Ansanella granifera gen. et sp. nov. (Dinophyceae), a new dinoflagellate from the coastal waters of Korea

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  • ABSTRACT

    A small dinoflagellate, Ansanella granifera gen. et sp. nov., was isolated from estuarine and marine waters, and examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. In addition, the identity of the sequences (3,663-bp product) of the small subunit (SSU), internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), and D1-D3 large subunit (LSU) rDNA were determined. This newly isolated, thin-walled dinoflagellate has a type E eyespot and a single elongated apical vesicle, and it is closely related to species belonging to the family Suessiaceae. A. granifera has 10-14 horizontal rows of amphiesmal vesicles, comparable to Biecheleria spp. and Biecheleriopsis adriatica, but greater in number than in other species of the family Suessiaceae. Unlike Biecheleria spp. and B. adriatica, A. granifera has grana-like thylakoids. Further, A. granifera lacks a nuclear fibrous connective, which is present in B. adriatica. B. adriatica and A. granifera also show a morphological difference in the shape of the margin of the cingulum. In A. granifera, the cingular margin formed a zigzag line, and in B. adriatica a straight line, especially on the dorsal side of the cell. The episome is conical with a round apex, whereas the hyposome is trapezoidal. Cells growing photosynthetically are 10.0-15.0 µm long and 8.5-12.4 µm wide. The cingulum is descending, the two ends displaced about its own width. Cells of A. granifera contain 5-8 peripheral chloroplasts, stalked pyrenoids, and a pusule system, but lack nuclear envelope chambers, a nuclear fibrous connective, lamellar body, rhizocysts, and a peduncle. The main accessory pigment is peridinin. The SSU, ITS regions, and D1-D3 LSU rDNA sequences differ by 1.2-7.4%, >8.8%, and >2.5%, respectively, from those of the other known genera in the order Suessiales. Moreover, the SSU rDNA sequence differed by 1-2% from that of the three most closely related species, Polarella glacialis, Pelagodinium bei, and Protodinium simplex. In addition, the ITS1-5.8S-ITS2 rDNA sequence differed by 16-19% from that of the three most closely related species, Gymnodinium corii, Pr. simplex, and Pel. bei, and the LSU rDNA sequence differed by 3-4% from that of the three most closely related species, Protodinium sp. CCMP419, B. adriatica, and Gymnodinium sp. CCMP425. A. granifera had a 51-base pair fragment in domain D2 of the large subunit of ribosomal DNA, which is absent in the genus Biecheleria. In the phylogenetic tree based on the SSU and LSU sequences, A. granifera is located in the large clade of the family Suessiaceae, but it forms an independent clade.


  • KEYWORD

    new genus , new species , protist , Suessiaceae , Symbiodiniaceae , taxonomy , ultrastructure

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  • [Fig. 1.] Cells of Ansanella granifera AGSW10 gen. et sp. nov. Micrographs taken by light microscopy (A-D) and epifluorescence microscopy (E & F). (A) Cells of various sizes and shapes. (B) Ventral view showing an eyespot (ES) and sulcus (SU). (C) Dorsal view showing the large nucleus (N) and yellowish-brown chloroplasts (C). (D) Lateral view. (E) Ventral view showing the nucleus extending from the anterior to the central part of the cell. (F) Dorsal view showing the chloroplasts located at the cell periphery. Scale bars represent: A, 20 μm; B-F, 5 μm.
    Cells of Ansanella granifera AGSW10 gen. et sp. nov. Micrographs taken by light microscopy (A-D) and epifluorescence microscopy (E & F). (A) Cells of various sizes and shapes. (B) Ventral view showing an eyespot (ES) and sulcus (SU). (C) Dorsal view showing the large nucleus (N) and yellowish-brown chloroplasts (C). (D) Lateral view. (E) Ventral view showing the nucleus extending from the anterior to the central part of the cell. (F) Dorsal view showing the chloroplasts located at the cell periphery. Scale bars represent: A, 20 μm; B-F, 5 μm.
  • [Fig. 2.] Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. taken by scanning electron microscopy. (A) Ventral view of the cell showing five rows (E1-E5) of amphiesmal vesicles (AVs) in the episome, and the elongated apical vesicle (EAV). (B) Left side view of the cell showing three rows (E3-E5) of AVs on the episome, three rows (C1-C3) in the cingulum, and four rows (H1-H4) on the hyposome. (C) Dorsal view of A. granifera showing five AV rows (E1-E5) including a small vesicle (asterisk). (D) Right-side view. FLP, finger-like protrusion; LF, longitudinal flagellum; TF, transverse flagellum. Scale bars represent: A-D, 2 μm.
    Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. taken by scanning electron microscopy. (A) Ventral view of the cell showing five rows (E1-E5) of amphiesmal vesicles (AVs) in the episome, and the elongated apical vesicle (EAV). (B) Left side view of the cell showing three rows (E3-E5) of AVs on the episome, three rows (C1-C3) in the cingulum, and four rows (H1-H4) on the hyposome. (C) Dorsal view of A. granifera showing five AV rows (E1-E5) including a small vesicle (asterisk). (D) Right-side view. FLP, finger-like protrusion; LF, longitudinal flagellum; TF, transverse flagellum. Scale bars represent: A-D, 2 μm.
  • [Fig. 3.] Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. taken by scanning electron microscopy (SEM). (A) Apical view. (B) Antapical view. (C) Apical view showing the elongated apical vesicle (EAV) (dashed box). (D) Drawing of the EAV-the ventral part of the EAV showing the long and narrow central plate ornamented with knobs. (E) Cingulum view showing a cell in which the outer membrane of some amphiesmal vesicles were removed (asterisks), showing the zigzag line of the lower cingular margin (arrows). (F) Ventral view showing 6-7 rows of amphiesmal vesicles in the sulcus. (G) SEM figure enlarged from Fig. 3F, showing the longitudinal flagellum (LF) and transverse flagellum (TF). FLP, finger-like protrusion. (H) Drawing of the sulcus. Scale bars represent: A, B & E, 2 μm; C & F-H, 1 μm; D, 0.5 μm.
    Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. taken by scanning electron microscopy (SEM). (A) Apical view. (B) Antapical view. (C) Apical view showing the elongated apical vesicle (EAV) (dashed box). (D) Drawing of the EAV-the ventral part of the EAV showing the long and narrow central plate ornamented with knobs. (E) Cingulum view showing a cell in which the outer membrane of some amphiesmal vesicles were removed (asterisks), showing the zigzag line of the lower cingular margin (arrows). (F) Ventral view showing 6-7 rows of amphiesmal vesicles in the sulcus. (G) SEM figure enlarged from Fig. 3F, showing the longitudinal flagellum (LF) and transverse flagellum (TF). FLP, finger-like protrusion. (H) Drawing of the sulcus. Scale bars represent: A, B & E, 2 μm; C & F-H, 1 μm; D, 0.5 μm.
  • [Fig. 4.] Drawings of Ansanella granifera AGSW10 gen. et sp. nov. showing the external morphology. (A) Ventral view. (B) Dorsal view showing zigzag line of the lower cingular margin (arrowheads). (C) Apical view. (D) Antapical view. EAV, elongated apical vesicle; FLP, finger-like protrusion. “E1-E5,” “C1-C3,” and “H1-H4” indicate episomal, cingular, and hyposomal amphiesmal vesicles, respectively. Scale bars represent: A-D, 2 μm.
    Drawings of Ansanella granifera AGSW10 gen. et sp. nov. showing the external morphology. (A) Ventral view. (B) Dorsal view showing zigzag line of the lower cingular margin (arrowheads). (C) Apical view. (D) Antapical view. EAV, elongated apical vesicle; FLP, finger-like protrusion. “E1-E5,” “C1-C3,” and “H1-H4” indicate episomal, cingular, and hyposomal amphiesmal vesicles, respectively. Scale bars represent: A-D, 2 μm.
  • [Fig. 5.] Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A-C) Micrographs showing several organelles inside the protoplasm: chloroplasts (C), eyespot (ES), fibrous vesicle (F), Golgi apparatus (G), mitochondria (M), nucleus (N), and pyrenoid (PY). Scale bars represent: A & C, 1 μm; B, 0.5 μm.
    Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A-C) Micrographs showing several organelles inside the protoplasm: chloroplasts (C), eyespot (ES), fibrous vesicle (F), Golgi apparatus (G), mitochondria (M), nucleus (N), and pyrenoid (PY). Scale bars represent: A & C, 1 μm; B, 0.5 μm.
  • [Fig. 6.] Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A) The stalked pyrenoid (PY) (dashed box) is surrounded by a starch sheath (S). (B) Same figure enlarged, showing the stalked PY, S, and thylakoids (arrow). (C) The chloroplasts are bounded by three membranes (arrows); each lamella possesses three thylakoids. (D-F) Chloroplast lobe, showing the grana-like thylakoids (GLT, dashed box) and thylakoids mostly in triplets. C, chloroplasts. Scale bars represent: A & D, 1 μm; B, C, E & F, 0.2 μm.
    Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A) The stalked pyrenoid (PY) (dashed box) is surrounded by a starch sheath (S). (B) Same figure enlarged, showing the stalked PY, S, and thylakoids (arrow). (C) The chloroplasts are bounded by three membranes (arrows); each lamella possesses three thylakoids. (D-F) Chloroplast lobe, showing the grana-like thylakoids (GLT, dashed box) and thylakoids mostly in triplets. C, chloroplasts. Scale bars represent: A & D, 1 μm; B, C, E & F, 0.2 μm.
  • [Fig. 7.] Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A) Transverse section through the cell showing the eyespot (ES) in the gap between the chloroplasts. (B) Transverse section of a cell showing the eyespot vesicles (ES) containing crystalline bricks and the R1 flagellar root. (C) The ES consists of seven layers of brick-containing cisternae. Scale bars represent: A, 1 μm; B & C, 0.2 μm.
    Micrographs of Ansanella granifera AGSW10 gen. et sp. nov. take by transmission electron microscopy. (A) Transverse section through the cell showing the eyespot (ES) in the gap between the chloroplasts. (B) Transverse section of a cell showing the eyespot vesicles (ES) containing crystalline bricks and the R1 flagellar root. (C) The ES consists of seven layers of brick-containing cisternae. Scale bars represent: A, 1 μm; B & C, 0.2 μm.
  • [Fig. 8.] Ansanella granifera AGSW10 taken by transmission electron microscopy. (A) Longitudinal section of the cell showing the basal bodies (longitudinal basal body [LB], transverse basal body [TB]) and the eyespot (ES). (B-E) Flagellar apparatus. Non-adjacent, nearly longitudinal serial sections proceeding from left to right. The encircled numbers are section numbers. Micrograph showing relative positions of the LB, TB, Root 1 (R1), putative Root 2 (R2), Root 4 (R4), striated root connective (SRC), basal body connectives (bbc), and longitudinal striated collar (LSC). Scale bars represent: A-E, 0.2 μm.
    Ansanella granifera AGSW10 taken by transmission electron microscopy. (A) Longitudinal section of the cell showing the basal bodies (longitudinal basal body [LB], transverse basal body [TB]) and the eyespot (ES). (B-E) Flagellar apparatus. Non-adjacent, nearly longitudinal serial sections proceeding from left to right. The encircled numbers are section numbers. Micrograph showing relative positions of the LB, TB, Root 1 (R1), putative Root 2 (R2), Root 4 (R4), striated root connective (SRC), basal body connectives (bbc), and longitudinal striated collar (LSC). Scale bars represent: A-E, 0.2 μm.
  • [Fig. 9.] Non-adjacent transverse serial sections of the flagellar apparatus taken by transmission electron microscopy. Sectioning is from anterior to posterior and the cell is seen from the anterior end. The encircled numbers are section numbers. (A) Micrograph showing the longitudinal basal body (LB) and Root 1 (R1). (B-D) The C1LB/R1 and C2LB/R1 interconnect the R1 root and one of the LB triplets. The dorsal fiber (DF) is present on the dorsal side of the R1. Scale bars represent: A-D, 0.2 μm.
    Non-adjacent transverse serial sections of the flagellar apparatus taken by transmission electron microscopy. Sectioning is from anterior to posterior and the cell is seen from the anterior end. The encircled numbers are section numbers. (A) Micrograph showing the longitudinal basal body (LB) and Root 1 (R1). (B-D) The C1LB/R1 and C2LB/R1 interconnect the R1 root and one of the LB triplets. The dorsal fiber (DF) is present on the dorsal side of the R1. Scale bars represent: A-D, 0.2 μm.
  • [Fig. 10.] Ansanella granifera AGSW10 taken by transmission electron microscopy. Nonadjacent longitudinal serial sections of the flagellar apparatus. The cell is seen from the outside, and the sectioning proceeds from right to left. The encircled numbers are section numbers. (A-D) Micrograph showing relative positions of the longitudinal basal body (LB), transverse basal body (TB), Root 1 (R1), putative Root 2 (R2), Root 3 (R3), Root 4 (R4), dorsal fiber (DF), and microtubular extension (transverse microtubular root extension [TMRE]). The striation pattern of the DF is very distinct. Notice also flagellar root R3 and its microtubular extension (TMRE). (E) Enlargement from Fig. 10C. Scale bars represent: A-D, 0.2 μm.
