Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2′, and G. belizeanus by the asymmetrical Plate 3′. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell⁻¹, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

Phylogeny of the Bacillariaceae with emphasis on the genus Pseudo-nitzschia (Bacillariophyceae) based on partial LSU rDNA

In order to elucidate the phylogeny and evolutionary history of the Bacillariaceae we conducted a phylogenetic analysis of 42 species (sequences were determined from more than two strains of many of the Pseudo-nitzschia species) based on the first 872 base pairs of nuclear-encoded large subunit (LSU) rDNA, which include some of the most variable domains. Four araphid genera were used as the outgroup in maximum likelihood, parsimony and distance analyses. The phylogenetic inferences revealed the Bacillariaceae as monophyletic (bootstrap support ≥90%). A clade comprising Pseudo-nitzschia, Fragilariopsis and Nitzschia americana (clade A) was supported by high bootstrap values (≥94%) and agreed with the morphological features revealed by electron microscopy. Data for 29 taxa indicate a subdivision of clade A, one clade comprising Pseudo-nitzschia species, a second clade consisting of Pseudo-nitzschia species and Nitzschia americana, and a third clade comprising Fragilariopsis species. Pseudo-nitzschia as presently defined is paraphyletic and emendation of the genus is probably needed. The analyses suggested that Nitzschia is not monophyletic, as expected from the great morphological diversity within the genus. A cluster characterized by possession of detailed ornamentation on the frustule is indicated. Eighteen taxa (16 within the Bacillariaceae) were tested for production of domoic acid, a neurotoxic amino acid. Only P. australis, P. multiseries and P. seriata produced domoic acid, and these clustered together in all analyses. Since Nitzschia navis-varingica also produces domoic acid, but is distantly related to the cluster comprising the Pseudo-nitzschia domoic acid producers, it is most parsimonious to suggest that the ability of species in the Bacillariaceae to produce domoic acid has evolved at least twice.     

Community structure of arbuscular mycorrhizal fungi in undisturbed vegetation revealed by analyses of LSU rDNA sequences

Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with plant roots and are found in most ecosystems. In this study the community structure of AMF in a clade of the genus Glomus was examined in undisturbed costal grassland using LSU rDNA sequences amplified from roots of Hieracium pilosella. Roots were sampled from May to November along eight 30-m transects, 30-120 m apart. Phylogenetic analysis of the sequences revealed 11 phylogenetic clusters within the clade of Glomus. The phylogenetic clusters were patchily distributed within the area; time had no influence on the distribution pattern. The dominant cluster covered up to 10 m along the transect, whereas other clusters formed what can be interpreted as small individual mycelia. Four of the phylogenetic clusters included known species; the other clusters, including the dominant sequence types, were unknown. The dominant phylogenetic cluster enclosed nine haplotypes, and analyses of genetic diversity of this phylogenetic cluster showed that the total diversity could be found within single root fragments, suggesting that the multiple sequences were derived from a single individual.     

商洛多花胡枝子根瘤菌16S rDNA-RFLP分析及系统发育研究

利用16S rDNA-RFLP和全序列测定方法,对分离自商洛地区5个分布点的59株多花胡枝子根瘤菌进行了RFLP分析和系统发育研究.结果表明:(1)42株供试菌株归属根瘤菌属(Rhizobium)、11株归属中华根瘤菌属(Sinorhizobium).其余6株非根瘤菌中3株是嗜麦芽黄单胞菌(Stenotrophomonas maltophilia)、3株是解淀粉类芽孢杆菌(Paenibacillus amylolyticus),说明胡枝子根瘤内生菌较为丰富且类型多样.(2)结合供试菌株的地理生境分析,发现来自不同采集点的菌株有些具有同样的遗传类型,而来自同一采集点的菌株遗传类型却有差异,证明胡枝子根瘤菌在分群类别上与地理环境之间没有明确的对应关系,地理环境并非根瘤菌多样性形成的主要因素.建议今后对根瘤菌多样性研究应从根瘤菌与寄主植物物之间的共生选择进化,特别是对共生体系中基因的横向转移方面进行深入探讨.     

PHYLOGENY OF THE EUGLENOID LORICATE GENERA TRACHELOMONAS AND STROMBOMONAS (EUGLENOPHYTA) INFERRED FROM NUCLEAR SSU AND LSU rDNA1

