Ultrastructure and molecular phylogenetic position of a new marine sand-dwelling dinoflagellate from British Columbia,Canada: Pseudadenoides polypyrenoides sp. nov. (Dinophyceae)

Two monospecific genera of marine benthic dinoflagellates, Adenoides and Pseudadenoides, have unusual thecal tabulation patterns (lack of cingular plates in the former; and no precingular plates and a complete posterior intercalary plate series in the latter) and are thus difficult to place within a phylogenetic framework. Although both genera share morphological similarities, they have not formed sister taxa in previous molecular phylogenetic analyses. We discovered and characterized a new species of Pseudadenoides, P. polypyrenoides sp. nov., at both the ultrastructural and molecular phylogenetic levels. Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated a close relationship between P. polypyrenoides sp. nov. and Pseudadenoides kofoidii, and Adenoides and Pseudadenoides formed sister taxa in phylogenetic trees inferred from LSU rDNA sequences. Comparisons of morphological traits, such as the apical pore complex (APC), demonstrated similarities between Adenoides, Pseudadenoides and several planktonic genera (e.g. Heterocapsa, Azadinium and Amphidoma). Molecular phylogenetic analyses of SSU and LSU rDNA sequences also demonstrated an undescribed species within Adenoides.     

<Emphasis Type="Italic">Prorocentrum rivalis</Emphasis> sp. nov. (Dinophyceae) and its phylogenetic affinities inferred from analysis of a mixed morphological and LSU rRNA data set

A new freshwater epiphytic Prorocentrum species, Prorocentrum rivalis, from the temperate region of the Haute-Vienne, France, is described. This species is the third freshwater species identified among approximately 60 marine Prorocentrum species. This new species is described using scanning electron microscope and phylogenetic analyses by a polyphasic approach (LSU rRNA sequences combined with 9 morphological characters). The phylogenetic analysis attests that P. rivalis is close to other planktonic freshwater species and the freshwater Prorocentrum clade is evolutionarily derived from an epiphytic freshwater prorocentroid ancestor. The unique marine species in the freshwater clade results from an ecophysiological reversion. P. rivalis differs from other epiphytic taxa by its rarity, its temperate distribution and its ecophysiological needs. The phylogeny confirms also that all planktonic Prorocentrum species are evolutionarily derived from epiphytic/benthic ancestors.     

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.     

PHYLOGENETIC ANALYSIS OF NINE SPECIES OF PROROCENTRUM (DINOPHYCEAE) INFERRED FROM 18S RIBOSOMAL DNA SEQUENCES, MORPHOLOGICAL COMPARISONS, AND DESCRIPTION OF PROROCENTRUM PANAMENSIS, SP. NOV.

Sequences of 18S rRNA genes were obtained from eight species of Prorocentrum Ehrenberg: P. minimum (Pavillard) Schiller, P. mexicanum Osorio Tafall, P. emarginatum Fukuyo, P. lima (Ehrenberg) Dodge, P. arenarium Faust, P. maculosum Faust, P. concavum Fukuyo, and P. panamensis, sp. nov. Prorocentrum panamensis is a new species of tropical dinoflagellate isolated from a benthic coral reef on the Pacific coast of Panama and described here using scanning electron microscopy. Cells are heart shaped, 46–52 μm long and 43–46 μm wide. The valve surfaces are areolate except in the central area. Pores of 0.15 μm in diameter are scattered in areolae, mainly around the periphery of the cell. The right valve has a specific ovoid depression with numerous appressed pores; we named this structure the sieve-like depression. The periflagellar area is nearly ovoid, located in a shallow depression, and almost equally set into both valves. It is unornamented (no apical expansion) but has numerous depressions in platelets. The flagellar and auxiliary pores are different in size and shape. The intercalary band is transversally striated. Phylogenetic relationships of gonyaulacoid, peridinioid, gymnodinioid, and prorocentroid dinoflagellates were inferred from complete 18S rDNA sequences. Two distinct phylogenetic analyses are presented for armored and unarmored Dinophyceae in an attempt to make the phylogenetic relationships between these different kinds of organisms clearer. The Prorocentrales appear to have a common origin, although two groups of Prorocentrum spp. are apparent. The first group includes benthic, symmetrical species (P. lima, P. arenarium, P. maculosum, and P. concavum). The second group contains planktonic and bentho-planktonic species (P. micans Ehrenberg, P. minimum, P. mexicanum, and P. panamensis sp. nov.). Genetic distances between species within these two groups were high; however, the divergence between the two groups seems to have occurred late in dinoflagellate evolution. In addition, the bentho-planktonic P. emarginatum appeared distantly related to both groups; however,its 18S rDNA sequence shares specific nucleotide substitutions with the two groups, suggesting an older origin of this species compared to the others. A morphological interpretation of this phylogenetic analysis is made on the basis of the specific structure of the periflagellar area. Finally, genetic data and morphological observations support the hypothesis that the genus Prorocentrum is rather heterogeneous; several species could be considered to constitute distinct genera.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF PROROCENTRUM CONSUTUM SP. NOV. (DINOPHYCEAE), A NEW BENTHIC DINOFLAGELLATE FROM SOUTH BRITTANY (NORTHWESTERN FRANCE)1

