Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

Biochemical Systematics and Ecology 36 (2008) 573–583

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Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives Jang-Seu Ki, Myung-Soo Han * Department of Life Science, College of Natural Sciences, Hanyang University, Seongdong-gu, Haengdang-dong 17, Seoul 133-791, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 September 2007 Accepted 14 March 2008

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the ‘‘GPP’’ complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Dinophyceae Cochlodinium polykrikoides Large subunit rDNA Secondary structure Phylogeny

1. Introduction The gymnodinioids are a major group of unarmored dinoflagellates in which some members, such as Akashiwo sanguinea, Cochlodinium polykrikoides and Karenia brevis, are responsible for a red tide that causes fish and shellfish mortality events (Botes et al., 2003; Kim et al., 2004). The cells are very small in size and easily disrupted or distorted during environmental samplings and fixation due to the fragile nature of the naked cell body. Furthermore, their morphoshapes are known to vary depending on different environmental conditions and growth stages, often resulting in difficulty in identification. Consequently, the gymnodinioid members have a very confusing taxonomic history, and also are considered as the subject of on-going taxonomical revisions. Recently, Iwataki et al. (2008) studied the phylogenetic relationship of Cochlodinium isolates

* Corresponding author. Tel.: þ82 2 2220 0956. E-mail address: [email protected] (M.-S. Han). 0305-1978/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2008.03.007

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collected from coastal waters worldwide. Nevertheless, the phylogenetic relationship of C. polykrikoides among the dinoflagellates has not been studied using molecular data. At present, it is irrefutable that the application of molecular techniques to unarmored dinoflagellate taxonomy is becoming increasingly important, and that rDNA sequence information is indispensable for molecular phylogeny and taxonomic description among cells having a similar appearance. As a member of the gymnodinioids, the ichthyotoxic Cochlodinium polykrikoides is a harmful dinoflagellate that is one of the most frequent causes of fish kills (Kim et al., 1999; Ahn et al., 2006). Over the past two decades, outbreaks of C. polykrikoides in Korean coastal waters have significantly increased in frequency, severity and duration (Kim et al., 1999). Red tides dominated by C. polykrikoides have recently been observed in several other countries, such as China (Huang and Dong, 2000), Japan (Kim et al., 2004), and North America (Whyte et al., 2001; Nuzzi, 2004), and have also caused economic losses to the aquacultures in the coastal waters. These causative organisms morphologically resemble (e.g. naked body, displacement of cingular band) the dinoflagellates Akashiwo sanguinea, Gymnodinium catenatum and G. impudicum. Therefore, they are considered taxonomically to reside in the same group as the order Gymnodiniales. To date, many studies have been conducted on the Gymnodiniales phylogeny (Daugbjerg et al., 2000; Hackett et al., 2004; Saldarriaga et al., 2004; Taylor, 2004), and all of these works have shown that the Gymnodiniales is polyphyletic. Up until now, phylogenetic studies on the dinoflagellates have been performed on the basis of many different gene molecules such as rDNA, actin, beta tubulin and heat shock protein (hsp) coding genes (Fast et al., 2002). In the cases of nuclear rDNA, 18S rDNAs shows low phylogenetic resolutions in dinoflagellate analyses; thus most recent attempts were based on the DNA sequences of large subunit (LSU) rDNA spanning the D1/D2 domains (Daugbjerg et al., 2000; Hackett et al., 2004; Saldarriaga et al., 2004; Taylor, 2004). However, the LSU rDNA consists of many regions, resulting in two different domains: one is the core DNA region, which is essential for ribosomal function (Raue´ et al., 1990), and the others are 12 hypervariable, divergent (D) domains (Hassouna et al., 1984). These properties offer various options for data constructions in phylogenetic analyses, since the core segments evolve at a slower rate, permitting phylogenies to be constructed for more distant divergences (Hillis and Dixon, 1991), whereas the D domain variability is useful for reconstructing relatively recent evolutionary events. However, these various molecular regions in the LSU rDNA have not yet been evaluated for dinoflagellate phylogeny as a consequence of the limited number of complete LSU DNA sequences. Recently, Ki and Han (2007a,b) attempted to characterize the complete LSU rDNA sequences of dinoflagellates, including the genus Alexandrium, Prorocentrum micans and gymnodinioid Akashiwo sanguinea. Besides these, the complete LSU sequences are unrevealed from other dinoflagellates, and thus they have not yet been compared widely on the basis of the complete LSU sequence of dinoflagellates, including typical members of both armored and unarmored groups. This study reported the complete nucleotide sequences of Cochlodinium polykrikoides LSU rDNA and, additionally, the secondary structure of the LSU and 5.8S rRNA genes for the unarmored dinoflagellate. In addition, the complete LSU rDNA sequences from nine typical dinoflagellate members, including both unarmored and armored species, were separated into different domains (e.g. core, 12 D domains), and evaluated individually using the structural information and phylogenetic tools to find informative genetic sites for subsequent molecular studies. Finally, this study discussed the phylogenetic relationship of the dinoflagellate species and the position of C. polykrikoides in the Gymnodiniales lineage, using a data matrix newly constructed from the present analysis. 2. Materials and methods 2.1. Cell culture, harvest and DNA purification Cultures of four Cochlodinium polykrikoides isolates (CcPk02, 03, 05, and 06) were provided by Dr. M. Chang of the Korean Ocean Research and Development Institute (KORDI). All cultures were maintained in f/2 medium at pH 8.2, 15  C, and a 12:12 h light/dark cycle, with a photon flux density of about 65 mmol photons m2 s1. For genomic DNA purification, approximately 200 ml of each culture were harvested by centrifugation at 2000 rpm for 10 min. The concentrated cells were transferred into 1.5-ml microtubes, resuspended in 100 ml of 1 TE buffer (10 mM Tris–HCl, pH 8.0 and 1 mM EDTA), and stored at 20  C until DNA extraction. Genomic DNA was isolated from the cells using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

