Taxonomy and phylogeny of marine unarmored dinoflagellates (and others)
Mitsunori Iwataki Asian Natural Environmental Science Center, The University of Tokyo Morphology of dinoflagellates Prorocentrales Dinophysiales Peridiniales Gonyaulacales Gymnodiniales
Noctilucales Armored and unarmored dinoflagellates
unarmored Woloszynskioids armored (athecate, naked) (thecate)
thecal plates
Amphiesmal vesicle (Taylor 1987) Taxonomy of armored dinoflagellates Armored dinoflagellates are classified based on number and arrangement of the thecal plates.
Scrippsiella Po, X, 4', 3a, 7", 6c, 4s, 5"', 2"” Heterocapsa Po, X, 5', 3a, 7", 6c, 4s, 5"', 2”” Previous taxonomy of unarmored dinoflagellates Since the cells are not covered by thecal plates, position and torsion of the cingulum was used for he crassic taxonomic criterion for genus of unarmored dinoflagellates.
Gymnodinium Gyrodinium Cochlodinium Amphidinium Katodinium Previous taxonomy of unarmored dinoflagellates
Gymnodinium Gyrodinium Cingular displacement <1/5 of cell length Cingular displacement >1/5 of cell length Phylogenetic positions of unarmored dinoflagellates Generic diagnoses of unaremod Gonyaulacales dinoflagellates were emended and several new genera were Peridiniales newly proposed. Peridiniales (Freshwater) (Daugbjerg et al. 2000)
Prorocentrales
Suessiales
Gymnodiniales Akashiwo Gymnodinium GPP complex GPP
Gymnodiniales
Gymnodiniales
Prorocentrales Peridiniales (Maine) Peridiniales (Marine) Karenia Karlodinium Apical groove Apical groove is visible under scanning electron microscope. Stable character in each genus, and used for taxonomic criterion of present taxonomy of unarmored dinoflagellates.
straight straight
curves in Horseshoe clockwise -shaped
Horseshoe elliptical sigmoid -shaped ? Apical groove of Cochlodinium fulvescens
C. polykrikoides C. fulvescens
(Iwataki et al. 2010) (Iwataki et al. in press) C. fulvescens Recently established genera (from Amphidinium) Amphidinium sp. Togula britannica
(Flø Jørgensen et al. 2004) Apicoporus glaber Ankistrodinium semilunatum
(Sparmann et al. 2008) (Hoppenrath et al. 2012) Testudodinium corrugatum Bispinodinium angelaceum
(Horiguchi et al. 2012) (Yamada et al. 2013) Recently established genera (Gymnodinium and Gyrodinium) Paragymnodinium shiwhaense Gyrodiniellum shiwhaense
(Kang et al. 2010) (Kang et al. 2011) Barrufeta bravensis Moestrupia oblonga
(Sanpedro et al. 2011) (Hansen et al. 2011) Ceratoperidinium falcatum Levanderina fissa
(Rene et al. 2013) (Moestrup et al. 2014) Phylogenetic position of unarmored dinoflagellates based on SSU rDNA sequences Photosynthetic Species55 samples belonging to 15 genera
C. convolutum Cochlodinium polykrikoides Cochlodinium fulvescens Gymnodinium catenatum Gymnodinium nolleri G. microreticulatum (Omura Bay) (Isahaya Bay, Malaysia) (Tachibana Bay, Indonesia) (Philippines) (Hiroshima Bay) (Sasebo, Obama)
G. cf. chlorophorum Gymnodinium sp. Gymnodinium sp. Gymnodinium sp. Gyrodinium instriatum Gyrodinium falcatum (Tokyo Bay) (East China Sea) (Yatsushiro Bay) (East China Sea) (Isahaya Bay) (Inokushi Bay, Tsushima)
D. pseudolunula Phaeobalechina catenata Karlodinium sp. Takayama sp. Spatulodinium pseudonoctiluca Torodinium teredo (Omura Bay) nom. nud. (Omura Bay) (Ariake Sound) (Ariake Sound) (Omura Bay) (Nagasaki) Heterotrophic Species
Achradina pulchra Balechina coerulea Erythropsidinium agile Gymnodinium cf. puniceum (East China Sea) (Nagasaki) (Nagasaki) (Nagasaki)
Gymnodinium gracile Gyrodinium spirale Gyrodinium rubrum Gymnodinium aureum (Nagasaki) (Nagasaki) (Nagasaki) (Nagasaki)