A new marine benthic Prorocentrum species from sandy habitats of South Brittany (northwestern France), P. consutum sp. nov., is described using LM and SEM and molecular phylogenetic analyses. Cells have a subcircular to broadly ovoid shape and are plainly flattened. They are 57–61 μm long and 52–55 μm wide. A central pyrenoid is present, and the kidney‐shaped nucleus is positioned in the posterior region. In right valve view, the periflagellar area is deeply excavated, and the left valve forms a prominent apical ridge. The periflagellar area consists of nine platelets, and a small narrow collar is present around the flagellar pore. The ornamentation of this new species is very peculiar and is characterized by a ring of round areolae located at the periphery of the valves, each areola containing three or four pores. Apart from this ring of areolae, the cell surface is smooth and with scattered pores. Pores are not present in the center of the right or left valve. The intercalary band is generally narrow and faintly striated horizontally. The molecular phylogenetic position of P. consutum sp. nov. was inferred using SSU and LSU rDNA. In both analyses, this species branched with high support in the clade comprising species with a symmetric shape and appeared to be a sister group to that formed by P. lima and other tropical benthic species, such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum.     

Morphology and Molecular Phylogeny of a New Marine,Sand-dwelling Dinoflagellate Genus,Pachena (Dinophyceae), with Descriptions of Three New Species

Marine benthic dinoflagellates are interesting not only because some epiphytic genera can cause harmful algal blooms but also for understanding dinoflagellate evolution and diversification. Our understanding of their biodiversity is far from complete, and many thecate genera have unusual tabulation patterns that are difficult to relate to the diverse known phytoplankton taxa. A new sand-dwelling genus, Pachena gen. nov., is described based on morphological and DNA sequence data. Three species were discovered in distant locations and are circumscribed, namely, P. leibnizii sp. nov. from Canada, P. abriliae sp. nov. from Spain, and P. meriddae sp. nov. from Italy. All species are tiny (about 9–23 μm long) and heterotrophic. Species are characterized by their tabulation (APC 4′ 3a 6′′ 5c 5s 5′′′ 2′′′′), an apical hook covering the apical pore, an ascending cingulum, and a sulcus with central list. The first anterior intercalary plate is uniquely “sandwiched” between two plates. The species share these features and differ in the relative sizes and arrangements of their plates, especially on the epitheca. The ornamentation of thecal plates is species-specific. The new molecular phylogenies based on SSU and LSU rDNA sequences contribute to understanding the evolution of the planktonic relatives of Pachena, the Thoracosphaeraceae.     

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.     

PROROCENTRUM BIMACULATUM SP. NOV. (DINOPHYCEAE,PROROCENTRALES), A NEW BENTHIC DINOFLAGELLATE SPECIES FROM KUWAIT (ARABIAN GULF)1

A new benthic dinoflagellate species, Prorocentrum bimaculatum sp. nov., is studied from Kuwait’s marine sediments, based on detailed morphological and molecular data. Cells are large, oblong oval in shape. They are 49.9–55.3 μm long and 38.4–43.2 μm wide. The ornamentation of this new species is peculiar, and characterized by smooth valves with large pores (0.32–0.50 μm) scattered on their surface, except in two circular patches of ～15 μm in diameter, devoid of ornamentation and located on both sides of the valve centers. The periflagellar area is widely triangular, located in a moderate excavation of the right valve, and comprises nine platelets. The intercalary band of P. bimaculatum is smooth. The molecular phylogenetic position of this new taxon was inferred from SSU and LSU rDNA genes. In both phylogenetic analyses, P. bimaculatum branched with high support with Prorocentrum consutum and formed a clade sister to the one including P. lima and related species such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum. From the phylogenetic study, since most species related to P. bimaculatum are known for their toxic effects and production of okadaic acid, this new species can be considered as a potential toxin producer, but this has to be analyzed.     

