and U. prolifera from a number of commercial green algal products.