Ultrastructure and phylogeny of Parvodinium cunningtonii comb. nov. (syn. Peridiniopsis cunningtonii) and description of P. cunningtonii var. inerme var. nov. (Peridiniopsidaceae, Dinophyceae).

Two strains of peridinioids were isolated from a flooded stream near Aveiro, central Portugal, and examined by light microscopy, scanning electron microscopy and serial-section transmission electron microscopy. The two strains showed the same tabulation and cell shape as Peridiniopsis cunningtonii. One of the strains had lightly reticulated plates and spines in most hypothecal plates, matching the features of typical P. cunningtonii. The other strain showed smooth plates and consistently lacked spines in the apiculate hypotheca. The strains were similar in fine structure and had a central pyrenoid with a starch sheath and perforated by cytoplasmic channels. Details of the flagellar apparatus matched those known from Parvodinium, as did the remarkably long microtubular strand leading to an extruded peduncle that was visible in serial sections. Phylogenetic analyses based on partial LSU rDNA and the concatenated ribosomal operon placed the strain with the smooth hypotheca in a clade with Parvodinium species. The two strains grouped as closely related sister taxa in the partial LSU rDNA phylogeny. A new combination is proposed, Parvodinium cunningtonii comb. nov. and a new variety, Parvodinium cunningtonii var. inerme var. nov., is described.

Fine-structural characterization and phylogeny of Sphaerodinium (Suessiales, Dinophyceae), with the description of an unusual type of freshwater dinoflagellate cyst.

Two strains of Sphaerodinium were established from two mountain areas in Portugal and examined by light microscopy, scanning and transmission electron microscopy, and sequence analyses of nuclear-encoded SSU, ITS1-5.8S-ITS2 and LSU rDNA. Both strains were identified as S. polonicum var. tatricum on the basis of comparison with the original taxonomic descriptions within the genus. The two strains were nearly identical in morphology and ultrastructure, except for the presence of pseudograna-like thylakoid stacks within more rounded chloroplast lobes in one of them. Sexual reproduction occurred in culture batches and resting cysts with single or grouped processes with wide bases and distal platforms with slightly recurved margins were seen to develop by sudden retraction of planozygote cytoplasm. Morphological, fine-structural and molecular characters were compared with previously available information from S. cracoviense, allowing for a more robust characterization of the genus. Important characters include a type F eyespot, a pusule canal linking the transverse flagellar canal to a collecting chamber connected to regular pusular tubes, a ventral fibre extending from the proximal-right side of the longitudinal basal body, and a membranous, lamellar body with a honeycomb pattern near the flagellar base area. The latter two features are shared with Baldinia anauniensis.

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.

Studies on Woloszynskioid Dinoflagellates X: Ultrastructure, Phylogeny and Colour Variation in Tovellia rubescens n. sp. (Dinophyceae).

The external morphology and internal cell fine structure of a new species of Tovelliaceae, Tovellia rubescens n. sp., is described. Phylogenetic analyses based on partial LSU rDNA sequences place the new species in a clade containing Tovellia species that accumulate red pigments and identify T. aveirensis as its closest known relative. Cells of T. rubescens n. sp. were mostly round and had the cingulum located near the middle, with its ends displaced about one cingular width. Small numbers of distinctly flat cells appeared in culture batches; their significance could not be determined. Cells of the new species in culture batches progressively changed from a yellowish-green, mainly due to chloroplast colour, to a reddish-brown colour that appeared associated with lipid bodies. The switch to a reddish colour happened earlier in batches grown in medium lacking sources of N or P. Pigment analyses by HPLC-MS/MS revealed the presence of astaxanthin and astaxanthin-related metabolites in the new species, but also in T. aveirensis, in which a reddish colour was never observed. The chloroplast arrangement of T. rubescens n. sp. resembled that of T. aveirensis, with lobes radiating from a central pyrenoid complex. The flagellar apparatus and pusular system fell within the general features described from other Tovelliaceae. A row of microtubules interpretable as a microtubular strand of the peduncle was present. Spiny resting cysts with red contents and an ITS sequence identical to that of cultured material of the new species were found in the original locality.

Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments.

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20-70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here.

Chaetoceros pauciramosus sp. nov. (Bacillariophyceae), a Widely Distributed Brackish Water Species in the C. lorenzianus Complex.

