Morphology and rDNA phylogeny of a Mediterranean Coolia monotis ( Dinophyceae ) strain from Greece *

Sequences of LSU and SSU ribosomal RNA genes and phylogeny have not been widely investigated for the dinoflagellate Coolia monotis Meunier, and no information is available on the small and large rDNA subunits of Mediterranean strains. A strain isolated from the Thermaikos Gulf in northern Greece was identified as C. monotis—a new record for the Greek algal flora—using thecal morphology by light, epifluorescence and scanning electron microscopy. The small subunit and partial (D1/D2) large subunit sequences were analyzed and compared to other strains of C. monotis and dinoflagellates from various regions. Thecal architecture showed that the Greek strain of C. monotis was phenotypically similar, but not identical, to other strains reported in literature. The partial LSU sequence (700 bp) was found to vary by 113 bp positions (16%) from the C. monotis strain from New Zealand, whereas the SSU (1757 bp) had 15 bp differences (0.85%) from the strain from Norway. Phylogenetic tree construction showed that the Greek strain fell within the Coolia clade and had a close relationship with the families Ostreopsidaceae and Goniodomaceae of the order Gonyaulacales. Preliminary findings suggest the existence of different genotype strains of C. monotis with large intraspecific genetic variability and minimal morphological differentiation (similar phenotypes). Certain ecological and evolutionary implications of these findings are discussed.


INTRODUCTION
Coolia monotis Meunier is a cosmopolitan dinoflagellate species previously reported in the Mediterranean (e.g.Tolomio and Cavolo, 1985;Riobó et al., 2002).There have been no records to date of its presence in Greece.It is a benthic and hardy species observed under variable environmental conditions, mainly in sandy biotopes but also as epiphytic or planktonic (Taylor and Pollingher, 1987;Faust, 1992;Steidinger and Tangen 1997).It is a taxon that appears to consist of several genetically distinct geographical strain groups (Penna et al., 2002), thus it exhibits significant intraspecific genetic variation (IGV).These strains occur both allopatrically due to geographical isolation and in sympatry in the same geographic region (Schluter, 2001;Coyne and Orr, 2004).The allopatric Mediterranean strains appear to be non-toxic (Rhodes et al., 2000;Riobó et al., 2002).
There are currently a limited number of available sequences in GenBank concerning the large subunit (LSU) and small subunit (SSU) of C. monotis, which do not include any from the Mediterranean region and are not presented in any phylogenetic analyses.The phylogeny of C. monotis using these rRNA genes and the ones read for a Mediterranean strain allows a phylogenetic investigation to be done concerning C. monotis and the relation of the family Ostreopsidaceae (e.g.Coolia, Ostreopsis) to Goniodomaceae (e.g.Alexandrium, Pyrodinium) and other families of the order Gonyaulacales.The phylogeny and morphology of a Mediterranean strain also serves to present a preliminary study into its intraspecific diversity and to discuss certain ecological and evolutionary implications.
Widely varying intraspecific genetic variability among populations of potentially harmful benthic dinoflagellates has been observed for taxa such as Ostreopsis spp.(Pin et al., 2001;Penna et al., 2002) and Gambierdiscus spp.(Babinchak et al., 1996;Chinain et al., 1999).The intraspecific variability of potentially harmful benthic dinoflagellate taxa has raised interest in phenotypes as compared to genotypes from a taxonomic and biogeographic point of view.Benthic species typing is growing in importance with increasing interest in microalgal monitoring due to the problems encountered with potentially harmful benthic or bentho-planktonic species that impact seafood consumption (Faust et al., 1996;ICES/IOC, 1999, 2000;Lenoir et al., 2004).
In phylogeny, using the LSU is imperative for lower level taxonomic analysis in order to discriminate between closely related taxa at the genus, species and even strain level (Baroin-Tourancheau et al., 1992;Buchheim et al., 2001;Harper and Saunders, 2001;John et al., 2003).D1-D6 are phylogenetically useful variable domains ("D" regions; Hassouna et al., 1984;Michot et al., 1984) within the LSU sequence of dinoflagellates (Daugbjerg et al., 2000;Edvardsen et al., 2003;Murray et al., 2004).The SSU sequences evolve much slower and therefore provide information for comparing groups at a higher taxonomic level than the LSU permits; however, they are also used for distinguishing between species (Saunders et al., 1997;Grzebyk et al., 1998;Saldarriaga et al., 2001).
Here we report on the morphology (light, epifluorescence and scanning electron microscopy) and rDNA phylogeny (D1/D2 LSU, SSU) of a strain of Coolia monotis isolated from a mussel farming area in the north Aegean Sea, an area that has experienced frequent and diverse harmful dinoflagellate blooms within the last four years.

