The effects of major environmental factors and nutrient limitation on growth and encystment of planktonic dinoflagellate Akashiwo sanguinea

The bloom-forming dinoflagellate Akashiwo sanguinea is commonly observed in estuarine and coastal waters around the world. Annually recurrent blooms of this species have been observed in the coastal waters of China, particularly in the Sishili Bay, Yantai since 2004. However, limited studies have been conducted on the recurrence mechanism of A. sanguinea other than periodical monitoring of its population dynamics and associated environmental variables. Thus, to further explore the bloom and succession mechanisms of A. sanguinea in the field, we studied the effects of major nutritional components on the growth and encystment of A. sanguinea, as well as the effects of key environmental factors on the growth of A. sanguinea through a series of laboratory trials. Our results indicated that A. sanguinea was able to grow well within the temperature range of 20–25 °C, salinity range of 20 - 30, with the maximum laboratory irradiance of 78.14 μE m⁻² s⁻¹, and was able to survive and grow in low nutrient. However, lower concentrations of nutrients (e.g., nitrate, phosphate) and higher ammonium exerted different degrees of limiting effects on the growth of A. sanguinea, and induced 2.3–21.24% of vegetative cells to form resting cysts simultaneously in laboratory cultures. On the other hand, very limited or no cyst formation was observed in nutrient-replete or extremely low nutrient cultures, indicating the threshold effect of nutritional stress on the encystment of A. sanguinea. The physiological strategy of encystment of A. sanguinea in nutrient-limiting environment facilitates the survival and succession of A. sanguinea species in fluctuating seawaters, and provides seed sources for reoccurring algal blooms under favorable environmental conditions.     

Efficiency of nitrogen and phosphorus removal by six macrophytes from eutrophic water

Abstract

Increased nitrogen and phosphorus pollution causes eutrophication in water bodies. Using aquatic plants to remove nutrients from water is an attractive phytoremediation. It is a cost-effective, environment-friendly, and efficient way that reduces water body eutrophication by the plant. It is important to choose suitable macrophytes to remove excess N and P under different nutrient conditions. In this study, six macrophyte species (Polygonum orientale, Juncus effuses, Iris pseudocorus, Phragmites australis, Iris sanguinea, Typha orientalis) were tested. Simulation experiment was conducted under five N and P levels. The removal rate, relative growth rate, and the dynamic nutrition concentration of cultivated solution were investigated. Of all the treatment, a 23–95% reduction in N removal and a 29–92% reduction in P removal were recorded. The results showed I. sanguinea is a promising species to treat various eutrophic waters and the other five species can be used specifically to treat certain types of water. The data provided a theoretical guidance to plant species selection for phytoremediation of polluted water bodies for the purpose of water quality improvement around the different reservoir in northern China.     

Diffuse competition for heterogeneous substrate in soil among six species of wood-decomposing basidiomycetes

Competition among six wood decay fungi was studied using 15×15 mm wood blocks placed in 250×250 mm plastic trays filled with unsterilized sand or clay. The wood blocks were preinoculated with Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm., Phanerochaete sanguinea (Fr.) Hjortstam, Coniophora sp. DC. ex Me"rat, Armillaria borealis Marxmuller and Korhonen and Hypholoma capnoides (Fr.) Kummer before they were combined in all possible combinations in the trays. Two methods were used, one with all wood blocks inoculated, and one with sterilized non-inoculated wood blocks distributed between the inoculated ones. Wood blocks preinoculated with the six species were also used in a pairwise competition test. Following incubation for 9 months in darkness at 21°C, mycelia were reisolated and identified. R. bicolor was most successful at invading through the soil and replacing other species in the wood blocks. P. sanguinea, Coniophora sp. and H. capnoides also had some success.     

Season and light affect constitutive defenses of understory shrub species against folivorous insects

Understory shrubs contribute to overall species diversity, providing habitat and forage for animals, influence soil chemistry and forest microclimate. However, very little is known about the chemical defense of various shrub species against folivorous insects. Using six shrub species, we tested how seasonal changes and light conditions affect their constitutive defense to insect damage. We monitored leaf perforation, concentrations of total phenols, condensed tannins, nitrogen (N), and total nonstructural carbohydrates (TNC). Leaf damage caused by insects was low in Sambucus nigra, Cornus sanguinea, and Frangula alnus, intermediate in Corylus avellana and Prunus serotina, and high in Prunus padus. Leaves of all the species, when growing in high light conditions, had high concentrations of defense metabolites. Except for C. avellana, leaves of the other shrub species growing in full sun were less injured than those in shade. This may be due to higher concentrations of defense metabolites and lower concentrations of nitrogen. Similar patterns of the effects of light on metabolites studied and N were observed for leaves with varying location within the crown of individual shrubs (from the top of the south direction to the bottom of the north), as for leaves from shrubs growing in full sun and shrubs in the shade of canopy trees. A probable cause of the greater damage of more sunlit leaves of C. avellana was the fact that they were herbivorized mostly by Altica brevicollis, a specialist insect that prefers plant tissues with a high TNC level and is not very sensitive to a high level of phenolic compounds.     

Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae (Pterospora andromedea and Sarcodes sanguinea) found in the Greater Yellowstone Ecosystem

Pterospora andromedea, a mycoheterotroph, has been shown to form obligate symbioses with only three species of Rhizopogon in section Amylopogon: R. salebrosus, R. arctostaphyli and an undescribed molecular taxon. Sarcodes sanguinea, another my coheterotroph in Ericaceae, and sister taxon to Pterospora andromedea, has been found to form symbioses with two species of Rhizopogon in section Amylopogon: R. ellenae and R. subpurpurascens. To date no overlap has been recorded between these two achlorophyllous plants and their associated mycobionts. Tissue from Pterospora andromedea rootballs and Rhizopogon spp. basidiocarps were collected from the Greater Yellowstone Ecosystem. The mycobionts were identified using sequence analysis of the ITS locus and compared with sequences of Rhizopogon spp. section Amylopogon from GenBank. Sequences of two additional loci, ATP6 and RPB2 were also generated and analyzed. In addition to Rhizopogon salebrosus, Pterospora andromedea was found for the first time in association with a fourth mycobiont, Rhizopogon ellenae, a known associate of Sarcodes sanguinea. The discovery of a new symbiont may provide evidence for an undiscovered lineage of Pterospora andromedea inhabiting the Greater Yellowstone Ecosystem. In addition, overlap in obligate mycobionts between closely related mycoheterotrophs provides interesting new information on the phylogenetic history and coevolution of the mycoheterotrophs in the Monotropoideae (Ericaceae).     

Diagnostic molecular markers of six lepidopteran insect pests infesting apples in Korea

Two molecular identification techniques for differentiating six lepidopteran pests infesting apples in Korea are presented. These six species include two internal fruit feeders (Grapholita molesta and Carposina sasakii), two leaf rollers (Adoxophyes sp. and Archips breviplicanus) and two leaf miners (Phyllonorycter ringoniella and Lyonetia prunifoliella). All species occur until near harvest and reduce apple production. A 489 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in these six species. The sequence was used to select species-specific restriction enzyme sites and to design diagnostic polymerase chain reaction (PCR) primers, resulting in the development of restriction fragment length polymorphism (RFLP)-PCR and diagnostic PCR. These methods were reliable and rapid in the identification of these six species.     

Morphophylogenetic study revealed that Erysiphe gracilis (powdery mildew of evergreen oaks,Erysiphales) is a species complex consisting of six different species

Erysiphe gracilis is a powdery mildew species that occurs on evergreen oak species belonging to Quercus subgen. Cyclobalanopsis in East Asia (China and Japan). In a previous report, we found that E. gracilis var. gracilis is divided into four genotypes each of them forming a separate clade with strong bootstrap support. In this study, we further investigated genotype speciation in E. gracilis var. longissima occurring on Q. acuta and Q. sessilifolia, and found that this variety is also divided into two distinct genotypes. These results suggested that E. gracilis represents a species complex consisting of six different species. Based on detailed morphological examinations correlating with results of molecular sequence analyses, we propose to divide E. gracilis into six species, encompassing three new species (E. uncinuloides, E. pseudogracilis, and E. longiappendiculata), one new name (E. longifilamentosa), and two known species (E. gracilis s. str. and E. hiratae). A key to the species concerned is provided.     