2.2. Isolation of entire LSU rDNA To amplify the complete LSU rDNA gene from Cochlodinium polykrikoides, conventional PCR methods were used to amplify the region with three sets of PCR primers (e.g. ITSF01: 50 -GAG GAA GGA GAA GTC GTA ACA AGG-30 , 28R1318: 50 -TCG GCA GGT GAG TTG TTA CAC AC-30 ; 28F2: 50 -AGGC TCG TAG CGA TAC TGA CGT GC-30 , 28R2219: 50 -CAG AGC ACT GGG CAG AAA TCA C-30 ; 28F1993: 50 -TTG GGG GAT TGG CTC TGA GG-30 ; and 28R2992: 50 -AAA CTA ACC TGT CTC ACG ACG GTC-30 ), which were designed based on a comparison with eukaryotic LSU rDNA sequences available from GenBank. Conventional PCR reactions were carried out in 1 PCR buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin), to which <0.1 mg genomic DNA template, 200 mM each of four dNTPs, 0.5 mM of each primer, and 0.2 U Taq polymerase (Promega, Madison, WI) per 25-ml reaction were added. Using an UNO-II Thermoblock (Biometra, Go¨ttingen, Germany), the PCR thermocycling parameters were as follows: initially, 95  C for 5 min, followed by 32 cycles of denaturation at 95  C for 20 s, annealing at 55  C for

