Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae,Stramenopiles)

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans‐neoxanthin, cis‐neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan‐thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well‐described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell‐specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.     

Baffinella frigidus gen. et sp. nov. (Baffinellaceae fam. nov., Cryptophyceae) from Baffin Bay: Morphology,pigment profile,phylogeny, and growth rate response to three abiotic factors

Twenty years ago an Arctic cryptophyte was isolated from Baffin Bay and given strain number CCMP 2045. Here, it was described using morphology, water‐ and non‐water soluble pigments and nuclear‐encoded SSU rDNA . The influence of temperature, salinity, and light intensity on growth rates was also examined. Microscopy revealed typical cryptophyte features but the chloroplast color was either green or red depending on the light intensity provided. Phycoerythrin (Cr‐PE 566) was only produced when cells were grown under low‐light conditions (5 μmol photons · m^?2 · s^?1). Non‐water‐soluble pigments included chlorophyll a , c ₂ and five major carotenoids. Cells measured 8.2 × 5.1 μm and a tail‐like appendage gave them a comma‐shape. The nucleus was located posteriorly and a horseshoe‐shaped chloroplast contained a single pyrenoid. Ejectosomes of two sizes and a nucleomorph anterior to the pyrenoid were discerned in TEM . SEM revealed a slightly elevated vestibular plate in the vestibulum. The inner periplast component consisted of slightly overlapping hexagonal plates arranged in 16–20 oblique rows. Antapical plates were smaller and their shape less profound. Temperature and salinity studies revealed CCMP 2045 as stenothermal and euryhaline and growth was saturated between 5 and 20 μmol photons · m^?2 · s^?1. The phylogeny based on SSU rDNA showed that CCMP 2045 formed a distinct clade with CCMP 2293 and Falcomonas sp. isolated from Spain. Combining pheno‐ and genotypic data, the Arctic cryptophyte could not be placed in an existing family and genus and therefore Baffinellaceae fam. nov. and Baffinella frigidus gen. et sp. nov. were proposed.     

MOLECULAR PHYLOGENY OF PHYCOCYANIN‐CONTAINING CRYPTOPHYTES: EVOLUTION OF BILIPROTEINS AND GEOGRAPHICAL DISTRIBUTION1

Of 34 strains assigned to the cryptophyte genera Chroomonas Hansg., Hemiselmis Parke, and Komma D. R. A. Hill, distribution patterns of biliproteins, habitats, and sampling sites across a phylogenetic tree have been examined. The combined data set assembled from nuclear SSU rDNA, partial nuclear LSU rDNA, and nucleomorph SSU rDNA sequences comprised 4,083 positions and yielded an almost completely resolved tree. Spectrophotometry of the biliproteins and mapping of the different types of biliproteins onto the phylogenetic tree unveiled a complex evolutionary history. Different from other cryptophyte clades, the types of biliproteins were not generally congruent with clades or subclades of the genera Chroomonas (paraphyletic, phycocyanins [PCs] 645 or 630), Hemiselmis (PCs 612, 630 or phycoerythrin [PE] 555), and Komma (PC 645). At least one putative character reversal took place in the genus Chroomonas. Several changes in biliproteins have been found in the genus Hemiselmis, including two new biliprotein variants that probably originated by slight modifications from PC 612 and PE 555, respectively (PC 577 and PE 545/555). Freshwater and marine/brackish taxa were intermingled across the tree without displaying a specific pattern. In four terminal clades, genetically identical strains have been found to occur both in Europe and in the USA. The Chroomonas/Hemiselmis/Komma clade proved to be the most diverse of all cryptophyte clades concerning types of biliproteins and distribution of clades across marine or freshwater habitats.     