CRYPTIC AND PSEUDO‐CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONS OF PSEUDO‐NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

A high degree of pseudo‐cryptic diversity was reported in the well‐studied diatom genus Pseudo‐nitzschia. Studies off the coast of Washington State revealed the presence of hitherto undescribed diversity of Pseudo‐nitzschia. Forty‐one clonal strains, representing six different taxa of the P. pseudodelicatissima complex, were studied morphologically using LM and EM, and genetically using genes from three different cellular compartments: the nucleus (D1–D3 of the LSU of rDNA and internal transcribed spacers [ITSs] of rDNA), the mitochondria (cytochrome c oxidase 1), and the plastids (LSU of RUBISCO). Strains in culture at the same time were used in mating studies to study reproductive isolation of species, and selected strains were examined for the production of the neurotoxin domoic acid (DA). Two new species, P. hasleana sp. nov. and P. fryxelliana sp. nov., are described based on morphological and molecular data. In all phylogenetic analyses, P. hasleana appeared as sister taxa to a clade comprising P. calliantha and P. mannii, whereas the position of P. fryxelliana was more uncertain. In the phylogenies of ITS, P. fryxelliana appeared to be most closely related to P. cf. turgidula. Morphologically, P. hasleana differed from most other species of the complex because of a lower density of fibulae, whereas P. fryxelliana had fewer sectors in the poroids and a higher poroid density than most of the other species. P. hasleana did not produce detectable levels of DA; P. fryxelliana was unfortunately not tested. In P. cuspidata, production of DA in offspring cultures varied from higher than the parent cultures to undetectable.     

Power law relationships among hierarchical taxonomic categories in algae reveal a new paradox of the plankton

Aim In this continental‐scale study, the biodiversity of benthic and planktonic algal communities was explored. A recent analysis of extinct and extant tree communities by Enquist et al. (2002) showed that richness of higher taxa was a power function of species richness, invariant across temporal and spatial scales. Here we examined whether the relationships between algal richness at hierarchical taxonomic levels conform to power laws as seen for trees, and if these relationships differ between benthic and planktonic habitats. Location Streams from more than 50 major watersheds in the United States. Method A total of 3698 samples were collected from 1277 locations by the National Water‐Quality Assessment Program. Three types of stream habitat were sampled: richest targeted habitats, depositional targeted habitats, and phytoplankton. The relationships between taxonomic richness at the species level vs. all higher categories from genus to phylum across the three habitats were examined by ordinary least squares (OLS) regressions after ln‐transformation of all variables. The slopes, b, of these regressions represent the exponents of the power functions that scaled the richness of higher taxonomic levels (T) to species richness (S) in the form: T∝S^b. Results Algal richness at hierarchical taxonomic categories (genus to phylum) is a power function of species richness. The scaling exponent of this function, which captures the diversification of higher taxa, i.e. the rate of increase of their richness with the increase of species richness, is significantly different across environments. Main conclusions The differential algal diversification in the three studied habitats emphasizes the fundamental role of the environment in structuring the communities of simple organisms such as algae. The finding that the diversification of higher taxa is greater in the seemingly homogeneous planktonic environment, when compared to benthic habitats, encompassing an array of ecological niches, poses a new paradox of the plankton.     

Morphology,phylogeny, dynamics,and ichthyotoxicity of Pheopolykrikos hartmannii (Dinophyceae) isolates and blooms from New York,USA

We report on morphological observations, phylogenetic analyses, bloom dynamics, and ichthyotoxicity of the common but poorly characterized dinoflagellate Pheopolykrikos hartmannii (Zimmermann) Matsuoka et Fukuyo. From 2008 to 2010 in the Forge River Estuary, NY, USA, P. hartmannii bloomed during summer and early fall, achieving densities exceeding 8,000 cells · mL^?1 and often dominating microphytoplankton communities. Large subunit (LSU) and small subunit (SSU) rDNA sequences demonstrated that NY isolates of P. hartmannii sequences were 99%–100% identical to P. hartmannii isolates from eastern US and Korea. In both the LSU and SSU rDNA phylogenies, the clades containing P. hartmannii sequences were distinct sister clades to those composed of Polykrikos schwartzii and P. kofoidii. In the LSU rDNA phylogeny, however, the clade composed of P. hartmannii and a sequence of the photosynthetic Polykrikos lebourae was well separated from the clade composed of 10 entries of Polykrikos schwartzii and P. kofoidii. In addition, a gap of ~180 bases was observed when the LSU rDNA sequences of P. hartmannii were aligned with P. schwartzii and P. kofoidii but was not observed in the alignment between P. hartmannii and P. lebourae. Using scanning electron microscopy, several morphological features previously not reported for P. hartmannii were observed: a ventral groove located in the sulcus, a deep arc‐like apical concavity within the area of apical groove, scale‐like vesicles, and a shallow, completely enclosed, loop‐like apical groove. Resting cysts with arrow‐like surface spines were produced heterothallically by crossing clonal isolates and germinated single gymnoid cells. Finally, filtered and unfiltered bloom water from the Forge River and clonal cultures of P. hartmannii exhibited acute ichthyotoxicity to juvenile sheepshead minnows (Cyprinodon variegates) and aeration did not mitigate this effect, suggesting P. hartmannii is an ichthyotoxic, harmful alga.     

