Morphology, molecular phylogeny, and pigment characterization of an isolate of the dinoflagellate Pelagodinium bei from Korean waters

  • cc icon
  • ABSTRACT

    The dinoflagellate genus Pelagodinium is genetically classified in distinct sub-clades and subgroups. However, it is difficult to determine whether this genetic diversity represents intra- or interspecific divergence within the genus since only the morphology of the type strain of the genus Pelagodinium, Pelagodinium bei, is available. An isolate associated with the genus Pelagodinium from Shiwha Bay, Korea, was recently cultured. This isolate was clustered with 3 to 4 strains from the Atlantic Ocean, Mediterranean Sea, and Indian Ocean. This cluster was distinct from the subgroup more closely associated with P. bei. The morphology of the isolate was analyzed using optical and scanning electron microscopy and was almost identical to that of P. bei except that this isolate had two series of amphiesmal vesicles (AVs) in the cingulum, unlike P. bei that has one series. When the pigment compositions of the isolate and P. bei were analyzed using high-performance liquid chromatography, these two strains had peridinin as a major accessory pigment and their pigment compositions were almost identical. In addition, the swimming behaviors of these two strains were very similar. The reexamination of the type culture of P. bei revealed two series in the cingulum as for the isolate. The new findings on the number of series of AVs in the cingulum, the pigment composition, and the swimming behaviors suggest that P. bei and the isolate are conspecific despite their genetic divergence. This study provides a basis to further understand the molecular classification within Pelagodinium combining genetic, morphological, pigment, and behavioral data.


  • KEYWORD

    foraminifera , Gymnodinium bei , pelagic symbiont , Suessiaceae , Suessiales

  • 1. Balzano S., Gourvil P., Siano R., Chanoine M., Marie D., Lessard S., Sarno D., Vaulot D. 2012 Diversity of cultured photosynthetic flagellates in the northeast Pacific and Arctic Oceans in summer [Biogeosciences] Vol.9 P.4553-4571 google doi
  • 2. Daugbjerg N., Andreasen T., Happel E., Pandeirada M. S., Hansen G., Craveiro S. C., Calado A. J., Moestrup Ø. 2014 Studies on woloszynskioid dinoflagellates VII. Description of Borghiella andersenii sp. nov.: light and electron microscopy and phylogeny based on LSU rDNA [Eur. J. Phycol.] Vol.49 P.436-449 google doi
  • 3. Decelle J., Siano R., Probert I., Poirier C., Not F. 2012 Multiple microalgal partners in symbiosis with the acantharian Acanthochiasma sp. (Radiolaria) [Symbiosis] Vol.58 P.233-244 google doi
  • 4. De Vargas C., Audic S., Henry N., Decelle J., Mahe F., Logares R., Lara E., Berney C., Le Bescot N., Probert I., Carmichael M., Poulain J., Romac S., Colin S., Aury J. -M., Bittner L., Chaffron S., Dunthorn M., Engelen S., Flegontova O., Guidi L., Horak A., Jaillon O., LimaMendez G., Luke? J., Malviya S., Morard R., Mulot M., Scalco E., Siano R., Vincent F., Zingone A., Dimier C., Picheral M., Searson S., Kandels-Lewis S., Acinas S. G., Bork P., Bowler C., Gorsky G., Grimsley N., Hingamp P., Iudicone D., Not F., Ogata H., Pesant S., Raes J., Sieracki M. E., Speich S., Stemmann L., Sunagawa S., Weissenbach J., Wincker P., Karsenti E. 2015 Eukaryotic plankton diversity in the sunlit ocean [Science] Vol.348 P.1261605 google doi
  • 5. Fujiki T., Takagi H., Kimoto K., Kurasawa A., Yuasa T., Mino Y. 2014 Assessment of algal photosynthesis in planktic foraminifers by fast repetition rate fluorometry [J. Plankton Res.] Vol.36 P.1403-1407 google doi
  • 6. Gast R. J., Caron D. A. 1996 Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria [Mol. Biol. Evol.] Vol.13 P.1192-1197 google doi