To understand the diversity of the diatom genus Chaetoceros Ehrenberg, the C. lorenzianus complex was previously chosen as a model and three new species were described. In the present study, yet another new species, Chaetoceros pauciramosus sp. nov., was discovered from Chinese tropical waters and the temperate south-eastern Baltic Sea. In the latter locality it has been known since 2003 and identified as C. cf lorenzianus, and now found to comprise a dominating component of the autumn phytoplankton community. Vegetative cells and resting spores were examined by light and electron microscopy. The LSU and SSU of the nuclear rDNA were sequenced. The characters suggesting that C. pauciramosus belongs to C. lorenzianus complex are: 1) the vegetative cells contain four or more chloroplasts, 2) the cells have stiff setae and form regular straight chains, 3) the terminal setae differ in the direction from the intercalary setae. The relationship was well-supported by molecular phylogenetic analyses inferred from both LSU and SSU markers. The primary valve of the resting spore has two conical elevations, each with dichotomously branching processes, similar to other species in the C. lorenzianus complex. Each process forms a tree-like structure with the pointed distal tips which possess one or two thin spikes, that distinguishes C. pauciramosus from allied taxa. In the phylogenetic trees, Chaetoceros pauciramosus clustered with C. elegans, in agreement with their morphologically similar resting spores.

Morphological, molecular and toxigenic characteristics of Namibian Pseudo-nitzschia species - including Pseudo-nitzschia bucculenta sp. nov.

A field study was undertaken to investigate the occurrence and toxin production of species in the diatom genus Pseudo-nitzschia in Namibian waters, in the extremely productive Benguela upwelling system. From surveys conducted on the R/V Mirabilis and the R/V !Anichab, 52 strains were morphologically determined to species level, supported by nuclear ITS rDNA data. Seven species were identified; P. australis, P. decipiens, P. dolorosa, P. fraudulenta, P. plurisecta, P. pungens var. cingulata, and the new species P. bucculenta F. Gai, C. K. Hedemand, N. Lundholm & Ø. Moestrup sp. nov. Molecular and morphological diversity of the Namibian Pseudo-nitzschia species is discussed. Most importantly, P. bucculenta is both morphologically and phylogenetically most similar to P. dolorosa differing mainly in valve width and densities of striae, poroids and band striae as well as by four hemi-compensatory base changes in the ITS2. Morphological and molecular differences among the strains of P. decipiens suggest a temperate and a warm water subdivision. The geographical and toxigenic characteristics of the identified Pseudo-nitzschia species are described and compared to previous studies. Initial tests of toxin production in all seven species revealed production of domoic acid (DA) in two species: one strain of P. australis (0.074 pg DA cell-1) and two strains of P. plurisecta (0.338 pg DA cell-1 and 0.385 pg DA cell-1).

The Rainwater Rock-pool Dinoflagellate Nottbeckia ochracea gen. et comb. nov. (syn.: Hemidinium ochraceum) - A Fine-structural and Molecular Study with Emphasis on the Motile Stage.

The rarely recorded dinoflagellate Hemidinium ochraceum Levander was isolated from a rainwater rock-pool in Finland and the motile stage studied by light- and electron microscopy and molecular sequencing. The ultrastructure of the motile stage revealed several features not previously observed in other dinoflagellates, e.g. thecal plates with inwards 'knobs' and a peculiar corrugated left sulcal plate. The apparent lack of the R2 and R4 flagellar roots was also unusual. The typical plate tabulation was 4', 2a, 8″, cx, 4c,?s, 3‴, cy, 1p, 2″″, cx and cy being extra plates associated with the cingulum, but intraspecific variations were observed in shape and number of plates. Phylogenetic analyses of nuclear-encoded LSU rDNA and concatenation of LSU and SSU rDNA sequences showed that H. ochraceum was only distantly related to Hemidinium nasutum (the type species of Hemidinium). The immotile coccoid stage resembled Gloeodinium montanum and a weakly supported relationship between the two was only inferred in the analyses based on LSU rDNA. Hemidinium ochraceum showed some resemblance to Rufusiella insignis sensu Richards, but the true identity of this species and of genus Rufusiella is uncertain. A new genus, Nottbeckia, is erected to accommodate H. ochraceum, as Nottbeckia ochracea gen. et comb. nov.

Ultrastructure and Phylogeny of Kirithra asteri gen. et sp. nov. (Ceratoperidiniaceae, Dinophyceae) - a Free-Living, Thin-Walled Marine Photosynthetic Dinoflagellate from Argentina.