Cryptic diversity within the harmful dinoflagellate Akashiwo sanguinea in coastal Chinese waters is related to differentiated ecological niches

Blooms of the harmful dinoflagellate Akashiwo sanguinea are responsible for the mass mortality of fish and invertebrates in coastal waters. This cosmopolitan species includes several genetically differentiated clades. Four clonal cultures were established by isolating single cells from Xiamen Harbour (the East China Sea) for morphological and genetic analyses. The cultures displayed identical morphology but were genetically different, thus revealing presence of cryptic diversity in the study area. New details of the apical structure complex of Akashiwo sanguinea were also found. To investigate whether the observed cryptic diversity was related to environmental differentiation, 634 cells were obtained from seasonal water samples collected from 2008 to 2012. These cells were sequenced by single-cell PCR. For comparison with Chinese material, additional large subunit ribosomal DNA sequences were obtained for three established strains from Malaysian and French waters. To examine potential ecological differentiation of the distinct genotypes, growth responses of the studied strains were tested under laboratory conditions at temperatures of 12 °C to 33 °C. These experiments showed four distinct ribotypes of A. sanguinea globally, with the ribotypes A and B co-occuring in Xiamen Harbour. Ribotype A of A. sanguinea was present year-round in Xiamen Harbour, but it only bloomed in the winter and spring, thus corresponding to the winter type. In contrast, A. sanguinea ribotype B bloomed only in the summer, corresponding to the summer type. This differentiation supports the temperature optimum conditions that were established for these two ribotypes in the laboratory. Ribotype A grew better at lower temperatures compared to ribotype B which preferred higher temperatures. These findings support the idea that various ribotypes of A. sanguinea correspond to distinct ecotypes and allopatric speciation occurred in different climatic regions followed by dispersal.     

Experimental Research on the In Vitro Antitumor Effects of Crataegus sanguinea

Crataegus sanguinea is a wild plant, which has been widely grown in the north and south of the Tianshan mountains in Xinjiang. In order to explore their anti-cancer properties, edible wild plants from Xinjiang have been tested for their antitumor properties. We used Ames tests, mouse bone marrow polychromatic erythrocytes micronucleus tests, and tumor cells cultured in vitro to study the anti-mutagenic and anti-tumor effects of C. sanguinea extract. We have shown that C. sanguinea has anti-mutagenic effect, but no mutagenicity. Cell culture in vitro experiments show that there is no inhibition of growth or increase in cell death on normal mouse fibroblasts, but a stronger inhibition of cell growth and an increase in cell death of Hep-2 and MGC-803 tumor cells. The results of this study illustrate that C. sanguinea extract has both anti-mutagenic and anti-tumor effects.     

Bacterial community dynamics during a bloom caused by Akashiwo sanguinea in the Xiamen sea area,China

Phytoplankton blooms are a worldwide ecological problem and one of the major algae that cause phytoplankton blooms is Akashiwo sanguinea. Though much research has addressed the abiotic causes (e.g. growth condition) of A. sanguinea blooms, few studies have examined the dynamics of microbial communities associated with these blooms. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA genes was used to document changes in the phylogenetic diversity of microbial communities associated with an A. sanguinea bloom that occurred in the Xiamen sea in May 2010. Surface sea water was sampled once a day within five consecutive days at four sites, and the microbial community composition was determined using DGGE. Sea water concentrations of chlorophyll a, nitrate and phosphate were also measured. The results indicated that the A. sanguinea bloom was probably stimulated by low salinity (26–30‰) and ended probably because inorganic nutrients were consumed and resulted in a N/P ratio unfavorable for this alga. Gammaproteobacteria populations increased significantly during bloom declines and then decreased post-bloom. Divergences in the microbial community composition during different bloom periods were the result of changes in Candidatus, Pelagibacter, Alteromonas, Rhodobacteraceae, Vibrio and Pseudoalteromonas populations. Sediminimonas qiaohouensis was the first bacterium shown to be significantly negatively correlated with A. sanguinea concentration. This study indicated that bacteria may play an important role in A. sanguinea–bloom regulation and provides a deeper insight into bacterial community succession during and after an A. sanguinea–bloom.     

Threat,Signal or Waste? Meaning of Corpses in two Dulotic Ant Species

Ant corpses, besides representing threat of infection by pathogens and parasites, can also be used during interspecific conflicts to inhibit the activity of the attacked colony, or they can be consumed as food. In the view of the former, the signal properties of corpses can be manifold. Besides discriminating nestmates and foes, the corpses of different ant species may act as cues for foragers, signaling the presence of other rival species, and triggering appropriate responses (e.g., alarm, retreat or foraging). In our study, we examined the responses of the facultative slave-maker Formica sanguinea and those of the obligate Polyergus rufescens towards corpses of nestmates, non-nestmate conspecifics, heterospecific slave-makers and their slaves, and corpses of non-enslaved host species under laboratory conditions. Both dulotic species responded differently to corpses of different origin. In F. sanguinea, the most intensive response was elicited by the corpses of P. rufescens and its slave, but also the corpses of non-nestmate conspecifics and their slaves elicited many adverse responses. In P. rufescens, the corpses of non-nestmate conspecifics and their slaves elicited the most adverse response. Both dulotic species distinguished corpses of their slaves from corpses of non-enslaved hosts. Based on our results, ant corpses are not meaningless objects scattered in the field, but cues carrying information that trigger different behavioral responses, and in F. sanguinea they can even represent an important food source.     