Samples, isolation and cultivation
Sampling was carried out near the Loudias River delta at Malgara, in the Thermaikos Gulf, north Aegean Sea (40:32:08 N, 22:41:32 E).The sampling area was adjacent to a mussel farm's 'clean-dry' posts where mussels are hung up and naturally exteriorly cleaned by drying in the sunlight.Samples were obtained with a 1L Ruttner plankton sampler from various depths and up to 2.5 m near the sandy bottom at midday in June 2002.The water temperature was 22°C and salinity 33 ppt.The samples were concentrated with 8 µm-pore polycarbonate Nuclepore membrane filters and back-washed into sterile test tubes containing 8-10 ml of L-1 medium (Guillard, 1995).Samples were cultivated at 23°C, 14/10 hours light/dark, illumination at 60-70 µmoles m -2 sec -1 without aeration, and studied 7 days later to identify and isolate dinoflagellate species of interest.Secondary isolations produced four clones (C1, C2 -C3, C4) from two initial isolations of C. monotis cells made from cultured samples from 2.0-2.5 m depths; these were placed in individual tubes with 5 ml of L-1 medium for 1 month and subsequently grown to 100 ml cultures.

Microscopy
Clones were examined under light microscopy and digitally photographed using a Sony CCD Hyper HAD digital camera.For SEM, 10 ml culture (exponential phase) were initially filtered using a 60 µm Nitex net, then placed in glass centrifuge tubes with 4 ml of clean seawater and fixed in 2% OsO 4 buffered with PBS in the dark for 1h at 4°C.The cells were then pelleted at 400 g, and washed with 3 decreasing concentrations of seawater then with filtered tap water.Following centrifugation, the cell pellet was left to stand overnight in 1:1 [30% H 2 O 2 ]:[glacial acetic acid], then rinsed 4 times with filtered tap water.An aliquot of the final pellet was mixed with acetone on a round cover slip and airdried overnight at 30°C.The sample was gold-plated and observed in a Jeol JSM-35 SEM.For epifluorescence microscopy, 2 ml exponential culture were fixed with 2.5% glutaraldehyde in the dark for 1h at 4°C, buffered in PBS, washed twice for 5 minutes in 10% Triton-X, then stained.Cellulose thecal plates were stained with calcofluor white (Sigma-Aldrich), using the method of Fritz and Triemer (1985).Samples were viewed using an Olympus AX epifluorescence microscope, using a Chroma 730 nm emission filter s/n 11000 (Rockingham, VT).Images were acquired with an Optronics DEI 750 camera (Goleta, CA).

DNA extraction
Cultures of approximately 2x10 6 cells in exponential phase of strains C1 and C3 (identical to C2 and C4 respectively since they were taken from the same primary clone) were filtered through 60 µm Nitex nets, then through 8 µm polycarbonate Nuclepore membrane filters, washed with 0.2 um filtered, sterilized seawater and transferred into 15 ml centrifuge tubes.The cells were pelleted at 400 g for 8 minutes and DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions.DNA quality and quantity was assessed by spectrophotometry, and by agarose gel electrophoresis.Approximately 20 to 25 pg of DNA was extracted per cell.

