Table 1.
Primers used in the present study.
Figure 1.
Light microscopic photographs of P. geminatum ( = C. geminatum) paired cells preserved in different fixatives.
a–b) Side view of cells fixed using Utermöhl's. Note that Utermöhl's fixation effectively preserved cell morphology, with visible cingulum and sulcus.
Figure 2.
Scanning electron microscopic (SEM) photographs of the paired cells of P. geminatum ( = C. geminatum) to show the apical groove (short thin arrow), connection pore (arrow head, in panel b), cingulum (short thick arrow), flagellum (long thin arrow), and sulcus (long hollow arrow) (a–g).
a) Apical views of the epitheca in the upper cell. b) Antapical views of the hypotheca in the upper cell. c) Vertical views of the lower cell. d) Antapical views of the hypotheca in the lower cell. e–g) Oblique ventral views of dual cells. Scale bar in all the photographs = 20 µm.
Figure 3.
Microscopic characterization of cell colony and cellular features of P. geminatum ( = C. geminatum).
a) Side-view light-microscopic image of a dual-cell colony fixed using the formaldehyde. The cells are packed with chloroplasts of golden-brown color (arrow). b) SEM image of a cell with the round shaped chloroplasts exposed (arrow) due to disrupted cell covering. c) Red fluorescence from Chl. a in the chloroplasts (arrow) in a dual-cell colony under blue light excitation. d) Light microscopic image of green fluorescence from SYBR Green I-stained DNA in the nuclei (arrow) in the dual-cell colony superimposed on the red fluorescence from Chl. a in the chloroplasts under blue excitation light. e) Transmission electron microscopic image showing two round-shaped chloroplasts (arrow) in a P. geminatum cell. f) Ultrastructure of a chloroplast showing thylakoids in stacks of three (thicker arrow) typical of peridinin-containing dinoflagellate chloroplasts and pyrenoid (thin arrow). Scale bars = 20 µm in (a–d), 3 µm in (e), and 1 µm in (f).
Figure 4.
Pigment characteristics of P. geminatum ( = C. geminatum).
a) HPLC chromatogram of the pigments extracted from a water sample during the P. geminatum bloom event. b) Absorption spectra of peak 3 shown in (a) (arrow), which was identified as peridinin.
Figure 5.
28S rDNA-based phylogeny of P. geminatum ( = C. geminatum) with other dinoflagellates.
Sequences obtained in this study are bold-typed. Support of nodes is based on bootstrap values of ML/NJ with 1000 and 500 resamplings, respectively. Only values greater than 60 are shown. If only one of the two phylogenetic methods yielded significant support, the other is shown with “–”. Oxyrrhis marina was used as the outgroup to root the tree. Note that P. geminatum is embedded within the Gymnodinium s. s. clade with strong support, grouped with Polykrikos hartmannii.
Figure 6.
Phylogram of P. geminatum ( = C. geminatum) with other dinoflagellates inferred from 18S rDNA (left) and drawing of apical groove shapes for major related lineages (right).
Sequences obtained in this study are bold-typed. Support of nodes is based on bootstrap values of ML/NJ with 1000 and 500 resamplings, respectively. Only values greater than 60 are shown. If only one of the two phylogenetic methods yielded significant support, the other is shown with “–”. Oxyrrhis marina was used as the outgroup to root the tree. Note that P. geminatum is affiliated within the Gymnodinium s. s. clade, grouped with Polykrikos with strong support. From bottom up on the right, C. polykrikoides has a U-shaped apical groove; the Gymnodinium s. s. clade has three major similar forms of anticlockwise apical grooves (apical views) documented so far: horseshoe-shaped apical groove for Lepidodinium spp./Gymnodinium spp., anticlockwise kidney bean-shaped open-ended loop for P. geminatum /Polykrikos spp., and closed horseshoe-shaped apical groove for P. lebourae.
Figure 7.
Maximum-likelihood (ML) phylogeny of P. geminatum ( = C. geminatum) with other dinoflagellates inferred from 18S+28S rDNA concatenated data.
Sequences obtained in this study are bold-typed. Support of nodes is based on bootstrap values of ML with 1000 resamplings. Only values greater than 60 are shown. Oxyrrhis marina was used as the outgroup to root the tree. Note that P. geminatum is grouped with the other species of Polykrikos with strong support.
Figure 8.
Comparison of historical and current morphological descriptions of the species.
(a) Ventral-view line drawing of P. geminatum cells showing kidney bean-shaped open-ended loop apical groove from this study. (b) Ventral-view line drawing of C. geminatum cells not showing apical groove from Schütt (1895). (c) Ventral-view micrograph of C. geminatum cells also showing kidney bean-shaped open-ended loop apical groove from Hallegraeff et al. (2010).