Morpho-molecular description of a new HAB species, Pseudocochlodinium profundisulcus gen. et sp. nov., and its LSU rRNA gene based genetic diversity and geographical distribution.

Harmful algal blooms (HABs) caused by an unknown dinoflagellate species have frequently occurred in the Pearl River Estuary, China Since 2006. These blooms were associated with severe water discoloration and economic losses, ranging from several km2 to 300 km2 with the maximum recorded cell density being 2.77 × 107 cells⋅L-1. This unknown dinoflagellate species was initially identified as Cochlodinium geminatum and subsequently reclassified as Polykrikos geminatus. However, after reviewing the original descriptions for Cochlodinium geminatum sensu Schütt (1895) and the genus Polykrikos, we considered this species is incongruent with their original descriptions. Further morphological examinations and particularly phylogenetic analyses based on the SSU and partial LSU rRNA genes of isolates and resting cysts from China and Japan prompted us to consider it a new species of a new genus. This new species was proposed to be Pseudocochlodinium profundisulcus gen. et sp. nov., based on its open comma-shaped apical structure complex (ASC), cingulum encircling the cell less than one and a half turns, a deep sulcus with a torsion of a half turn, either single cell or cell chain consisting of two cells with the same number of nuclei and zooids, the resting cyst bearing lobed ornaments, and the evolutionary distances from Polykrikos (and others) on the phylogenetic trees constructed using the concatenated SSU and partial LSU rRNA gene sequences. Metabarcoding investigation of surface sediment samples collected in China revealed that the species to be widely present along the entire Chinese coast with the highest abundance in the South China Sea. Further re-analysis of the Tara Oceans metabarcoding dataset targeting the SSU rRNA gene V9 domain suggested a global distribution of this new genus. Phylogenetic analyses on 46 OTUs (average length: ∼552 bases) of its LSU rRNA gene sequences (mainly D1-D2 domains) obtained from surface sediment samples revealed intraspecific genetic diversity of this species. Interestingly, based on the different distributions and the abundance of these OTUs along the coast of China, this species appeared to have expanded its distribution from the South China Sea to the northern Yellow Sea, or preferred a warm water habitat. We consider that the present work improves the taxonomy and provides important insights into the biogeography of Pseudocochlodinium profundisulcus.⋅.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Prorocentrum shikokuense Hada and P. donghaiense Lu are junior synonyms of P. obtusidens Schiller, but not of P. dentatum Stein (Prorocentrales, Dinophyceae).

In Japanese, Chinese and Korean coastal waters, recurrent blooms of a small, elongate Prorocentrum species have been observed in recent years. In previous studies, this species has been respectively identified as P. shikokuense, P. donghaiense and P. dentatum, despite morphological similarity and identical rDNA sequences. To resolve the confusion, morphological features, including the architectural details of the periflagellar area, were examined and ribosomal DNA (rDNA) sequences were obtained from specimens collected from the East China Sea and Korean coast, and a strain established in the waters off the Canary Islands of Spain. In addition, the descriptions of the three species and allied species were reviewed. Morphological observations and a phylogeny based on the SSU, ITS region and LSU rDNA sequences revealed that the previously confused species and our studied strains are conspecific and that the morphology of strains identified as P. dentatum in the phylogenetic trees does not coincide with P. dentatum sensu stricto. The confusion can be traced back to Dodge (1975, p. 116), who considered P. veloi, P. monacense and P. obtusidens as junior heterotypic synonyms of P. dentatum. However, Dodge's P. dentatum are closer to P. obtusidens sensu stricto, rather than P. dentatum sensu stricto. P. obtusidens sensu stricto can be distinguished from P. dentatum sensu stricto by its relatively small size, parallel sides towards the anterior and a blunt anterior extension on one side. This indicates that P. obtusidens should not be considered a synonym of P. dentatum sensu stricto. In addition, a comparison of the original descriptions of P. obtusidens and allied species allowed to conclude that small, elongate Prorocentrum from Japanese, Chinese and Korean coastal waters previously identified as P. shikokuense, P. donghaiense, P. dentatum sensu Yoo and Lee (1986) and the specimens studied herein, which share identical rDNA sequences, morphologically coincide with P. obtusidens. Therefore, it is proposed that P. shikokuense and P. donghaiense should be regarded as junior synonyms of P. obtusidens.

