Ultrastructure of calcareous dinophytes (Thoracosphaeraceae, Peridiniales) with a focus on vacuolar crystal-like particles.

Biomineralization in calcareous dinophytes (Thoracosphaeracaea, Peridiniales) takes place in coccoid cells and is presently poorly understood. Vacuolar crystal-like particles as well as collection sites within the prospective calcareous shell may play a crucial role during this process at the ultrastructural level. Using transmission electron microscopy, we investigated the ultrastructure of coccoid cells at an early developmental stage in fourteen calcareous dinophyte strains (corresponding to at least ten species of Calciodinellum, Calcigonellum, Leonella, Pernambugia, Scrippsiella, and Thoracosphaera). The shell of the coccoid cells consisted either of one (Leonella, Thoracosphaera) or two matrices (Scrippsiella and its relatives) of unknown element composition, whereas calcite is deposited in the only or the outer layer, respectively. We observed crystal-like particles in cytoplasmic vacuoles in cells of nine of the strains investigated and assume that they are widespread among calcareous dinophytes. However, similar structures are also found outside the Thoracosphaeraceae, and we postulate an evolutionarily old physiological pathway (possibly involved in detoxification) that later was specialized for calcification. We aim to contribute to a deeper knowledge of the biomineralization process in calcareous dinophytes

Taxonomic clarification of the dinophyte <i>Rhabdosphaera erinaceus</i> Kamptner, ≡ <i>Scrippsiella erinaceus</i> comb. nov. (Thoracosphaeraceae, Peridiniales)

<div><p>The <i>Scrippsiella trochoidea</i> species complex (Thoracosphaeraceae, Peridiniales) consists of a cryptic diversity and multiple species, for which established scientific names are not available at present. Previously, the name <i>Scrippsiella trochoidea</i> has been taxonomically clarified, leaving a reliable determination of morphologically similar, but only distantly related species impossible. We isolated and cultivated <i>Scrippsiella erinaceus</i> comb. nov. (strains GeoM*533 and GeoM*534) from material collected near the type locality off Rovinj, Republic of Croatia (Adriatic Sea). We barcoded the species of the Thoracosphaeraceae using rRNA sequences (including 22 new sequences) and investigated the morphology of the strains using light and electron microscopy. The <i>Scrippsiella trochoidea</i> species complex was composed of three primary clades, and the Adriatic strains were reliably assigned to one of them being distinct from the true <i>Scrippsiella trochoidea</i>. We designate an epitype for the basionym <i>Rhabdosphaera erinaceus</i> prepared from the strain GeoM*534. The unambiguous links between a scientific species name, its protologue, genetic characterization and spatial distribution have particular importance for character-poor, unicellular organisms such as the Dinophyceae. </p></div

Species list of TEM investigations (abbreviations: n.i., not indicated).

<p>Species list of TEM investigations (abbreviations: n.i., not indicated).</p

Data from: Delimitation of the Thoracosphaeraceae (Dinophyceae), including the calcareous dinoflagellates, based on large amounts of ribosomal RNA sequence data

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage apparently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Ensiculifera and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future

General ultrastructure.

<p>(TEM). A. Large thecate cell of <i>Scrippsiella bicarinata</i> (GeoB 411, note the peripheral chloroplast connected to a multiply-stalked pyrenoid). B. Early coccoid cell of <i>Pernambugia tuberosa</i> (GeoB*61, note the numerous peripheral lipid droplets, the two matrices surrounding the cell and the protrusions highlighted by arrows). C. Large thecate cell (longisection) of <i>Scrippsiella bicarinata</i> (GeoB 411, note the multiply-stalked pyrenoids and the numerous mitochondria in the center of the cell). D. Early coccoid cell of <i>Scrippsiella</i> aff. <i>trochoidea</i> (GeoB 283, note the chloroplasts with stalked pyrenoids and the lipid droplets). E. Mature coccoid cell of <i>Leonella granifera</i> (GeoB 38, note the numerous starch grains and lipid droplets and that the cell is surrounded by a single layer containing large, regularly arranged calcareous crystals). Abbreviations: ab, accumulation body; ch, chloroplast; cs, chromosomes; ix, inner matrix; lp, lipid droplet; nu, nucleus; mt, mitochondrion; ox, outer matrix; py, pyrenoid; st, starch grain. Scale bars: 2 µm.</p

Ultrastructural traits in detail.