    Ansanella granifera AGSW10 taken by transmission electron microscopy. Nonadjacent longitudinal serial sections of the flagellar apparatus. The cell is seen from the outside, and the sectioning proceeds from right to left. The encircled numbers are section numbers. (A-D) Micrograph showing relative positions of the longitudinal basal body (LB), transverse basal body (TB), Root 1 (R1), putative Root 2 (R2), Root 3 (R3), Root 4 (R4), dorsal fiber (DF), and microtubular extension (transverse microtubular root extension [TMRE]). The striation pattern of the DF is very distinct. Notice also flagellar root R3 and its microtubular extension (TMRE). (E) Enlargement from Fig. 10C. Scale bars represent: A-D, 0.2 μm.
  • [Fig. 11.] Ansanella granifera AGSW10 taken by transmission electron microscopy. (A-F) Adjacent longitudinal serial sections. The cell is seen from the outside, and the sectioning moves from posterior to anterior. The encircled numbers are section numbers. The striated pattern of transverse striated root (TSR) is evident. Notice the Root 4 (R4) root with its single microtubule transverse striated root microtubule (TSRM). Pusule vesicles (PU) and the transverse striated collar (TSC) are also visible. TB, transverse basal body. Scale bars represent: A-F, 0.2 μm.
    Ansanella granifera AGSW10 taken by transmission electron microscopy. (A-F) Adjacent longitudinal serial sections. The cell is seen from the outside, and the sectioning moves from posterior to anterior. The encircled numbers are section numbers. The striated pattern of transverse striated root (TSR) is evident. Notice the Root 4 (R4) root with its single microtubule transverse striated root microtubule (TSRM). Pusule vesicles (PU) and the transverse striated collar (TSC) are also visible. TB, transverse basal body. Scale bars represent: A-F, 0.2 μm.
  • [Fig. 12.] Ansanella granifera AGSW10 taken by transmission electron microscopy. Nonadjacent serial sections. The sulcus region in transverse section, the sectioning moves from posterior to anterior. The encircled numbers are section numbers. (A-D) Micrograph showing relative positions of the longitudinal basal body (LB), transverse basal body (TB), Root 1 (R1), Root 4 (R4, transverse striated root [TSR] + transverse striated root microtubule [TSRM]), dorsal fiber (DF), transverse striated collar (TSC), and eyespot (ES). The R1 flagellar root is located in the narrow space between the eyespot and the cell surface. Scale bars represent: A-D, 0.2 μm.
    Ansanella granifera AGSW10 taken by transmission electron microscopy. Nonadjacent serial sections. The sulcus region in transverse section, the sectioning moves from posterior to anterior. The encircled numbers are section numbers. (A-D) Micrograph showing relative positions of the longitudinal basal body (LB), transverse basal body (TB), Root 1 (R1), Root 4 (R4, transverse striated root [TSR] + transverse striated root microtubule [TSRM]), dorsal fiber (DF), transverse striated collar (TSC), and eyespot (ES). The R1 flagellar root is located in the narrow space between the eyespot and the cell surface. Scale bars represent: A-D, 0.2 μm.
  • [Fig. 13.] Diagrammatic reconstruction of the flagellar apparatus of Ansanella granifera AGSW10 gen. et sp. nov., based mainly on transverse serial sections (70-nm-thick sections). LB, longitudinal basal body; TB, transverse basal body; LSC, longitudinal striated collar; TSC, transverse striated collar; R1, root 1, longitudinal microtubular root; R2, root 2, single-stranded microtubular root; R3, root 3, transverse microtubular root; R4, root 4, transverse striated root (TSR) + transverse striated root microtubule (TSRM); SRC, striated root connective; C1LB/R1, connective 1 linking LB and R1; C2LB/R1, connective 2 linking LB and R1; bbc, basal body connective; TMRE, transverse microtubular root extension; DF, dorsal fiber.
    Diagrammatic reconstruction of the flagellar apparatus of Ansanella granifera AGSW10 gen. et sp. nov., based mainly on transverse serial sections (70-nm-thick sections). LB, longitudinal basal body; TB, transverse basal body; LSC, longitudinal striated collar; TSC, transverse striated collar; R1, root 1, longitudinal microtubular root; R2, root 2, single-stranded microtubular root; R3, root 3, transverse microtubular root; R4, root 4, transverse striated root (TSR) + transverse striated root microtubule (TSRM); SRC, striated root connective; C1LB/R1, connective 1 linking LB and R1; C2LB/R1, connective 2 linking LB and R1; bbc, basal body connective; TMRE, transverse microtubular root extension; DF, dorsal fiber.
