Nicolas Chomérat
email: nicolas.chomerat@ifremer.fr

Gwenael Bilien

Amélie Derrien

Kévin Henry

André Ung

Hélène Jérôme Viallon

Taiana Darius

Clémence Mahana

Iti Gatti

Mélanie Roué

Fabienne Hervé

Hélène Taiana

Clémence Mahana Iti Gatti

Damien Réveillon

Zouher Amzil

Mireille Chinain

O Cf Siamensis

French Polynesia

CAWD184, Cook isl. Ab674877

Mk227180

Moorea Mk227229

Nuku Hiva

MRP23 Tahiti Mk227182

Mk227228

Mangareva Mk227213

Mangareva Mk227212

TPV9 Mangareva Mk227226

Tahiti Mk227207

Tubuai Tub28

Mk227206

Mk227187

MRP25 Tahiti Mk227183

MRP26 Moorea Mk227184

Moorea Ab674900

Japan Kohama

Ab674916

MRP26 Australia Mk227244

Moorea Mk227246

Tahiti Mk227247

Tahiti Mk227248

ATH6, Moorea MK227241, HPT6, Moorea MK227242, MRP23, Moorea MK227243, MRP25 Tahiti Mk227240

Moorea Mk227230

Raivavae Mk227231

Tubuai Tub10

Mk227232

Mangareva Mk227233

Mangareva Mk227234

ANH33 Tahiti Mk227235

Mh844088

Jx065585

Viet Nam Af218465

Jx065584

Viet Nam

CAWD184, Cook LT220222 Ab674914

OvPR03 Malaysia Af076218

Af218457

Malaysia Ovpr01

TE4OS Thailand Ab841222

Af218455

Malaysia Ovpd06

Af076217

Malaysia Ovpd04

Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean)

Keywords: ITS-5.8S rDNA, LSU rDNA, microscopy, Ostreopsis lenticularis, taxonomy, toxins O. cf. ovata Ostreopsis sp

Introduction

First described from the Gulf of Thailand in 1901 [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF], the genus Ostreopsis Johs.

Schmidt has become a well-studied dinoflagellate due to its recurrent toxic blooms in several places around the world and their deleterious impacts on both marine ecosystem functioning and human health [START_REF] Penna | A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt[END_REF][START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF]Accoroni and Totti, 2016;[START_REF] Verma | Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon[END_REF].

Originally limited to tropical locales, this genus is now found in temperate areas where they cause severe health problems (e.g. [START_REF] Tichadou | Health impact of unicellular algae of the Ostreopsis genus blooms in the Mediterranean Sea: experience of the French Mediterranean coast surveillance network from 2006 to 2009[END_REF][START_REF] Del Favero | Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach[END_REF]Accoroni and Totti, 2016;[START_REF] Berdalet | Harmful Algal Blooms in Benthic Systems: Recent Progress and Future Research[END_REF]. Ostreopsis species have been shown to produce a variety of toxic compounds [START_REF] Lassus | Toxic and harmful microalgae of the world ocean[END_REF], that can bio-accumulate in marine organisms such as molluscs, herbivorous echinoderms or fishes [START_REF] Amzil | Ovatoxin-a and Palytoxin Accumulation in seafood in relation to Ostreopsis cf. ovata blooms on the French Mediterranean coast[END_REF][START_REF] Brissard | Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification[END_REF]. The toxins produced by Ostreopsis species are analogs of palytoxin (PlTX), a toxin primarily isolated from the zoanthid Palythoa toxica [START_REF] Moore | Palytoxin: a new marine toxin from a Coelenterate[END_REF]. So far, several toxin classes which share both similar structure and site of action with PlTX have been described, such as ostreocins -B and -D (OSTs) [START_REF] Usami | Palytoxin analogs from the dinoflagellate Ostreopsis siamensis[END_REF][START_REF] Ukena | Structure elucidation of Ostreocin D, a palytoxin analog isolated from the dinoflagellate Ostreopsis siamensis[END_REF][START_REF] Terajima | Structure elucidation of ostreocin-A and ostreocin-E1, novel palytoxin analogs produced by the dinoflagellate Ostreopsis siamensis , using LC/Q-TOF MS[END_REF], mascarenotoxins-a, b and c (McTXs) [START_REF] Lenoir | First evidence of palytoxin analogues from an Ostreopsis mascarenensis (Dinophyceae) benthic bloom in southwestern Indian Ocean[END_REF][START_REF] Rossi | New palytoxin-like molecules in Mediterranean Ostreopsis cf. ovata (dinoflagellates) and in Palythoa tuberculosa detected by liquid chromatography-electrospray ionization time-of-flight mass spectrometry[END_REF], and ovatoxins (OvTxs) [START_REF] Ciminiello | Putative palytoxin and its new analogue, ovatoxin-a, in Ostreopsis ovata collected along the ligurian coasts during the 2006 toxic outbreak[END_REF][START_REF] Ciminiello | Complex palytoxin-like profile of Ostreopsis ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry[END_REF][START_REF] Sanchez | Phylemon 2.0: a suite of web-tools for molecular evolution, phylogenetics, phylogenomics and hypotheses testing[END_REF][START_REF] Ciminiello | Isolation and structure elucidation of ovatoxin-a, the major toxin produced by Ostreopsis ovata[END_REF][START_REF] Suzuki | LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan[END_REF][START_REF] Uchida | Structural elucidation of palytoxin analogs produced by the dinoflagellate Ostreopsis ovata IK2 strain by complementary use of positive and negative ion liquid chromatography/quadrupole time-of-flight mass spectrometry: Structural elucidation of ovatoxin-a, -d, -e IK2 by LC/QTOFMS[END_REF][START_REF] Brissard | Characterization of ovatoxin-h, a new ovatoxin analog, and evaluation of chromatographic columns for ovatoxin analysis and purification[END_REF][START_REF] García-Altares | The novel ovatoxin-g and isobaric palytoxin (so far referred to as putative palytoxin) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MS[END_REF]Accoroni et al., 2016;[START_REF] Tartaglione | Chemical, molecular, and eco-toxicological investigation of Ostreopsis sp. from Cyprus Island: structural insights into four new ovatoxins by LC-HRMS/MS[END_REF][START_REF] Tartaglione | Variability in toxin profiles of the Mediterranean Ostreopsis cf. ovata and in structural features of the produced ovatoxins[END_REF]. Other compounds called ostreotoxins (OTXs) were reported but not yet structurally elucidated and, as they have a different mode and site of action than the other Ostreopsis toxins, their classification as PlTX analogues remains uncertain [START_REF] Mercado | An extraction procedure modification changes the toxicity, chromatographic profile and pharmacologic action of Ostreopsis lenticularis extracts[END_REF][START_REF] Meunier | Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis[END_REF]. To date, the genus Ostreopsis comprises eleven species, of which nine have been described by their morphology only, namely O. siamensis Johs. Schmidt [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF], O. lenticularis Y. Fukuyo [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], O. ovata Y. Fukuyo [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], O. heptagona D.R.Norris, J.W.Bomber & Balech [START_REF] Norris | Benthic dinoflagellates associated with ciguatera from the Florida Keys. I. Ostreopsis heptagona sp. nov[END_REF], O. mascarenensis Quod [START_REF] Quod | Ostreopsis mascarenensis sp. nov. (Dinophyceae), dinoflagellé toxique associé à la ciguatera dans l'océan Indien[END_REF], O. labens M.A. Faust & S.L. Morton [START_REF] Faust | Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae)[END_REF], O. belizeana M.A. Faust, O. caribbeana M.A. Faust and O. marina M.A. Faust (Faust, 1999), whereas the two most recent descriptions of O. fattorussoi [START_REF] Accoroni | Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea[END_REF] and O. rhodesiae Verma, Hoppenrath & S.A. Murray [START_REF] Verma | Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon[END_REF] include genetic data. As already reviewed in detail by [START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF], the identification of Ostreopsis species based solely on morphology is extremely difficult and many confusions have occurred. Although the generic characters are very peculiar among dinoflagellates and readily identify the genus Ostreopsis, the criteria used to delineate species are based primarily on variations in cell size, outline and some slight differences of certain thecal plates [START_REF] Faust | Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov., O. belizeanus sp. nov., and O. caribbeanus sp[END_REF][START_REF] Penna | Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western mediterranean sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences[END_REF][START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF]. However, most of these features have been shown to vary within a given species (e.g. [START_REF] Penna | Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western mediterranean sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences[END_REF]. This significant morphological plasticity thus makes the species identification by morphology often ambiguous [START_REF] Rhodes | Toxic marine epiphytic dinoflagellates, Ostreopsis siamensis and Coolia monotis (Dinophyceae), in New Zealand[END_REF][START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF][START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF].

