PICES 14 (Vladivostok)

PhylogenyPhylogeny andand geographicalgeographical distributiondistribution ofof CochlodiniumCochlodinium polykrikoidespolykrikoides populationpopulation (Gymnodiniales,(Gymnodiniales, )Dinophyceae) collectedcollected fromfrom JapaneseJapanese andand KoreanKorean coastscoasts

Mitsunori Iwataki1, Hisae Kawami1, Kazumi Matsuoka1, Takuo Omura2 & Yasuwo Fukuyo2 1. Institute for East China Sea Research, Nagasaki University 2. Asian Natural Environmental Science Center, the University of Tokyo A harmful Cochlodinium polykrikoides

An unarmored dinoflagellate Cochlodinium polykrikoides has formed red tides responsible for mass mortalities of caged and wild fishes in Japanese and Korean coasts. Occurrences of C. polykrikoides in Japan and Korea Red tides of C. polykrikoides have continuously occurred along coastal waters, does this imply that the Japanese and Korean population are identical?

Miyahara et al. 2005

Sep. 2003

?

Jun-Aug 2002

Matsuoka & Iwataki 2004 The genus Cochlodinium

The genus Cochlodinium has been established by Schütt (1894), circumscribing unarmored possessing the cingulum encircling the cell >1.5 times (Kofoid & Swezy 1921).

Unarmored dinoflagellates have been classified C. citron C. lebourae based mainly on the position and torsion of the cingulum, however, this classification was C. catenatum Kofoid & Swezy 1921 incongruent with phylogenetic relationship based on recent molecular analyses.

Cochlodinium is still classified based on characteristics of the cingulum, because their phylogenetic relationships have not been examined.

Objectives

To clarify the phylogenetic positions of Cochlodinium species among dinoflagellates.

To distinguish populations of a harmful red tide forming species C. polykrikoides, and investigate distributions of each C. polykrikoides population. MaterialsMaterials andand MethodsMethods

Morphology Light and fluorescence microscopy Molecular phylogeny SSU rDNA LSU rDNA (D1-D3) Trees constructed by weighted NJ method

Manila Bay, Philippines

Sabah, Malaysia

Lampung Bay, Indonesia Red tide occurred in Malaysian, Brunei and Philippine waters

na hi C h ut a So Se

Palawan

Malaysia

Cochlodinium sp. ThreeThree CochlodiniumCochlodinium speciesspecies

Three morphotypes assigned to the genus Cochlodinium, with the cingulum encircling the cell more than 1.5 times, have so far been observed and analyzed rDNAs.

C. convolutum C. polykrikoides Cochlodinium sp. CochlodiniumCochlodinium convolutumconvolutum

Cingulum

Sulcus

Cingulum CochlodiniumCochlodinium polykrikoidespolykrikoides

Single cell Eye spot Cingulum August 2002, Kamigoto, Japan Sulcus

Cingulum August 1999, Imari Bay, Japan

June 2003, Shikamachi, Japan Chain-forming cell

January 2004, Sabah, Malaysia

July 2003, Tachibana Bay, Japan

October 2003, Usuka Bay, Japan

May 2004, Inokushi Bay, Japan

August 2003, Mishima Is, Japan

August 2003, Isahaya Bay, Japan CochlodiniumCochlodinium sp.sp. Resembling C. polykrikoides, but different in the position of the sulcus and chloroplasts.

Eye spot

Cingulum

Sulcus

Cingulum Morphological characters of Cochlodinium spp. C. convolutum C. polykrikoides Cochlodinium sp.