The affiliation of Hexasterias problematica and Halodinium verrucatum sp. nov. to ciliate cysts based on molecular phylogeny and cyst wall composition.

Species in the genera Hexasterias and Halodinium have been recorded over the last decades as acritarchs in palynological and/or plankton studies. In paleoenvironmental studies, these resting stages are often interpreted as indicators of freshwater input. The biological affinity of these genera has never been definitely established. Here, a new species, Halodinium verrucatum sp. nov., is described and molecular evidence (single specimen SSU and LSU rDNA sequencing) reveals that both this new species and Hexasterias problematica, collected from sediment samples in the Skagerrak and Baltic Sea, are resting stages of prorodontid ciliates. Additionally, infrared spectroscopic analysis (micro-FTIR) of Hexasterias problematica and Halodinium spp. specimens indicates a carbohydrate-based composition of the cyst wall with evidence for nitrogen-containing functional groups. A similar composition is recorded for tintinnid loricae, which further supports the placement of Hexasterias and Halodinium as ciliate cysts, and the composition is consistent with the heterotrophic nature of ciliates. The morphologically similar species Radiosperma corbiferum has a comparable composition, suggesting a similar ciliate affinity and indicating the utility of micro-FTIR in understanding acritarch affinity. Hexasterias problematica typically occurs in coastal waters from temperate to arctic regions. Halodinium verrucatum sp. nov. is observed in temperate estuarine sediments in the northern hemisphere.

Repercussions of the Great East Japan Earthquake tsunami on ellipsoidal Alexandrium cysts (Dinophyceae) in Ofunato Bay, Japan.

Shellfish aquaculture in Ofunato Bay, Northeast Japan, was seriously damaged by a tsunami generated by the Great East Japan Earthquake, March 11th, 2011, accompanied by paralytic shellfish poisoning (PSP) outbreaks caused by Alexandrium tamarense (Dinophyceae). To understand longer future trends of PSP, an investigation was made of the historical occurrence and causes of Alexandrium outbreaks after the tsunami. Vertical distributions of Alexandrium cysts in two sediment-cores from Ofunato Bay revealed that the sediments above ca. 25 cm were eroded, re-suspended and re-deposited, and they included unusually abundant Alexandrium cysts. This abundance of cysts was due to re-deposition of older sediments by the tsunami. The first Ofunato Bay PSP incident was in 1961 after the Chilean Earthquake tsunami and was probably caused by similar unusual blooms of Alexandrium germinated from older sediments as the Great East Japan tsunami, together with nutrient enrichment because of population increase at the start of shellfish aquaculture.

Pentaplacodinium saltonense gen. et sp. nov. (Dinophyceae) and its relationship to the cyst-defined genus Operculodinium and yessotoxin-producing Protoceratium reticulatum.

Strains of a dinoflagellate from the Salton Sea, previously identified as Protoceratium reticulatum and yessotoxin producing, have been reexamined morphologically and genetically and Pentaplacodinium saltonense n. gen. et sp. is erected to accommodate this species. Pentaplacodinium saltonense differs from Protoceratium reticulatum (Claparède et Lachmann 1859) Bütschli 1885 in the number of precingular plates (five vs. six), cingular displacement (two widths vs. one), and distinct cyst morphology. Incubation experiments (excystment and encystment) show that the resting cyst of Pentaplacodinium saltonense is morphologically most similar to the cyst-defined species Operculodinium israelianum (Rossignol, 1962) Wall (1967) and O. psilatum Wall (1967). Collections of comparative material from around the globe (including Protoceratium reticulatum and the genus Ceratocorys) and single cell PCR were used to clarify molecular phylogenies. Variable regions in the LSU (three new sequences), SSU (12 new sequences) and intergenic ITS 1-2 (14 new sequences) were obtained. These show that Pentaplacodinium saltonense and Protoceratium reticulatum form two distinct clades. Pentaplacodinium saltonense forms a monophyletic clade with several unidentified strains from Malaysia. LSU and SSU rDNA sequences of three species of Ceratocorys (C. armata, C. gourreti, C. horrida) from the Mediterranean and several other unidentified strains from Malaysia form a well-supported sister clade. The unique phylogenetic position of an unidentified strain from Hawaii is also documented and requires further examination. In addition, based on the V9 SSU topology (bootstrap values >80%), specimens from Elands Bay (South Africa), originally described as Gonyaulax grindleyi by Reinecke (1967), cluster with Protoceratium reticulatum. The known range of Pentaplacodinium saltonense is tropical to subtropical, and its cyst is recorded as a fossil in upper Cenozoic sediments. Protoceratium reticulatum and Pentaplacodinium saltonense seem to inhabit different niches: motile stages of these dinoflagellates have not been found in the same plankton sample.