Gyrodinium sp. Katodinium glaucum Polykrikos cf. kofoidii Nematodinium sp. Nematodinium sp. Nematodinium sp. (East China Sea) (Omura Bay, Ariake Sound) (Nagasaki) (Nagasaki) (Nagasaki) (Nagasaki) Perkinsus sp. Perkinsus marinusPhylogenetic Tree Based on SSU rDNA Sequences Noctiluca scintillans Spatulodinium pseudonoctiluca Spatulodinium pseudonoctiluca Gamma corrected NJ tree, evolutionary model: Tamura & Nei (1993), Phaeobalechina catenata Phaeobalechina catenata Proportion of invariable site: 0.2608, Shape parameter: 0.6499 Balechina coerulea Balechina coerulea Gyrodinium dorsum Gyrodinium uncatenum Gyrodinium instriatum N. scintillans Alexandrium tamarense Alexandrium minutum Pyrocystis noctiluca Pyrocystis lunula Spatulodinium pseudonoctiluca Symbiodinium corculorum Symbiodinium microadriaticum Gymnodinium gracile Gymnodinium gracile Gymnodinium gracile Achradina pulchra Achradina pulchra Achradina pulchra Gymnodinium sp. Gymnodinium sp. Gyrodinium galatheanum Gymnodinium galatheanum Karlodinium micrum Takayama sp. Karenia + Karlodinium + Takayama Gymnodinium breve Karenia brevis Karenia mikimotoi Karenia mikimotoi Amphidinium carterae Amphidinium massartii Akashiwo sanguinea Akashiwo sanguinea Gymnodinium cf. puniceum Erythropsidinium agile Gyrodinium sp. Polykrikos kofoidii Pheopolykrikos hartmannii Gymnodinium fuscum Nematodinium sp. Erythropsidinium + Nematodinium + Polykrikos etc. Nematodinium sp. Nematodinium sp. Gyrodinium impudicum Dissodinium pseudolunula Gymnodinium sensu stricto + Lepidodinium Lepidodinium viride Gymnodinium chlorophorum Gymnodinium catenatum Gymnodinium catenatum Gymnodinium nolleri Gymnodinium microreticulatum Gymnodinium microreticulatum Gyrodinium falcatum Gyrodinium falcatum Gyrodinium falcatum Gymnodinium sp. C. convolutum + G. falcatum + Gymnodinium spp. Cochlodinium convolutum Cochlodinium convolutum Gymnodinium sp. Gyrodinium spirale Gyrodinium fusiforme Gyrodinium spirale Gyrodinium sp. Gyrodinium rubrum Gymnodinium rubrum Gyrodinium sensu stricto Gyrodinium aureum Gymnodinium helveticum Katodinium glaucum Katodinium glaucum Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides C. polykrikoides + C. fulvescens Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium sp. Cochlodinium sp. Torodinium teredo Related Species Unknown
Akashiwo sanguinea Levanderina fissa Achradina pulchra (Isahaya Bay) (East China Sea)
Gymnodinium sp. Gymnodinium gracile (East China Sea) (Nagasaki, Omura Bay)
Torodinium teredo Katodinium glaucum Balechina coerulea Balechina sp. (Nagasaki) (Omura Bay, Ariake Sound) (Nagasaki) (Omura Bay) Gymnodinium sensu stricto - Heterotrophic species possessing nematocyst, ocellus eyespot and piston are related to Gymnodinium sensu stricto.
88 Gymnodinium cf. puniceum Nagasaki Erythropsidinium agile Nagasaki
Gyrodinium sp. 80 Polykrikos cf. kofoidii Nagasaki Polykrikos hartmannii Gymnodinium fuscum
Nematodinium sp. Nagasaki
100 Nematodinium sp. Ariake Sound Nematodinium sp. East China Sea Gymnodinium impudicum Dissodinium pseudolunula Omura Bay 100 Lepidodinium viride Gymnodinium cf. chlorophorum Tokyo Bay 100 Gymnodinium catenatum Gymnodinium catenatum Philippines 95 Gymnodinium cf. nolleri Hiroshima Bay 100 Gymnodinium microreticulatum Sasebo Bay 100 Gymnodinium microreticulatum Obama Bay Gyrodinium sensu stricto
- Gyrodinium species characterized by possessing an elliptical apical groove and cell surface striations are monophyletic.
Gyrodinium spirale 77
100 Gyrodinium fusiforme
Gyrodinium spirale (Nagasaki) 90 Gyrodinium sp. 100 (East China Sea) Gyrodinium cf. nasutum 100 (Nagasaki) Gyrodinium rubrum 100 Gyrodinium cf. lineatum 100 (Nagasaki) Gyrodinium helveticum Karenia/Karlodinium/Takayama - Apical groove is strait or sigmoid, containing chloroplast derived from a haptophyte.