干旱胁迫对青藏高原6种植物生理指标的影响

以青藏高原6种植物:红花岩黄芪(Hedysarum multijugum Maxim.)、西北沼委陵菜(Comarum salesovianum(Steph.)Asch.et Graebn.)、鲜黄小檗(Berberis diaphana Maxim.)、鲜卑花(Sibiraea laevigata(Linn.)Maxim.)、树锦鸡儿(Caragana arborescens(Amm.)Lam.)、砂生槐(Sophora moorcroftiana(Benth.)Baker)为试材进行干旱胁迫模拟盆栽试验,通过对比6种植物叶片中丙二醛(MDA)、脯氨酸、可溶性蛋白质、叶绿素、可溶性糖、淀粉含量的变化,对供选植物进行抗旱性能分析与评价,为在青藏高原地区筛选优良耐旱植物提供参考依据。设置土壤含水量占田间持水量80%以上为正常水平,50%—70%为轻度干旱,30%—50%为中度干旱,低于30%为重度干旱。研究表明,6种供试植物叶片MDA含量逐渐升高,当土壤含水量占田间持水量12%左右时达到最大值,树锦鸡儿的增幅较另5种苗木最小,而峰值最大。脯氨酸含量都呈增加趋势,其中砂生槐最高,为9981.67mg/g,峰值与对照值相比,砂生槐增加最多为37.43倍,鲜卑花最小为0.49倍。苗木叶片中可溶性蛋白质含量总体表现出现增大后减小的规律,且所有测定值都高于正常水分条件下的含量。6种苗木的叶绿素含量在干旱胁迫下总体呈现先降低再升高最后再降低的趋势,24 d时均低于对照值,下降最多的为西北沼委陵菜(15.43 mg/g)。由于干旱下苗木的光合产物转化受抑,以淀粉形式的累积量减少,所以6种苗木的可溶性糖含量呈上升趋势,而淀粉含量呈下降趋势,通过积累可溶性糖以提高细胞的渗透调节能力。砂生槐、树锦鸡儿的可溶性糖含量增幅与淀粉含量的减少明显高于其余苗木,使其在较低叶水势下仍保持较高的细胞膨压,以减缓水分胁迫的不利影响。主成分分析表明,6种苗木抗旱性由强到弱依次为:树锦鸡儿、红花岩黄芪、砂生槐、鲜黄小檗、鲜卑花、西北沼委陵菜。     

Interspecific Associations between Cycloneda sanguinea and Two Aphid Species (Aphis gossypii and Hyadaphis foeniculi) in Sole-Crop and Fennel-Cotton Intercropping Systems

Aphids cause significant damage to crop plants. Studies regarding predator-prey relationships in fennel (Foeniculum vulgare Mill.) and cotton (Gossypium hirsutum L.) crops are important for understanding essential ecological interactions in the context of intercropping and for establishing pest management programs for aphids. This study evaluated the association among Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae), Aphis gossypii Glover (Hemiptera: Aphididae) and Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) in cotton with coloured fibres, fennel and cotton intercropped with fennel. Association analysis was used to investigate whether the presence or absence of prey and predator species can indicate possible interactions between aphids and ladybugs. Significant associations among both apterous and alate H. foeniculi and C. sanguinea were observed in both the fennel and fennel-cotton intercropping systems. The similarity analysis showed that the presence of aphids and ladybugs in the same system is significantly dependent on the type of crop. A substantial amount of evidence indicates that the presence of the ladybug C. sanguinea, is associated with apterous or alate A. gossypii and H. foeniculi in fennel-cotton intercropping system. We recommend that future research vising integrated aphid management taking into account these associations for take decisions.     

