Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp. nov. from Southern Coastal Waters in Korea and Japan

Accepted Manuscript Title: Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp. nov. from Southern Coastal Waters in Korea and JapanProrocentrum micans s. str. and Prorocentrum koreanum sp. nov.–> Author: Myung-Soo Han Pengbin Wang Jin Ho Kim Soo-Yeon Cho Bum Soo Park Joo-Hwan Kim Toshiya Katano Baik-Ho Kim PII: DOI: Reference:

S1434-4610(15)00121-2 http://dx.doi.org/doi:10.1016/j.protis.2015.12.001 PROTIS 25513

To appear in: Received date: Revised date: Accepted date:

20-3-2015 2-12-2015 5-12-2015

Please cite this article as: Han, M.-S., Wang, P., Kim, J.H., Cho, S.-Y., Park, B.S., Kim, J.-H., Katano, T., Kim, B.-H.,Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp. nov. from Southern Coastal Waters in Korea and JapanProrocentrum micans s. str. and Prorocentrum koreanum sp. nov.–>, Protist (2015), http://dx.doi.org/10.1016/j.protis.2015.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ORIGINAL PAPER

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Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp. nov.

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from Southern Coastal Waters in Korea and Japan

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Myung-Soo Hana,1,2, Pengbin Wanga,1, Jin Ho Kima,1, Soo-Yeon Choa,3, Bum Soo Parka, Joo-Hwan Kima, Toshiya Katanob,4, and Baik-Ho Kima a

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Department of Life Science, College of Natural Sciences, Hanyang University,

222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, Korea b

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Institute of Lowland and Marine Research, Saga University, Honjo 1, Saga City 840-8502, Japan

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Submitted March 20, 2015; Accepted November 30, 2015

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Monitoring Editor: Mona Hoppenrath

Running title: Prorocentrum micans s. str. and Prorocentrum koreanum sp. nov. 1

These authors contributed equally as co-first authors

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Corresponding author; fax +82-2-2220-1171

e-mail [email protected] (M-S. Han). 3

Present address: Marine Ecosystem Management Team, Korea Marine

Environment Management Corporation, Haegong Bldg., Samseong-ro 610, Gangnam-gu, Seoul 135-870, Korea 4

Present address: Graduate School of Marine Science and Technology, Tokyo

University of Marine Science and Technology, Tokyo 108-8477, Japan

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Prorocentrum micans is an extremely variable dinoflagellate species, with many different local forms reported worldwide. Because of this

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morphological diversity, it is important to establish whether these various

forms belong to P. micans sensu stricto. For this study, P. micans-like

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specimens were isolated from several localities in the southern coastal

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waters of Korea and Japan. The morphological characteristics and the molecular signatures of P. micans were re-examined. Moreover, a new Prorocentrum species, Prorocentrum koreanum sp. nov. was established

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through detailed light microscopy and scanning electron microscopy observations. Examination of the periflagellar platelets revealed that P.

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koreanum sp. nov. differs from P. micans. Furthermore, P. koreanum and P. micans exhibited different distribution patterns of trichocyst pores.

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Through molecular phylogeny analysis of small subunit (SSU) rRNA,

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internal transcribed spacer (ITS), and large subunit (LSU) rRNA sequence, we found P. koreanum to be more closely related to P. mexicanum and P.

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rhathymum than to P. micans. Additionally, ITS2 compensatory base changes also provide strong evidence to support P. koreanum and P. micans being separate species.

Key words: Prorocentrum; molecular phylogeny; morphology; new species; southern coastal waters of Korea and Japan.

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Introduction

The dinoflagellate genus Prorocentrum is one group of red tide organisms,

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which was established by Ehrenberg (1834), with Prorocentrum micans

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Ehrenberg as the type species. Prorocentrum is a cosmopolitan genus; most of the species are marine organisms, although some freshwater species have also

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been reported (Croome and Tyler 1987). At least eighty accepted species have

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been reported in this genus to date (Guiry and Guiry 2014). Taxonomic research on this harmful algal bloom organism has been continually conducted worldwide

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(Dickey et al. 1990; Dodge and Bibby 1973; Dodge 1975; Han and Furuya 2000;

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Herrera-Sepúlveda et al. 2015; Hoppenrath 2000; Hoppenrath et al. 2013; Lu

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and Goebel 2001; Wood 1954), including in Korea (Han et al. 1991; Han and Yoo 1983; Lee and Han 2007; Lim et al. 2013; Yoo and Lee 1986). The invention of new sampling tools and the increased application of molecular techniques to the study of Prorocentrum have allowed the revision of species and the description of new species in this genus. However, many morphological and molecular characteristics have not yet been well matched with species in Prorocentrum (Henrichs et al. 2013). Morphologically, P. micans is similar to P. gracile, P. sigmoides, and P.

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texanum, although P. sigmoides has been treated as a synonym of P. gracile

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(Cohen-Fernandez et al. 2006). Because the morphological characteristics of P. micans overlap with those of P. gracile, it is often difficult to differentiate these

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species from one another, except by observing the posterior mucron of P. gracile

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(Cohen-Fernandez et al. 2006). Moreover, P. micans shows considerable morphological variation at the intraspecific level (Cohen-Fernandez et al. 2006;

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Steidinger and Tangen 1997). Even early monograph drawings of individual P.

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micans organisms show varying cell shapes (Schiller 1933). The extremely variable morphology of this species has been previously described (Dodge

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1975), and many local types appear to have been described as unique species.