Length ca. 60-70 µm ca. 30-40 µm ca. 45-50 µm Eye spot Absent Dorsal, epicone Dorsal, epicone Cingulum ca. 1.5 times ca. twice ca. twice Deeper Shallow, immediately Shallow, intermediate of Sulcus below the cingulum the cingulum Nucleus Rectangular Spherical, anterior Spherical, anterior Reticulate (?) Rod-like, aligned Granulate Chloroplast longitudinally Others Forming hyaline cyst Cell-chains (<16 cells) Cell-chains (<4 cells) Phylogenetic positions of Cochlodinium (SSU rDNA) Gamma weighted NJ tree, Substitution model: Tamura & Nei (1993), sp. Perkinsus marinus Proportion of invariable site: 0.3834, Shape parameter: 0.5580 100 Spatulodinium pseudonoctiluca 100 carterae Amphidinium massrtii Amphidinium 100 Gyrodinium dorsum 100 Gyrodinium uncatenum Gyrodinium instriatum Gyrodinium instriatum 100 Akashiwo sanguinea Akashiwo sanguinea Protoceratium reticulatum Akashiwo sanguinea 84 100 Alexandrium tamarense 100 55 Alexandrium minutum Pyrocystis lunula 100 corculorum Symbiodinium microadriaticum 100 Nematodinium sp. Nematodinium sp. 100 Nematodinium sp. 81 Polykrikos kofoidii sensu stricto 92 Pheopolykrikos hartmannii Gymnodinium fuscum Gyrodinium impudicum 100 Dissodinium pseudolunula Lepidodinium viride Gymnodinium catenatum 93 Gymnodinium cf. nolleri 100 Gymnodinium microreticulatum 76 Gyrodinium falcatum 93 Gyrodinium falcatum 63 Gyrodinium falcatum Gymnodinium 97 Gymnodinium sp. 100 80Cochlodinium convolutum Gyrodinium Cochlodinium convolutum Gymnodinium sp. Gymnodinium sp. Cochlodinium convolutum 98Takayama sp. micrum brevis 100Karenia mikimotoi Karenia + Karlodinium Gyrodinium spirale 94100 Gyrodinium sp. Gyrodinium rubrum Gyrodinium sensu stricto 100 Gyrodinium aureum 85 Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides 96 Cochlodinium polykrikoides Cochlodinium sp. Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides 100 Cochlodinium polykrikoides Cochlodinium polykrikoides 100Cochlodinium polykrikoides Cochlodinium polykrikoides 100 Cochlodinium polykrikoides 100 Cochlodinium polykrikoides Cochlodinium sp. 100 Cochlodinium sp. 0.01 substitutions/site Cochlodinium sp. Cochlodinium sp. Phylogenetic relationship in C. polykrikoides (SSU rDNA)

Cochlodinium polykrikoides Geoje, Korea Cochlodinium polykrikoides Saryang-do, Korea Cochlodinium polykrikoides Busan, Korea Cochlodinium polykrikoides Yoesu, Korea Cochlodinium polykrikoides Yoesu, Korea 96 Cochlodinium polykrikoides Kamigoto, Japan Cochlodinium polykrikoides Hirado, Japan Cochlodinium polykrikoides Mishima, Japan Cochlodinium polykrikoides Inokushi Bay, Japan Cochlodinium polykrikoides Isahaya Bay, Japan 100 Cochlodinium polykrikoides Tachibana Bay, Japan Cochlodinium polykrikoides Imari Bay, Japan Cochlodinium polykrikoides Omura Bay, Japan 100 Cochlodinium polykrikoides Manila Bay, Philippines Cochlodinium polykrikoides Sabah, Malaysia <50 100 100 Cochlodinium polykrikoides Sabah, Malaysia Cochlodinium sp. East China Sea, Japan 100 Cochlodinium sp. Lampung Bay, Indonesia Cochlodinium sp. Lampung Bay, Indonesia Cochlodinium sp. Lampung Bay, Indonesia Phylogeny of Cochlodinium (LSU rDNA, D1-D3)