Owing to the ambiguities in defining the morphological characters, there have been several attempts in revising the description of Ostreopsis and species therein, using molecular data [START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF]. To this end, sequences of the Internal Transcribed Spacers (ITS-5.8S rDNA region), or more recently the hypervariable D8-D10 domains of the ribosomal DNA large subunit (LSU rDNA) have been made available for species identification, in combination with morphometrics. One of the first molecular studies on Ostreopsis was by [START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF] in Malaysia who showed that within O. cf. ovata, the isolates clustered in two distinct clades, corresponding to geographically distinct groups. Later, [START_REF] Penna | Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western mediterranean sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences[END_REF][START_REF] Penna | A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt[END_REF] obtained similar results and found a clade of O. cf. ovata corresponding to Mediterranean/Atlantic strains, well separated from Indo-Pacific strains. These findings strongly suggest that a given morphospecies can actually encompass a wide genetic cryptic diversity. Studying the genetic diversity of the genus Ostreopsis in the western Pacific, [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] identified 8 clades corresponding to eight putative species, based on two different genetic markers (LSU D8-D10 and ITS-5.8S rDNA sequences). Their molecular data revealed the existence of a new, cryptic species (Ostreopsis sp. 1) possessing the same morphology as O. cf. ovata and therefore very difficult to describe as a separate species.

Moreover, in the absence of reliable genetic references for most of the clades, the authors deliberately did not assign them taxonomically and the different genetic entities were thus named Ostreopsis sp. 1-6. Later, [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF] used the same approach in their analysis of strains from Thailand, and they identified a new genetic clade, Ostreopsis sp. 7, for which morphological features cannot be distinguished from O. cf. ovata. They concluded that it may represent a yet undescribed cryptic species, but distinctive characters needed to be found to support its description as a separate taxon [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF].

To date, the taxonomy of Ostreopsis is controversial and morphological and molecular data cannot be easily combined in order to delimit species [START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF]. Several genotypes

are not yet associated with a morphospecies, leading to a situation of a dual-taxonomy based either on morphospecies or genotypes. In order to unify them, reference molecular data acquired from unambiguously identified specimens are absolutely necessary. Ideally, the genetic data should include type material (holotype, lectotype, neotype), which is the reference point for the application of the species name, according to the International Code of Nomenclature for Algae, Fungi and Plants [START_REF] Turland | International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress[END_REF]. For recently described species (Accoroni et al., 2016;[START_REF] Verma | Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon[END_REF], sequences have been acquired from the same strains as used for the descriptions and thus are taxonomically reliable. In the case of older descriptions without genetic data associated, the situation is more complex since in most cases, the type material has not been designated, is no more available, or, if still extant, has been conserved in an improper way to obtain reliable sequences. Thus, in modern day molecular studies, it is not usually possible or practical to obtain reference sequences from the types, and therefore fresh collections or cultures must be used [START_REF] Ariyawansa | Epitypification and neotypification: guidelines with appropriate and inappropriate examples[END_REF]. This creates the problem that sequence data may come from incorrectly named isolates, which can make the whole resulting taxonomy unsound [START_REF] Ariyawansa | Epitypification and neotypification: guidelines with appropriate and inappropriate examples[END_REF]. For Ostreopsis species, the intraspecific genetic variability is often linked to geographical origins of strains.

Therefore, in order to provide reliable information and ensure a correct association between phenotype/genotype of a given species, new investigations and acquisition of reference genetic data should always be made in the type locality.

The species Ostreopsis lenticularis was first described from Tahiti Island in the Society Archipelago, (French Polynesia), the Gambier Archipelago as well as New Caledonia, by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], in the framework of a study of benthic dinoflagellates from coral reefs in the Pacific. This description was based on light microscopy observations. Morphologically, it displays the same size and outline to O. siamensis from which it differs by the absence of a body undulation and the presence of two sizes of thecal pores [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF][START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF]. [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] did not designate a holotype, but he indicated that the type locality was Tahiti Island, and that this species was absent in Ryukyu Islands (Japan). Since then, it has been found associated with various benthic habitats in almost all tropical regions in the world [START_REF] Faust | Observation of sand-dwelling toxic dinoflagellates (Dinophyceae) from widely differing sites, including two new species[END_REF][START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF][START_REF] Gárate-Lizárraga | Occurrence of Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) from the Archipiélago de Revillagigedo, Mexican Pacific[END_REF]. In some instances, authors identified this species with morphological features different than the ones reported in the original description, which not only makes these reports doubtful but also the species delimitation unclear (e.g. [START_REF] Faust | Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae)[END_REF][START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF][START_REF] Faust | Identifying harmful marine dinoflagellates[END_REF]. In addition, the toxicity status of this species needs to be clarified since no toxicity has been clearly demonstrated in the species described from French Polynesia [START_REF] Bagnis | The dynamics of three toxic benthic dinoflagellates and the toxicity of ciguateric surgeonfish in French Polynesia[END_REF] whereas several studies from the Caribbean Sea mentioned it as a toxic species (e.g. [START_REF] Mercado | An extraction procedure modification changes the toxicity, chromatographic profile and pharmacologic action of Ostreopsis lenticularis extracts[END_REF]. Moreover, from a molecular point of view, the genetic identity of O. lenticularis appears ambiguous since a sequence ascribed to O. lenticularis was identical to another sequence identified as O. labens, causing a taxonomic confusion [START_REF] Penna | A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt[END_REF][START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF]. More recently, in the study by [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF], sequences ascribed to O. lenticularis cluster into two separate clades (Ostreopsis sp. 5 and Ostreopsis sp. 6), making the genetic identity of this species even more elusive. Moreover, [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] demonstrated that these two clades were genetically divergent enough to support two distinct species and showed that whereas Ostreopsis sp. 5 was not toxic to mice, Ostreopsis sp. 6 produced ostreocin-D [START_REF] Suzuki | LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan[END_REF].