  • 7. Guillard R. R. L., Ryther J. H. 1962 Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran [Can. J. Microbiol.] Vol.8 P.229-239 google doi
  • 8. Hall T. A. 1999 BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT [Nucleic Acids Symp. Ser.] Vol.41 P.95-98 google
  • 9. Hansen G., Daugbjerg N. 2009 Symbiodinium natans sp. nov.: a “free-living” dinoflagellate from Tenerife (Northeast-Atlantic Ocean) [J. Phycol.] Vol.45 P.251-263 google doi
  • 10. Hansen G., Daugbjerg N., Henriksen P. 2007 Baldinia anauniensis gen. et sp. nov.: a ‘new’ dinoflagellate from Lake Tovel, N. Italy [Phycologia] Vol.46 P.86-108 google doi
  • 11. Jakobsen H. H., Everett L. M., Strom S. L. 2006 Hydromechanical signaling between the ciliate Mesodinium pulex and motile protist prey [Aquat. Microb. Ecol.] Vol.44 P.197-206 google doi
  • 12. Jang S. H., Jeong H. J., Moestrup Ø., Kang N. S., Lee S. Y., Lee K. H., Lee M. J., Noh J. H. 2015 Morphological, molecular and ecophysiological characterization of the phototrophic dinoflagellate Biecheleriopsis adriatica from Korean coastal waters [Eur. J. Phycol.] Vol.50 P.301-317 google doi
  • 13. Jeong H. J., Jang S. H., Moestrup Ø., Kang N. S., Lee S. Y., Potvin E., Noh J. H. 2014 Ansanella granifera gen. et sp. nov. (Dinophyceae), a new dinoflagellate from the coastal waters of Korea [Algae] Vol.29 P.75-99 google doi
  • 14. Jeong H. J., Lee S. Y., Kang N. S., Yoo Y. D., Lim A. S., Lee M. J., Kim H. S., Yih W., Yamashita H., LaJeunesse T. C. 2014 Genetics and morphology characterize the dinoflagellate Symbiodinium voratum, n. sp., (Dinophyceae) as the sole representative of Symbiodinium clade E [J. Eukaryot. Microbiol.] Vol.61 P.75-94 google doi
  • 15. Jeong H. J., Lim A. S., Franks P. J. S., Lee K. H., Kim J. H., Kang N. S., Lee M. J., Jang S. H., Lee S. Y., Yoon E. Y., Park J. Y., Yoo Y. D., Seong K. A., Kwon J. E., Jang T. Y. 2015 A hierarchy of conceptual models of red-tide generation: nutrition, behavior, and biological interactions [Harmful Algae] Vol.47 P.97-115 google doi
  • 16. Jeong H. J., Yoo Y. D., Kang N. S., Lim A. S., Seong K. A., Lee S. Y., Lee M. J., Lee K. H., Kim H. S., Shin W., Nam S. W., Yih W., Lee K. 2012 Heterotrophic feeding as a newly identified survival strategy of the dinoflagellate Symbiodinium [Proc. Natl. Acad. Sci. U. S. A.] Vol.109 P.12604-12609 google doi
  • 17. Jeong H. J., Yoo Y. D., Kim J. S., Seong K. A., Kang N. S., Kim T. H. 2010 Growth, feeding, and ecological roles of the mixotrophic and heterotrophic dinoflagellates in marine planktonic food webs [Ocean Sci. J.] Vol.45 P.65-91 google doi
  • 18. Kang N. S., Jeong H. J., Yoo Y. D., Yoon E. Y., Lee K. H., Lee K., Kim G. 2011 Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration [J. Eukaryot. Microbiol.] Vol.58 P.152-170 google doi
  • 19. Kok S. P., Tsuchiya K., Komatsu K., Toda T., Kurosawa N. 2014 The protistan microplankton community along the Kuroshio Current revealed by 18S rRNA gene clone analysis: a case study of the differences in distribution interplay with ecological variability [Plankton Benthos Res.] Vol.9 P.71-82 google doi
  • 20. Kremp A., Elbrachter M., Schweikert M., Wolny J. L., Gottschling M. 2005 Woloszynskia halophila (Biecheler) comb. nov.: a bloom-forming cold-water dinoflagellate co-occurring with Scrippsiella hangoei (Dinophyceae) in the Baltic Sea [J. Phycol.] Vol.41 P.629-642 google doi
  • 21. LaJeunesse T. C., Lee S. Y., Gil-Agudelo D. L., Knowlton N., Jeong H. J. 2015 Symbiodinium necroappetens sp. nov. (Dinophyceae): an opportunist ‘zooxanthella’ found in bleached and diseased tissues of Caribbean reef corals [Eur. J. Phycol.] Vol.50 P.223-238 google doi