Previous studies using the nuclear SSU rDNA and partial LSU rDNA have demonstrated that the euglenoid loricate taxa form a monophyletic clade within the photosynthetic euglenoid lineage. It was unclear, however, whether the loricate genera Trachelomonas and Strombomonas were monophyletic. In order to determine the relationships among the loricate taxa, SSU and LSU nuclear rDNA sequences were obtained for eight Strombomonas and 25 Trachelomonas strains and combined in a multigene phylogenetic analysis. Conserved regions of the aligned data set were used to generate maximum‐likelihood (ML) and Bayesian phylogenies. Both methods recovered a strongly supported monophyletic loricate clade with Strombomonas and Trachelomonas species separated into two sister clades. Taxa in the genus Strombomonas sorted into three subclades. Within the genus Trachelomonas, five strongly supported subclades were recovered in all analyses. Key morphological features could be attributed to each of the subclades, with the major separation being that all of the spine‐bearing taxa were located in two sister subclades, while the more rounded, spineless taxa formed the remaining three subclades. The separation of genera and subclades was supported by 42 distinct molecular signatures (33 in Trachelomonas and nine in Strombomonas). The morphological and molecular data supported the retention of Trachelomonas and Strombomonas as separate loricate genera.     

西北地区天蓝苜蓿根瘤菌16S rDNA RFLP分析

利用RFLP和序列测定方法,对分离自西北地区的67株天蓝苜蓿根瘤菌16S rDNA进行了分析研究。结果表明:所有供试菌株分别归属于中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobac-terium)。以CCNWNX0042-2为代表的大部分天蓝苜蓿根瘤菌属于草木樨中华根瘤菌(Sinorhizobium meliloti),其余菌株在分群上表现出了较为明显的地域特征。     

Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics

Views on myxosporean phylogeny and systematics have recently undergone substantial changes resulting from analyses of SSU rDNA. Here, we further investigate the evolutionary trends within myxosporean lineages by using 35 new sequences of the LSU rDNA. We show a good agreement between the two rRNA genes and confirm the main phylogenetic split between the freshwater and marine lineages. The informative superiority of the LSU data is shown by an increase of the resolution, nodal supports and tree indexes in the LSU rDNA and combined analyses. We determine the most suitable part of LSU for the myxosporean phylogeny by comparing informative content in various regions of the LSU sequences. Based on this comparison, we propose the D5–3′-end part of the LSU rRNA gene as the most informative region which provides in concatenation with the complete SSU a well resolved and robust tree. To allow for simple amplification of the marker, we design specific primer set for this part of LSU rDNA.     

Studies on woloszynskioid dinoflagellates VII. Description of Borghiella andersenii sp. nov.: light and electron microscopy and phylogeny based on LSU rDNA

Freshwater woloszynskioid dinoflagellates were collected independently in Scotland and Portugal and found to belong to a previously unknown species of the genus Borghiella, here described as B. andersenii. The new species differs in morphology and nuclear-encoded LSU rDNA and ITS sequences from B. dodgei and B. tenuissima, the two species presently comprising the genus Borghiella. Unusual features of the new species were observed particularly during asexual reproduction, which took place in the motile stage – as in many other dinoflagellates – or in a so-called division cyst, recalling cell division in the family Tovelliaceae. Such diversity in cell division is rarely reported in dinoflagellates. Morphologically Borghiella andersenii differs from B. tenuissima in being only slightly compressed dorsoventrally whereas the latter species is flat. The slight compression is also visible in lateral view. Borghiella andersenii and B. dodgei are more challenging to discriminate but the apical structure complex is only half the length in B. andersenii compared with B. dodgei (3–4 vs 6 µm). This difference can only be accounted for in the scanning electron microscope. At the light microscopy level the epicone in B. andersenii is rounded whereas it is conical in B. dodgei. Sexual reproduction in Borghiella andersenii was homothallic by formation of planozygotes, followed by apparent resting cysts. Phylogenetic studies on woloszynskioids have recently shown that they comprise a polyphyletic assemblage, which has been divided into the three families Borghiellaceae, Tovelliaceae and Suessiaceae. New species of the three families are now being found rapidly in many parts of the world, proving that the techniques required to investigate these small, morphologically similar dinoflagellates are now in place and proving that such ‘gymnodinioids’ or ‘woloszynskioids’ comprise an often overlooked biological entity in both marine and freshwater biotopes. Based on LSU rDNA, B. andersenii is most closely related to B. tenuissima.     

Ultrastructure of Gyrodinium spirale, the type species of Gyrodinium (Dinophyceae), including a phylogeny of G. dominans, G. rubrum and G. spirale deduced from partial LSU rDNA sequences

A detailed ultrastructural analysis of the type species of Gyrodinium, G. spirale, was made based on cells collected from Skagerrak and southern Kattegat (Denmark). This material is considered very similar to the type material studied by Bergh from southern Kattegat. The analysis revealed many characters typical for dinoflagellates as well as a number of previously undescribed features. Here, emphasis was given to a three-dimensional configuration of the flagellar apparatus, the surface ridges, and the nuclear capsule. The latter had a rather complex ultrastructure consisting of two wall-like layers surrounded by membranes, with nuclear pores restricted to globular invaginations of these layers. To overcome difficulties with culturing of many auto- and heterotrophic dinoflagellates, we designed a specific reverse primer to amplify ca. 1800 base pairs of nuclear-encoded LSU rDNA. Using this approach, LSU rDNA sequences were determined from three heterotrophic species of Gyrodinium, including the type species. Using other alveolates (i.e. ciliates and Apicomplexa) as outgroup species, phylogenetic analyses based on Maximum Likelihood, Maximum Parsimony, and Neighbor-Joining supported Gyrodinium as a separate lineage. Unfortunately, the nearest sister group to Gyrodinium could not be established due to low bootstraps support for the deep branching pattern.     