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30 s and extension at 72  C for 2 min. After the cycles, a final extension was completed at 72  C for 5 min. The PCR products (2 ml) were analyzed by 1% agarose gel electrophoresis according to general methods. 2.3. PCR walking of 30 ETS rDNA For the PCR amplification of genome walking, two conserved primers (28-F2864: 50 -TTA CCA CAG GGA TAA CTG GCT TG-30 ; 28F2879: 50 -CTT GTG GCA GCC AAG CGT TC-30 ) were developed as described previously. The regions 30 to the LSU rDNA were sequenced by the external transcribed spacer (ETS) walking PCR technique (Ki and Han, 2005). In brief, the unknown 30 ETS region was amplified from genomic DNA using a conserved universal primer (28F2864), four walking primers, and a universal M13F primer. The PCR reactions were performed as described above with changes in the primers and reactions parameters. PCR parameters were as follows: 95  C for 4 min followed by 5 cycles of denaturation at 95  C for 20 s, annealing at 60  C for 30 s, and extension at 72  C for 1 min; 2 cycles of denaturation at 95  C for 20 s, annealing at 36  C for 30 s, increasing to 72  C at a rate of about 0.5  C s1, and extension at 72  C for 1 min; 30 cycles of denaturation at 95  C for 20 s, annealing at 55  C for 30 s and extension at 72  C for 1 min; and a final extension at 72  C for 5 min. The PCR fragments were diluted in 1 TE buffer at a 1:100 ratio for the next amplification without visualization on an agarose gel. The diluted fragments were used as the DNA template in the second amplification using nested (28F2879) and universal M13F primers. These PCR reactions followed the standard conditions. After the reaction, the PCR products (3 ml) were thoroughly mixed with 3 ml distilled water (DW) and 2 ml of 6 blue/orange loading dye (Promega), heated at 65  C for 5 min to separate, and cooled on ice. After 5 min, the samples were loaded onto a 1.5% agarose gel in 1 TBE buffer and electrophoresed at 8 V cm1 for 1–2 h. Agarose gels were stained with ethidium bromide and destained with D.W. for 30 min. The fragments were visualized at 254 nm UV light, and the core band was collected with an aerosol barrier pipette tip as quickly as possible. The tip was soaked with 100 ml DW in a 1.5-ml microtube, and the gel was separated from the tip with a pipette and heated at 95  C for 5 min, followed immediately by vortexing the tube to release the DNA fragments. The purified eluate was re-amplified with the same PCR primers under identical conditions. The third PCR product was analyzed as described previously. 2.4. DNA sequencing DNA sequencing reactions were performed in a ThermoSequenaseÔ version 2.0 Cycle Sequencing Kit (USB, Cleveland, OH) using the PCR products (3–6 ml) as the template and 1.5 pmol of the primers (Table 2). All the primers were labeled with near infrared dye (IRD) at the 50 end. The DNA sequencing reactions were performed as previously described (Ki and Han, 2005), and the DNA fragments were separated using a Dual Dye Automated Sequencer (LONG READIR 4200, Li-cor, NE). Editing and contig assembly of the rDNA sequences were carried out with Sequencher 4.1.4 (Gene Codes, MI, USA). All DNA sequences determined here have been deposited in the GenBank database as accession numbers AY347309 and DQ779984–DQ779986. 2.5. Secondary structure of Cochlodinium LSU rRNA The complete C. polykrikoides LSU rDNA nucleotide sequences, including 5.8S rDNA, were aligned using the Dedicated Comparative Sequence Editor (DCSE) (De Rijk and De Wachter, 1993), taking the Prorocentrum micans primary and secondary structures into account (GenBank accession no. M14649). The C. polykrikoides LSU rRNA secondary structure was constructed based on the general LSU rRNA model (Gutell et al., 1993; Ben Ali et al., 1999; Wuyts et al., 2001). The helices in the LSU rRNA secondary structural elements were located and labeled based on the LSU rRNA secondary structure database (De Rijk et al., 1998). In addition, the hypervariable areas (D1–12) were located on this predicted structure. Secondary structure models were drawn with the software RNAViz (De Rijk and De Wachter, 1997), a versatile program developed to draw secondary structures of molecules in a fast and user-friendly manner. 2.6. LSU rDNA characteristics For comparisons of each LSU domain, alignment was performed with one Cochlodinium LSU rDNA sequence revealed in the present study, due to the identical genotype of all the Cochlodinium isolates, together with eight other LSU sequences of typical dinoflagellates (e.g. Akashiwo sanguinea, Alexandrium affine, A. minutum, A. tamarense, Pfiesteria piscicida, Prorocentrum micans, P. donghaiense, and Gonyaulax polyedra), using Clustal W ver.1.83 (Thompson et al., 1994). The aligned sequences were partitioned into different domains according to previous studies (Lenaers et al., 1989; Ki and Han, 2007b). Sequence characteristics of the various D domains were measured separately, based on parsimony analysis, in PAUP 4.10b (Swofford, 2002). Genetic distance was calculated using the Kimura-2 parameter model (Kimura, 1980). Neighbor-joining (NJ) analysis (Saitou and Nei, 1987) was performed to compute trees. 2.7. Phylogenetic analysis of dinoflagellates For the dinoflagellate phylogeny, more than 1300 bp of LSU rDNA sequences were retrieved from the DDBJ/EMBL/GenBank. A matrix of LSU rDNAs (37 dinoflagellates and one outgroup) was aligned using Clustal W. The D1 and D5 regions