  • 22. Larkin M. A., Blackshields G., Brown N. P., Chenna R., Mc-Gettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A., Lopez R., Thompson J. D., Gibson T. J., Higgins D. G. 2007 Clustal W and Clustal X version 2.0 [Bioinformatics] Vol.23 P.2947-2948 google doi
  • 23. Lee S. K., Jeong H. J., Jang S. H., Lee K. H., Kang N. S., Lee M. J., Potvin E. 2014 Mixotrophy in the newly described dinoflagellate Ansanella granifera: feeding mechanism, prey species, and effect of prey concentration [Algae] Vol.29 P.137-152 google doi
  • 24. Lee S. Y., Jeong H. J., Kang N. S., Jang T. Y., Jang S. H., Lajeunesse T. C. 2015 Symbiodinium tridacnidorum sp. nov., a dinoflagellate common to Indo-Pacific giant clams, and a revised morphological description of Symbiodinium microadriaticum Freudenthal, emended Trench & Blank [Eur. J. Phycol.] Vol.50 P.155-172 google doi
  • 25. Litaker R. W., Vandersea M. W., Kibler S. R., Reece K. S., Stokes N. A., Steidinger K. A., Millie D. F., Bendis B. J., Pigg R. J., Tester P. A. 2003 Identification of Pfiesteria piscicida (Dinophyceae) and Pfiesteria-like organisms using internal transcribed spacer-specific PCR assays [J. Phycol.] Vol.39 P.754-761 google doi
  • 26. Luo Z., Yang W., Xu B., Gu H. 2013 First record of Biecheleria cincta (Dinophyceae) from Chinese coasts, with morphological and molecular characterization of the strains [Chin. J. Oceanol. Limnol.] Vol.31 P.835-845 google doi
  • 27. Moestrup Ø., Hansen G., Daugbjerg N. 2008 Studies on woloszynskioid dinoflagellates III: on the ultrastructure and phylogeny of Borghiella dodgei gen. et sp. nov., a cold-water species from Lake Tovel, N. Italy, and on B. tenuissima comb. nov. (syn. Woloszynskia tenuissima) [Phycologia] Vol.47 P.54-78 google doi
  • 28. Moestrup Ø., Lindberg K., Daugbjerg N. 2009 Studies on woloszynskioid dinoflagellates IV: The genus Biecheleria gen. nov [Phycol. Res.] Vol.57 P.203-220 google doi
  • 29. Moestrup Ø., Lindberg K., Daugbjerg N. 2009 Studies on woloszynskioid dinoflagellates V. Ultrastructure of Biecheleriopsis gen. nov., with description of Biecheleriopsis adriatica sp. nov [Phycol. Res.] Vol.57 P.221-237 google doi
  • 30. Montresor M., Lovejoy C., Orsini L., Procaccini G., Roy S. 2003 Bipolar distribution of the cyst-forming dinoflagellate Polarella glacialis [Polar Biol.] Vol.26 P.186-194 google
  • 31. Pochon X., Gates R. D. 2010 A new Symbiodinium clade (Dinophyceae) from soritid foraminifera in Hawai’i [Mol. Phylogenet. Evol.] Vol.56 P.492-497 google doi
  • 32. Posada D., Buckley T. R. 2004 Model selection and model averaging in phylogenetics: advantages of Akaike Information Criterion and Bayesian approaches over likelihood ratio tests [Syst. Biol.] Vol.53 P.793-808 google doi
  • 33. Posada D., Crandall K. A. 1998 MODELTEST: testing the model of DNA substitution [Bioinformatics] Vol.14 P.817-818 google doi
  • 34. Ronquist F., Huelsenbeck J. P. 2003 MrBayes 3: Bayesian phylogenetic inference under mixed models [Bioinformatics] Vol.19 P.1572-1574 google doi
  • 35. Rowan R., Powers D. A. 1992 Ribosomal RNA sequences and the diversity of symbiotic dinoflagellates (zooxanthellae) [Proc. Natl. Acad. Sci. U. S. A.] Vol.89 P.3639-3643 google doi
  • 36. Shaked Y., De Vargas C. 2006 Pelagic photosymbiosis: rDNA assessment of diversity and evolution of dinoflagellate symbionts and planktonic foraminiferal hosts [Mar. Ecol. Prog. Ser.] Vol.325 P.59-71 google doi
  • 37. Siano R., Kooistra W. H. C. F., Montresor M., Zingone A. 2009 Unarmoured and thin-walled dinoflagellates from the Gulf of Naples, with the description of Woloszynskia cincta sp. nov. (Dinophyceae, Suessiales) [Phycologia] Vol.48 P.44-65 google doi