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In addition, many previous studies have provided unclear descriptions and

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blurred images of P. micans, which might have led to erroneous identifications of this species (Taylor 1976; Yamaji 1962, 1966). For example, the illustration of P. micans shown by Taylor (1976) depicts a rounded posterior, whereas P. micans sensu stricto should exhibit an acute posterior, at least according to the first description of this species (Ehrenberg 1834). The illustrations of P. micans shown by Yamaji (1962, 1966) depicted a thin organism whose cell shape is mostly drop-like, rather than an oval as described earlier by Ehrenberg (1834). These various illustrations of P. micans have added an element of confusion to

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the true description of P. micans. To resolve this confusion, it was proposed that

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the various forms be assessed to determine whether they truly belong to P. micans sensu stricto (Dodge 1975). Although Cohen-Fernandez et al. (2006)

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described the external morphology of several P. micans organisms and provided

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several micrographs of these, they reported neither the morphology of the periflagellar region nor any molecular-based phylogenetic information.

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P. micans organisms have typically been identified based on early

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descriptions. This approach is of limited use when distinguishing different species from one another. In addition, P. micans-like species could occupy

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different phylogenetic positions and be part of a different clade (Henrichs et al.

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2013). More detailed morphological information is needed to differentiate the

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various P. micans-like species from one another. As one example, the periflagellar region of P. texanum exhibits a uniquely distinguishing morphology (Henrichs et al. 2013).

The majority of Prorocentrum-related phylogenetic studies have been based

on rRNA-encoding gene sequences (Chomérat et al. 2010; Grzebyk et al. 1998; Herrera-Sepúlveda et al. 2015; Hong et al. 2008; Murray et al. 2009; Saldarriaga et al. 2004). In studies of small subunit (SSU) rRNA gene phylogenetic trees, Prorocentrum was often found to form two clades; however, the SSU region

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exhibits low variability and is sometime not that useful for differentiating different

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species, especially on planktonic species (Henrichs et al. 2013; Murray et al. 2009). In contrast, the large subunit (LSU) and internal transcribed spacer (ITS)

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rRNA regions of Prorocentrum show high variation and thus enable sensitive

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discrimination of different species (Chomérat et al. 2010; Henrichs et al. 2013; Murray et al. 2009). Some phylogenetic studies have used the cox 1 and cob

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markers for species identification. However, the lack of sequence data for those

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markers has meant that most studies could not use them. With the aim of conducting a comprehensive morphological study and

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genomic DNA analysis of P. micans and P. micans-like species, specimens were

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collected from several localities in the southern coastal waters of Korea and

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Japan. We examined the morphological characteristics and molecular signatures of P. micans and P. micans-like species and here report a new planktonic Prorocentrum species, Prorocentrum koreanum sp. nov. The new species was characterized using light microscopy and scanning electron microscopy, and its

phylogenetic position was established using molecular

techniques.

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Results

Species Descriptions

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Specimens of Prorocentrum were collected from natural water at several stations

in coastal waters ranging from Korea to Japan (Fig. 1). From these specimens, a

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number of different Prorocentrum strains were established, among which P.

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micans and P. koreanum sp. nov. were investigated in this study (Supplementary

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Material Table S1).

Prorocentrum micans Ehrenberg 1834 (Fig. 2)

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LOCALITY: Masan, Korea.

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MOLECULAR CHARACTERIZATION: The nucleotide sequences of the SSU, ITS1–5.8S–ITS2, and D1–D3 regions of the LSU rDNA of strain “HYMS-1211-1”

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were deposited in GenBank under the accession numbers KP711341, KP711342, and KP711343, respectively. HABITAT: Marine, planktonic. MORPHOLOGY: Cells are pyriform to heart-shaped, 32.6–38.1 μm in length, and 23.5–29.1 μm in width (n = 40) (Table 1). Each cell has asymmetrical valve margins with a rounded anterior and a pointed posterior and is broadest at the middle or towards the anterior end (Fig. 2A). Yellow-brown chloroplasts containing a large internal pyrenoid were found below the two valves and can presumably conduct photosynthesis for the cell (Fig. 2A). Pusules and fibrous

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vesicles are not visible by light microscopy. The cell is dorsoventrally flattened (Fig. 2B-C). The right valve forms a moderately excavated, V-shaped triangular

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depression in the periflagellar region (Fig. 2B). A strong winged spine is present in the apical region with length 3.1–5.4 μm (Fig. 2B-C). The posterior angle is

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93.8–124.4°. The valve surface is covered with numerous thecal pores and

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trichocyst pores (Fig. 2B-C). Trichocyst pores form radiating tangential rows; each valve has 7–9 rows (Fig. 5A-B). A posterior semicircle formed by posterior

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trichocyst pores is present at the posterior end of the valves, but in some cells the pores are more irregular (Fig. 2B-C). Above the posterior pores on each side

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of the valves are 3–4 rows of trichocyst pores, each of which is lined with 3–6 pores on each side of both valves. A few trichocyst pores are found at the center

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of the valve, with several pores lining the margin and some forming small rows of

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2–4 pores. At the anterior end of each valve, several pores are found along the

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margin, some of which are in pairs or form small rows of 2–3 pores. Approximately two recessed trichocyst pores are found on the right valve below the periflagellar area, whereas on the left valve only one trichocyst pore is found beneath the periflagellar area (Fig. 2B-C). The periflagellar area is covered by 8 platelets (Fig. 6A, C, Supplementary Material Fig. S1A-J). The flagellar pore (FP) is nearly oblong, and is adjacent to platelets 3, 5, 6, and 8, whereas the accessory pore (AP) is similar in size to the FP and is surrounded by platelets 2, 7, and 8. Two flagella emerge from the flagellar pores (Fig. 6A, C). The winged spine, supported by periflagellar platelet 1, and an opposing short flange are

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evident on platelet 4 when viewed from the right valve. A large heart-shaped nucleus is present in the center to the posterior part of the cell (Fig. 2D). Young

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cultures of P. micans swim actively, like other planktonic dinoflagellates, and also

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produce a large amount of mucus that remains embedded in old cultures.