Polymerase Chain Reaction (PCR) amplification
Primers used for PCR (Saiki et al., 1988) amplification of gene sequences were made by Invitrogen (Carlsbad, CA) and are listed in Table 1.LSU primer D1R-forward (Domain "D1") was targeted towards conserved core sequence positions 24 to 43, while primer D2C-reverse was for positions 733 to 714 relative to Prorocentrum micans LSU rRNA (Lenaers et al., 1989).The SSU eukaryotic universal forward primer EUF was targeted towards conserved core sequences 1 to 22 relative to the 18S sequence of dinoflagellates while the eukaryotic universal reverse primer EUR was for position 1795 to 1819.PCR was done using an Invitrogen AccuPrime kit following the manufacturer's instructions.An MJ Research (Waltham, MA) PTC-225 Peltier Thermal Cycler (DNA Engine Tetrad) was used for 50 µl PCR reactions with thin-walled 0.2 ml Eppendorf tubes.Approximately 40 ng of DNA template per 700 bp fragment was used with 100 pmol each of forward and reverse primer.Invitrogen AccuPrime (Carlsbad, California) and Roche (Basel, Switzerland) core kits were used.PCR cycles were: denaturation at 94°C for 4 min., 35 cycles of 94°C for 1 min., 41 to 45°C for 2.5 min., and 72°C MORPHOLOGY AND PHYLOGENY OF COOLIA MONOTIS 69

Sequencing
Amplified fragments were gel purified using a MinElute Gel Extraction Kit (Qiagen) according to the manufacturer's instructions.Purified fragments were sequenced by dye terminator cycle sequencing using a DTCS Quick Start Kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions.The sequencing primers used are listed in Table 1.Following ethanol precipitation, sequencing reactions were analyzed on a Beckman Coulter CEQ 2000.

Phylogenetic tree construction
Sequences were initially evaluated using the Basic Length Alignment Searching Tool (BLAST; Altschul et al., 1990) against deposited sequences (GenBank).Using Prorocentrum micans as an outgroup, sequences of 21 and 18 taxa of dinoflagellates (Table 2) were used to produce the LSU and SSU trees respectively.For the partial LSU, 626 characters were considered unambiguous and used for the alignment (see also John et al., 2003).The LSU genotype from Malaysia deposited as C. malayense (AF244942) was used as Coolia sp.(C.monotis ?) (see also Usup et al., 2002) in the absence of morphologic validation and as it exhibited 99 % similarity with C. monotis from New Zealand.Multiple sequence alignments were carried out using ClustalX (Thomson et al., 1997).Alignments were checked and improved using BioEdit manually (Hall, 1999).Using the PHYLIP 3.63 software package (Felsenstein, 2004), distance matrices were produced using the DNADIST module assuming Kimura's two-parameter model (Kimura, 1980) and then used to construct the phylogenetic tree topologies with the NEIGHBOR module (NJ, neighbor-joining algorithm of Saitou and Nei, 1987).In order to confirm robustness of the Ostreopsidaceae clades, the NJ topology was checked against the tree produced using the FITCH module (Fitch-Margoliash Least-Squares Distance method of Fitch and Margoliash, 1967) and the modules DNAPARS (Fitch, 1971) and DNAML (Felsenstein and Churchill, 1996) for Parsimony and Maximum Likelihood (ML) respectively.One thousand bootstrap resamplings (Felsenstein, 1985) were performed using the SEQ-BOOT module and the consensus tree was generated using CONSENSE.Trees were viewed with the Treeview software (Page, 1996).