  • 38. Siano R., Montresor M., Probert I., Not F., De Vargas C. 2010 Pelagodinium gen. nov and P. beii comb. nov., a dinoflagellate symbiont of planktonic Foraminifera [Protist] Vol.161 P.385-399 google doi
  • 39. Spero H. J. 1987 Symbiosis in the planktonic foraminifer, Orbulina universa, and the isolation of its symbiotic dinoflagellate, Gymnodinium beii sp. nov [J. Phycol.] Vol.23 P.307-317 google doi
  • 40. Stamatakis A. 2006 RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models [Bioinformatics] Vol.22 P.2688-2690 google doi
  • 41. Swofford D. L. 2002 PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4 google
  • 42. Takahashi K., Sarai C., Iwataki M. 2014 Morphology of two marine woloszynskioid dinoflagellates, Biecheleria brevisulcata sp. nov. and Biecheleriopsis adriatica (Suessiaceae, Dinophyceae), from Japanese coasts [Phycologia] Vol.53 P.52-65 google doi
  • 43. Venn A. A., Wilson M. A., Trapido-Rosenthal H. G., Keely B. J., Douglas A. E. 2006 The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium [Plant Cell Environ.] Vol.29 P.2133-2142 google doi
  • 44. Zapata M., Rodriguez F., Garrido J. L. 2000 Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases [Mar. Ecol. Prog. Ser.] Vol.195 P.29-45 google doi
  • [Fig. 1.] Micrographs of the isolate taken using optical microscopy. (A) Ventral view illustrating the round to elliptical nucleus (N). (B) Side view. The arrowhead indicates the orange to red eyespot located in the sulcal area. (C) Side view. The arrowheads indicate pyrenoids. Scale bars represent: A-C, 1 μm.
    Micrographs of the isolate taken using optical microscopy. (A) Ventral view illustrating the round to elliptical nucleus (N). (B) Side view. The arrowhead indicates the orange to red eyespot located in the sulcal area. (C) Side view. The arrowheads indicate pyrenoids. Scale bars represent: A-C, 1 μm.
  • [Fig. 2.] Micrographs of the isolate taken using scanning electron microscopy. (A) Hypoconal flange. (B & C) Apical furrow. (D-G) Apical and ventral views. (H) Left view. (I) Right view. (J) Sulcal view. (K & L) Antapical views. X, small squared vesicle. The amphiesmal vesicles were numbered and assigned to their respective series. Scale bars represent: A-L, 1 μm.
    Micrographs of the isolate taken using scanning electron microscopy. (A) Hypoconal flange. (B & C) Apical furrow. (D-G) Apical and ventral views. (H) Left view. (I) Right view. (J) Sulcal view. (K & L) Antapical views. X, small squared vesicle. The amphiesmal vesicles were numbered and assigned to their respective series. Scale bars represent: A-L, 1 μm.
  • [Fig. 3.] Micrographs of the type culture of Pelagodinium bei (RCC #1491) taken using scanning electron microscopy and a schematized view of the sulcal area. (A) Ventral view. (B) Dorsal view. (C-E) Sulcal views. The arrowhead indicates a small amphiesmal vesicle located at the left side of the longitudinal flagellar pore. (F) Drawing of the sulcus. The black circles indicate the location of the flagellar pores of the transversal and longitudinal flagella. The amphiesmal vesicles were numbered and assigned to their respective series. Scale bars represent: A-E, 1 μm.
    Micrographs of the type culture of Pelagodinium bei (RCC #1491) taken using scanning electron microscopy and a schematized view of the sulcal area. (A) Ventral view. (B) Dorsal view. (C-E) Sulcal views. The arrowhead indicates a small amphiesmal vesicle located at the left side of the longitudinal flagellar pore. (F) Drawing of the sulcus. The black circles indicate the location of the flagellar pores of the transversal and longitudinal flagella. The amphiesmal vesicles were numbered and assigned to their respective series. Scale bars represent: A-E, 1 μm.