Hence, the definition of O. lenticularis is still unclear and in a context of toxic risks associated with increasing proliferations of Ostreopsis species, it is therefore of utmost importance to revisit the taxonomic identity of this species by unambiguously associating phenotypic and genotypic data, and also clarifying its toxic status [START_REF] Hoppenrath | Dinoflagellate taxonomy -a review and proposal of a revised classification[END_REF].

The aim of the present study is to re-investigate O. lenticularis in the type locality (Tahiti Island) and other sites in French Polynesia, including the Gambier Archipelago mentioned in the original description by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF]. Following recent sampling campaigns conducted in four different archipelagos of French Polynesia, clonal cultures of 47 distinct strains were obtained in the laboratory. The morphology and degree of genetic variation between French Polynesian O. lenticularis strains were assessed using microscopy analysis and molecular data derived from LSU rDNA (D8-D10) and ITS-5.8S sequences. In addition, the toxic status of 19 of these strains was investigated using the neuroblastoma cell-based assays and, for 4 strains from Tahiti Island, using liquid chromatography coupled with tandem mass spectrometry. All together, these morphological, phylogenetic and toxicological data allowed to refine the descriptive characters of O. lenticularis.

Material and methods

Sampling techniques

Wild samples of Ostreopsis were collected from different sites in the Society, Marquesas, Gambier and Australes archipelagos (Fig. 1), using both the natural (i.e., macroalgae) and artificial (i.e., window screens, WSs) substrate methods. Briefly, ≈200 g of turf-like macroalgae were collected at water depths of 1-5 m and examined for the presence of Ostreopsis cells, following the protocol described by [START_REF] Chinain | Ciguatera risk management in French Polynesia: The case study of Raivavae Island (Australes Archipelago)[END_REF]. To this end, macroalgal samples were sealed within plastic bags underwater and shaken and kneaded vigorously to dislodge dinoflagellate cells. The detrital suspension was then successively filtered through 125, 40 and 20 µm mesh sieves and the 40 and 20 µm fractions preserved in 50 mL of 5% formalin-seawater. The artificial substrate method used 150 cm² WS devices assembled and deployed for 24 h following the protocol proposed by [START_REF] Tester | Sampling harmful benthic dinoflagellates: Comparison of artificial and natural substrate methods[END_REF]. After 24 h, WSs were collected with 250 mL of ambient sea water and shaken to dislodge the cells. The entire volume was filtered through 10 µm polycarbonate filters that were replaced as the filters became obstructed. Then, all filters used to process individual samples were transferred to 15 mL tubes with 8 mL of sterile filtered sea water. Before removing the filters, the tubes were shaken to dislodge Ostreopsis cells.

In vitro culturing of Ostreopsis

Ostreopsis clonal cultures were established from single cells isolated from wild samples, using an inverted microscope. They were routinely maintained in 1 L Fernbach flasks in f10k enriched natural sea water (NSW) medium [START_REF] Holmes | Strain dependent production of ciguatoxin precursors (gambiertoxins) by Gambierdiscus toxicus (Dinophyceae) in culture[END_REF] at 26°C, a salinity of 36, with 7500 lux of light and a 12:12 h light:dark photoperiod. After approximately 21 days of growth, cells were harvested by filtration and centrifugation. Cell counts were achieved using a Coulter counter (Beckman) and the resulting cell pellets lyophilized and weighted. This step was repeated several times for each studied strains, in order to accumulate sufficient cell biomass for further toxicity analysis.

A total of 47 strains originating from various locales in French Polynesia (except form the Tuamotu archipelago for which no culture could be established) were analyzed in the present study.

All the information relative to these strains, e.g. strain codes, species, year of isolation and location, are summarized in Table 1. These isolates are part of the algal collection of the Laboratory of Toxic Micro-algae of the Institut Louis Malardé (Tahiti, French Polynesia), where cultures are deposited.

All the following experiments were conducted on non-axenic acclimated batch cultures.

For comparisons with wild cells, subsamples of the WS samples collected in November 2016 from Anaho Bay (WS 51.2, 08°49.194'S -140°03.800'W), Taiohae (WS 55.24, 08°56.009'S -140°05.581'W) and Agapaa (WS 56.14, 08°53.777'S -140°03.008'W) in Nuku Hiva Island, (Marquesas archipelago) were fixed with acidic Lugol's (for molecular analysis) and 2.5 % glutaraldehyde (for scanning electron microscopy, SEM).

Microscopy observations

Light microscopy (LM) observations of live cells were conducted using a Leica DMLB microscope (Leica, Wetzlar, Germany) equipped with a D850 DSLR camera (Nikon, Tokyo, Japan).

The shape and location of the nucleus were studied in epifluorescence microscopy (EM) after staining cells fixed in ethanol with 1:100,000 SYBR Safe (Thermo Fisher, Waltham, MA, USA). In order to study the thecal plate pattern of the different strains, EM was used after staining Lugol-fixed cells with Solophenyl Flavine 7GFE 500 (Ciba Specialty Chemicals, High Point, NC, USA) according to the method described in [START_REF] Chomérat | Studies on the benthic genus Sinophysis (Dinophysales, Dinophyceae) II. S. canaliculata from Rapa Island (French Polynesia)[END_REF]. The observations were done with a Zeiss Universal microscope fitted with epifluorescence cubes (335WB50 excitation filter, FT395 dichromatic beam splitter and D510/80M emission filter for Sybr Green and 443AF40 excitation filter, 475DCLP dichromatic beam splitter, and 500DF25 emission filter for Solophenyl Flavine, Omega Optical, Brattleboro, VT, USA), Nikon CF Fluor optics (Nikon, Tokyo, Japan), an HBO 50-W mercury lamp and a EOS-M digital camera (Canon, Tokyo, Japan). Cells were also observed using field-emission scanning electron microscopy (FE-SEM). Prior to scanning electron microscopy, cells fixed in 2.5% glutaraldehyde from the cultures in early exponential growth phase or the field sample WS 51.2 were first isolated using a micropipette, rinsed in distilled water and processed according to [START_REF] Chomérat | Protoperidinium bolmonense sp. nov. (Peridiniales, Dinophyceae), a small dinoflagellate from a brackish hypereutrophic lagoon (South of France)[END_REF]. Dehydration was carried out in ethanol baths of 15%, 30%, 50%, 70%, 95% vol. ethanol, and several baths of absolute ethanol (100%) and then cells were critical point dried using an EMS 850 (Electron Microscopy Sciences, Hatfield, PA, USA) critical point drier. Dried filters were then mounted onto 12 mm SEM stubs using carbon adhesive and coated with gold using a Cressington 108Auto (Cressington, Watford, UK) sputter coater. Cells were then observed using a FE-SEM Zeiss SIGMA 300 (Carl Zeiss Microscopy GmbH, Jena, Germany) at the Marine Biology station in Concarneau (France). Measurements were realized directly with the microscope or on digital images using ImageJ software [START_REF] Rasband | ImageJ[END_REF]. For measurements of the curved apical pore plate (Po), the arc length was measured in apical view.