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Prorocentrum koreanum M.-S. Han, S. Y. Cho et P. Wang sp. nov. (Fig. 3 and Fig. 4)

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DIAGNOSIS: Photosynthetic prorocentroid dinoflagellate. Drop shaped cell, length 30.4–45.2 μm and width 21.8–31.5 μm. Valve convex with a rounded

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anterior, pointed posterior, and numerous trichocyst pores. The broad, shallow, V-shaped periflagellar area has 8 platelets: 1, 2, 3, 4, 5, 6, 7, and 8. The straight,

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long, and strong winged spine extends from periflagellar platelet 1, and an

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opposing short flange is evident on platelet 4.

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TYPE LOCALITY: Jangmok, Korea.

HOLOTYPE: A slide of isolate “LMBEV9” was deposited in the Laboratory for Water Environmental Ecology and Restoration of Hanyang University (slide number HY-PK-S1).

ISOTYPE: Fixed material of “LMBEV9” was deposited at the Laboratory for Water Environmental Ecology and Restoration of Hanyang University (slide number HY-PK-F1). MOLECULAR CHARACTERIZATION: The nucleotide sequences of SSU rDNA, ITS1–5.8S–ITS2, and D1–D3 of LSU rDNA of the “LMBEV9” strain were

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deposited in GenBank under the accession numbers KP711350, KP711351, and KP711352, respectively.

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HABITAT: Marine, planktonic.

ETYMOLOGY: Latin; refers to the geographic locality of the dinoflagellate, which

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is off the coast of Korea.

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MORPHOLOGY: Cells are solitary and drop shaped, 30.4–45.2 μm in length, and 21.8–31.5 μm in width (n = 105) (Table 1). The cell has asymmetrical valve

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margins with a convex ventral side, a rounded anterior, and a pointed posterior (Figs 3A, I, 4A). Two yellow-brown chloroplasts are present (Fig. 3A), but no

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pyrenoids, pusules, or fibrous vesicles are visible by light microscopy. The cell is dorsoventrally flattened (Figs 3B-C, J-K, 4B-C) and widest in the middle of the

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valve. The right valve forms a moderately excavated, U-shaped triangular

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depression in the periflagellar region (Figs 3B, J, Fig. 4B). A long, straight, and

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strong winged spine is present in the apical region, and a small collar is evident by scanning electron microscopy (Figs 3B-C, F, J-K, M, Fig. 4B-C). The posterior angle is 58.7–101.7°.The valve surface is covered with numerous thecal pores and trichocyst pores (Figs 3B-C, J-K, 4B-C). The trichocyst pores form radiating tangential rows, and each valve has 5–8 rows (Fig. 5C-H). At the posterior end of each valve, a posterior semi-circle of trichocyst pores is present (Figs 3B-C, E, G, J-K, N, P, 4B-C, E). A row of 4–7 trichocyst pores is located above these posterior pores on each side of each valve (Figs 3B-C, E, G, J-K, N, P, 4B-C, E). There nearly not exist the trichocyst pore at the valve center; moreover, several

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pores line the margin, with some forming short rows. Several pores are located along the margin at the anterior end of each valve. Some are found in pairs or

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form small rows consisting of 3 pores. The right valve has 2–4 recessed

trichocyst pores below the periflagellar area, whereas the left valve has only one

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trichocyst pore below the periflagellar area (Figs 3B-C, F, H, J-K, M, O, 4B-C, F).

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The U-shaped periflagellar area is covered by 8 platelets (Fig. 6B, D, Supplementary Material Fig. S1K-1B). The FP is irregular-form and is located

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adjoining platelets 3, 5, 6, and 7; the oblong-form AP is adjacent to platelets 2, 7, and 8. Two flagella emerge from the FP (Fig. 6B, D). The straight, long, and

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strong winged spine extends from periflagellar platelet 1, and an opposing short flange is evident on platelet 4 when viewed from the right valve. A heart-shaped

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nucleus is present in the central to posterior part of the cell (Figs 3D, L, 4G). In

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young cultures, P. koreanum cells swim actively and produce large amounts of

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mucus that remain embedded in old cultures. Molecular Analysis

The SSU, ITS/5.8S, and LSU rRNA gene regions of P. koreanum and P. micans were amplified and sequenced. Sequences were deposited in GenBank under the accession numbers listed in Table 2. An initial BLAST search revealed that the sequences of P. koreanum have very high degrees of similarity to those of P. micans. The ITS and LSU genes of strain CCMP2794 were sequenced and found them to be identical to those of P. koreanum. The CCMP2794 isolate, kept at the Provasoli-Guillard National Center for Marine Algae and Microbiota

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derived from the Gulf of Mexico and had been identified as P. micans. The phylogenetic tree for the SSU rDNA regions (Fig. 7) exhibited a

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topology similar to those reported in previous studies. Specifically, it is difficult to

distinguish P. koreanum from the P. micans clade based on this tree. However, P.

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koreanum and P. micans were clearly distinguished as different species

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according to the ITS and LSU phylogenetic trees (Fig. 8 and Fig. 9), which show them to be in different clades. On those trees, P. koreanum is closely related to P.

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mexicanum and P. rhathymum and located far from the P. micans clade. In addition, P. koreanum and the CCMP2794 isolate occupy the same phylogenetic

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position in the ITS phylogenetic tree.

The uncorrected genetic distances (p)

between different Prorocentrum

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species calculated based on the ITS1–5.8S–ITS2 (ITS region) rDNA sequences

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revealed that P. koreanum was significantly different from P. mexicanum, P.

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micans, P. minimum, P. rhathymum, and P. texanum (Table 3). All distances were p>0.04, whereas, within-species differences exhibited p values <0.02. That P. koreanum and P. micans are different taxa is further supported by

the presence of compensatory base changes (CBCs) in the ITS2 region (Fig. 10, Supplementary Material Table S2). There is a CBC in helix II, which provides strong evidence supporting that P. koreanum and P. micans are separate species (Fig. 10).