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were removed, including their up- and down-stream regions from the alignment matrix, and further aligned manually, while regions that could not be aligned unambiguously were excluded from the analysis. Only those positions that could be unambiguously aligned were used in the analysis. This resulted in 908 sites out of the 958 alignment positions for the subsequent analysis. Bayesian phylogenetic analyses were conducted using MrBayes 3.12 (Huelsenbeck and Ronquist, 2001). The general time reversible model, with some sites assumed to be invariable and with variable sites assumed to follow a discrete gamma distribution (GTRþIþG), was selected as the best-fit model of nucleotide substitution (ModelTest version 3.07; Posada and Crandall, 1998). The maximum likelihood parameters in MrBayes were set as follows: ‘‘lset nst ¼ 6’’ (GTR), ‘‘rates ¼ invgamma’’, and ‘‘basefreq ¼ estimate’’ (estimated proportion of base types from the data). The Markov chain Monte Carlo process was set so that four chains (three heated and one cold) ran simultaneously. Two independent runs were conducted for 1 million generations, with trees being sampled every 100 generations, each of which started from a random tree. After analysis, the first 1000 trees were deleted as burn-in processes and the consensus tree was constructed. Bayesian posterior probabilities (>0.50) were indicated at each branch node. The apicomplexa Toxoplasma gondii (GenBank accession no. X75429) was used as the outgroup. The phylogenetic tree was visualized with TreeView version 1.66 (Page, 1996). 3. Results 3.1. Entire LSU rDNA sequences Putative Cochlodinium polykrikoides LSU rRNA coding regions were recorded to be 3433 bp in length (Table 1). Upon comparison with other dinoflagellates, the nucleotide sequences differed in length considerably from the gymnodinioid Akashiwo sanguinea. Interestingly, they were nearly identical in length to the armored Prorocentrum micans. Furthermore, the sequences were compared with those from the LSU rDNA project database (http://www.psb.ugent.be/rRNA/) and it was found that no relationship in length existed with other organisms such as plants, fungi and animals. An intron-like sequence (group I intron) was not found within the LSU rDNA gene. The LSU GC content of C. polykrikoides was recorded at 49.0% with only slight variations depending on the isolates. Interestingly, the value is identical to Akashiwo sanguinea, while the sequence lengths are quite different. Overall, it was high when compared to other dinoflagellates (e.g. 44.0% in Alexandrium affine, 47.6% in P. micans, 47.7% in Pfiesteria piscicida). In addition, it is considerably high in comparison with those of apicomplexans (approximately 44.5%) and ciliates (45.5%), which belong, along with dinoflagellates, to the alveolates. The value was highly variable within other alveolata groups and among the higher taxonomic levels. Some examples include the ciliate Tetrahymena thermophila (X54512; 44.8%), apicomplexan Toxoplasma gondii (X75429; 47.8%), and rhodophyta Gracilaria gracilis (Y11508; 50.9%). This suggests that the GþC content may not be related with the LSU length in dinoflagellates; rather, it is instead species-dependent. Intraspecific variations in the C. polykrikoides LSU rDNA were investigated. Here, C. polykrikoides samples included four strains isolated at different seasons and from geographically segregated Korean coastal waters (see Table 1). Upon comparison, their sequences were 100% identical to one another. In addition, BLAST analyses showed that they were almost completely identical to the partial C. polykrikoides sequences (e.g. AY725423) deposited previously into GenBank. 3.2. Secondary structure of C. polykrikoides LSU rRNA The primary and secondary structures of C. polykrikoides LSU rDNA are shown in Fig. 1. For construction of the structure, the complete LSU rDNA of the dinoflagellate P. micans, for which the secondary structure was previously reported (Lenaers et al., 1989), was used as a reference sequence for C. polykrikoides. Using the helix numbering method established by De

Table 1 Origin of dinoflagellate strains and complete LSU rDNA sequence GenBank accession numbers Species

GenBank access. no.

Strain

Isolation locality

Akashiwo sanguinea Alexandrium affine Alexandrium minutum Alexandrium tamarense Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Cochlodinium polykrikoides Pfiesteria piscicida Prorocentum micans Prorocentrum donghaiense Gonyaulax polyedra

AY831412 AY831409 AY831408 AY831406 DQ779984a DQ779985a DQ779986a AY347309a AY112746 X16108 AY822610 AF377944

CCMP1321 CCMP112 CCMP113 HY970328M CcPk02 CcPk03 CcPk05 CcPk06 – – – –

Great South Bay, USA Ria de Vigo, Spain North Atlantic Ocean Masan Bay, Korea Tongyoung, Korea Narodo, Korea Hakdong, Korea Sarangdo, Korea – – – –

a

DNA sequences revealed in the present study.

Complete LSU rDNA Length (bp)

GþC (%)

3339 3398 3409 3393 3433 3433 3433 3433 >2763 3408 3376 >3221

49.0 44.0 43.8 43.8 49.0 49.0 49.0 49.0 47.7 47.6 48.0 47.5

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Fig. 1. A secondary structure model for the LSU rRNA of Cochlodinium polykrikoides (accession no. AY347309). The sequence is written clockwise from 50 to 30 ‘‘D’’ represents divergent domains.