DNA amplification and sequencing

For DNA amplification, direct cell PCR approach was used using one or a few cells from the cultures in early exponential growth phase preserved in ethanol 70%. Under the inverted microscope, fixed cells were pipetted and rinsed in several drops of nuclease-free distilled water, and then transferred into a 0.2 ml PCR tube. A similar process was used for isolation of single-cells from the Lugol-fixed samples from Nuku Hiva.

Due to the low amount of DNA, a first round of PCR was realized using ITS-FW and RB primers [START_REF] Chinain | Morphology and molecular analyses of three toxic species of Gambierdiscus (Dinophyceae): G. pacificus, sp. nov., G. australes, sp. nov., and G. polynesiensis[END_REF][START_REF] Nézan | Genetic diversity of the harmful family Kareniaceae (Gymnodiniales, Dinophyceae) in France, with the description of Karlodinium gentienii sp. nov.: A new potentially toxic dinoflagellate[END_REF], allowing the amplification of the ITS1-5.8S-ITS2 and D8-D10 regions. A second round of PCR (nested PCR) was realized using 1 µl of the amplicon produced in the first step as template. Primers used for the second amplification are given in [START_REF] Chinain | Morphology and molecular analyses of three toxic species of Gambierdiscus (Dinophyceae): G. pacificus, sp. nov., G. australes, sp. nov., and G. polynesiensis[END_REF] and [START_REF] Nézan | Genetic diversity of the harmful family Kareniaceae (Gymnodiniales, Dinophyceae) in France, with the description of Karlodinium gentienii sp. nov.: A new potentially toxic dinoflagellate[END_REF], and two internal reverse primers were specifically designed for PCR and sequencing: OstD1R (5ʹ-GTAGCAGCATGCCATGATCA-3ʹ) and OstD10R (5ʹ-GCACTGAAAATGAAAATCAAGC-3ʹ). PCR reactions were realized in 20 µl using KOD Hot Start Master Mix (Novagen-Merck KgaA, Darmstadt, Germany), according to the manufacturer's instructions. The PCR cycling comprised an initial 2 min heating step at 95°C to activate the polymerase, followed by 35 cycles of 95°C for 20 sec, 56°C for 20 s, and a final extension at 70°C for 20 min. Prior to sequencing, amplicons were visualized on an agarose gel after electrophoresis and the positive samples were purified using the ExoSAP-IT PCR Product Cleanup reagent (Affymetrix, Cleveland, OH, USA).

The Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) was used for sequencing of the amplicon generated at the second PCR round. Primers and excess dye-labeled nucleotides were first removed using the Big Dye X-terminator purification kit (Applied Biosystems, Foster City, CA, USA). Sequencing products were run on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Forward and reverse reads were obtained. All the information of strains, including origin of sample and accession numbers, are listed in Table 1.

Alignment and phylogenetic analyses

For both the D8-D10 and ITS-5.8S rDNA datasets, the 5′ and 3′ ends were manually aligned to truncate and refine the ends. The D8-D10 dataset was aligned using Clustal Ω v. 1.2 implemented in SeaView v. 4.7 [START_REF] Gouy | SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building[END_REF], while the ITS-5.8S rDNA dataset was aligned using MAFFT algorithm with selection of the q-ins-i strategy [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. Poorly aligned positions in both alignments were removed using Gblocks algorithm, using less stringent parameters than default [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF].

Prior to phylogenetic analyses, the search for the most appropriate model of sequence evolution has been performed using jModeltest2 v. 2.1.7 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF]. Two methods of phylogenetic reconstruction were used. Maximum Likelihood analysis (ML) was performed using PHY-ML v. 3 [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF], and a bootstrap analysis (1000 pseudoreplicates) was used to assess the relative robustness of branches of the ML tree. Bayesian Inference analysis (BI) was realized using MrBayes 3.1.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Initial Bayesian analyses were run with a GTR model (nst = 6) with rates set to gamma. The number of generations used in these analyses was 5,000,000 for the LSU rDNA D8-D10 alignment, and 4,000,000 for the ITS-5.8S rDNA alignment, with sampling every 100 th generations. The burnin values were set so that the first 10,000 trees were discarded for the LSU rDNA D8-D10, while it was set to 12,000 for the ITS-5.8S rDNA alignment. Therefore, the posterior probabilities of each clade were calculated from the remaining 40,000 trees in the LSU analysis, and the remaining 28,000 trees in the ITS-5.8S rDNA analysis.

Genetic distance (uncorrected genetic p distance) calculations among and within the Ostreopsis clades were estimated from the LSU D8-D10 and ITS-5.8S rDNA matrices used for phylogenetic analyses, using the p-distance model in MEGA X: Molecular Evolutionary Genetic Analysis across Computing Platforms v. 10.0.5 [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF].

Toxicity analysis

Extraction procedures

A total of 19 strains were extracted for further toxicity analysis (Table 1). For each strain, 50 mg of freeze-dried cells (corresponding to a cell biomass >10 6 cells) were sampled and extracted under sonication in 670 µL of methanol (MeOH)/water (1/1, v/v) for 15 min while cooling the solution in ice. Once cells disruption was completed, the sample was centrifuged at 10,000 g at 4°C for 10 min. The resulting supernatant was carefully recovered and the cell pellet re-extracted twice in 670 µL of methanol/water (1/1, v/v). All supernatants were then combined (total volume recovered: ≈2 mL) and centrifuged at 10,000 g at 4°C for 15 min. Finally, a 1.6 mL aliquot was sampled and stored at -20°C until tested for its toxicity using the neuroblastoma cell-based assay.

Neuroblastoma cell-based assay (CBA-N2a)

The Ostreopsis lenticularis cell extracts were analyzed for their toxicity using the neuroblastoma cell-based assay (CBA-N2a), a test designed to detect the presence of PlTX-like molecules acting on Na + /K + -ATPase [START_REF] Ledreux | Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)[END_REF][START_REF] Pawlowiez | Evaluation of seafood toxicity in the Australes archipelago (French Polynesia) using the neuroblastoma cell-based assay[END_REF]. The procedure for CBA-N2a followed the method previously described by [START_REF] Darius | Toxicological investigations on the sea urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the presence of Pacific ciguatoxins[END_REF] except that only ouabain was used instead of a mixture of ouabain and veratridine. This assay was first calibrated using a PlTX standard purchased from Wako (Ref. 161-26141): the neuroblastoma (neuro-2a) cells were exposed to a serial dilution 1:3 of 9 concentrations of PlTX (ranging from 1.5 to 9,524 pg mL -1 ), in the absence (O -conditions) versus the presence of 250 µM ouabain (O + conditions) to generate a full dose-response curve. Ten (10) µL of each concentration were tested in triplicate in O -and O + conditions in three independent experiments. Following 20-22 h incubation period, cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay according to [START_REF] Darius | Toxicological investigations on the sea urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the presence of Pacific ciguatoxins[END_REF]. Resulting coloration was measured at 570 nm on a plate reader (iMark Microplate Absorbance Reader, BioRad, Marnes la Coquette, France). Absorbance data were fitted to a sigmoidal dose-response curve (variable slope) based on the 4-parameter logistic model (4PL), allowing the calculation of the concentration causing 50% of maximum cytotoxicity on cell viability (EC50) values using Prism v7.04 software (GraphPad, San Diego, CA, USA).