  • [Fig. 14.] Chromatogram of Ansanella granifera gen. et sp. nov. derived by using high-performance liquid chromatography.
    Chromatogram of Ansanella granifera gen. et sp. nov. derived by using high-performance liquid chromatography.
  • [Table 1.] Comparison of the sequences of Ansanella granifera AGSW10 (GenBank accession Nos. HG529978-HG529980) with other genera included in the order Suessiales
    Comparison of the sequences of Ansanella granifera AGSW10 (GenBank accession Nos. HG529978-HG529980) with other genera included in the order Suessiales
  • [Fig. 15.] Consensus Bayesian tree of the order Suessiales based on 1,534 aligned positions of nuclear small subunit rDNA. Cystodinium phaseolus and Phytodinium sp. comprised the outgroup. The parameters were as follows: assumed equal nucleotide frequency; substitution rate matrix with A-C substitutions = 0.0750, A-G substitutions = 0.2798, A-T substitutions = 0.0934, C-G substitutions = 0.0403, C-T substitutions = 0.4600, and G-T substitutions = 0.0515; proportion of sites assumed to be invariable = 0.5026; and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.0929. The branch lengths are proportional to the amount of character change. The numbers above the branches indicate the Bayesian posterior probability (left) and maximum-likelihood bootstrap values (right). A filled black circle is used to indicate the highest possible support value for the two phylogenetic methods applied. Posterior probabilities ≥0.5 are shown.
    Consensus Bayesian tree of the order Suessiales based on 1,534 aligned positions of nuclear small subunit rDNA. Cystodinium phaseolus and Phytodinium sp. comprised the outgroup. The parameters were as follows: assumed equal nucleotide frequency; substitution rate matrix with A-C substitutions = 0.0750, A-G substitutions = 0.2798, A-T substitutions = 0.0934, C-G substitutions = 0.0403, C-T substitutions = 0.4600, and G-T substitutions = 0.0515; proportion of sites assumed to be invariable = 0.5026; and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.0929. The branch lengths are proportional to the amount of character change. The numbers above the branches indicate the Bayesian posterior probability (left) and maximum-likelihood bootstrap values (right). A filled black circle is used to indicate the highest possible support value for the two phylogenetic methods applied. Posterior probabilities ≥0.5 are shown.
  • [Fig. 16.] Consensus Bayesian tree of the order Suessiales based on 676 aligned positions of nuclear large subunit rDNA (including the highly divergent domain D2). Baldinia anauniensis was chosen as the outgroup taxon. The parameters were as follows: assumed equal nucleotide frequency; substitution rate matrix with A-C substitutions = 0.0551, A-G substitutions = 0.2047, A-T substitutions = 0.0829, C-G substitutions = 0.0452, C-T substitutions = 0.5090, G-T substitutions = 0.1031; proportion of sites assumed to be invariable = 0.2291; and rates for variable sites assumed to follow a gamma distribution with shape parameter = 1.0076. The branch lengths are proportional to the amount of character change. The numbers above the branches indicate the Bayesian posterior probability (left) and maximum-likelihood bootstrap values (right). A filled black circle is used to indicate the highest possible support value for the two phylogenetic methods applied. Posterior probabilities ≥0.5 are shown.
    Consensus Bayesian tree of the order Suessiales based on 676 aligned positions of nuclear large subunit rDNA (including the highly divergent domain D2). Baldinia anauniensis was chosen as the outgroup taxon. The parameters were as follows: assumed equal nucleotide frequency; substitution rate matrix with A-C substitutions = 0.0551, A-G substitutions = 0.2047, A-T substitutions = 0.0829, C-G substitutions = 0.0452, C-T substitutions = 0.5090, G-T substitutions = 0.1031; proportion of sites assumed to be invariable = 0.2291; and rates for variable sites assumed to follow a gamma distribution with shape parameter = 1.0076. The branch lengths are proportional to the amount of character change. The numbers above the branches indicate the Bayesian posterior probability (left) and maximum-likelihood bootstrap values (right). A filled black circle is used to indicate the highest possible support value for the two phylogenetic methods applied. Posterior probabilities ≥0.5 are shown.
  • [Table 2.] Comparison of the morphology of Ansanella granifera and other genera included in the family Suessiaceae
    Comparison of the morphology of Ansanella granifera and other genera included in the family Suessiaceae