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Discussion

Morphology

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Morphological analysis clearly indicates that P. koreanum belongs to the genus Prorocentrum. We did not find an apparent difference between different strains

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from Korea, America, and Japan (Figs 3, 4). In this study, we did not find additional variability except for some abnormal cells being reported in old

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cultures. Thus, in the present study, we used healthy cultures in the exponential growth stage to prevent the appearance of abnormal cells. The morphology of P.

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koreanum is very similar to that of P. mexicanum, P. rhathymum, P. texanum, and P. micans, with the similarity being greatest with the latter. P. mexicanum has an

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anterior serrated winged spine with 2–3 crests and a large round nucleus

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(Cortés-Altamirano and Sierra-Beltrán 2003). In contrast, P. rhathymum has a

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simple, non-serrated winged spine, no trichocyst pores around the periflagellar area on the right valve, and a smaller posterior nucleus. P. koreanum has a long conspicuous winged spine, a greater number of trichocyst pores around the periflagellar area, and a heart-shaped nucleus in the central to the posterior part of the cell. Although P. texanum and P. micans are similar to P. koreanum, those species differ in their shape, size, features of the periflagellar plates and winged spine, valve pore patterns, and surface ornamentation (Ehrenberg 1834; Henrichs et al. 2013; Steidinger and Tangen 1997). P. texanum cells are round to oval shaped and have a short serrated winged spine and a U-shaped nucleus.

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In contrast, P. micans is medium-sized, has a pyriform to heart-shaped cell with a short winged spine extending from the valve edge, a large heart-shaped

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nucleus, and a more prominent arched side compared with P. koreanum. Indeed, P. mexicanum, P. rhathymum, and P. texanum (Henrichs et al. 2013) all have

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rounded posterior ends (Cortés-Altamirano and Sierra-Beltrán 2003). In contrast,

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the posterior end of P. micans is almost an obtuse angle (Fig. 5A-B), whereas the posterior end of P. koreanum is pointed (Fig. 5C-H). ANOVA analysis did not

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reveal any significant differences with respect to cell length or cell width between P. koreanum and P. micans; however, the spine length and posterior angle can

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distinguish P. koreanum from P. micans (Table 1). In addition, the trichocyst pore patterns around the periflagellar area and on the valve surface are very different

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between P. koreanum and P. micans (Figs 2B-C, 3B-C, J-K, 4B-C). Furthermore,

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the distinct periflagellar plates differ between the two species (Fig. 6).

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The morphological characteristics of P. arcuatum, P. gracile, P. triestinum, and P. scutellum are also similar to those of P. koreanum, and P. micans. P. arcuatum is elongated and lanceolate, with a rounded anterior end and tapering posterior end that terminates in a short point. The cells are broadest at a third of the length from the anterior end, and the anterior spine is very long with a wide base. In contrast, P. gracile is elongated and lanceolate, with a rounded anterior end and a pointed posterior end. The anterior spine is long, sharp, and narrow in the plate view, whereas it is broad-lanceolate in the side view. P. triestinum is rounded at the anterior end, pointed at the posterior end, and about twice as

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long as it is wide. The anterior end exhibits a short apical spine. P. scutellum is broadly heart-shaped with a rounded or pointed posterior end; the anterior end

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has a slight indentation and two spines, to the larger of which is attached a delicate broad wing.

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Phylogenetic and CBC Analysis

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The cultured strain CCMP2794 was found to have high sequence and morphological similarity to P. koreanum, suggesting that this isolate is P.

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koreanum rather than P. micans. We confirmed this finding by phylogenetic analyses using ITS (Fig. 8) and the LSU rRNA gene (Fig. 9). In addition, the P.

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micans AY863008 isolate had the same phylogenetic position as P. koreanum in the LSU phylogenetic tree (Fig. 9). Although we did not have access to a sample

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of the P. micans AY863008 isolate for morphological assessment, the LSU

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phylogenetic tree reported by Howard et al. (2009) and data from the present

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study (Fig. 9) indicate that P. micans AY863008 is not a P. micans species. Rather, P. micans AY863008 is likely to be a strain of P. koreanum; P. micans AY863008 was present in a P. mexicanum clade and thus separate from the P. micans clade (AF260377, AY032654, and AF042814) in both the Neighbor joining and maximum parsimony phylogenetic trees reported by Howard et al. (2009). In a similar manner, we found in the present study that P. micans AY863008 was separate from other clades containing P. micans, P. mexicanum, P. texanum, and P. triestinum (Fig. 9). Thus, many identification errors have presumably occurred because of the similar and easily confused shapes of P.

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koreanum and P. micans sensu stricto. Although several studies have reported the polyphyletic characteristics of P. micans species (Howard et al. 2009; Munir

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et al. 2013; Murray et al. 2009), this information was perhaps insufficient to

demonstrate that different P. micans specimens exhibiting similar morphologies

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and different phylogenetic positions were not P. micans sensu stricto. These

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species have often been treated as local forms of P. micans and have even been raised to specific ranks, which complicates the literature (Dodge 1975).