As for the O. lenticularis cell extracts, the maximum concentration of extract (MCE) that does not induce unspecific mortality in neuro-2a cells in O -conditions was established at 23,810 cells equiv. mL -1 . As a first screening step, all extracts were tested at this MCE and, if toxic, a full dose-response curve was generated by testing a serial dilution 1:2 of 8 concentrations in the same conditions as for the PlTX standard.

Finally, the limits of detection (LOD) and quantification (LOQ) of the CBA-N2a test were estimated using the following formula: LOD = (PlTX EC80/MCE) and LOQ = (PlTX EC50/MCE).

Liquid Chromatography coupled with tandem Mass Spectrometry

Four strains from Tahiti Island (Table 1), the type locality of O. lenticularis were screened for the presence of PlTX and related known structural analogues at Ifremer Phycotoxins Laboratory (Nantes, France). Freeze-dried pellets were extracted with methanol (ratio 1:25, weight/volume) using glass beads (250 mg) in a mixer mill (Retsch MM400, Germany) for 20 min at 30 Hz. After centrifugation at 8000 g, supernatants were ultrafiltered (0.20 µm, Nanosep MF, Pall, Mexico) before LC-MS/MS analyses. Liquid chromatography was performed on a Poroshell 120 EC-C18 column (100 × 2.1 mm, 2.7 µm, Agilent, France) equipped with a guard column (5 × 2.1 mm, 2.7 µm, same stationary phase) using a Nexera Ultra-Fast Liquid Chromatography system (Prominence UFLC-XR, Shimadzu, France). Gradients of water (A) and acetonitrile 95% (B) both containing 0.2% of acetic acid were used at a flow rate of 0.2 mL min -1 . Injection volume was 5 µL and column temperature 25 °C. MS/MS analyses were performed with an API 4000QTRAP (AB Sciex, France) in positive ion mode and using MRM (Multiple Reaction Monitoring) acquisition. UV detection at 220, 233, 263 and 220-360 nm was performed with a diode array detector (Prominence, SPD-M20A, Shimadzu, France). In total, two LC-MS/MS and one LC-UV-MS/MS methods (Table S1) were used to detect PlTX, 42-OH-PlTX, 12 OvTXs (-a to -k), [START_REF] Cf | siamensis Ostreopsis sp. 8 Ostreopsis sp. 7 Ostreopsis sp[END_REF] McTXs (A to C) and OTX-1 and -3 (Table S1). Quantification was performed relative to Palytoxin standard (Wako Chemicals GmbH, Germany) with a 6-point calibration curve. Limit of detection and quantification were 20 and 30 ng mL -1 for PlTX standard.

Results

Microscopy observations

Observations in light and epifluorescence microscopy of the 47 strains analyzed in the present study revealed similar morphological features. The cells were broadly oval in shape, lenticular and photosynthetic, as shown for the strain THT16-4 (Fig. 2A). The oval nucleus was located dorsally (Fig. 2B). A similar thecal plate pattern has been observed in all the strains. For this reason, only the epifluorescence micrographs of one strain from each archipelago and wild specimens from a WS sample are presented in Fig. 3, for comparison purpose. No morphological difference could be observed among the strains and field specimens from the different archipelagos (Figs 3A-O), and all the features identify the species Ostreopsis lenticularis.

For a detailed morphological analysis, the strain THT16-4 from Tahiti Island (Society Archipelago) has been chosen for further investigations using FE-SEM. Scanning electron micrographs of one strain from Australes, Marquesas and Gambier archipelagoes are given in supplementary figure S1. Additionally, for comparison, cells from the field sample WS 51.2 from Nuku Hiva Island (Marquesas Archipelago) were also used for a detailed observation of wild specimens corresponding to the same morphotype. 4D, 5A-B). The third apical plate (3′) plate was hexagonal in shape, in contact with 1′, 2′, 3′ ′, 4′ ′, 5′ ′ and 6′ ′ but also had a very short suture with Po (Figs 4A, 5A-B). In the precingular series, 1′ ′ was the smallest while 6′ ′ was the largest (Figs 3A,4A). All precingular plates were four-sided except 2′ ′ and 6′ ′ that were pentagonal (Fig. 4A). The cingulum was narrow and straight ( Figs 4C-D). The postcingular plate series comprised 5 plates (Figs 3B, 4B-C), 1′ ′ ′ being small and more conspicuously visible in ventral view than in antapical view (Fig. 4C). The remaining four postcingular plates were large (Fig. 4B). Among postcingular plates, 1′ ′ ′ was three-sided (Fig. 4C), 2′ ′ ′ five-sided, and 3′ ′ ′, 4′ ′ ′ and 5′ ′ ′ four-sided (Figs 4B, 6B). The two antapical plates were unequal in size, 1′′′′ being relatively small and in contact with the cingulum and the left side of the posterior sulcal plate (Sp), while 2′′′′ was elongated, with its sutures with 2′ ′ ′ and 5′ ′ ′ nearly parallel (Figs 3B,4B).

The cingulum consisted of 6 distinct plates (supplementary Fig. S2). The sulcus was not studied in detail and only four plates were observed, with Sp being roughly pentagonal, Sda having a conspicuous list, Ssa partially hidden by overlapping 1′′′′ (Fig. 5C). Another plate, only partially visible, was present between Sp and Sda (Fig. 5C). The presence of other platelets could not be revealed from the observations. The thecal surface was smooth and plates possessed numerous pores of two kinds. Large pores were round, 0.29 -0.59 µm in diameter (mean 0.41 µm; s.d. 0.08 µm; n = 60), ) and scattered all over the plates. Rarely, they were oblong to elongated in some plates of the theca, as in the 1′′′′, while other plates have round pores (Fig. 5C). Small pores, 75-120 nm in diameter (mean 102 nm; s.d. 13 nm; n = 70) were abundant and scattered on the surface of thecal plates (Fig. 5D) but due to their small size, they could be observed only at high magnifications in LM and epifluorescence, and with SEM (Figs 3C, 5D).

Wild specimens collected from window-screen samples

Cells were large, subcircular in shape, slightly pointing ventrally (Figs 3N-O The thecal pattern was APC 3′ 7′ ′ 6c 4?s 5′ ′ ′ 2′ ′′ ′ (Figs 3N-O, 6A-F), and all plates were found to possess the same characteristics than previously described for the strain THT16-4 (Figs 6A-G).

As for the cells in culture, specimens from the field sample were smooth and possessed two kinds of thecal pores: large pores were round, 0.4 µm in diameter, and smaller pores (ca. 0.1 µm) were abundant and scattered all over the surface of the theca (Fig. 6G). With high resolution SEM, no morphological difference was found among field specimens and those from the culture THT16-4.