A gymnodinioid photosynthetic dinoflagellate was isolated from Argentina and examined by light and electron microscopy and analysis of nuclear-encoded LSU rDNA. Kirithra asteri gen. et sp. nov. was proposed as morphology and molecular phylogeny separated this dinoflagellate from others within the family Ceratoperidiniaceae. Cells were surrounded by a hyaline amphiesma comprising polygonal vesicles. Each vesicle contained a honeycomb and a trilaminar structure. An anterior sulcal extension ending in a complete circle formed the apical structure complex (ASC), which characterizes Ceratoperidiniaceae. The ASC comprised three rows of vesicles. The nucleus was located in the hypocone, and several large, irregularly shaped vesicles were present in the epi- and hypocone. Chloroplasts were surrounded by three membranes, and grana-like arrangements of thylakoids were observed in one strain used for ultrastructural study. The cell centre contained 1-3 multiple-stalked pyrenoids and membrane-bound vesicles containing tile-like structures surrounded each pyrenoid. Two pusules with collecting chambers and associated vesicles branched off each of the flagellar canals. The flagellar apparatus featured a ventral connective between the amphiesma and the R1 root, and almost opposite basal bodies, rarely seen in dinoflagellates. This was the first ultrastructural study of a species within Ceratoperidiniaceae.

Effects of irradiance and prey deprivation on growth, cell carbon and photosynthetic activity of the freshwater kleptoplastidic dinoflagellate Nusuttodinium (= Gymnodinium) aeruginosum (Dinophyceae).

The freshwater dinoflagellate Nusuttodinium aeruginosum lacks permanent chloroplasts. Rather it sequesters chloroplasts as well as other cell organelles, like mitochondria and nuclei, from ingested cryptophyte prey. In the present study, growth rates, cell production and photosynthesis were measured at seven irradiances, ranging from 10 to 140 µmol photons m-2s-1, when fed the cryptophyte Chroomonas sp. Growth rates were positively influenced by irradiance and increased from 0.025 d-1 at 10 µmol photons m-2s-1 to maximum growth rates of ~0.3 d-1 at irradiances ≥ 40 µmol photons m-2s-1. Similarly, photosynthesis ranged from 1.84 to 36.9 pg C cell-1 h-1 at 10 and 140 µmol photons m-2s-1, respectively. The highest rates of photosynthesis in N. aeruginosum only corresponded to ~25% of its own cell carbon content and estimated biomass production. The measured rates of photosynthesis could not explain the observed growth rates at high irradiances. Cultures of N. aeruginosum subjected to prey starvation were able to survive for at least 27 days in the light. The sequestered chloroplasts maintained their photosynthetic activity during the entire period of starvation, during which the population underwent 4 cell divisions. This indicates that N. aeruginosum has some control of the chloroplasts, which may be able to replicate. In conclusion, N. aeruginosum seems to be in an early stage of chloroplast acquisition with some control of its ingested chloroplasts.

Dactylodinium pterobelotum gen. et sp. nov., a new marine woloszynskioid dinoflagellate positioned between the two families Borghiellaceae and Suessiaceae.

A new marine woloszynskioid dinoflagellate Dactylodinium pterobelotum gen. et sp. nov., collected from a southern Vietnamese estuary, was described on the basis of LM, SEM, and TEM, and molecular phylogeny inferred from rDNA sequences. This species had the smallest number of amphiesmal vesicles (5 latitudinal series) in woloszynskioid dinoflagellates assigned to the Suessiaceae and Borghiellaceae. The eyespot was of type B, composed of osmiophilic globules and brick-like material, located in- and outside of the chloroplast respectively. An apical structure comprised a pair of elongate anterior vesicles (PEV). A large peduncle was conspicuous, located in the sulcal extension in the epicone, and supported by a microtubular strand of ~140 microtubules. Ultrastructural features of trichocysts represent a novel type in the Dinophyceae, bearing lateral hairs besides anterior fibers. The molecular phylogeny based on partial LSU rDNA showed the species in a basal position in the family Suessiaceae; this indicates the eyespot type B and PEV of the Borghiellaceae are ancestral states of the eyespot comprising brick-like material (type E) and an elongate apical vesicle of the Suessiaceae.

Diversity in the Globally Distributed Diatom Genus Chaetoceros (Bacillariophyceae): Three New Species from Warm-Temperate Waters.

