Flagellar apparatus.

<p>5A. Longitidinal section of <i>Picomonas</i> from ventral to dorsal (sections are oblique from anterior part). Anterior flagellum (AF) and posterior flagellum (PF). The anterior flagellum locates close to the ventral surface followed more interiorly by the posterior flagellum. The basal bodies of both flagella are connected by a proximal connecting fiber (pcf) and exhibit two transitional regions (the proximal is termed ‘tr1’ and the distal ‘tr2’). 5B, C. Consecutive serial cross sections and two serial longitudinal sections of the anterior flagellum. Serial sections of 5B correspond to 5C, denoted in 5B_a-j at the left lower end. a. The axoneme with 9outer doublet and 2 central pair microtubules. b. The distal trasitional region (tr2) is involved in flagellar shedding (* indicates electron dense material near outer doublets). f. the central pair of microtubules orginate. h. the transitional region 1 (tr1), C-tubules added (arrowhead indicates a microtubulular triplet and arrow indicates a microtubular doublet. i,j. cross sections through basal body with microtubular triplets arranged in the clockwise direction, the basal body lumen is filled with electron dense material. AF/PF (anterior−/posterior flagellum); tr₁, tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); Ab/Pb (anterior−/posterior basal body); pcf (proximal connecting fiber); G (Golgi body); N (nucleus); Pr2 (posterior microtubular flagellar root 2). Numbers at the top right indicate the serial section. Scale bar: 100 nm.</p

<i>Picomonas judraskeda</i> Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes, Formerly Known as ‘Picobiliphytes’

<div><p>In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe <i>Picomonas judraskeda</i> gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for <i>Picomonas judraskeda</i>, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’.</p> </div

Flagellar root system in <i>Picomonas judraskeda</i>.

<p>7A. Non-consecutive serial sections from dorsal to ventral (sections are oblique to cell’s right). Each basal body is connected to two microtubular flagellar roots. The anterior root 1 (Ar1) runs anteriorly to cell’s left. The Ar2 runs posteriorly, at the right side of the cell and passes the cleft (cl). The other two flagellar roots originate from the posterior basal body and extend towards the posterior part of the cell. One of the posterior flagellar roots (Pr1) runs on the left side of the cell. The other broader posterior flagellar root (Pr2), runs between the Ar1 and Pr1. 7B. The Pr2 with 6 microtubules (arrowheads) obliquely sectioned. 7C. A cell with the Pr2 passing the vacuolar cisterna and mitochondrion. 7D. Consecutive serial cross sections through the Pr2 located in a depression of the mitochondrion. AF/PF (anterior−/posterior flagellum); Ab/Pb (anterior−/posterior basal body); Ar1/Ar2 (Anterior microtubular flagellar roots 1 and 2); Pr1/Pr2 (posterior microtubular flagellar roots 1 and 2); G (Golgi body); M (mitochondrion); vc (vacuolar cisterna); P (posterior digestive body); CMT (secondary cytoplasmic microtubule). Numbers at the top right indicate the number of the serial section. Scale bar: 200 nm.</p

Ultrastructure of the mitochondrion of a <i>Picomonas</i> cell.

<p>Single mitochondrion with tubular cristae, membrane bound (single and double) electron dense material (edsm1 and 2 respectively) and regular projections of the outer envelope membrane. 3A. a double membrane bound edsm2 near the ventral surface; serial sections (1–4) of the edsm2 displayed some tube-like structures in the lumen (thin lines); also note that the edsm2 is a branched structure. 3B. A membrane invagination of edsm2 into the mitochondrion that reveals continuity between the mitochondrial envelope membranes and the two membranes encircling the edsm2. 3C. Oblique section of a cell from dorsal right to ventral left with edsm1 and edsm2 displayed. The edsm1 (on the left) is positioned between the outer (black arrow) and inner (white arrow) mitochondrial envelope membrane and the edsm2 (on the right) with a double membrane (higher magnifications of the two edsms on the right. A large number of short cylindrical membrane protrusions termed ‘mitovilli’ (arrow heads) extend from the outer mitochondrial membrane towards posterior digestive body (P), they terminate in granular material covering the posterior digestive body. A vacuolar cisterna (vc) in the ‘cleft’ region separates the anterior from posterior part of the cell and is only absent (i.e. contains a large hole) in the area of the mitovilli (3C). Towards the right are shown higher magnifications of two serial sections from the cell depicted in 3C revealing details of the mitovilli-posterior digestive body junction. F (feeding apparatus with basket fibers; G (Golgi body); M (mitochondrion); N (nucleus); P (posterior digestive body); vc (vacuolar cisterna). Numbers at the top right indicate the section number of a series. Scale bar: 100 nm.</p