Molecular phylogenies

LSU rDNA D8-D10 regions

In the phylogenetic analysis inferred from LSU D8-D10 sequences, 50 new sequences acquired from French Polynesia (from 47 strains in culture and 3 single-cells isolated from a field sample from Nuku Hiva Island), with reference sequences retrieved from GenBank, were used. The final alignment comprised 110 sequences and had a length of 696 base pairs, with 174 variable sites, of which 114 were parsimony informative. The best-fit model of LSU D8-D10 sequences was found to be TN93 + I + G model with the following parameters: Ti/Tv for purines = 2.312, Ti/Tv for pyrimidines = 5.531, base frequencies of A = 0.28009, C = 0.17427, G = 0.24939, T = 0.29625; assumed with invariable sites (I = 0.503) and gamma distribution shape (G = 0.512).

Both analyses performed with ML and BI gave the same tree topology and the relationships among Ostreopsis clades were identical. Hence, only the majority-rule consensus tree of the ML analysis is shown (Fig. 7). The tree shows that there are ten distinct clades (O. cf. ovata, O. cf. siamensis, O. rhodesiae and Ostreopsis spp. 1-7 clades). All sequences of O. lenticularis acquired in this study from various sites in French Polynesia cluster with a strong support in a monophyletic group comprising also three strains from Okinawa and Iriomote Islands in Japan (s0577, s0578 and IkeOst2) and previously assigned to Ostreopsis sp. 5 by [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] (Fig. 7). This clade appears to be sister with a group of ten sequences from Shikoku Island (Japan) also previously ascribed to Ostreopsis sp. 5 (Fig. 7). Because of their low divergence (p-distance between the groups of 0.010, Table 2), these two groups are considered as two subclades of O. lenticularis (= Ostreopsis sp. 5).

Results of the phylogenetic analysis showed that O. lenticularis is a sister to Ostreopsis sp. 6 with maximal support (ML = 100, BI = 1.00). The clade Ostreopsis sp. 6 is divided into three subclades, one including four sequences from Japan (IR33, IR49, OU8, OU11), one including two sequences from Japan (s0587 and s0595) and one two sequences from Thailand (TF25OS, TF29OS) (Fig. 7). The p-distance between these subclades in Ostreopsis sp. 6 ranged from 0.014 to 0.020 (Table 2).

ITS-5.8S rDNA phylogeny

In the phylogenetic analysis inferred from ITS-5.8S rDNA sequences, we used 19 new sequences acquired from French Polynesian strains with other sequences retrieved from GenBank.

The final alignment comprised 92 sequences and had a length of 329 base pairs, with 222 variable sites, of which 175 were parsimony informative. The best-fit model of ITS-5.8S rDNA sequences was found to be TN93 + G model with the following parameters: Ti/Tv for purines = 9.128, Ti/Tv for pyrimidines = 0.148, base frequencies of A = 0.27149, C = 0.19120, G = 0.18559, T = 0.35172; assumed with a gamma distribution shape (G = 0.636).

Both analyses performed with ML and BI gave the same tree topology and the relationships among Ostreopsis clades were identical. Hence, only the majority-rule consensus tree of the ML analysis is shown (Fig. 8). The tree shows that there are twelve distinct clades (O. cf. ovata, O. cf. siamensis, O. fattorussoi, O. rhodesiae, and Ostreopsis spp. 1-8 clades). All sequences of O. lenticularis acquired in this study from various sites in French Polynesia cluster with a strong support in a monophyletic group comprising also eight sequences from Réunion Island, China, Hawaii and Galapagos Islands, and a subclade of three sequences from Japan (Shikoku Island) (Fig. 8). These sequences were previously ascribed to Ostreopsis sp. 5 (strains MB80828-4, O70421-1, O70421-2), Ostreopsis sp. (isolates P-079.1L, P-079.2L, P-0107, P-0108, P-0109, strain CBA0203), O. cf. lenticularis (isolate 17G) and only the strain 2S1E10 was identified as O. lenticularis in GenBank. The sequences from the Pacific Ocean, Indian Ocean and South China Sea are genetically very closely related (p-distance within the clade 0.001, Table 3) but they are slightly divergent from the three sequences from Japan (p-distance between the subclades of 0.068, Table 3).

In the phylogenetic analysis, O. lenticularis is a sister to Ostreopsis sp. 6 with good support (ML = 84, BI = 0.96, Fig. 8). Nine sequences from various localities of Southeastern Asia and Japan cluster within this clade. The two sequences FM244728 from Malaysia (ascribed to O. labens) and AF218465 (ascribed to O. lenticularis) cluster together and are identical (Table 3). Two sequences from the Gulf of Thailand AB841254 and AB842255 (ascribed to Ostreopsis sp.) and two sequences from Japan (IR33 and OU11) form a sister clade to the Malaysian sequences (Fig. 8).

The sequence s0587 is basal to these sequences and its distance varied from 0.110 to 0.161 (Table 3). Two sequences from Vietnam diverge earlier in the Ostreopsis sp. 6 clade and their genetic distances with all other sequences of the clade varied from 0.057 to 0.135 (Table 3).

Toxicity assessment

CBA-N2a analysis

The EC50 values for PlTX in O -and O + conditions were 1,191 ± 175 pg mL -1 (n = 3) and 107 ± 36 pg/mL (n = 3), respectively. The LOD was estimated at 44.3 ± 4.7 and 3.4 ± 1.5 fg PlTX equiv./cell in O -and O + conditions, respectively, whereas LOQ was estimated at 50 ± 7.3 and 4.5 ± 1.5 fg PlTX equiv./cell in O -and O + conditions, respectively. For all the samples tested, no toxicity was detected at the MCE.

LC-UV-MS/MS analysis

In the four tested strains originating from Tahiti Island analyzed, all the 21 toxic compounds that were targeted were below detection levels.

Discussion

Morphological features

All the strains observed in this study displayed a similar morphology and no difference was observed between specimens from cultures and from field samples. Morphologically, the broadly oval shape, size range, and thecal plate pattern displayed by all the specimens from field samples and strains from widely distant islands were in perfect agreement with the original description of O. lenticularis by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], who reported it from French Polynesia (Tahiti Island as type locality). As mentioned by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], the cingulum was not undulated and cells possess two different sizes of thecal pores. The presence of both small and large pores on thecal plates was conspicuous in all specimens examined, even at high magnification with epifluorescence microscopy. Interestingly, in O. lenticularis, the smaller pores were much more abundant than larger pores, a feature not reported from any other known species. Indeed, the presence of smaller pores less abundant that larger pores has been reported in O. rhodesiae, but they were rare [START_REF] Verma | Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species, Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon[END_REF]. [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF] reported the presence of small, large and also oblong to kidney-shaped pores as a third type of pores present in the strains from Hainan Island (China). In the present study, oblong pores were observed in some O. lenticularis specimens in culture, but this was uncommon and only located on some plates of the theca. Such pores were not found in any of the specimens from the field samples. It should be noted that [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF] reported very large sizes for some specimens (up to 121.3 µm in DV) that were larger than in the original description, and the presence of oblong pores may be related to the size and age of the specimens. This feature should be carefully checked in further studies as it likely results from morphological plasticity and an extreme variation of the large pores. Since the specimens from China were genetically almost identical to those from French Polynesia, this feature may not be taxonomically significant.