Light Microscopy
In light microscopy (Fig. 1a, b) Coolia monotis cells from cultures were observed and the oblique axis seen when viewed from the side, and a compressed spherical shape when viewed ventrally with a strongly-lipped and defined cingulum and sulcus.Cells were variable in size, 30 to 40 µm in length.Smaller-sized cells were observed during reproduction.Cleaned cells under phase contrast microscopy (Fig. 2a, b  revealed a pentagonal and partly wedge-shaped 3' plate touching the APC.At the antapex, the pentagonal 2'''' plate was observed at the end of the sulcus. Staining Coolia monotis with calcofluor revealed the general thecal architecture of 3 apical ('), 7 precingular (''), 5 postcingular (''') and 2 antapical ('''') plates (Fig. 3a-d).The existence of posterior intercalaries (p) could not be confirmed.Six cingulum (6c) plates were recorded.The APC was surrounded by plates 1', 2' and 3' (Fig. 3a, b).The lipped cingulum and sulcus were evident in the ventral view (Fig. 3c), while the antapical plates were seen dorsal-posteriorly (Fig. 3d).

Scanning Electron Microscopy
The plates of C. monotis Meunier (Fig. 4a perforated by a clear row of small pores and containing the 7-9 µm, slit-like apical pore opening.Apical plate 2'' was wider than its neighboring precingular plates.The lists of the cingulum and sulcus were very prominent, with plate 1'''' extending into a sulcus list.The cingulum and sulcus were deep and wide in all specimens.The interior view of the collapsed epicone showed a characteristic wedge-shaped 3' plate (Fig. 4b).

DNA
Sequences of clones C1 and C3 were compared and found to be identical for both the partial LSU and SSU sequences.
For the LSU, a 700 bp fragment was generated and sequenced.This sequence aligned with the reported LSU sequences of C. monotis from New Zealand and Coolia sp.(C.malayense?)from Malaysia, both at 84% nucleotide similarity.The sequences of the Greek strain were highly similar in the D1 region (93% nucleotide similarity), but greater variation was observed after nucleotide 400, the D2 region (ca.74% nucleotide similarity).Other LSU sequence similarity values ranged between 60% and 61% with Alexandrium spp., Pyrodinium bahamense var.compressum, and Gonyaulax baltica, 55 and 57% with Fragilidium subglobosum, and 54 and 55% with Ostreopsis spp..
A 1757 bp SSU sequence was generated and read.This sequence aligned with the reported SSU sequences of C. monotis from Norway at 99% nucleotide similarity.BLAST sequence alignment showed the closest related dinoflagellates to be taxa  of Ostreopsis, Alexandrium and Pyrodinium.Nucleotide similarity values were between 88% and 89% with Alexandrium spp., 87% with Pyrodinium bahamense, 84 % with Gonyaulax spinifera, 83 % with Fragilidium subglobosum, and 81% with Ostreopsis ovata.

Phylogeny
In both the LSU and SSU neighbor phylogenetic trees, Coolia strains clustered at 100% bootstrap values, while Coolia and Ostreopsis species (Ostreopsidaceae) formed a discrete cluster with decreasing affiliation with Fragilidium (Pyrophacaceae), and Alexandrium and Pyrodinium strains (Goniodomataceae).The tree determined from the LSU sequences showed that the Coolia cluster is related to that of Ostreopsis at a 100% bootstrap value (see Fig. 5), compared to a 99% bootstrap value for the SSU sequence tree (see Fig. 6).All other phylogenetic trees constructed (Fitch, Parsimony, ML; not shown here), for both the LSU and SSU sequences, gave very similar tree topology and bootstrap results.The differences were the positioning of Fragilidium with either the Coolia/Ostreopsis or the Alexandrium clade and the topology of Ceratium in relation to Gonyaulax and Protoceratium.The Greek C. monotis, however, was repeatedly confirmed in the Coolia clade at 100% bootstrap values, with a close relation to the Ostreopsis clade (99-100%).