ON THE IDENTITY OF KARLODINIUM VENEFICUM AND DESCRIPTION OF KARLODINIUM ARMIGER SP. NOV. (DINOPHYCEAE), BASED ON LIGHT AND ELECTRON MICROSCOPY,NUCLEAR‐ENCODED LSU RDNA,AND PIGMENT COMPOSITION1

An undescribed species of the dinoflagellate genus Karlodinium J. Larsen (viz. K. armiger sp. nov.) is described from Alfacs Bay (Spain), using light and electron microscopy, pigment composition, and partial large subunit (LSU) rDNA sequence. The new species differs from the type species of Karlodinium (K. micrum (Leadbeater et Dodge) J. Larsen) by lacking rows of amphiesmal plugs, a feature presently considered to be a characteristic of Karlodinium. In K. armiger, an outer membrane is underlain by a complex system of cisternae and vacuoles. The pigment profile of K. armiger revealed the presence of chlorophylls a and c, with fucoxanthin as the major carotenoid. Phylogenetic analysis confirmed K. armiger to be related to other species of Karlodinium; thus forming a monophyletic genus, which, in the LSU tree, occupies a sister group position to Takayama de Salas, Bolch, Botes et Hallegraeff. The culture used by Ballantine to describe Gymnodinium veneficum Ballantine (Plymouth 103) was examined by light and electron microscopy and by partial LSU rDNA. Ultrastructurally, it proved identical to K. micrum (cultures Plymouth 207 and K. Tangen KT‐77D, the latter also known as K‐0522), and in LSU sequence, differed in only 0.3% of 1438 bp. We consider the two taxa to belong to the same species. This necessitates a change of name for the most widely found species, K. micrum, to K. veneficum. The three genera Karlodinium, Takayama, and Karenia constitute a separate evolutionary lineage, for which the new family Kareniaceae fam. nov. is suggested.     

The phylogeny and taxonomy of genera Cystoderma and Cystodermella (Agaricales) based on nuclear ITS and LSU sequences

Species delimitation in Cystoderma and Cystodermella was evaluated based on ITS and LSU rDNA sequences as well as morphological data. Two species of Cystoderma are synonymised with C. carcharias and three species with C. jasonis, distinguishing the synonymised taxa as varieties of these accepted species. Analyses of partial LSU rDNA sequences revealed Cystoderma and Cystodermella as distinct monophyletic genera, with Ripartitella representing a well-supported sister group of the latter. Phaeolepiota aurea represents either an unsupported sister group or member of Cystoderma in the phylogenies based on LSU and ITS sequences rDNA data, respectively. The tribe Cystodermateae sensu Singer did not appear monophyletic according to analyses of LSU sequences. On the basis of these data, the phylogenetic relationships among most of the analyzed genera could not be resolved unequivocally.     

PHYLOGENETICS OF RHINODINIUM BROOMEENSE GEN. ET SP. NOV., A PERIDINIOID,SAND‐DWELLING DINOFLAGELLATE (DINOPHYCEAE)1

The phylogeny of Rhinodinium broomeense, a new genus and species of heterotrophic peridinioid dinoflagellates, has been studied based on morphological and molecular genetic data. The genus was found in tidal marine sand habitats in Broome, north‐western Australia, and from three marine sand habitats in Japan. The thecal plate formula is Po 3′ 1^a 5″ 4c ?s 5″′ 1″″. A large apical hook points toward the dorsal side. Its plate pattern is similar to species of the genus Roscoffia; however, it differs from that genus in its much larger epitheca, narrow cingulum, which could be interpreted as incomplete, the narrow sulcus without sulcal lists on both sides, and the strong oblique lateral compression. Phylogenetic analyses using partial LSU rDNA sequences, as well as plate pattern information, support the placement of this genus in the Peridiniales; however, it is sufficiently different from other genera that the family affinity remains unclear.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.