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Rijk et al. (1998), eight helical groups (B–I) were found in the structure. These helices were generally congruent with the predicted structure of P. micans LSU (Lenaers et al., 1989), and therefore the potential tertiary interactions are well conserved. Beside these identical structures, seven helices (C1-1, C1-2, C1-3, D10, D20-1, H1-2, and I1) were missing from the C. polykrikoides LSU structure. The C1-1/-2/-3 areas, which correspond to the D2 domain, are extremely reduced due to nucleotide deletion. Comparative analyses showed that the overall secondary structure of the LSU rRNA was generally similar to those of P. micans, A. tamarense and A. sanguinea, whereas the secondary structures of the D2 regions predicted by the thermodynamicsbased method showed different configurations between distantly related taxa, implying that these structural differences can provide phylogenetically informative features. 3.3. Characteristics of 12 divergent domains The different LSU rDNA segments of C. polykrikoides, as well as other dinoflagellate members, were characterized individually with respect to their length and GC content (Table 2). The core DNA sequence was recorded at 1876 bp in length and 46.5% GþC content, which are almost identical to the values in P. micans. In addition, the core sequence was nearly identical in length to A. tamarense, although their GþC content was significantly different from that of A. tamarense (44.4%). This indicates that the sequence may have many numbers of transversion mutations among the dinoflagellate species. On the other hand, total nucleotide sequences of the 12 D domains were recorded at 1557 bp in length and 52.2% GþC content. The sequence was relatively variable when compared with those of Akashiwo sanguinea (1460 bp, 51.5% GC) and the genus Alexandrium (approximately 1520 bp, 42.8% GC). Parsimony analyses showed that the core LSU rDNA sequence from nine typical species contained most conserved nucleotides (around 1880 bp) and relatively low parsimony-informative sites (7.9%) within the dinoflagellates, whereas those of the D domains were significantly variable and informative (30.6%) (Table 2). Also, additional sequence characteristics such as conserved, variable sites, transitional and transversional pairs suggested that the D domains were highly divergence when compared with the core region. On the other hand, the C. polykrikoides LSU rDNA D domains were 1557 bp in combined length. This difference is primarily due to variation in the D domains. Among 1607 sites of the aligned 12 D domains, variable sites and informative nucleotides were recorded at 758 (47.2%) and 492 (30.6%), respectively. Indeed, the D domain nucleotide variation is approximately 3.87 times higher than that of the core sequence, judged by a comparison of the parsimonyinformative sites. 3.4. Entropy plot of dinoflagellate LSU rDNAs The new genetic information allows for the construction of a map of LSU rDNA organization and an entropy plot corresponding to the core and D domains (Fig. 2). The plot showed a clear distribution of both variable and conserved positions in the complete LSU rRNA molecules. The D2, D7 and D12 domains are relatively long. Overall, when sequences are aligned, the core regions are conserved, and the D domains are variable within them. In addition, the variations were relatively frequent at the two ends of the LSU rDNA sequence, and in the core areas adjacent to D1 domain. The core areas between D8 and D11, which corresponds to E22 to G20 in the secondary structure, are highly conserved. Ben Ali et al. (1999) reported that the

Table 2 Nucleotide length (bp) and GþC content (%) of different segments in the LSU rDNA of C. polykrikoides (CcPk06) C. polykrikoides (CcPk06)

Entire Core Total D D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12

Sequences compareda

Characteristics of LSU DNA sequences

bp

GþC

sa

cs

vs

ii

si

sv

P-i (%)

3433 1876 1557 143 327 107 16 52 105 286 139 12 106 28 236

49.0 46.5 52.2 56.7 58.1 50.5 43.8 40.3 55.2 51.8 55.4 58.3 40.5 42.8 48.3

3491 1884 1607 144 360 109 16 52 105 289 142 12 108 28 242

2485 1609 807 66 65 47 10 22 65 184 79 4 50 17 85

959 270 758 76 293 61 6 30 40 103 62 8 56 11 153

2896 1759 1121 108 150 84 14 39 87 244 114 9 81 22 148

262 65 205 25 55 13 1 7 10 26 17 2 15 3 38

192 46 154 9 61 10 1 6 5 14 7 1 10 2 38

632 149 492 51 187 30 1 20 23 63 36 3 38 6 91

(18.1) (7.9) (30.6) (35.4) (51.9) (27.5) (6.3) (38.5) (21.9) (21.8) (25.4) (25.0) (35.2) (21.4) (37.6)

7 8 9 9 9 9 9 9 9 9 9 9 9 8 7

Individual segments were characterized based on nine sequences from dinoflagellates, including a single C. polykrikoides strand, as described in Table 1. Abbreviations: sa, site aligned; cs, conserved site; vs, variable site; ii, identical pairs; si, transitional pairs; sv, transversional pairs; P-i, Parsimony-informative site. a DNA sequences in Table 1 (9, all strands included; 8, not including P. piscicida; 7, not including G. polyedra and P. piscicida).

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1

D1

D2

D6

D3

D8

D7

579

D12

D10 D9

D4 D5

D11

0.5

0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4 (kb)

Fig. 2. Organization of both the core and D domains within the LSU rDNAs, and an entropy plot calculated from the amount of variability among seven dinoflagellate LSU rDNAs through a column in an alignment of the sequences. Mean variability for successive windows of 60 nucleotide positions in line was represented.

large multibranched loop in area G and the helices surrounding it are conserved both in bacterial and eukaryotic LSU rDNA. This is likely due to the fact that this structure is considered to be the major element of the ribosomal peptidyl transferase center (Raue´ et al., 1990). They also reported that helices E21–E28 are conserved, which is in agreement with the results of our study. 3.5. Phylogenetic resolution of each LSU domain The phylogenetic resolution of the segmented LSU D domains within the dinoflagellates was investigated (Fig. 3). Of the nine species compared here, genetic distance was slightly lower in the combined core segment, although it was significantly higher over the entire LSU rDNA or combined segments of the D domains. Furthermore, this study investigated the

A.tamarense A.affine A.minutum G.polyedra P.micans P.donghaiense P.piscicida A.sanguinea C.polykrikoides T.gondii

[Complete LSU]