Feeding apparatus.

<p>4A. Longitudinal sections of a <i>Picomonas</i> cell from ventral to dorsal with the feeding apparatus in cross section. Sections 2 to section 7 depict a recently formed food vacuole inside the ‘basket’ of the feeding apparatus. 4B & C. Cross sections through two cells of <i>Picomonas</i> from anterior to posterior; sections begin in the central part of the cell, posterior to the basal bodies. 4B. Cell, in the non-feeding mode, without a food vacuole within the basket. 4C. Cell during active feeding with a large food vacuole within the basket (black triangles); the food vacuole contains irregularly-shaped, ‘fuzzy’ material, presumably taken up by endocytosis through the cytostome (white triangles). Two rows of fibers representing the left (LF) and right (RF) margins of the basket accompany the food vacuole. 4D. An SEM image of <i>Picomonas</i> visualizing the left side of the cell. Note that the posterior flagellum has been shed at the tr2; white triangles indicate the cytostome region of the feeding apparatus. 4E. Longitudinal section through the basket near the cytostome. It shows approx. 60 rows of fibers arranged in parallel (white arrows depict fibers in the right margin of the basket). 4F. 3D model of the feeding apparatus with rows of fibers in parallel arrangement (white arrows) forming a basket (arrangement of the cell as in 4D). Note that the fibers are interconnected by thin filaments. The basket is open towards the top and right (i.e. towards the anterior and dorsal direction of the cell respectively), while it is closed at the bottom (the posterior end of the cell). On the left side of the basket (representing the cell’s ventral surface) the fibers are attached to the plasma membrane thus forming the narrow, slit-like cytostome. AF/PF (anterior−/posterior flagellum); G (Golgi body); LF/RF (left and right row of fibers of the basket); M (mitochondrion); MB (microbody); N (nucleus); P (posterior digestive body); Ar2 (anterior microtubular flagellar root 2); Pr1 (posterior microtubular flagellar root 1); Pr2 (posterior microtubular flagellar root 2); FV, (food vacuole); vc (vacuolar cisterna); cy (cytostome). Numbers at the top right indicate the serial section. Scale bar: 200 nm; except Fig. 4A, section 2 (100 nm).</p

Identification and isolation of ‘(pico)biliphytes’ by fluorescence-activated cell sorting (<i>MitoTracker®</i> Green FM [FL1] vs. Forward Scatter [FSC]) and PCR amplification using specific primers.

<p>1A. Approximately 100 cells from each of three regions with higher fluorescence/forward scatter (R1–R3) were sorted into PCR tubes. Cells marked in ‘red’ in the cytogram corresponded to ‘(pico)biliphytes’ by PCR amplification and sequencing. 1B. Sorted cells used for primary amplification with ‘picobiliphyte’-specific primers (PICOBI01F/P01ITS1R) and reamplified with PICOBI01F and 1055rev. Amplicons in region R1 were confirmed by DNA sequencing to correspond to ‘(pico)biliphytes’. Arrow 650 base pairs; M 1 Kb ladder.</p

A <i>Picomonas</i> cell.