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Overall, the phylogenetic trees of Prorocentrum spp. (Figs 7–9) based on SSU, ITS/5.8S, and LSU sequence analysis are similar to those in previous

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reports (Chomérat et al. 2010; Henrichs et al. 2013; Hong et al. 2008; Murray et al. 2009). The SSU phylogenetic tree (Fig. 7) reveals the difficulty in

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distinguishing P. koreanum, P. micans, and P. texanum from one another based

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on this marker alone. However, the ITS/5.8S and LSU sequences provide

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sufficient information to distinguish these P. micans-like species from one another. In addition, the ITS and LSU phylogenetic trees (Figs 8–9) indicate that P. koreanum is more closely related to P. mexicanum and P. rhathymum than to P. micans and P. texanum. Also, the genetic distances obtained from the ITS1–5.8S–ITS2 dataset demonstrate that P. koreanum is clearly different from P. micans, P. texanum, P. mexicanum, P. rhathymum, and P. minimum. Specifically, all differences were > 0.04 substitutions per site (Table 3), and p ≥ 0.04 can be used to delineate most free-living dinoflagellate species (Litaker et al. 2007). Furthermore, CBCs were detected in helix II of P. koreanum and P. micans that

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can prove that they are different species based on previous studies (John et al. 2014; Pröschold et al. 2011). Thus, genetic differences were clearly observed

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between P. koreanum and P. micans in this study.

In conclusion, the data presented here show that P. micans-like species are

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not a single species. A combination of morphological analysis and genetic

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sequence information supports the designation of P. koreanum as a new species

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of dinoflagellate.

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Methods

Isolation and cultures: Several strains of Prorocentrum were established from

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coastal water in various geographical regions of Korea and Japan (Fig. 1). The

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clonal cultures we used in this study were derived either from a net sample (20 µm mesh size) or from surface seawater samples that were gently concentrated

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with a 20 µm mesh filter. Single cells were isolated by the capillary method

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(Andersen 2005) using a Zeiss Axioplan 100 inverted microscope (Carl Zeiss, Jena, Germany) and cultured them in 96-well plates containing 200 μl of f/2-Si

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medium (Guillard and Ryther 1962). Cultures of isolated strains were

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subcultured into fresh f/2-Si medium at approximately 20-day intervals to

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maintain culture health. All isolates and cultures were maintained at 20C on a 12:12-h light:dark cycle (100–150 μmol m–2 s–1; cool white fluorescent tubes). All strains are available upon request.

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Light microscopy: Observations of live and fixed cells were made using an

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inverted microscope (IX 71, Olympus, Japan) and a stereomicroscope (Axioplan microscope, Zeiss, Germany) equipped with epifluorescence and Nomarski

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differential interference contrast (DIC) optics. Natural and cultured samples were

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fixed for at least 8 h at 4 C in Lugol's solution (Throndsen 1978; final concentration approximately 2%) or glutaraldehyde solution (Sigma-Aldrich, St.

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Louis, MO, USA; final concentration approximately 1%). The fixed samples were

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either observed directly or rinsed with distilled water to completely remove all fixation reagents and sea salt. Samples were mounted in glycerol gelatin

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(Sigma-Aldrich) for slide preparation. All slides are archived in the Laboratory for

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Water Environmental Ecology and Restoration at Hanyang University. Light

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microscopy images were recorded using a cooled charge-coupled device (CCD) camera (XC10, Olympus, Japan) and analyzed using cellSens Standard 1.8 software (Olympus, Japan). The shape and position of the nucleus was determined

by

staining

glutaraldehyde-fixed

cells

for

10

min

in

4’-6-diamidino-2-phenylindole (DAPI; 0.1 μg mL–1 final concentration).

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Scanning electron microscopy (SEM): For SEM examination of the pore

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patterns on the valves and the periflagellar plate structures of Prorocentrum, cells from growing cultures were fixed in glutaraldehyde solution (Sigma-Aldrich)

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or osmium tetroxide (Ted Pella, St. Louis, CA, USA) at final concentrations of

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approximately 1%. The specimens were rinsed with distilled water to completely remove all fixation reagents and sea salt and then dehydrated them with a

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graded ethanol series treatment (30, 50, 70, 90 and 100%; 30 min per

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concentration). Samples (50 μl) of the dehydrated specimens were directly mounted onto a 3 μm TSTP Millipore filter membrane (Millipore Filter

d

Corporation, Cork, Ireland), dried them at room temperature for 12 h (Wang et al.

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2014), coated them with gold for 150 s under a 40 mA current (BAL-TEC SCD

Ac ce p

005 Super Coater, Liechtenstein, Germany), and examined them using a scanning electron microscope (Hitachi S-2380n, Japan, and Nova NanoSEM 450, FEI, Netherlands).

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Identification of organisms and statistical analysis: Cell length, width,

ip t

spine length, and posterior angle measurements were determined by light microscopy. All results are based on measurements of more than 30 randomly

cr

selected cells. Cell length was estimated from the anterior end to the posterior

us

end in the valve view, and cell width was estimated as the trans-diameter in the lateral view (Faust et al. 2008). Trichocyst distribution and periflagellar area

an

structure from at least 10 cells were observed under a SEM. The periflagellar

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area numbering system followed the labeling system proposed by Hoppenrath et al. (2013). All tested strains were examined at the exponential growth stage.

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Species groupings based on cell length, cell width, spine length, and posterior

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angle were analyzed by ANOVA with the SPSS software program (IBM). A p

Ac ce p

value < 0.05 was considered significant. DNA extraction, PCR amplification, and sequencing: Clonal cultures in

the mid-logarithmic growth phase (3 mL) were harvested by centrifugation at 8000 × g for 5 min. Pelleted cells were transferred to 1.5 ml Eppendorf tubes containing 100 μl of TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM ethylenediaminetetraacetic acid] and stored at –20 C until DNA extraction (Wang et al. 2014). Genomic DNA was extracted from cells using the DNeasy Plant Mini Kit (Qiagen, USA).