Chaetoceros is one of the most species rich, widespread and abundant diatom genera in marine and brackish habitats worldwide. It therefore forms an excellent model for in-depth biodiversity studies, assessing morphological and genetic differentiation among groups of strains. The global Chaetoceros lorenzianus complex presently comprises three species known to science. However, our recent studies have shown that the group includes several previously unknown species. In this article, 50 strains, mainly from high latitudes and from warm-temperate waters, were examined morphologically and genetically and the results compared with those of field studies from elsewhere. The strains clustered into five groups, two of which are formed by C. decipiens Cleve and C. mitra (Bailey) Cleve, respectively. Their species descriptions are emended based on samples collected close to the type localities. The three other groups are formed by new species, C. elegans sp. nov., C. laevisporus sp. nov. and C. mannaii sp. nov. Characters used to distinguish each species are: orientation of setae, shape and size of the apertures, shape, size and density of the poroids on the setae and, at least in some species, characters of the resting spores. Our aim is to cover the global species diversity in this complex, as correct species delineation is the basis for exploring biodiversity, distribution of organisms, interactions in the food web and effects of environmental changes.

Yihiella yeosuensis gen. et sp. nov. (suessiaceae, dinophyceae), a novel dinoflagellate isolated from the coastal waters of Korea.

A small (7-11 µm long) dinoflagellate with thin amphiesmal plates was isolated into culture from a water sample collected in coastal waters of Yeosu, southern Korea, and examined by LM, SEM, and TEM, and molecular analyses. The hemispheric episome was smaller than the hyposome. The nucleus was oval and situated from the central to the episomal region of the cell. A large yellowish-brown chloroplast was located at the end of the hyposome, and some small chloroplasts extended into the periphery of the episome. The dinoflagellate had a single elongated apical vesicle (EAV) and a type E eyespot, which are key characteristics of the family Suessiaceae. Unlike other genera in this family, it had two long furrow lines, one on the episome and the other on the hyposome, and encircling the dorsal, and lateral sides of the cell body. The pyrenoid lacked starch sheaths, but tubular invaginations into the pyrenoid matrix from the cytoplasm were observed. In the TEM, the dinoflagellate was observed to have cable-like structures (CLSs) near the eyespot but so far not observed in other dinoflagellates. The SSU rDNA sequences examined were 1.2%-5.1% different from those of other genera in the family Suessiaceae, whereas the LSU (D1-D3) rDNA sequences of this dinoflagellate were 15.1%-31.5% different. The dinoflagellate lacked a 51-bp fragment in domain D2 of the LSU rDNA, but it had an ~100-bp fragment in domain D2. This feature has been found previously only in the genera Leiocephalium and Polarella, two other genera of the Suessiaceae. The molecular phylogeny and sequence divergence based on SSU, and LSU rDNA indicate that the Korean dinoflagellate holds a taxonomically distinctive position and we consider it to be a new species in a new genus in the family Suessiaceae, named Yihiella yeosuensis gen. et sp. nov.

Why Plants Were Terrestrial from the Beginning.

The current hypothesis is that land plants originated from a charophycean green alga and that a prominent feature for adaptation to land was their development of alternating life cycles. Our work on cell wall evolution and morphological and physiological observations in the charophycean green algae challenged us to reassess how land plants became terrestrial. Our hypothesis is simple in that the charophycean green algae ancestors were already living on land and had been doing so for some time before the emergence of land plants. The evolution of alternate life cycles merely made the ancestral land plants evolutionary successful and had nothing to do with terrestrialization per se.

Two New Freshwater Woloszynskioids Asulcocephalium miricentonis gen. et sp. nov. and Leiocephalium pseudosanguineum gen. et sp. nov. (Suessiaceae, Dinophyceae) Lacking an Apical Furrow Apparatus.

Two new woloszynskioid dinoflagellates, Asulcocephalium miricentonis gen. et sp. nov. and Leiocephalium pseudosanguineum gen. et sp. nov., are described from Japanese freshwater ponds on the basis of bright field and fluorescence light microscopy, scanning and transmission electron microscopy, and molecular phylogeny inferred from rDNA sequences. Asulcocephalium miricentonis has a spherical anterior nucleus and chloroplast with a pyrenoid penetrated by the cytoplasm. This species has 9-12 latitudinal series of amphiesmal vesicles (AVs), including an apparently large AV on the right ventral side of the epicone. Leiocephalium pseudosanguineum has a U-shaped nucleus in the epicone and chloroplasts without a pyrenoid. This species has at least 24 latitudinal series of AVs. The characteristic features of both species were brick-like material (type E) in the eyespot and the lack of an apical furrow. These features coincide with those of Polarella glacialis, but the two species differ in cell shape, number and arrangement of AVs, shape of resting cysts, and habitats; i.e., P. glacialis has been reported only from marine cold waters. Molecular phylogeny revealed that A. miricentonis and L. pseudosanguineum were positioned in the Suessiaceae and closely related to Piscinoodinium sp., but their relationship to Polarella and other reported taxa was not supported.