- Relationship between Karenia and Karlodinium/Takayama is not highly supported in phylogenetic tree SSU rDNA. Karlodinium micrum 93 (as Gyrodinium galatheanum) Karlodinium micrum 95 (as Gymnodinium galatheanum) Karlodinium sp. Ariake Sound 59
Takayama sp. Omura Bay <50 Karenia brevis 98 （as Gymnodinium breve） Karenia brevis 100 （as Gymnodinium breve） Karenia mikimotoi 72 （as Gymnodinium mikimotoi） Karenia mikimotoi (as Gyrodinium aureolum) Noctiluca scintillans/Spatulodinium pseudonoctiluca
- Noctiluca scintillans and Spatulodinium pseudonoctiluca formed a monophyletic group, which branched out the basal position of all dinoflagellates.
- Two forms of Spatulodinium pseudonoctiluca, Noctiluca-form (S. pseudonoctiluca) and Gymnodinium-form (Gymnodinium lebourae) are the same species.
Noctiluca scintillans 100 Spatulodinium pseudonoctiluca 100 (Omura Bay) Spatulodinium pseudonoctiluca (Omura Bay) The clade related to Ceratoperidinium Consisted of Cochlodinium convolutum, Gyrodinium falcatum and Gymnodinium spp. Photosynthetic, with a circle shaped apical groove (?) and forming hyaline cyst.
Ceratoperidinium falcatum 77 (Tsushima Is) 87 Ceratoperidinium falcatum (Inokushi Bay) 71 Ceratoperidinium falcatum (Indonesia) 98 Gymnodinium sp. (Ariake Sound) Cochlodinium convolutum 100 79 (Omura Bay) Cochlodinium convolutum (Omura Bay) Gymnodinium sp. (Yatsushiro Bay) Recent our works on taxonomy and phylogeny of small species including unarmored, woloszynskioid and armored dinoflagellates Small dinoflagellates occurred in the Western Pacific Amphidinium
Woloszynskioids
Armored dinoflagellates Identified Amphidinium spp. Scale bars = 10 µm (LM), 100 nm (TEM)
Amphidinium carterae Amphidinium massarti
Amphidinium cupulatisquama
Amphidinium gibbosum Amphidinium mootonorum Identified Amphidinium spp.
Length Other Species Locality Cell shape Chloroplast （μm） characters Turtle Cove, 12.0-18.0 Ellipsoid Membranous Palau A. carterae Ngeruktabel, 12.5-22.5 Ellipsoid Membranous Palau Tsuruoka, Eye spot, 32.5-45.0 Ellipsoid Rod-like Japan Body scale A. cupulatisquama Tsuruoka, Eye spot, 23.0-37.0 Ellipsoid Rod-like Japan Body scale Pari Island, Asymmetrical, 25.0-37.5 Rod-like Indonesia Heart-shaped A. gibbosum Turtle Cove, Asymmetrical, 18.0-32.0 Rod-like Palau Heart-shaped Okinawa, A. massartii 13.5-19.5 Ellipsoid Rod-like Japan Yamagata, Nucleus located A. mootonorum 32.5-50.0 Ellipsoid Rod-like Japan in the middle Cell shape, chloroplasts, presence of eyespot and body scales, and position of nucleus are recognized as reliable character for species identification of some species. Difficulty of Amphidinium identification
Species Length（µm） Shape Pyrenoid Chloroplast Pusule Immotile cell A. steinii 20-38 ellipsoid spherical rod-like 2 present A. trulla 18-31 ellipsoid spherical rod-like 0 absent
A. operculatum A. gibbosum A. steinii A. trulla
(Murray et al. 2004) Species in middle cell size range are difficult to identify… A. massarti A. carteriae Unidentified Amphidinium spp. Scale bars = 10 µm
Amphidinium sp. 1 (Indonesia) Amphidinium sp. 2 (Japan)
Amphidinium sp. 3 (Australia) Amphidinium sp. 4 (Japan)
Amphidinium sp. 5 (Vietnam) Amphidinium sp. 6 (Malaysia) Unidentified Amphidinium spp.