Contrary to [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF] conclusions, it is suggested that the presence of two sizes of thecal pores is actually a stable character in O. lenticularis, as it was conspicuous in all the specimens examined in French Polynesia and Tahiti (type locality).

Comparison of O. lenticularis to other broadly oval species

As previously reported by [START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF], O. lenticularis shares most of its morphological features (e.g. size, outline shape, and thecal plate pattern) with O. siamensis. The more or less elongated or round shape cannot be used to differentiate these species [START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF]. In his description of the genus and type species O. siamensis, [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF] emphasized morphological characters such as flattening of the cell, oyster-shape, short sulcus, and thecal plate pattern [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF] which define well the genus but are shared by almost all the species [START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF]. The size of O. siamensis (dorso-ventral length) is about 90 µm, the theca possesses conspicuous pores and, from the illustration, O. siamensis has a body undulation, visible in lateral view [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF]. According to [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], O. lenticularis differs from O. siamensis by the absence of a body undulation and by the presence of fine pores densely scattered all over the thecal plates, which is also observed for all the specimens examined herein. Some authors (e.g. [START_REF] Penna | Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western mediterranean sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences[END_REF][START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF] claimed that Fukuyo used the difference in cell shape to distinguish O. siamensis from O. lenticularis, but this statement is erroneous since Fukuyo (1981, pp. 970-971) clearly indicated a similar size and outline for these two species, and mentioned the shape only to distinguish O. lenticularis from Gambierdiscus toxicus. In the Ryukyu Islands, [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] identified specimens with a body undulation and only one type of thecal pores, and ascribed them to O. siamensis. The author mentioned the absence of this morphotype in the French Polynesian and New Caledonian samples that he studied [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], a statement that could be confirmed in the present study. Despite Fukuyo's very clear interpretation [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF], the distinction between O. lenticularis and O. siamensis based on thecal pores and undulation of the cingulum did not gather consensus among taxonomists. For instance, [START_REF] Norris | Benthic dinoflagellates associated with ciguatera from the Florida Keys. I. Ostreopsis heptagona sp. nov[END_REF] questioned this interpretation and regarded O. lenticularis as being conspecific with O. siamensis rather than as an independent species. At the same period, a series of confusing identifications were made in the Caribbean area. [START_REF] Carlson | The distribution, periodicity, and culture of benthic/epiphytic dinoflagellates in a ciguatera endemic region of the Caribbean[END_REF] identified O. siamensis as a relatively rare species around Virgin Islands, although dominant in some stations, but he also emphasized that the distinction between this species and O. lenticularis was questionable for some specimens. On the following year, [START_REF] Carlson | Distribution and periodicity of toxic dinoflagellates in the Vrigin Islands[END_REF] changed this identification to O. lenticularis using the same dataset, which added to the confusion. Working on the south west coast of Puerto Rico, [START_REF] Ballantine | Seasonal abundance of Gambierdiscus toxicus and Ostreopsis sp. in coastal waters of Southwest Puerto Rico[END_REF] initially mentioned Ostreopsis sp., but it was then renamed Ostreopsis cf. lenticularis [START_REF] Tosteson | Comparative toxicity of Gambierdiscus toxicus, Ostreopsis cf. lenticuiaris, and associated microflora[END_REF] and later O. lenticularis [START_REF] Ballantine | Population dynamics and toxicity of natural populations of benthic dinoflagellates in southwestern Puerto Rico[END_REF][START_REF] Tosteson | Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus[END_REF], thus emphasizing the doubt regarding the identification of this species. Later, [START_REF] Faust | Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae)[END_REF] confused even more the taxonomy by reporting the presence of O. lenticularis with a morphological description which does not support this identification (only one size of thecal pores). Conversely, in the same study, O. lenticularis was likely misidentified as "O. siamensis" since the morphology (size and two sizes of thecal pores) was in agreement with the original description by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF]. As already pointed out by several authors [START_REF] Penna | Genetic diversity of the genus Ostreopsis Schmidt: Phylogeographical considerations and molecular methodology applications for field detection in the Mediterranean Sea[END_REF][START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF][START_REF] Gárate-Lizárraga | Occurrence of Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) from the Archipiélago de Revillagigedo, Mexican Pacific[END_REF], these erroneous interpretations were a major source of confusion in the delineation of both O. siamensis and O. lenticularis for subsequent workers. For instance, reports of O. lenticularis showing different features than the ones provided in the original description (e.g. [START_REF] Chang | Three recently recorded Ostreopsis spp[END_REF][START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF] appear doubtful and likely correspond to misidentifications of other Ostreopsis species rather than reflecting a true variability in O. lenticularis.

Confusions with other broadly oval species may also have occurred with two additional species with a similar shape to O. siamensis and O. lenticularis, and overlapping sizes. [START_REF] Faust | Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae)[END_REF] described O. labens from Belizean and Japanese samples. This species possesses only one type of pore (trichocyst pores) conspicuously visible in LM, showing an average diameter of 0.3 µm [START_REF] Faust | Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae)[END_REF]. Later, [START_REF] Faust | Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov., O. belizeanus sp. nov., and O. caribbeanus sp[END_REF] described O. marina, another species in the same size-range and a roughly oval shape. It is described with a longer Po plate (24 µm vs. 18 µm in O. labens) and only "minute pores visible only by SEM or epifluorescence microscopy" whose size (0.33 µm, Faust 1999) is larger than in O. labens (0.3 µm, Faust and Morton 1995) for which pores were described as conspicuous in LM [START_REF] Faust | Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae)[END_REF]. These contradictory statements added to the taxonomic confusion, and additionally, O. marina has not been compared with O. labens in the description [START_REF] Faust | Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov., O. belizeanus sp. nov., and O. caribbeanus sp[END_REF].

Although [START_REF] Gárate-Lizárraga | Occurrence of Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) from the Archipiélago de Revillagigedo, Mexican Pacific[END_REF] consider that these four large and oval species are 'readily distinguishable by their plate pattern', their delimitation remains unclear owing to the existing variability in the thecal plates pattern of Ostreopsis species along with their poor descriptions [START_REF] Hoppenrath | Marine benthic dinoflagellatesunveiling their worldwide biodiversity[END_REF]. The length of certain thecal plates such as Po might be a weak character to distinguish among them. To date, O. labens and O. marina should be considered as doubtful species for which further studies are necessary not only to prove their existence but also to provide reliable morphological and molecular characters useful for their discrimination from O.

siamensis. In all cases, these three species were all described showing only one kind of thecal pore, and therefore no confusion is possible with O. lenticularis which can be easily distinguished from all other known Ostreopsis species.