Fine-structural characterization and phylogeny of Peridinium polonicum, type species of the recently described genus Naiadinium (Dinophyceae).

Peridinium polonicum is a freshwater peridinioid with an unusual tabulation that includes one or two anterior intercalary plates in the mid-dorsal axis, and in such a low position that it seems inset in precingular Plate 4. Although the species has been classified in both Peridinium and Peridiniopsis, evidence from nucleotide sequences consistently shows that its closest relatives are within the Scrippsiella group. The genus Naiadinium Carty has been recently described with P. polonicum as its type species. However, Naiadinium was separated from other peridinioids only on the basis of shape and plate arrangements and these characters do not allow reliable determination of its closest phylogenetic relatives. Serial section fine-structural analysis revealed the presence of a small peduncle supported by a conspicuous microtubular basket that extended far into the cell; a complex pusular system that included a collecting chamber from which about 70 pusular tubes radiated; a flagellar apparatus with general peridinioid characters but with an unusually large distance of nearly 700 nm between basal bodies. An ITS1-5.8S-ITS2 rDNA-based phylogenetic analysis grouped, with high statistical support, Naiadinium polonicum with three species currently placed in Scrippsiella, viz. S. irregularis, S. precaria and S. ramonii.

Recent radiation in a marine and freshwater dinoflagellate species flock.

Processes of rapid radiation among unicellular eukaryotes are much less studied than among multicellular organisms. We have investigated a lineage of cold-water microeukaryotes (protists) that appear to have diverged recently. This lineage stands in stark contrast to known examples of phylogenetically closely related protists, in which genetic difference is typically larger than morphological differences. We found that the group not only consists of the marine-brackish dinoflagellate species Scrippsiella hangoei and the freshwater species Peridinium aciculiferum as discovered previously but also of a whole species flock. The additional species include Peridinium euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient Lake Baikal, Russia, and freshwater S. hangoei from Lake Baikal. These species are characterized by relatively large conspicuous morphological differences, which have given rise to the different species descriptions. However, our scanning electron microscopic studies indicate that they belong to a single genus according to traditional morphological characterization of dinoflagellates (thecal plate patterns). Moreover, we found that they have identical SSU (small subunit) rDNA fragments and distinct but very small differences in the DNA markers LSU (large subunit) rDNA, ITS2 (internal transcribed spacer 2) and COB (cytochrome b) gene, which are used to delineate dinoflagellates species. As some of the species co-occur, and all four have small but species-specific sequence differences, we suggest that these taxa are not a case of phenotypic plasticity but originated via recent adaptive radiation. We propose that this is the first clear example among free-living microeukaryotes of recent rapid diversification into several species followed by dispersion to environments with different ecological conditions.

Strains of the Morphospecies Ploeotia costata (Euglenozoa) Isolated from the Western North Pacific (Taiwan) Reveal Substantial Genetic Differences.

Two phagotrophic euglenid strains (Strains Pac and Tam) were isolated from coastal locations in Taiwan. Ultrastructural characteristics of the strains included five pellicle strips joined at the posterior end. The strips were formed by major grooves with bifurcated edges. At the cell anterior, the feeding structure formed a lip. Underneath the lip was a comb composed of layers of microtubules. Farther back, two supporting rods tapered toward the posterior end, and a number of vanes with attached microtubules were present between the rods. The morphological characteristics agree with Ploeotia costata Strain CCAP 1265/1. However, the 18S rDNA sequences of Strains Pac/Tam lacked a group I intron and possessed three extra insertions of 116, 67, and 53 bp. Phylogenetic analysis indicated low sequence similarity between Strains Pac/Tam and CCAP 1265/1 (92%). The morphospecies P. costata apparently includes a substantial level of DNA sequence divergence, and likely represents multiple molecular species units.