Locality Length (µm) Cell shape Pyrenoid Chloroplast Pusule Immotile cell
Pari Island, Indonesia 17.5-27.5 ellipsoid 1 rod-like - absent Amphidinium Pari Island, Indonesia 15.0-25.0 ellipsoid 1 rod-like - present sp. 1 Pari Island, Indonesia 17.5-30.0 ellipsoid 1 rod-like - -
Sendai, Japan 17.5-27.5 ellipsoid 1 rod-like - present Amphidinium sp. 2 Sendai, Japan 17.5-27.5 ellipsoid 1 rod-like - - Amphidinium Melbourne, Australia 17.5-32.5 ellipsoid 1 reticulate - - sp. 3 Tsuruoka, Japan 17.5-30.0 ellipsoid 1 rod-like - - Amphidinium Tsuruoka, Japan 17.5-32.5 ellipsoid 1 rod-like - - sp. 4 Tsuruoka, Japan 17.5-28.0 ellipsoid 1 rod-like 2 absent
Nha Trang, Vietnam 20.0-35.5 ellipsoid >10 rod-like 2 absent Amphidinium sp. 5 Nha Trang, Vietnam 17.0-31.0 ellipsoid 1-4 rod-like 2 absent
Port Dickson, Malaysia 15.0-25.0 ellipsoid 1 reticulate 1 - 2 present Amphidinium sp. 6 Port Dickson, Malaysia 13.0-23.0 ellipsoid 1 reticulate 2 present Port Dickson, Malaysia 14.0-22.0 ellipsoid 1 reticulate 2 present Molecular phylogeny based on LSU rDNA
Gamma corrected maximum likelihood (ML) tree, evolutionary model: GTR+I+G
A. carterae
A. massartii
Amphidinium sp. 5 (Vietnam) Not related
Amphidinium sp. 2 (Japan) Not related A. gibbosum Amphidinium sp. 3 (Australia) A. trulla
Amphidinium sp. 1 (Indonesia) Amphidinium sp. 4 (Japan) A. thermaeum Amphidinium sp. 6 (Malaysia)
A. herdmanii / mootonorum
A. steinii
Bootstrap support values: ML/MP/NJ, >50%, ●=100% Distribution of Amphidinium in East and Southeast Asia
Distribution of Amphidinium species in the East and Southeast Asia
Amphidinium carterae
Amphidinium cupulatisquama
Amphidinium gibbosum
Amphidinium massartii
Amphidinium mootonorum
Amphidinium thermaeum
Amphidinium sp. 2
Amphidinium sp. 6
A. thermaeum is widely distributed in East and SE Asia. Woloszynskioid dinoflagellates
unarmored Woloszynskioids armored (athecate, naked) (thecate)
thecal plates
Amphiesmal vesicle (Taylor 1987) Biecheleriopsis adriatica
Biecheleriopsis adriatica (Japan and Palau)
10μm
E1 E2
E3 E4
2μm
Present study Original （Moestrup et al. 2009b） Pelagodinium bei
Pelagodinium bei (Japan and Vietnam)
10μm
1-5 1-4
2-2 1-3
1-6 1-2 1-1
2μm Present study Original （Siano et al. 2009） Biecheleria brevisulcata Takahashi et Iwataki 2014
Biecheleria brevisulcata (Japan)
10μm
2μm Small armored dinoflagellates
・7 cultures of small armored dinoflagellates from the western Pacific Peridinioid sp.1 From Palau, Guam, Philippines, Malaysia, Peridinioid sp.2 From Indonesia, Philippines
Peridinioid sp.1 Peridinioid sp.2 Peridinioid sp.1
Motile cell Immotile cell
Mushroom-shaped Outline Mushroom-shaped
Posteriorly Nucleus (N) Anteriorly N
Posteriorly Eyespot (E) Anteriorly Py NN Pyrenoid Anteriorly Posteriorly (Py) E Py 17.1 μm Cell length 17.5 μm E (14.0–21.5) n=31 (15.0–20.0)
Scale bar=10 μm Location of organelles change in motile and immotile Peridinioid sp.1
2μm
Scale bars=2 μm (SEM), 10 μm (LM) Thecal plates: Po,cp,4’,3a,7”,6c,5’’’,2”” A conspicuous sulcal list in the sulcus Peridinioid sp.2
Motile cell Immotile cell
Ellipsoidal, conical epitheca Outline Spherical
Nucleus Posterior Central (N) Eyespot Posterior Peripheral (E) Pyrenoid Anterior Central (Py)
Absent Red body Central Py N Cell E N 17.5 μm 18.7 μm length E (16.5‐19.0) (12.5‐28.8) n=31 Scale bars=10 μm Different cell shape in motile and immotile cells Peridinioid sp.2
Scale bar=10μm
Thecal plates: Po,cp,4’,2a,6”,6c,5’’’,2”” Two anterior intercalary plate (2a) (Peridinioids have 3a typically) Phylogenetic tree
Peridinioid sp.1 formed a monophyletic group related species was uncertain
Peridinioid sp.2 formed a monophyletic group related to Peridiniopsis borgei, Scrippsiella hexapraecingula
Peridinioid sp.2
Peridinioid sp.1
ML Tree（D1-D3, 579 bp） BS values>50%: ML/NJ Summary
New genera have being newly proposed based on the phylogenetic position and combination of morphological characters different from those of previouly reported genera.
Levanderina fissa (Levander) Moestrup et al. 2014 = Gyrodinium instriatum = Gyrodinium uncatenum = Gyrodinium fissum
Many of small undescribed dinoflagellates are commonly found in the Western Pacific, these species can be be distinguished from unarmored dinoflagellates, - Woloszynskioid: an eyespot (difficult to observe by light microscopy), sigmoid sulcus in the ventral
- Small peridinioids: thecal plates and an eyespot (difficult to observe by light microscopy), mushroom-shaped or coccoid immotile stage.