<p>(TEM). A. Multiply-stalked pyrenoid covered by a starch shed of <i>Calciodinellum</i> aff. <i>operosum</i> (GeoB 34, note the large, vacuolar crystal-like particles). B. Different chloroplast types of <i>Scrippsiella bicarinata</i> (GeoB 411, note that chloroplasts could be connected to multiply-stalked pyrenoids covered by a starch shed, or have interlamellar pyrenoids, with thylakoid lamellae leading through the pyrenoid. C. Two to four thylakoid lamellae (arrows) of <i>Scrippsiella bicarinata</i> (GeoB 411). D. Mitochondria with tubular cristae of <i>Scrippsiella</i> aff. <i>trochoidea</i> (GeoB 283). Abbreviations: ch, chloroplast; cb, crystal-like particle; me, membrane; mt, mitochondrion; py, pyrenoid; st, starch grain; tr, trichocyst. Scale bars: A and B 1 µm, C and D 0.1 µm.</p

Ultrastructural traits in detail.

<p>(TEM). A. ‘Apical furrow’ system of <i>Scripsiella trochoidea</i> (GeoB 377, note the numerous vesicles under the cell surface). B. Overlapping thecal plates of <i>Scripsiella trochoidea</i> (GeoB 188, note the outer protrusions of overlapping theca plates). C. Overlapping theca plates in the sulcal region of “<i>Calciodinellum</i>” spec. (tub*2). D. Strand of peripheral microtubules in <i>Scrippsiella bicarinata</i> (GeoB 411, note the multiple membranes under the cell surface). E. Thecal plate boundary of <i>Scripsiella trochoidea</i> (GeoB 377, note the detached outer unit membrane). F. Trichocysts of <i>Scrippsiella trochoidea</i> var. <i>aciculifera</i> (GeoB 228). G. Subcellular organization of <i>Scrippsiella bicarinata</i> (GeoB 411, note the longisection of a trichocyst and the rough endoplasmatic reticulum indicated by an arrow). H. Golgi apparatus of <i>Scrippsiella trochoidea</i> (M34*25/5). J. Pusule of <i>Scrippsiella bicarinata</i> (GeoB 411, note the multiple membranes under the cell surface). Abbreviations: cb, crystal-like particle; ch, chloroplast; cp, cover plate; d, dictyosome, fb, microtubular fiber; gv, Golgi-derived vesicle; me, unit membrane; mt, mitochondrion; po, pore plate; pv, pusular vesicle; py, pyrenoid; re, rough endoplasmatic reticulum; st, starch grain; tr, trichocyst; th, thecal plate. Scale bars: A to C and E to G 1 µm, D, H, J 0.1 µm.</p

Delimitation of the Thoracosphaeraceae (Dinophyceae), Including the Calcareous Dinoflagellates, Based on Large Amounts of Ribosomal RNA Sequence Data

The phylogenetic relationships of the Dinophyceae (Alveolata) are not sufficiently resolved at present. The Thoracosphaeraceae (Peridiniales) are the only group of the Alveolata that include members with calcareous coccoid stages; this trait is considered apomorphic. Although the coccoid stage appar- ently is not calcareous, Bysmatrum has been assigned to the Thoracosphaeraceae based on thecal morphology. We tested the monophyly of the Thoracosphaeraceae using large sets of ribosomal RNA sequence data of the Alveolata including the Dinophyceae. Phylogenetic analyses were performed using Maximum Likelihood and Bayesian approaches. The Thoracosphaeraceae were monophyletic, but included also a number of non-calcareous dinophytes (such as Pentapharsodinium and Pfiesteria) and even parasites (such as Duboscquodinium and Tintinnophagus). Bysmatrum had an isolated and uncertain phylogenetic position outside the Thoracosphaeraceae. The phylogenetic relationships among calcareous dinophytes appear complex, and the assumption of the single origin of the potential to produce calcareous structures is challenged. The application of concatenated ribosomal RNA sequence data may prove promising for phylogenetic reconstructions of the Dinophyceae in future

Gottschling_etal_Protist_2011

Gottschling_etal_Protist_201