Zooming on dynamics of marine microbial communities in the phycosphere of Akashiwo sanguinea (Dinophyta) blooms

Characterizing ecological relationships between viruses, bacteria and phytoplankton in the ocean is critical to understanding the ecosystem; however, these relationships are infrequently investigated together. To understand the dynamics of microbial communities and environmental factors in harmful algal blooms (HABs), we examined the environmental factors and microbial communities during Akashiwo sanguinea HABs in the Jangmok coastal waters of South Korea by metagenomics. Specific bacterial species showed complex synergistic and antagonistic relationships with the A. sanguinea bloom. The endoparasitic dinoflagellate Amoebophrya sp. 1 controlled the bloom dynamics and correlated with HAB decline. Among nucleocytoplasmic large DNA viruses (NCLDVs), two Pandoraviruses and six Phycodnaviruses were strongly and positively correlated with the HABs. Operational taxonomic units of microbial communities and environmental factors associated with A. sanguinea were visualized by network analysis: A. sanguinea–Amoebophrya sp. 1 (r = .59, time lag: 2 days) and A. sanguinea–Ectocarpus siliculosus virus 1 in Phycodnaviridae (0.50, 4 days) relationships showed close associations. The relationship between A. sanguinea and dissolved inorganic phosphorus relationship also showed a very close correlation (0.74, 0 day). Microbial communities and the environment changed dynamically during the A. sanguinea bloom, and the rapid turnover of microorganisms responded to ecological interactions. A. sanguinea bloom dramatically changes the environments by exuding dissolved carbohydrates via autotrophic processes, followed by changes in microbial communities involving host‐specific viruses, bacteria and parasitoids. Thus, the microbial communities in HAB are composed of various organisms that interact in a complex manner.     

Transformation of 3-(3-carboxyphenyl)alanine in iris species An example of diversity in catabolism of secondary plant products

3-(3-Carboxyphenyl)-DL-[2-¹⁴C]alanine has been incorporated into four species of iris. In all species extensive metabolization takes place. In Iris × hollandica, in which both the alanine derivative and 3′-carboxyphenylglycine occur, the products identified are the glycine derivative, 3′-carboxyphenylacetate acid, 3′-carboxymandelic acid, and 3′-carboxyphenylglyoxylic acid. In I. sanguinea, in which the alanine and glycine derivatives also occur, the products identified are the glycine and acetic acid derivatives but the major product is 3-(3-hydroxymethylphenyl)alanine, a naturally occurring amino acid in this species. In I. tectorum, in which only the carboxy-substituted alanine derivative occurs, the products identified are the acetic acid and glyoxylic acid derivatives. In I. pallida, not containing any of the meta-substituted amino acids, the products identified are again the acetic acid and glyoxylic acid derivatives. The results have been further substantiated by incorporation of labelled 3′-carboxyphenylacetic acid and 3′-carboxymandelic acid into I. × hollandica and I. sanguinea.The results demonstrate three different metabolic patterns for the alanine derivative and confirm previous results on the pathway from the alanine to the glycine derivative. Furthermore, the results may be of significance for the elucidation of the catabolism of phenylalanine.     

Sesquiterpenes of six Porella species (hepaticae)

The distribution in six Porella species of drimane-, aromadendrane- and pinguisane-type sesquiterpenes and norsesquiterpenes is described. The sharp pungent substance of P. gracillissima, P. fauriei and P. macroloba is (+)-tadeonal.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Efficient 5' ETS walking from conserved 18S rDNA sequences of the dinoflagellates <Emphasis Type="Italic">Alexandrium</Emphasis> and <Emphasis Type="Italic">Akashiwo sanguinea</Emphasis> (Dinophyceae)

An external transcribed spacer (ETS) walking PCR technique was developed for the isolation of unknown sequences adjacent to the 18S rDNA. This strategy relied on four "walking primers", which were designed to bind unknown sequences upstream from the 18S rDNA, and a specially programmed series of thermocycles. This method was successful in the isolation of the 5' ETS regions from harmful dinoflagellates, including Alexandrium affine, A. catenella, A. minutum, A. tamarense, and Akashiwo sanguinea. Mono-directional sequencing reactions revealed the PCR products to be 392–962 nucleotides in length, and the 5' ETS in these products were longer than 362 bp. These are the first such sequences available for A. sanguinea and the Alexandrium. In comparisons of the ETS sequences, genetic distance was considerably high within the Alexandrium. Furthermore, the sequences were significantly variable among the different strains of identical species: genetic distance was recorded at 0.0420 for A. tamarense strains and as less than 0.7841 within strains of A. sanguinea. The 5'-start nucleotide of 18S rDNA was variable between the two genera: the five species of Alexandrium contained a T base, and A. sanguinea contained an A base. These results demonstrate the effectiveness of the ETS walking PCR method. This method will be valuable in directional ETS walking from known regions to unknown regions, particularly concerning the boundary sequences of rRNA genes.