The 24 hour recovery kinetics from n starvation in Phaeodactylum tricornutum and Emiliania huxleyi

The response of N (nitrate) starved cells of the diatom Phaeodactylum tricornutum and the coccolithophore Emiliania huxleyi to a pulse of new N were measured to investigate rapid cellular and photosynthetic recovery kinetics. The changes of multiple parameters were followed over 24 h. In P. tricornutum, the recovery of F_v/F_m (the maximum quantum yield of PS II) and σ_PSII (the functional absorption cross‐section for PSII) started within the first hour, much earlier than other parameters. Cellular pigments did not recover during the 24 h but the chlorophyll (chl) a/carotenoid ratios increased to levels measured in the controls. Cell division was independent of the recovery of chl a. In E. huxleyi, the recovery of F_v/F_m and σ_PSII started after an hour, synchronous with the increase in cellular organic N and chl a with pigments fully recovered within 14 h. P. tricornutum prioritized the recovery of its photosynthetic functions and cell divisions while E. huxleyi did not follow this pattern. We hypothesize that the different recovery strategies between the two species allow P. tricornutum to be more competitive when N pulses are introduced into N‐limited water while E. huxleyi is adapted to N scarce waters where such pulses are infrequent. These findings are consistent with successional patterns observed in coastal environments. This is one of only a few studies exploring recovery kinetics of cellular functions and photosynthesis after nitrogen stress in phytoplankton. Our results can be used to enhance ecological models linking phytoplankton traits to species diversity and community structure.     

DETECTION OF CHLOROPHYLL C1 AND MAGNESIUM-2,4-DIVINYLPHEOPORPHYRIN A5 MONOMETHYLESTER IN CRYPTOPHYTES1

Three different chlorophyll (chl) c-type pigments were isolated from two cryptophyte species by silica thin-layer chromatography or polyethylene high-performance liquid chromatography. Chroomonas sp. Hansgirg contained chl c₁ and magnesium-2,4-divinylpheoporphyrin a, mono-methylester; chl c₂ and magnesium-2,4-divinylpheoporphyrin a₅ monomethylester were found in Cryptomonas maculata (syn. Rhodomonas maculata Butcher). These identifications were based on spectral characteristics and on comparison with reference pigments isolated from the synurophycean Synura petersenii Korshikov and the prasinophyte Mantoniella squamata Manton & Park. Neither of the cryptophyte species contained chl c₁ and chl c₂. The significance of chl c₁ as a major pigment and the occurrence of magnesium-2,4-divinylpheoporphyrin a₅ monomethylester in cryptophytes are discussed.     

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c₂ and c₃.     

Validation and description of Symbiodinium microadriaticum,the type species of Symbiodinium (Dinophyta)

It has been 55 years since Hugo Freudenthal described Symbiodinium microadriaticum (Dinophyceae), the type species of this large and important dinoflagellate genus found commonly in mutualistic symbiosis with cnidarians, other invertebrates, and certain protists. However, no type specimen was designated by Freudenthal, thus S. microadriaticum was invalid, as was Symbiodinium and every species subsequently assigned to the genus. The original culture was lost, but since 1979, a different culture, CCMP2464/rt‐061, had been considered to represent S. microadriaticum. From this culture, a preserved specimen is herein designated the holotype of S. microadriaticum, validating the binomial and Symbiodinium. All binary designations previously considered to belong in Symbiodinium also are validated herein.     

Growth and Chloroplast Replacement of the Benthic Mixotrophic Ciliate Mesodinium coatsi

While the ecophysiology of planktonic Mesodinium rubrum species complex has been relatively well studied, very little is known about that of benthic Mesodinium species. In this study, we examined the growth response of the benthic ciliate Mesodinium coatsi to different cryptophyte prey using an established culture of this species. M. coatsi was able to ingest all of the offered cryptophyte prey types, but not all cryptophytes supported its positive, sustained growth. While M. coatsi achieved sustained growth on all of the phycocyanin‐containing Chroomonas spp. it was offered, it showed different growth responses to the phycoerythrin‐containing cryptophytes Rhodomonas spp., Storeatula sp., and Teleaulax amphioxeia. M. coatsi was able to easily replace previously ingested prey chloroplasts with newly ingested ones within 4 d, irrespective of prey type, if cryptophyte prey were available. Once retained, the ingested prey chloroplasts seemed to be photosynthetically active. When fed, M. coatsi was capable of heterotrophic growth in darkness, but its growth was enhanced significantly in the light (14:10 h light:dark cycle), suggesting that photosynthesis by ingested prey chloroplast leads to a significant increase in the growth of M. coatsi. Our results expand the knowledge of autecology and ecophysiology of the benthic M. coatsi.     

Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach

The genera Elliptochloris and Pseudochlorella were erected for Chlorella‐like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS‐2 Barcodes using the ITS‐2/CBC approach.     

The pigment composition of Phaeocystis antarctica (Haptophyceae) under various conditions of light,temperature, salinity,and iron

The pigment composition of Phaeocystis antarctica was monitored under various conditions of light, temperature, salinity, and iron. 19′‐Hexanoyloxyfucoxanthin (Hex‐fuco) always constituted the major light‐harvesting pigment, with remarkably stable ratios of Hex‐fuco‐to‐chl a under the various environmental conditions. Increased pigment‐to‐chl a ratios at low irradiance confirmed the light‐harvesting function of Fucoxanthin (Fuco), 19′‐Hexanoyloxy‐4‐ketofucoxanthin (Hex‐kfuco), 19′‐butanoyloxyfucoxanthin (But‐fuco), and chl c2 and c3. Increased pigment‐to‐chl a ratios at high irradiance, low iron concentrations, and to a lesser extent at high salinity confirmed the photoprotective function of diadinoxanthin, diatoxanthin, and ß,ß‐carotene. Pigment ratios were not always according to expectations. The consistent increase in But‐fuco/chl at high temperature, high salinity, and low iron suggests a role in photoprotection rather than in light harvesting. Low Hex‐kfuco/chl ratios at high salinity were consistent with a role as light harvester, but the high ratios at high temperature were not, leaving the function of Hex‐kfuco enigmatic. Dedicated experiments were performed to test whether or not the light‐harvesting pigment Fuco could be converted into its structural relative Hex‐fuco, and vice versa, in response to exposure to light shifts. Rapid conversions could not be confirmed, but long‐term conversions cannot be excluded. New pigment ratios are proposed for chemotaxonomic applications. The ratios will improve pigment‐based diagnosis of algal species in waters dominated by P. antarctica.     

Key sites residues involved in interacting with chemicals of pheromone‐binding proteins from Lymantria dispar

Pheromone‐binding proteins (PBPs) are distributed widely on the antennae of insects, and they are believed to be involved in the process of chemical signal transduction, but their interaction with chemicals is largely unknown. Here, we present our findings on the key amino acid residues of PBPs in the gypsy moth, Lymantria dispar. Potential key residues were screened with the Calculate Mutation Energy program and molecular docking methods. Mutated proteins were obtained by mutating residues to alanine via site‐directed mutagenesis. Circular dichroism (CD) spectroscopy showed that the mutated proteins formed α‐helix, and the stability of protein structure was influenced due to mutations. Fluorescence binding assays were further conducted with the mutated proteins, sex pheromones and analogues. Results showed that to PBP 1, tyrosine at position 41 and phenylalanine at position 76 could be the key amino acid residues influencing the stability of structure; in addition, phenylalanine at 36 and lysine at position 94 could be key amino acid residues interacting with chemicals. To PBP 2, glycine at position 49, phenylalanine at position 76 and lysine at position 121 could be the key amino acid residues in the structural stability. These results shed light on the relationship between the specific amino acids and functions of PBPs in transmitting the chemical signals.     

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)‐choline to diacylglycerol. Phosphocholine, the direct precursor to CDP‐choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [³²P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [³²P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.     