<p>2A. Differential interference contrast of a chemically fixed cell. Inset shows phase contrast image of a live cell from tissue culture flask photographed with an inverted microscope (Scale bar 5 µm). 2B. Fluorescence and phase contrast overlay, nucleus (blue), mitochondrion (red). 2C. SEM image. 2D. A longitudinal section through a cell in the plane of the flagella, viewed from the cell’s left. 2E. A 3 D serial section reconstruction of the cell depicted in 2D. AF/PF (anterior−/posterior flagellum); AP/PP (anterior/posterior part of the cell); G (Golgi body); M (mitochondrion); MB (‘microbody’); N (nucleus); tr₁,tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); P (posterior digestive body); Cl (cleft separating the anterior from the posterior part of the cell); vc (vacuolar cisterna).</p

Basal apparatus and probasal bodies.

<p>6A. Electron micrograph shows consecutive serial sections of <i>Picomonas judraskeda</i> from left to right (Sections are angled about 30 degree towards left from central axis). Each probasal body diverges from its parental basal body at right angles, both probasal bodies are oriented towards the ventral surface of the cell and are attached to the plasma membrane. 6B. 3D- scheme of the arrangement of basal bodies with probasal bodies. 6C. A schematic presentation of the flagellar basal apparatus seen from the ventral surface of the cell with proximal parts of the axonemes, basal bodies and four microtubular flagellar roots (for details see text). Numbers in brackets indicate the number of microtubules in each root. AF/PF (anterior−/posterior flagellum); tr₁,tr₂ (distal [tr2] and proximal [tr1] flagellar transitional regions); Ab/Pb (anterior−/posterior basal body); Apb/Ppb (anterior/posterior probasal body); pcf (proximal connecting fiber); Ar1/Ar2 (Anterior microtubular flagellar roots 1 and 2); Pr1/Pr2 (posterior microtubular flagellar roots 1 and 2); G (Golgi body); N (nucleus); M (mitochondrion); cy (cytostome); P (posterior digestive body). Numbers at the top right indicate the number of the serial section. Scale bar: 200 nm.</p

Unrooted randomized accelerated maximum likelihood (RAxML) phylogenetic tree of partial nuclear SSU rDNA of Picozoa.

<p>The phylogeny is based on 1253 aligned characters of the SSU rDNA and includes 201 sequences of Picozoa. Most sequences are database entries derived from clone libraries (nine environmental sequences generated from a sample taken at Helgoland Roads and one sequence from <i>Picomonas judraskeda</i> are new sequences; accession numbers of the newly determined sequences are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059565#pone.0059565.s002" target="_blank">Fig. S2</a>). Bootstrap values >60% and posterior probabilities >0.80 for the three methods of analyses used (RAxML/NJ by PAUP/MrBayes) are shown on the respective branches. Branches in bold show maximal (100%/1.00) support. Labeling of clades (‘BP1–3′) followed [Cuvelier et al. 2008], 12 novel clades (‘P1–P13’) are recognized (for details see Results). The sequence of <i>Picomonas judraskeda</i> (in bold) was positioned in clade ‘P3’ (shaded).</p

New Insights into Plagiogrammaceae (Bacillariophyta) Based on Multigene Phylogenies and Morphological Characteristics with the Description of a New Genus and Three New Species

<div><p>Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: <i>Plagiogramma</i>, <i>Dimeregramma</i>, <i>Neofragilaria</i>, <i>Talaroneis</i>, <i>Psammogramma</i> and <i>Psammoneis</i>. In this paper, we describe a new plagiogrammoid genus, <i>Orizaformis</i>, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded <i>rbc</i>L and <i>psb</i>C). Also included in the new phylogenies is <i>Glyphodesmis</i>. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of <i>Talaroneis</i>, <i>Orizaformis</i> and <i>Psammoneis</i>, and the second of <i>Glyphodesmis</i>, <i>Psammogramma</i>, <i>Neofragilaria</i>, <i>Dimeregramma</i> and <i>Plagiogramma</i>. In addition, we describe three new species within established genera: <i>Psammoneis obaidii</i>, which was collected from the Red Sea, Saudi Arabia; and <i>Neofragilaria stilus</i> and <i>Talaroneis biacutifrons</i> from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: <i>Neofragilaria anomala</i> and <i>Neofragilaria lineata</i>. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.</p></div