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For PCR amplification, primers (Table 4) were combined with nuclear SSU, ITS, and LSU rDNA. PCR reactions were performed using TaKaRa EX Taq™

ip t

(TaKaRa, Japan) in a total volume of 50 μl. Positive bands were excised following gel electrophoresis and purified with a QIAquick PCR Purification Kit

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(Qiagen, Germany) according to the manufacturer’s instructions. DNA

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sequencing reactions were performed using an ABI PRISM® BigDye™ Terminator v 3.1 Kit (Applied Biosystems, Foster City, CA, USA) with the primers

an

listed in Table 4. Labeled DNA fragments were analyzed by capillary electrophoresis on an ABI 3730xl Genetic Analyzer (Applied Biosystems).

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Editing and contig assembly of rDNA sequence fragments were carried out using Sequencher 4.7 (Gene Codes, USA).

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DNA sequence comparisons: Full multiple alignments of the sequences obtained in our study (Table 2) with NCBI sequences were generated using the

Ac ce p

Clustal W1.8 (Thompson et al. 1994) portion of the Bioedit program v7.0.9.0 (North Carolina State University). All aligned nuclear SSU rDNA sequences were trimmed to the same length, and the gaps were deleted. DNA similarities were calculated using Bioedit. For maximum likelihood (ML) phylogenetic tree analysis, the best model

was selected by MrModelTest2.3 and PAUP*4.0b10 (Swofford 2003). The best-fit model for the PhyML 3.0 settings (Guindon et al. 2010) was selected from 24 tested models. Bootstrap values (branch support) were obtained from

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1000 replicates. Bootstrap values > 50 are indicated at each branch node. For Bayesian inference (BI) analysis, the optimal model of nucleotide

ip t

substitution was selected and used it in the ML analysis. The best-fit model was selected from 24 tested models using MrBayes 3.2.1 (Ronquist et al. 2012). The

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Markov Chain Monte Carlo (MCMC) process was set at two chains and

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5,000,000 generations were performed. The sampling frequency was 100 generations. Following analysis, the standard deviation of frequencies was

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confirmed to be < 0.01, the first 25% of all trees were deleted as burn-ins, and a

indicated at each branch node.

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consensus tree was constructed. Bayesian posterior probabilities (BI) > 0.50 are

Maximum parsimony (MP) branch and bound searches were performed in

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PAUP*4.0b10 (Swofford 2003) using the hierarchical likelihood ratio tests

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(hLRTs) settings. Bootstrap values (branch support) were based on 1000

Ac ce p

replicates. Bootstrap values > 50 are indicated at each branch node. The uncorrected genetic distances (p) between different Prorocentrum species were analyzed with PAUP*4.0b10 (Swofford 2003) using the ITS1–5.8S–ITS2 (ITS region: 491 bp) rDNA sequences.

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ITS2 rRNA secondary structure modeling: The beginning and end of the

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ITS2 region in these strains were identified via the homology with P. micans given in the ITS2 database (Koetschan et al. 2010). The ITS2 region consisted

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of approximately 182 bp in our Prorocentrum strains. The ITS2 secondary

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structure was predicted using the existing structural model of the type strain P. micans (CCMP1589) ITS2 region in the ITS2 database (Koetschan et al. 2010).

an

Structures were visualized using VARNA (Darty et al. 2009). The compensatory

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base changes were identified using the software 4SALE (Seibel et al. 2006).

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Acknowledgements

This research was supported by a grant from the Marine Biotechnology Program

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Funded by the Ministry of Oceans and Fisheries. We thank Dr. Øjvind Moestrup (Biological Institute, Section of Phycology, University of Copenhagen) and Dr. R. Wayne Litaker (NOAA, Center for Coastal Fisheries and Habitat Research) for their critical comments and revision of this manuscript.

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Andersen RA (2005) Algal culturing techniques. Academic Press, USA, 578p

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Prorocentrum consutum sp. nov.(Dinophyceae), a new benthic dinoflagellate from South Brittany (Northwestern France). J Phycol 46:183-194

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Cohen-Fernandez EJ, Meave Del Castillo E, Salgado Ugarte I, Pedroche FF (2006) Contribution of external morphology in solving a species complex: The case of Prorocentrum

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micans, Prorocentrum gracile and Prorocentrum sigmoides (Dinoflagellata) from the Mexican Pacific Coast. Phycol Res 54:330-340

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Cortés-Altamirano R, Sierra-Beltrán AP (2003) Morphology and taxonomy of Prorocentrum mexicanum and reinstatement of Prorocentrum rhathymum (Dinophyceae). J Phycol 39:221-225

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Croome R, Tyler P (1987) Prorocentrum playfairi and Prorocentrum foveolata, two new

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dinoflagellates from Australian freshwaters. Brit Phycol J 22:67-75 Darty K, Denise A, Ponty Y (2009) VARNA: Interactive drawing and editing of the RNA

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secondary structure. Bioinformatics 25:1974–1975 Dickey RW, Bobzin SC, Faulkner DJ, Bencsath FA, Andrzejewski D (1990) Identification of okadaic acid from a Caribbean dinoflagellate, Prorocentrum concavum. Toxicon 28:371-377 Dodge JD (1975) The Prorocentrales (Dinophyceae). II. Revision of the taxonomy within the genus Prorocentrum. Bot J Linn Soc 71:103-125 Dodge J, Bibby B (1973) The Prorocentrales (Dinophyceae): I. A comparative account of fine structure in the genera Prorocentrum and Exuviaella. Bot J Linn Soc 67:175-187 Ehrenberg CG (1834) Dritter Beitrag zur Erkenntniss grosser Organisation in der Richtung des kleinsten Raumes. Abhandlungen der Königlichen Akademie der Wissenschaften zu Berlin 1833:145–336

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performance of PhyML 3.0 performance of PhyML 3.0. Systematic Biology 59: 307-321. performance of PhyML 3.0. Syst Biol 59:307-321

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Guiry, Guiry (2014) AlgaeBase, World-wide electronic publication, National University of Ireland,