Noctilucascintillans Spatulodinium pseudonoctiluca Gamma weighted NJ tree, Substitution model: Tamura & Nei (1993), 100 elongata Proportion of invariable site: 0.1580, Shape parameter: 0.7286 99 Gonyaulax cf. spinifera 65 Gonyaulax membranacea 100 Protoceratium reticulatum 96 Protoceratium reticulatum Pyrodinium bahamense 94 Alexandrium leei 100 Alexandrium tamiyavanichii Alexandrium hiranoi cinctum 100 Karenia brevis Karenia mikimotoi 100 Karenia + Karlodinium 97 Karenia mikimotoi Karlodinium micrum 100 Karlodinium micrum 100 Gyrodinium dominans Gyrodinium spirale Gyrodinium sensu stricto 95 Gyrodinium rubrum Pheopolykrikos hartmannii 100 Gymnodinium aureolum 72 58 Gymnodinium aureolum 95 Gymnodinium impudicum 69 Gymnodinium chlorophorum Gymnodinium sensu stricto 99 Gymnodinium fuscum 100 Gymnodinium catenatum Gymnodinium nolleri 93 Gymnodinium cf. nolleri 100 Gymnodinium microreticulatum 100 Togula jolla 75 Togula compacta Togula Togula britannica Prorocentrum minimum 100 Prorocentrum minimum 100 Heterocapsa triquetra Heterocapsa pseudotriquetra Peridinium willei Cochlodinium polykrikoides Cochlodinium polykrikoides 94 Cochlodinium polykrikoides 100 Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides 100 Cochlodinium polykrikoides Cochlodinium polykrikoides 100 Cochlodinium polykrikoides Cochlodinium sp. 100 Cochlodinium polykrikoides 92 Cochlodinium polykrikoides 100 100 Cochlodinium polykrikoides Cochlodinium sp. 100 Akashiwo sanguinea Akashiwo sanguinea Amphidinium klebsii Akashiwo sanguinea 100 90 Amphidinium carterae 100 Amphidinium carterae Amphidinium massartii 97 Amphidinium trulla 100 Amphidinium steinii Amphidinium 100 Amphidinium mootonoroum Amphidinium herdmanii Phylogenetic relationship in C. polykrikoides (LSU rDNA)

Cochlodinium polykrikoides Busan, Korea C. polykrikoides Inokushi Bay, Japan C. polykrikoides Tachibana Bay, Japan 100 C. polykrikoides Hirado, Japan C. polykrikoides Kamigoto, Japan C. polykrikoides Mishima, Japan 100 C. polykrikoides Isahaya Bay, Japan

100 C. polykrikoides Manila Bay, Philippines 100 C. polykrikoides Omura Bay, Japan 92 100 C. polykrikoides Sabah, Malaysia C. polykrikoides Sabah, Malaysia Cochlodinium sp. East China Sea, Japan DistributionDistribution ofof C.C. polykrikoidespolykrikoides populationpopulation

C. polykrikoides Cochlodinium sp.

Manila Bay, Philippines

Sabah, Malaysia

Lampung Bay, Indonesia

C. polykrikoides Japan, Korea Other sequences SummarySummary

Taxonomy and phylogeny of Cochlodinium

o Cochlodinium convolutum could be distinguished from other chain- forming Cochlodinium species based on, 1) shape of nucleus, 2) the deeper sulcus, and 3) formation of hyaline cyst; molecular data also indicates phylogenetic separation among them.

o Cochlodinium sp. is superficially similar to C. polykrikoides, but they could be distinguished by 1) cell size, 2) position of the sulcus, and 3) shape of chloroplast; their affinity was supported by molecular phylogeny.

Population and distribution of C. polykrikoides in southeastern Asia

o Majority of C. polykrikoides red tides occurred in Japanese and Korean coasts are derived from the same population; it could be distinguished from other populations distributed in SE Asia.

o SE Asian populations of C. polykrikoides scarcely exist in Japanese coastal waters. AcknowledgementsAcknowledgements

Mr. Toshifumi Yamatogi (Nagasaki Prefectural Institute of Fisheries, Japan) Mr. Yoshiyuki Kuroki (Kumamoto Prefectural Fisheries Research Center, Japan) Mr. Toshinori Baba (Yamaguchi Prefectural Fisheries Research Center, Japan) Mr. Kazuyoshi Miyamura (Oita Institute of Marine and Fisheries Science, Japan) Dr. Haruyoshi Takayama (Hiroshima Fisheries and Marine Technology Center, Japan) Mr. Koichiro Mizushima (Nagasaki University, Japan) Mr. Juan Relox Jr. (National Fisheries Research and Development Institute, Philippines) Dr. Ann Anton (University of Malaysia Sabah, Malaysia) Dr. Keun-Yong Kim (Pukyong University, Korea)

ThankThank youyou

Phylogeny and geographical distribution of Cochlodinium polykrikoides population (Gymnodiniales, Dinophyceae) collected from Japanese and Korean coasts Mitsunori Iwataki1, Hisae Kawami1, Kazumi Matsuoka1, Takuo Omura2 & Yasuwo Fukuyo2 1. Institute for East China Sea Research, Nagasaki University 2. Asian Natural Environmental Science Center, the University of Tokyo