Cyst-Theca Relationship and Phylogenetic Position of Impagidinium caspienense Incubated from Caspian Sea Surface Sediments: Relation to Gonyaulax baltica and Evidence for Heterospory within Gonyaulacoid Dinoflagellates.

We investigate the cyst-theca relationship of Impagidinium caspienense. Through an incubation experiment, we succeeded in examining the motile stage. Additional molecular analysis of single-cyst PCR (LSU and SSU rDNA) reveal that the cyst is related to the species Gonyaulax baltica Ellegaard et al. (). The ability of this species to belong to two types of cyst-based genera (spiniferate and impagidinioid) suggests that environmental (particularly salinity) and not genetic factors explain the formation of both morphotypes by G. baltica, which provides evidence for heterospory in this species. The affiliation to G. baltica demonstrates that I. caspienense is not endemic to the Caspian Sea. The phylogenetic position of several other gonyaulacoid species is also documented: Impagidinium pallidum, Ataxiodinium choane, Pyxidinopsis psilata, Spiniferites belerius, and Spiniferites ramosus. The LSU and SSU rDNA based phylogenies suggest that the genera Impagidinium and Spiniferites are not monophyletic, and that P. psilata and A. choane are close to Gonyaulax verior and Gonyaulax polygramma, respectively. In addition, this study accentuates the importance of cyst morphology in the classification of the Gonyaulacales.

Identification of the resting cyst of Cochlodinium polykrikoides Margalef (Dinophyceae, Gymnodiniales) in Korean coastal sediments.

This study provides the first morphological features of resting cysts of Cochlodinium polykrikoides collected from Korean coastal sediments. Evidence for the existence of resting cysts of C. polykrikoides is based on the morphological and molecular phylogenetic data of the germinated cells and a resting cyst. The morphology of the resting cysts differed from that reported previously in sediments and culture experiments. The distinct feature is that the cyst body was covered by the reticulate ornaments and spines.

Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay, Turkey, and molecular characterization.

Here, we established the cyst-motile stage relation-ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.

A new heterotrophic dinoflagellate from the North-eastern Pacific, Protoperidinium fukuyoi: cyst-theca relationship, phylogeny, distribution and ecology.

The cyst-theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown-coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst-defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North-east Pacific, its distribution reflecting optimal summer sea-surface temperatures of ~14-18 °C and salinities of 22-34 psu.

Ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with reference to the apical groove and Flagellar apparatus.

The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three-dimensional structure of the flagellar apparatus. The apical groove is U-shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5-1.0 microm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.

Reconstruction of historical nutrient levels in Korean and Japanese coastal areas based on dinoflagellate cyst assemblages.

Dinoflagellate cysts acquired from sediment cores were analyzed in order to reconstruct historical nutrient levels in Gamak Bay, Korea and Ariake Bay, Japan. Dinoflagellate cyst assemblages in Gamak Bay were characterized by high proportions of heterotrophic cysts such as Brigantedinium spp., Protoperidinium americanum and Polykrikos cysts, which suggested that nutrients levels may have already been high before 1970s, and then increased further to the hypertrophic conditions of the 1990s. In contrast, dinoflagellate cyst assemblages in Ariake Bay were characterized by high relative abundances of Lingulodinium machaerophorum and Spiniferites spp., which suggested that nutrient levels in Ariake Bay had increased gradually since the mid 1960s, and may have been significantly enhanced by the mid 1980s. Dinoflagellate cyst assemblages reflecting environmental changes in the two bays are contrasting, perhaps due to different nutrient enrichment mechanisms. This suggests that the indicators of nutrient levels encoded in dinoflagellate cyst assemblages may exhibit site-specific information.

Detection of nitric oxide (NO) in marine phytoplankters.

In raphydophyte cell suspensions such as those of Chattonella marina, C. ovata and Heterosigma akashiwo, a gradual increase in NO-specific fluorescence intensity was observed, and the increase in the fluorescence intensity of each of these phytoplankters was completely inhibited in the presence of carboxy-PTIO, a specific NO scavenger. However, no such significant changes were observed in the case of other phytoplankter species.