DISCUSSION
Morphologically the Greek strain of C. monotis Meunier (Meunier, 1919;Balech, 1956;Fukuyo, 1981;Faust, 1992Faust, , 1995;;Steidinger and Tangen, 1997;Ten-Hage et al. 2000), exhibits slight variations from other C. monotis strains of different geographical origins.The apical plate 1' is comparatively narrower with straight edges and the 2'' plate is much wider, similar only to Balech's (1956) findings.The APC is wider and the sulcus more curved than that of the Belizian C. monotis strain (Faust 1992).The characteristic partly wedge-shaped 3' plate is unlike any other reported in the literature, resembling more that observed for the species C. tropicalis (Faust, 1995).The cingulum and sulcus are wider in the Greek strain, resembling more those observed for the species C. areolata by Ten-Hage et al. (2000).
The partial LSU nucleotide sequence of the Greek C. monotis exhibited relatively low pairwise similarity (84%) to the LSU of C. monotis from New Zealand or Coolia sp.(C.monotis?) from Malaysia.Although it has nearly identical morphologically, the Greek strain's sequence varied by a very high number of 113 bp positions from the New Zealand one, mostly in the D2 region.This large number of character differences, however, is also observed when comparing the ITS rDNA sequences for various geographic-origin C. monotis strains deposited at GenBank (Penna et al., 2002).The SSU sequence of the Greek C. monotis had 15 base pair differences from the Norwegian C. monotis's SSU sequence, which resulted in a very high sequence similarity between them (99%).The large similarity among the compared dinoflagellate SSU sequences contrasted with the lower similarity observed between the LSU sequences.However, using both sequences provided better phylogenetic analyses of C. monotis as has been done with Dinophysis (Edvardsen et al., 2003) and other algae (Huelsenbeck et al., 1996;Buchheim et al., 2001;Harper and Saunders, 2001).Phylogenies inferred from both LSU and SSU neighbor-joining trees were similar, agreeing with the Dinophyceae phylogeny produced by others (e.g.Daugbjerg et al., 2000;Ellegaard et al., 2003;Saldarriaga et al., 2004).In overall, topologies in both of the phylogenetic trees support the morphologybased taxonomic system (Taylor, 1980;Taylor, 1987;Fensome et al., 1993;Steidinger and Tangen, 1997).The phylogenetic clade containing Coolia and Ostreopsis (see Figure 5 and Figure 6) was found to be very robust and in agreement with the identical plate formulae of these two genera (Balech, 1956;Besada et al., 1982;Faust et al., 1996;Steidinger and Tangen, 1997).Although these two Ostreopsidaceae taxa fall within the order Peridiniales in the NCBI taxonomic database (http://www.ncbi.nih.gov/Taxonomy/), Taylor (1987), Fensome et al., (1993) and Steidinger and Tangen (1997) place these genera, classically, in the order Gonyaulacales (Taylor, 1980).Our findings support the latter taxonomic structure in which a close relationship appears between the investigated Gonyaulacales families (Lindemann, 1928) such as Ostreopsidaceae (Coolia, Ostreopsis), Goniodomataceae (Alexandrium, Pyrodinium), Pyrophacaceae (Fragilidium), Ceratiaceae (Ceratium) and Gonyaulacaceae (Gonyaulax).This also agrees with the distant relationship observed in our phylogenetic analyses of the Peridiniales (e.g.Heterocapsa, Pentapharsodinium, Scrippsiella) to the Gonyaulacales, the latter also consistently forming a very robust phylogenetic group in the majority of extensive phylogenies carried out by other researchers.Genetic information concerning Gambierdiscus and other Coolia species could further elaborate Gonyaulacales phylogeny and structures of Ostreopsidaceae and Goniodomataceae families.