  • [Fig. 4.] Maximum likelihood (ML) phylogenetic tree based on 595 aligned nucleotides of the nuclear internal transcribed spacer rDNA using the GTR + G model with Polarella glacialis as an outgroup taxon. Alignment length includes gaps. The parameters were as follows: assumed nucleotide frequencies A = 0.1893, C = 0.2283, G = 0.2600, and T = 0.3224; substitution rate matrix with G-T = 1.0000, A-C = 0.5967, A-G = 1.7246, A-T = 0.9218, C-G = 0.3420, C-T = 3.2104; proportion of invariable sites = 0.0000 and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.2878. The numbers at the nodes of the branches indicate the ML bootstrap (left) and Bayesian posterior probability (right) values; only values ≥ 50% or 0.5 are shown.
    Maximum likelihood (ML) phylogenetic tree based on 595 aligned nucleotides of the nuclear internal transcribed spacer rDNA using the GTR + G model with Polarella glacialis as an outgroup taxon. Alignment length includes gaps. The parameters were as follows: assumed nucleotide frequencies A = 0.1893, C = 0.2283, G = 0.2600, and T = 0.3224; substitution rate matrix with G-T = 1.0000, A-C = 0.5967, A-G = 1.7246, A-T = 0.9218, C-G = 0.3420, C-T = 3.2104; proportion of invariable sites = 0.0000 and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.2878. The numbers at the nodes of the branches indicate the ML bootstrap (left) and Bayesian posterior probability (right) values; only values ≥ 50% or 0.5 are shown.
  • [Fig. 5.] Maximum likelihood (ML) phylogenetic tree based on 558 aligned nucleotides of the nuclear large subunit rDNA using the TIM + I + G model with Alexandrium tamarense, A. catenella, Ceratium fusus, and C. lineatum as outgroup taxa. Alignment length includes gaps. The parameters were as follows: assumed nucleotide frequencies A = 0.2404, C = 0.1639, G = 0.2942, and T = 0.3014; substitution rate matrix with G-T = 1.0000, A-C = 1.0000, A-G = 2.2082, A-T = 0.7906, C-G = 0.7906, C-T = 6.5313; proportion of invariable sites = 0.1258 and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.7930. The numbers at the nodes of the branches indicate the ML bootstrap (left) and Bayesian posterior probability (right) values; only values ≥50% or 0.5 are shown.
    Maximum likelihood (ML) phylogenetic tree based on 558 aligned nucleotides of the nuclear large subunit rDNA using the TIM + I + G model with Alexandrium tamarense, A. catenella, Ceratium fusus, and C. lineatum as outgroup taxa. Alignment length includes gaps. The parameters were as follows: assumed nucleotide frequencies A = 0.2404, C = 0.1639, G = 0.2942, and T = 0.3014; substitution rate matrix with G-T = 1.0000, A-C = 1.0000, A-G = 2.2082, A-T = 0.7906, C-G = 0.7906, C-T = 6.5313; proportion of invariable sites = 0.1258 and rates for variable sites assumed to follow a gamma distribution with shape parameter = 0.7930. The numbers at the nodes of the branches indicate the ML bootstrap (left) and Bayesian posterior probability (right) values; only values ≥50% or 0.5 are shown.
  • [FIG. 6.] Pigment composition of the isolate from Korea and Pelagodinium bei derived by high performance liquid chromatography. (A) Chromatogram of the isolate from Korea. (B) Chromatogram of the type culture of P. bei (RCC #1491). 1, Chlorophyll c2; 2, Peridinin; 3, Diadinoxanthin; 4, Diatoxanthin; 5, Zeaxanthin; 6, Alloxanthin; 7, Chlorophyll a; 8, β-carotene.
    Pigment composition of the isolate from Korea and Pelagodinium bei derived by high performance liquid chromatography. (A) Chromatogram of the isolate from Korea. (B) Chromatogram of the type culture of P. bei (RCC #1491). 1, Chlorophyll c2; 2, Peridinin; 3, Diadinoxanthin; 4, Diatoxanthin; 5, Zeaxanthin; 6, Alloxanthin; 7, Chlorophyll a; 8, β-carotene.
  • [Table 1.] Comparison between the isolate from Korea and Pelagodinium bei from the Caribbean Sea
    Comparison between the isolate from Korea and Pelagodinium bei from the Caribbean Sea