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Galway, p. http://www.algaebase.org; accessed on 22 December 2014 Han M-S, Furuya K (2000) Size and species-specific primary productivity and community

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structure of phytoplankton in Tokyo Bay. J Plankton Res 22:1221-1235 Han M-S, Yoo K (1983) A taxonomical study on the dinoflagellates in Jinhae Bay. 1. Armored and unarmored dinoflagellates. Bull KORDI 5:37-47 Han M-S, Kim S-W, Kim Y-O (1991) Influence of discontinuous layer on plankton community structure and distribution in Masan Bay, Korea. Bull Korean Fish Soc 24:459-471 Henrichs DW, Scott PS, Steidinger KA, Errera RM, Abraham A, Campbell L (2013) Morphology and phylogeny of Prorocentrum texanum sp. nov.(dinophyceae): a new toxic dinoflagellate from the gulf of mexico coastal waters exhibiting two distinct morphologies. J Phycol 49:143-155 Herrera-Sepúlveda A, Medlin LK, Murugan G, Sierra-Beltrán AP, Cruz-Villacorta AA,

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Hernández-Saavedra NY (2015) Are Prorocentrum hoffmannianum and Prorocentrum belizeanum (Dinophyceae, Prorocentrales), the same species? An integration of morphological

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Hoppenrath M (2000) A new marine sand-dwelling Prorocentrum species, P. clipeus sp. nov. (Dinophyceae, Prorocentrales) from Helgoland, German Bight, North Sea. Eur J Protistol

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of Prorocentrum donghaiense Lu sp. nov. from the East China Sea. Chin J Oceanol Limn 19:337-344

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Pröschold T, Darienko T, Silva PC, Reisser W, Krienitz L (2011) The systematics of Zoochlorella revisited employing an integrative approach. Environ Microbiol 13:350-364 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice across a Large Model Space. Syst Biol 61:539-542

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Yamaji I (1966) Illustrations of the marine plankton of Japan. Hoikusha, Osaka, Japan, 369 p Yoo K, Lee J (1986) Taxonomical studies on dinoflagellates in Masan Bay. 1. Genus

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Figure Legends

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Figure 1. Sampling sites for collection of Prorocentrum spp.

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Figure 2. Prorocentrum micans (Strain: HYMS-1211-1). (A) General view. (B) Right valve. (C) Left valve. (D) DAPI-stained posterior heart-shaped nucleus

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(blue) with UV excitation. A: light microscopy; B–C: scanning electron

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microscopy; D: fluorescence microscopy. Scale bars = 10 μm (A–C). Figure 3. Prorocentrum koreanum (Korean strain: LMBEV9; A–H). (A) General

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view. (B) Right valve. (C) Left valve. (D) DAPI-stained posterior heart-shaped nucleus (blue) under UV excitation. (E) Posterior part of the right valve. (F)

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Anterior part of the right valve and winged spine. (G) Posterior part of the left valve. (H) Anterior part of the left valve and winged spine. Prorocentrum

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koreanum (American strain: CCMP 2794; I–P). (I) General view. (J) Right valve.

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(K) Left valve. (L) DAPI-stained posterior heart-shaped nucleus (blue) under UV excitation. (M) Anterior part of the right valve. (N) Posterior part of the right valve and winged spine. (O) Anterior area of the left valve. (P) Posterior part of the left valve and winged spine. A, I: Light microscopy; B, C, E–H, J, K, M–P: Scanning electron microscopy; D, L: Fluorescence microscopy. Scale bars = 10 μm (A–F, I–K) or 5 μm (G–H, M–P).

Figure 4. Prorocentrum koreanum (Japanese strain: HYSG-1311-1). (A) General view of a cell fixed with Lugol's solution. (B) Right valve. (C) Left valve. (D) Dorsal view. (E) Posterior area of the left valve. (F) Anterior part of the left

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valve and winged spine. (G) DAPI-stained posterior heart-shaped nucleus (blue) under UV excitation. A: Light microscopy; B–F: Scanning electron microscopy; G:

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Fluorescence microscopy. Scale bars = 10 μm (A–D, F), 5 μm (E), or 20 μm (G).

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Figure 5. Schematic diagrams of the right and left valves of Prorocentrum spp.

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showing the various trichocyst pore fields. All drawings are to scale. (A) P. micans (Korean strain: HYMS-1211-1), left valve. (B) P. micans (Korean strain:

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HYMS-1211-1), right valve. (C) P. koreanum (Korean strain: LMBEV9), left valve. (D) P. koreanum (Korean strain: LMBEV9), right valve. (E) P. koreanum (American strain: CCMP 2794), left valve. (F) P. koreanum (American strain:

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CCMP 2794), right valve. (G) P. koreanum (Japanese strain: HYSG-1312-1), left valve. (H) P. koreanum (Japanese strain: HYSG-1312-1), right valve. More than

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20 cells were observed for each species.

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Figure 6. (A–B) Schematic diagram of the periflagellar area of Prorocentrum; the periflagellar area numbering system follows the labeling system of Hoppenrath et al. (2013). (A) Periflagellar platelets of P. micans (drawing based on Korean strain: HYMS-1211-1). (B) Periflagellar platelets of P. koreanum (drawing based on Korean strain: LMBEV9). (C–D) Scanning electron microscope images of the periflagellar area of Prorocentrum spp. (C) P. micans (Korean strain: HYMS-1211-1). (D) P. koreanum (Korean strain: LMBEV9). FP: flagellar pore; AP: accessory pore.

Figure 7. Maximum likelihood tree of the SSU dataset (1590 bp). Alexandrium tamarense was included as an out-group. The best model, as chosen by MrModel-Test2.3, was GTR+I+G. Support values shown are maximum

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likelihood/Bayesian inference/maximum parsimony values. Only values > 50%

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(MP, ML) and 0.50 (BI) are shown.