A.tamarense A.affine A.minutum G. polyedra

A.tamarense [12Ds] A.affine A.minutum G. polyedra P.donghaiense P.micans P.piscicida C.polykrikoides A.sanguinea T.gondii

[Core]

P.micans P.donghaiense P.piscicida A.sanguinea C.polykrikoides

T.gondii

0.1

[D1] II

II P.micans P.donghaiense P.piscicida A.sanguinea

A.minutum C.polykrikoides G. polyedra

A.affine A.tamarense

T.gondii

[D2] G. polyedra

P.donghaiense P.micans

P.piscicida A.sanguinea

A.affine A.tamarense

C.polykrikoides T.gondii

A.affine A.tamarense A.minutum [D6] P.micans P.micans P.donghaiense P.piscicida II C.polykrikoides P.piscicida II A.sanguinea A.sanguinea C.polykrikoides G. polyedra P.donghaiense T.gondii

A.minutum A.affine A.tamarense

G. polyedra

II

[D3]

A.minutum

II

[D7] II P.piscicida II P.donghaiense P.micans C.polykrikoides A.sanguinea

[D10]

A.tamarense A.affine A.minutum G. polyedra

A.sanguinea P.piscicida P.micans P.donghaiense II C.polykrikoides II

[D8] T.gondii

A.affine A.tamarense A.minutum G. polyedra P.donghaiense P.micans C.polykrikoides A.sanguinea II P.piscicida T.gondii

T.gondii A.affine A.tamarense A.minutum G. polyedra

T.gondii A.affine A.tamarense

[D12]

A.minutum II P.micans P.donghaiense T.gondii

A.sanguinea

C.polykrikoides

Fig. 3. NJ trees of segmented D domains in the dinoflagellate LSU rDNAs. Each NJ tree was constructed from domains longer than 100 bp, including the core, 12 D domains and complete LSU, using the Kimura 2-parameter model (Kimura, 1980). Branch lengths are proportional to the scale given. ‘‘II’’ on the branch lines represents conflict branches that differ from the core NJ tree.