The Effect of Starvation on Plastid Number and Photosynthetic Performance in the Kleptoplastidic Dinoflagellate Amylax triacantha

The dinoflagellate Amylax triacantha is known to retain plastids of cryptophyte origin by engulfing the mixotrophic ciliate Mesodinium rubrum, itself a consumer of cryptophytes. However, there is no information on the fate of the prey's organelles and the photosynthetic performance of the newly retained plastids in A. triacantha. In this study, we conducted a starvation experiment to observe the intracellular organization of the prey's organelles and temporal changes in the photosynthetic efficiency of acquired plastids in A. triacantha. The ultrastructural observations revealed that while the chloroplast‐mitochondria complexes and nucleus of cryptophyte were retained by A. triacantha, other ciliate organelles were digested in food vacuoles. Acquired plastids were retained in A. triacantha for about 1 mo and showed photosynthetic activities for about 18 d when measured by a pulse‐amplitude modulation fluorometer.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Sensitive phylogenetics of Clematis and its position in Ranunculaceae

Ranunculaceae are a nearly cosmopolitan plant family with the highest diversity in northern temperate regions and with relatively few representatives in the tropics. As a result of their position among the early diverging eudicots and their horticultural value, the family is of great phylogenetic and taxonomic interest. Despite this, many genera remain poorly sampled in phylogenetic studies and taxonomic problems persist. In this study, we aim to clarify the infrageneric relationships of Clematis by greatly improving taxon sampling and including most of the relevant subgeneric and sectional types in a simultaneous dynamic optimization of phenotypic and molecular data. We also investigate how well the available data support the hypothesis of phylogenetic relationships in the family. At the family level, all five currently accepted subfamilies are resolved as monophyletic. Our analyses strongly imply that Anemone s.l. is a grade with respect to the Anemoclema + Clematis clade. This questions the recent sinking of well‐established genera, including Hepatica, Knowltonia and Pulsatilla, into Anemone. In Clematis, 12 clades conceptually matching the proposed sectional division of the genus were found. The taxonomic composition of these clades often disagrees with previous classifications. Phylogenetic relationships between the section‐level clades remain highly unstable and poorly supported and, although some patterns are emerging, none of the proposed subgenera is in evidence. The traditionally recognized and horticulturally significant section Viorna is both nomenclaturally invalid and phylogenetically unsupported. Several other commonly used sections are likewise unjustified. Our results provide a phylogenetic background for a natural section‐level classification of Clematis.     

Global diversity of two widespread,colony‐forming diatoms of the marine plankton,Chaetoceros socialis (syn. C. radians) and Chaetoceros gelidus sp. nov.

Marine phytoplankton samples containing diatoms of the Chaetoceros socialis group were collected from Thailand, China, Denmark, and Greenland, and cells were isolated into culture for light and electron microscopy and DNA sequencing of D1–D3 of the LSU rDNA. Species of this lineage are characterized by three short and one long setae extending from each cell, the long setae from several cells joining into a common center to form large colonies, which are sometimes visible with the naked eye. Phylogenetic analyses including sequences from other parts of the world revealed segregation into three groups. Most sequences fell into two large clades, one comprising material from cold waters, whereas the other contained material from warmer waters. Strain CCMP 172 from the Strait of Georgia, Washington State, USA, formed a separate group. The warm‐water species included Chinese and Thai material and therefore probably also material from the type locality of C. socialis, Hong Kong. It is characterized by all setae being covered by spines and the setae extending from the valve at some distance from the margin. In the resting spores, both valves are ornamented with spines. The cold‐water material is characterized by three spiny and one mostly smooth long setae, and the setae extend from the valve near the margin. Both valves of the resting spore are smooth. This material is described as C. gelidus sp. nov. C. radians, described from the Baltic in 1894, is considered a synonym of C. socialis. CCMP172 is in many ways intermediate and probably constitutes a separate species. The published evidence on this globally distributed and sometimes bloom‐forming group of species indicates higher species diversity than presently thought.