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Figure 8. Maximum likelihood tree of the ITS1–5.8S–ITS2 dataset (491 bp).

Karenia brevis was included as an out-group. The best model, as chosen by was

GTR+G.

Support

values

shown

are

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MrModel-Test2.3,

maximum

likelihood/Bayesian inference/maximum parsimony values. Only values > 50%

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(MP, ML) and 0.50 (BI) are shown. *: It was named as "P. micans" in GenBank.

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Figure 9. Maximum likelihood tree of the LSU dataset (475 bp). Alexandrium tamarense was included as an out-group. The best model, as chosen by was

GTR+G.

Support

values

shown

are

maximum

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MrModel-Test2.3,

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likelihood/Bayesian inference/maximum parsimony values. Only values > 50%

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(MP, ML) and 0.50 (BI) are shown.

Figure 10. Secondary structure of ITS2 region of rRNA for Prorocentrum micans (Korean strain: HYMS-1211-1), based on model of type strain P. micans CCMP1589. Helices I–IV are shown. The boxed part: highlighted partial sequence and the compensatory base change detected in helix II for P. micans and P. koreanum.

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Table 1. Morphological characteristics of Prorocentrum spp.

Trichocyst pore No.of observations Cell Length (μm)

Cell Width μm)

Spine Length (μm)

Posterior Angle (°)

Korean strain

CCMP 2794

HYSG-1312-1

texanum

Pyriform to heart-shaped

Drop shaped

Drop shaped

Drop shaped

Spheroid to pyriform

One convex side and one arched

One convex side and one

One convex side and one

One convex side and one little

One convex side and

side

little one arched side

little one arched side

one arched side

one arched side

Obvious

Obvious

Obvious

Obvious

not Obvious

Point

Point

Point

Point

Round to point

Exist the pore at the valve

No pore at the valve

No pore at the valve

Nearly not exist the pore at the

central

central

central

valve central

36

35

40

34

32.6-38.1 (35.1) *

a

23.5-29.1 (27.1)

b

3.1-5.4 (4.4)

a

93.8-124.4 (111.2)

c

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Korean strain

Prorocentrum koreanum

M an

Posterior

Prorocentrum

ed

Anterior winged spines

Prorocentrum koreanum

30.4-44.6 b

(39.0)

ce pt

Valve

Prorocentrum koreanum

Ac

Shape

Prorocentrum micans

39.5-45.2 (42.31)

c

21.8-31.5

24.1-29.0

a, b

a, b

(26.7)

4.9-9.4 (6.9)

b

71.5-101.7 (87.4)

b

(26.4)

3.8-8.3 (6.9)

b

70.8-95.0 (82.0)

a

-

35.6-44.6 (41.5)

b

23.3-28.9 (26.0)

a

5.0-8.5 (7.0)

b

58.7-93.8 (81.6)

divers type

a

-

-

-

-

*Value in ( ) is average of all data of each item on different species. a, b, c were marked as different groups from the results of ANOVA (P<0.05)

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Table 2. GenBank accession numbers of Prorocentrum spp. Species Strain DNA Region Accession number Prorocentrum micans HYMS-1211-1 SSU KP711341 Prorocentrum micans HYMS-1211-1 ITS1-5.8S-ITS2 KP711342 Prorocentrum micans HYMS-1211-1 D1-D3 of LSU KP711343 Prorocentrum koreanum HYSG-1312-1 SSU KP711344 Prorocentrum koreanum HYSG-1312-1 ITS1-5.8S-ITS2 KP711345 Prorocentrum koreanum HYSG-1312-1 D1-D3 of LSU KP711346 Prorocentrum koreanum HYSG-1312-2 SSU KP711347 Prorocentrum koreanum HYSG-1312-2 ITS1-5.8S-ITS2 KP711348 Prorocentrum koreanum HYSG-1312-2 D1-D3 of LSU KP711349 Prorocentrum koreanum LMBEV9 SSU KP711350 Prorocentrum koreanum LMBEV9 ITS1-5.8S-ITS2 KP711351 Prorocentrum koreanum LMBEV9 D1-D3 of LSU KP711352 Prorocentrum koreanum CCMP 2794 SSU KP711353 Prorocentrum koreanum CCMP 2794 ITS1-5.8S-ITS2 KP711354 Prorocentrum koreanum CCMP 2794 D1-D3 of LSU KP711355

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Table 4. Primers sequences used to amplify SSU, ITS and LSU rDNA regions in Prorocentrum species Primer name* Target rDNA Nucleotide sequence (5’ to 3’) Reference EUKA-F SSU AACCTGGTTGATCCTGCCAGT (Medlin et al., 1988) EUKB-R SSU TGATCCTTCTGCAGGTTCACCTAC (Medlin et al., 1988) SR4-F 548-566 SSU AGGGCAAGTCTGGTGCCAG (Hong et al., 2007) SR5kaw-R 630-611 SSU ACTACGAGCTTTTTAACCGC (Hong et al., 2007) SR6-F 891-910 SSU GTCAGAGGTGAAATTCTTGG (Hong et al., 2007) SR7-R 951-932 SSU TCCTTGGCAAATGCTTTCGC (Hong et al., 2007) SR9-R 1286-1267 SSU AACTAAGAACGGCCATGCAC (Hong et al., 2007) ITS1-F ITS TCCGTAGGTGAACCTGCGG (White et al., 1990) ITS4-R ITS TCCTCCGCTTATTGATATGC (White et al., 1990) LSU D1R-F LSU ACCCGCTGAATTTAAGCATA (Scholin et al., 1994) LSU D2C-R LSU CCTTGGTCCGTGTTTCAAGA (Scholin et al., 1994) * F is the forward primer and R is the reverse primer.

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Figure 3

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Figure 4

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Figure 5

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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