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phylogenetic utility of LSU D domain comparison on the neighbor-joining (NJ) trees constructed from the Kimura twoparameter model (Kimura, 1980). A NJ tree, predicted from the assembled core sequences, showed the generally accepted branch topology among the dinoflagellate species with relatively low genetic distance (Fig. 3). In contrast, the D domains showed a higher genetic distance and lower sequence similarity than the core. Among them, the tree inferred that D2 was generally identical in branch pattern to the core sequence, whereas the branch patterns constructed from individual D1, D3, D6, D7, D8, D10 and D12 domains were quite different from those both from the core and D2. This suggests that the D2 domain may be useful to resolve closely related dinoflagellate species or strains with high phylogenetic resolution. 3.6. Phylogeny of dinoflagellate with new data set of LSU molecules From the above structural information and segmented NJ analyses, it was found that the core DNA sequences are highly conserved and should be suitable for a long-term evolutional history of dinoflagellates. In addition, of the 12 D domains, branch topology of the D2 domain was nearly identical to that inferred from the core DNA. The genetic distance among the species was considerably higher in D2 sequences than in the assembled core. Considering these informative characteristics, herein D2 and the adjacent core regions were used for a dinoflagellate phylogenetic analysis, particularly the phylogenetic position of C. polykrikoides. This study utilized the DNA sequences from domains D2–D4 and their up- and down-stream core regions, however not including both D1/D5 domains due to their high variability. Their influence could result in a biased tree topology (see Fig. 3). A Bayesian tree was inferred from the available dinoflagellate LSU rDNA sequences (37 strands and a new LSU rDNA sequence of C. polykrikoides) obtained from GenBank. In the phylogeny, armored and unarmored dinoflagellates were never clustered with each other, whereas they were divided into two groups: the GPP complex (term for a group of the gymnodinioid, peridinioid, and prorocentroid; Saunders et al., 1997), including Suessiales and Dinophysidae, and Gonyaulacales (Fig. 4). The order Gymnodiniales appeared to be a paraphyletic group from which other dinoflagellate orders such as Peridiniales and Dinophysiales originated. Since taxon samplings were limited due to a deficiency of complete LSU sequences, the lineages of Peridiniales and Dinophysiales had not yet been clearly determined. However, the Bayesian tree showed that individual dinoflagellates included 17 genera were clearly grouped into the same genus-cluster, and apparently separated from each other, as judged by the branch topology. C. polykrikoides apparently belonged to the GPP complex; however, it clearly formed an identical clade with both Pfiesteria piscicida and Akashiwo sanguinea, rather than that of both Gymnodinium and Gyrodinium. 4. Discussion This study reported the complete LSU rRNA gene sequence from the causative dinoflagellate C. polykrikoides and evaluated their segmented domains and secondary structure with other dinoflagellates. When comparing C. polykrikoides to other dinoflagellates, variations of LSU rDNA in sequence length were recorded at nearly all 12 D domains, whereas core sequences were conserved considerably. Sequence divergence between a pair of D domains ranged from 6.3% (D4, 16 sites) to 51.9% (D2, 360 sites). Among the 12 D domains, the D2 and D12 domains have relatively more parsimony-informative sites in comparison with the others. In a previous study, the D2 domain, however, is conserved in varying geographic origins of Akashiwo sanguinea strains (Ki and Han, 2007b) and the genus Alexandrium (Ki and Han, 2007a). In the present study, it was also found that four isolates of C. polykrikoides had completely identical DNA sequences, which was well-supported by previous data regarding molecular comparison of internal spacer (ITS) regions (Ki and Han, 2007c). In the dinoflagellate LSU, the variations in the D domains are mainly responsible for the differences in LSU rDNA among the species (Table 2 and Figs. 2, 3). In a view of the secondary structure (Fig. 1), a reason for this is that the areas enclosed by helix C1 and E20, the entire area H1_n and, to a lesser extent, helix D20 are hot spots for extremely variable insertions (Ben Ali et al., 1999; Ki and Han, 2007a). The hot areas were generally in accordance with the present results. For example, the D2, D8, and D12 areas, which corresponded approximately to C, E20_1/_2, and the entire area of H1-n, were the most highly variable areas among the dinoflagellate LSU. In addition, these regions were predicted to contain a single-stranded structure rather than a double helix. Moreover, the single-stranded region in the D2 domain contained the two deleted areas of sequence, possibly because it may be rather easy to insert or delete DNA fragments in a single-stranded region, rather than in a double-stranded helix. Taking into the LSU characteristics, this study used a different data set of LSU rDNA for a Bayesian phylogenetic analysis. To date, only 16 strands of complete LSU rDNA from three genera of dinoflagellates (Alexandrium, Akashiwo, Cochlodinium and Prorocentrum) have been available from public sources. The sequence samplings, therefore, were limited for a study of the broad phylogeny of the dinoflagellates. Alternatively, a data matrix was constructed, consisting of domains D2–D4, including their up- and down-stream flanking core regions, which included 17 typical genera belonging to six families. Bayesian analysis showed that the new LSU data set could resolve several major dinoflagellate clades (Gonyaulacales and GPP complex). In addition, individual dinoflagellates (e.g. Alexandrium, Akashiwo, Cochlodinium, Gyrodinium Karlodinium, Peridinium, Pfiesteria, and Prorocentum) were clearly grouped into the same genus-cluster (>0.99 of posterior probability), and apparently separated from each other, as judged from the predicted branch topology. As noted previously, this was caused by the nature of D2, which is conserved within the species-level and is highly variable among the relative genera (Ki and Han, 2006, 2007a,b). In addition, the Bayesian tree and branch topology were generally congruent with those inferred from complete LSU and core sequences (Fig. 3), suggesting it represented the relationships of the dinoflagellates with high resolution.

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1.00

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0.94 0.97

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Toxoplasma gondii P. bahamense A. pseudogonyaulax A. affine Gonyaulacales A. tamarense A. minutum "GPP A. margalefeii complex" G. baltica G. spinifera G. elongata G. digitale G. cf. spinifera G. membranacea A. incoloratum A. herdmanii A. steinii P. bipes P. willei D. norvegica T. britannica T. compacta T. jolla P. glacialis W. tenuissima P. donghaiense P. micans P. piscicida A. sanguinea C. polykrikoides H. arctica H. triquetra K. armiger K. veneficum G. venator G. rubrum G. dominans G. spirale

Su e Di ssial n Gy ophyes m Pe nod sidae rid ini Pro inia ale roc les s ent ral es

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Fig. 4. A phylogenetic relationship of dinoflagellate species inferred from partial large subunit rDNA sequences, including D2 to D4 and their flanking core regions, and using a consensus ML analysis, computed with MrBayes version 3.1.2. The numbers at the nodes are posterior probabilities greater than 0.50. The apicomplexan Toxoplasma gondii (GenBank accession no. X75429) was used as the outgroup. Thick branch lines represent armored dinoflagellate lineages. Dinoflagellate taxa sampled here are as follows: Alexandrium affine (AY831409), A. minutum (AY831408), A. sanguinea (AY831412), A. tamarense (AY831406), A. margalefeii (AY154958), A. pseudogonyaulax (AY154957), Amphidinium herdmanii (AY455675), A. incoloratum (AY455677), A. steinii (AY455673), Cochlodinium polykrikoides (AY347309), Dinophysis norvegica (AY571375), Gonyaulax baltica (AY154962), Gonyaulax cf. spinifera (AY154960), G. digitale (AY154963), G. elongata (AY154964), G. membranacea (AY154961), G. polyedra (AF377944), G. spinifera (AF260388), Gymnodinium cf. placidum (AF260383), G. venator (AY455681), Gyrodinium dominans (AY571370), G. rubrum (AY571369), G. spirale (AY571371), Heterocapsa arctica (AY571372), H. triquetra (AF260401), Karlodinium armiger (DQ114467), K. veneficum (DQ114466), Peridinium bipes (AY733012), P. willei (AB232669), Pfiesteria piscicida (AY112746), Polarella glacialis (AY036081), Prorocentrum micans (X16108), P. donghaiense (AY822610), Pyrodinium bahamense var. compressum (AY154959), Togula britannica (AY455679), T. compacta (AY568562), T. jolla (AY568559) and Woloszynskia tenuissima AY571374), respectively.