Our initial observations indicate the probable existence of intraspecific genetic variability (IGV, expressed as nucleotide differences) with significantly different LSU and SSU genotypes for C. monotis.High genetic variability with only slight morphological variations do not warrant new species designations, whereas the possible existence of cryptic species within populations cannot be ruled out (Montresor et al., 2003).Likewise, the possible existence of dominant strain types of C. monotis in various geographic populations (as with Prorocentrum micans; see Shankle, 2001;Shankle et al., 2004) needs to be assessed.Species genotypes from different geographical origins (populations) that have a high degree of conserved morphological features are also expected (Taylor, 1993;Medlin et al, 2000;Coyne and Orr, 2004).As the criteria used for species differentiation based on genetic information are continuously debated (Manhart and McCourt, 1992;Taylor, 1993;Coyne and Orr, 2004), it will continue to be necessary to use both morphotypes and genotypes when defining strains, especially when ascertaining the extent of speciation and strain development of an organism.Some taxonomical methods will probably need to be upgraded by using new genetic regions for strain identification, as proof exists that morphologically or toxically variable dinoflagellate strains such as Karenia brevis and Pfiesteria piscida (Loret et al., 2002;Tengs et al., 2003) have identical rDNA sequences.Open questions also remain as to how sexual reproduction and polyploidy of dinoflagellates (Pfiester and Anderson, 1987;Faust, 1992) compound genetic variability and speciation.
Intraspecific genetic variability observed in distantly-related Dinophyceae such as Karenia, Gymnodinium, Amphidinium and Dinophysis spp.appears to be minimal compared to that observed for Gonyaulacalean dinoflagellates (~10 to 50% IGV depending on the rDNA gene) such as Alexandrium (Scholin et al., 1994;Adachi et al., 1996;Guillou et al., 2002;John et al., 2003), Coolia (Penna et al., 2002), Ostreopsis (Pin et al., 2001;Penna et al., 2002), and Gambierdiscus (Babinchak et al., 1996;Chinain et al., 1999).This fact seems true even for the highly variable and quick evolving ITS region, but less evident for the slow evolving SSU.For example, compared to the 16% IGV found in this study for the D1/D2 LSU (700 bp) and 0.85 % for the SSU of Coolia monotis, intraspecific genetic variability for Amphidinium spp. was only 0 to 4.4% for the larger D1-D6 LSU fractions (ca.1400 bp) (Murray et al., 2004), for Karenia and Gymnodinium spp. it was 0 to 0.3% for the D1/D2/D3 LSU fraction (ca.700-1000 bp) (Adachi et al., 1997;Sako et al., 1998;Hansen et al. 2000;Guillou et al., 2002), and for Dinophysis spp. it was 0 to 2.0% for the D1/D2 LSU fraction, the SSU and ITS1-ITS2 regions (Guillou et al., 2002;Edvardsen et al., 2003).As numerous biological and ecological parameters affect intraspecific diversity (Medlin et al., 2000;Schluter, 2001;Montresor, 2003;Coyne and Orr, 2004), the above-observed large variations in species' IGV still remain to be confirmed and explained.Moreover, the evolution and the biogeography of strains need to be assessed in order to arrive at reasonable conclusions concerning dinoflagellate IGV.For example, the closely-related Alexandrium genus, like Coolia, apart from demon-strating high IGV also seems to have evolved late in dinoflagellate history (see John et al., 2003;Saldarriaga et al., 2004), in comparison to the distantly-related and "older" dinoflagellates such as those of the order Gymnodiniales which coincidentally seem to have low IGV.Such work could also shed light on any relation of IGV with other dinoflagellate classification systems such as plate evolution models (see Saunders et al., 1997;Saldarriaga et al., 2004).
FIG. 1. -Coolia monotis cells (fixed) under light microscopy showing: a) Variability in cell size with smaller cells during reproduction (white arrowheads), and a strongly-lipped and defined sulcus (black arrowheads); b) The oblique axis when viewed from the side with strongly-lipped and defined cingulum.Scale = 10 µm.

TABLE 1 .
-Primers used for PCR and sequencing.

TABLE 2 .
-Dinoflagellates used for sequence comparisons and phylogenetic tree construction.