First Report of the Epiphytic Dinoflagellate Gambierdiscus caribaeus in the Temperate Waters off Jeju Island,Korea: Morphology and Molecular Characterization

Gambierdiscus spp. are epiphytic, benthic dinoflagellates. Some species have been shown to be toxic and cause ciguatera fish poisoning. We report, for the first time, the occurrence of Gambierdiscus caribaeus isolated from the waters off Jeju Island in Korea. Its morphology was similar to that of the original Belize strains of G. caribaeus. Gambierdiscus caribaeus has been reported in the tropical and subtropical waters of the Pacific, Gulf of Mexico, Caribbean Sea, and Floridian coast. Our report extends its range to the North Pacific Ocean. The plates of the Korean strain were arranged in a Kofoidian series of Po, 3′, 7′′, 6c, 6s, 5′′′, 1p, and 2′′′′, morphologically closer to other strains of G. caribaeus than to G. carpenteri. When properly aligned, its small subunit (SSU) rDNA was 0.5% different from those of Gambierdiscus sp. C‐1, a strain that was isolated from the waters off eastern Japan, but was 2.4–4.0% different from those of the NOAA strains of G. caribaeus and 3.1–3.4% different from those of the NOAA strains of G. carpenteri. Additionally, the D1–D3 large subunit (LSU) rDNA sequence of the Korean strain of G. caribaeus was 4.7–5.3% different from those of the NOAA strains of G. caribaeus and 7.1–7.5% different from those of all reported G. carpenteri strains, including the NOAA strains. In phylogenetic trees based on SSU and LSU rDNA sequences, our Korean strain was basal to the clade consisting of the NOAA strains of G. caribaeus, which in turn was sister clade to all reported G. carpenteri strains.     

Gambierdiscus species exhibit different epiphytic behaviors toward a variety of macroalgal hosts

Ciguatera fish poisoning is a common form of seafood poisoning caused by toxins (ciguatoxins) that accumulate in demersal (reef) food webs. The precursors of ciguatoxins are produced by dinoflagellates of the genus Gambierdiscus, and enter the food web via herbivory and detritivory. The Gambierdiscus genus was recently revised and new research on the physiology and ecology of the revised species is needed. While it has been demonstrated that Gambierdiscus spp. are predominately epiphytic, the variability in epiphytic behavior among the various Gambierdiscus species is not known. Five Gambierdiscus species isolated from the Greater Caribbean Region were the focus of this study (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, and G. yasumotoi). Cells of Gambierdiscus were grown in wells with algae fragments from eight different macroalgal host genera (Acanthophora, Caulerpa, Dasya, Derbesia, Dictyota, Laurencia, Polysiphonia, and Ulva) where the epiphytic behavior and growth of the different Gambierdiscus species were monitored over 29 days. The results of this experiment demonstrate that epiphytic behavior (growth and attachment) differs among the Gambierdiscus species toward the various macroalgal hosts. Results tended to be specific to Gambierdiscus – host pairings with few commonalities in the way a particular Gambierdiscus species interacted across hosts or how the various Gambierdiscus species responded to a particular host. The Gambierdiscus – host pairings that resulted in the highest growth and attachment combinations were examined in terms of known cellular toxicity and host palatability to determine which pairings could represent the most likely vectors for the transfer of ciguatoxins (or precursors) into the demersal food web. Two pairings, Gambierdiscus belizeanus – Polysiphonia and G. belizeanus – Dictyota, best met these criteria, providing a hypothetical approach to better focus sampling and monitoring efforts on such potential vectors in the benthic environment.     

香蕉束顶病植原体16S rDNA片段的RFLP和序列分析

香蕉束顶病（ＢＢＴ）是一种发生在蕉类作物的严重病害。从带有典型香蕉束顶病症状的香蕉植株中按照检测植原体的方法提取ＤＮＡ,扩增患病植株中植原体１６ＳｒＤＮＡ片段,证明香蕉束顶病中有植原体存在。对此扩增片段进行限制性酶切片段长度多态性（ＲＦＬＰ）分析和核酸序列分析,并与已知植原体的序列进行同源性比较,构建进化树。结果显示该片段与Ｇｒ１的亲缘关系最近。     

Growth and epiphytic behavior of three Gambierdiscus species (Dinophyceae) associated with various macroalgal substrates

Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.     

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.