Furthermore, the present phylogenetic findings were generally accordance with the relationships determined from morphological characteristics (Fensome et al., 1999; Daugbjerg et al., 2000). Within the GPP complex clade, the unarmored dinoflagellates having relationships within the gymnodinioid, peridinioid, and prorocentroid groups still remain ambiguous, and the gymnodinioid and peridinioid groups were polyphyletic, with several lineages. Of the unarmored dinoflagellates included here, the earliest divergence was found to be Amphidinium, with 0.86 of posterior probability. Recently, Hackett et al. (2004), Saldarriaga et al. (2004) and Taylor (2004) tried to explain the complicated phylogeny and evolution of dinoflagellates with different aspects and angles. With regard to morphological comparative studies, the genus Cochlodinium is similar to Gyrodinium as a member of the Gymnodiniales (Taylor, 1980). They can be distinguished by the degree of displacement of the cingulum (more than one fifth of the body length; Steidinger et al., 1996) separating the epicone and hypercone, whereas Gymnodinium was less than one fifth. However, the morphological characteristics are quite variable depending on the life stage, environmental conditions, as well as fixation procedures used during species sampling. In many species, cingular displacement is approximately equal to one fifth of the cell length, making the generic affiliation ambiguous. For these reasons, morphological classification of the three members remains a controversial subject. Recently, Daugbjerg et al. (2000) re-described the typical dinoflagellate members, including armored and unarmored groups, with comparisons of ultrastructual features and molecular D1/D2 LSU rDNA sequences. Since then, the unarmored dinoflagellates have been diversified into several new genera, such as Akashwo, Togula, Karlodinium, Karenia, and Takayama. With regard to comparative studies between Cochlodinium and other dinoflagellates, Cho et al. (2001) attempted to compare the fine-scale features of transverse sections, biochemical analysis, and ITS rDNA sequences between C. polykrikoides and G. impudicum. Besides the data, comparative studies on morphology have not been attempted to reveal the ultra- and fine-structural differences between Cochlodinium and other dinoflagellates. Later,

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considering the present molecular phylogeny such as the close relationship between A. sanguinea and C. polykrikoides, morphological comparisons will be necessary for a better understanding of the phylogenetic lineage of both species, as well as sometimes for their taxonomic revisions. In order to construct hypothetical phylogenetic trees, molecular phylogeneticists must carefully consider several factors such as homologous genes, evolutionary models, alignment algorithms, samplings involving the number and selection of taxa, and the choice of outgroups. Incorrect choices for these factors can sometimes greatly influence the resultant trees (Taylor, 2004). For example, Murray et al. (2005) examined the dinoflagellate phylogeny in an analyses based on SSU, partial LSU rDNA and both concatenated molecules, considering an adequate evolutionary model and phylogenetically informative sites in the compared DNA molecules. They found that the Gonyaulacales was monophyletic, while the Gymnodiniales was polyphyletic. In this study, the LSU rDNA was evaluated phylogenetically and the secondary structure according to the segmented core and D domains in order to properly select phylogenetically informative LSU rDNA regions. The data in this study showed that the D domains were highly variable among the dinoflagellate species, affecting greatly their branch topologies constructed in the data sets. Particularly, the D1 domain, which has been included in the previous phylogenetic analyses, was relatively long in nucleotide length (approximately 140 bp), and has many variable sites, possibly greatly influencing the tree branch patterns. Although the complete LSU rDNA sequences from typical dinoflagellate members were still limited, this study attempted first to evaluate the segmented dinoflagellate LSU rDNAs from nine typical members, and found that D2 and core LSU sequences might be considered as valuable molecules in the construction of the evolutionary history of dinoflagellates, as well as recent divergent lineages of dinoflagellate members. A data matrix constructed newly from the LSU DNA molecules produced a new phylogenetic tree, as described previously (see Fig. 4). This study builds on these and discusses aspects not considered previously. Also, this study highlights proper selection of molecular signatures within the LSU rDNA molecules. Acknowledgments We would like to thank Dr. J. Wuyts (University of Antwerp, Belgium) and Richard P. Brown (Liverpool John Moores University, UK) for their assistance with the secondary structure. We wish to thank the two anonymous reviewers for very helpful comments. 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