Molecular phylogenies and taxonomic implications

The molecular phylogenies inferred from this study reveal that all the large and oval specimens identified as O. lenticularis in all the study sites from French Polynesia are genetically identical and belong to a unique genotype, previously found in the Pacific close to Japan and named 'Ostreopsis sp. 5' by [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] and subsequent authors. This finding of only one genotype in all study sites confirms that the same species is widespread in the different archipelagos of French Polynesia and supports Fukuyo's former observations of O. lenticularis in the Society and Gambier Archipelagos [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF]. This situation corresponds to the scenario 1 described in [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF], and since morphological and molecular data are congruent, it is straightforward to assign taxonomically the genotype Ostreopsis sp. 5 to O. lenticularis. This result confirms the identification by [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF] of specimens from Hainan Islands, but it also highlights the misidentification of some sequences annotated as O. lenticularis in GenBank. To date, about fifteen sequences of various genetic markers (rDNA, ITS regions, cox1) have been ascribed to Ostreopsis lenticularis in GenBank, coming from various areas: Portugal, Malaysia, China and Viet Nam but none was acquired in the type locality (Tahiti Island) or in French Polynesia. As previously shown by [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF], some of these sequences clustered with Ostreopsis sp. 6, which is genetically divergent enough to be regarded as a separate species from Ostreopsis sp. 5 [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF]this study). For instance, the sequences AF218465 (strain O1PR01 from Malaysia) ascribed to O. lenticularis and the sequence FM244728 annotated as O. labens cluster with Ostreopsis sp. 6, proving an identification issue [START_REF] Penna | A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt[END_REF][START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF]. For the strain OPR01 from Malaysia, morphological features such as 'trichocysts pores equal in size' reported by Leaw at al. marina (Faust andMorton 1995, Faust et al. 1996).

In order to resolve the taxonomic assignation of Ostreopsis sp. 6, reference sequences from the type localities of these species are necessary for comparisons, but none are currently available.

Nevertheless, [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF] recently sampled several sites in the Gulf of Thailand, including one site (TF) located on the north coast of the small Koh Wai Island, off the south of Koh Chang Island. Interestingly, this location is close to the sites where O. siamensis was originally described by [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF] (stations 3 and 6, south of Koh Chang, while the station 2, between Koh Kahdat and Koh Kut was about 15-20 km southeast). Because of this close proximity, this area could be considered as the type locality of O. siamensis even if it was not formally designated as such. In contrast with [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF] who found only one morphotype (large and oval cells) in plankton samples, [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF] found two different species in the site TF, O. cf. ovata (South China subclade) which is ovate and rather small, and large oval cells with a typical undulation of the cingulum (Ostreopsis sp. 6, strain TF29OS). When observed with epifluorescence microscopy, these large specimens were found to have only one size of thecal pores [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF] but this was not further confirmed by SEM observations. Genetically, the strains TF29OS and TF25OS are closely related to the sequence of the Malaysian strain O1PR01, possessing similar morphological features and only one type of thecal pores [START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF]. Results of the phylogenetic analysis conducted in the present study clearly confirm that these three strains rather belong to Ostreopsis sp. 6, and are not related to O. lenticularis as speculated by [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF], in absence of reference sequence from Tahiti Island for this species. The morphological and molecular studies by [START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF] and [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF] are congruent to associate specimens with only one size of thecal pores and a body undulation with Ostreopsis sp. 6. Interestingly, these morphological features are in perfect agreement with Schmidt's description and illustrations of O. siamensis [START_REF] Schmidt | Preliminary report of the botanical results of the Danish expedition to Siam (1899-1900). Part IV[END_REF], and the area where Ostreopsis sp. 6 has been found (TF site), coincides with the type locality of this species. Hence, it can be hypothesized that Ostreopsis sp. 6 likely corresponds to O. siamensis as it was the unique large oval species with all the morphological characters of this taxon found in the type locality. The environment and species composition in this area could have changed during more than a century, but in the absence of Schmidt's original material available, a complete re-investigation of specimens from this locality in the Gulf of Thailand and the designation of an epitype of O. siamensis would indisputably stabilize the taxonomy of this complicated genus for which several confusions occurred in the past [START_REF] Ariyawansa | Epitypification and neotypification: guidelines with appropriate and inappropriate examples[END_REF].

As emphasized by [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF], no sequence from the Gulf of Thailand clustered in the clade 'O. cf. siamensis' in the phylogenies. A similar result has been found in the present study, but since all the sequences of this clade were obtained from subtropical regions of Europe (Portugal, Spain, Italy) and New Zealand (Kerikeri, northern part of North Island), all very distant from the type locality (Gulf of Thailand) located in the tropical area, their taxonomic assignation should be considered with care. As already suggested by [START_REF] Penna | A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt[END_REF], the specimens of the 'O. cf.

Biogeography of O. lenticularis

In his description, [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] reported the presence of O. lenticularis not only in Tahiti Island, but also Gambier Archipelago and New Caledonia, which indicates a wide distribution within the tropical Pacific Ocean. This study confirms the wide presence of this species in four archipelagos of French Polynesia, and the phylogenetic analysis also confirms the presence of O. lenticularis in other locations of the Pacific including Japan (Iriomote, Okinawa, Shikoku Islands), Hawaii and Galapagos Islands. Interestingly, [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] and may be more restricted to the subtropical area, as suggested by [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF]. Despite the lack of molecular support, the identification of O. lenticularis in Revillagigedo archipelago by [START_REF] Gárate-Lizárraga | Occurrence of Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) from the Archipiélago de Revillagigedo, Mexican Pacific[END_REF] is well supported by morphological features, and consistent with the finding of this species in Galapagos Islands in the eastern Pacific.

In the western Pacific, molecular data confirm the presence of this species in the China Sea [START_REF] Zhang | 3, C4 and C5 (arrowheads point to the sutures); (D) right lateral view showing plates C5 and C6 (arrowheads point to the sutures). Scale bars: 1µm in A and 5 µm in B[END_REF] but not in Malaysia and Thailand where previous reports probably correspond to misidentifications of Ostreopsis sp. 6 (e.g. [START_REF] Leaw | Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia[END_REF]. Unambiguous report of O. lenticularis in Vietnamese waters, based on detailed morphological identifications showing the two types of thecal pores was also provided by [START_REF] Larsen | Potentially toxic microalgae of Vietnamese waters[END_REF]. These authors also found another large oval species with one type of thecal pores, and a long Po plate (24 µm) which they ascribed to O. marina and did not report O. siamensis [START_REF] Larsen | Potentially toxic microalgae of Vietnamese waters[END_REF].

Molecular studies would be necessary for a better characterization of the latter species.

In the Indian Ocean, the presence of O. lenticularis around La Réunion Island was mentioned by [START_REF] Hansen | Poentially harmful microalgae of the western Indian Ocean, Manual and guides[END_REF]. In their study, [START_REF] Carnicer | Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): molecular, morphologic and toxicity characterization[END_REF] recorded large specimens difficult to assign to a species by morphology because they appeared close to O. siamensis and O lenticularis, although their size that better fit Ostreopsis marina. Unfortunately, no detailed observations with SEM were performed and the presence of one or two kinds of pores was not clearly demonstrated by epifluorescence microscopy. Genetically, these specimens were found to belong to Ostreopsis sp. 5 [START_REF] Carnicer | Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): molecular, morphologic and toxicity characterization[END_REF] and, based on the present study, are genetically identical to Ostreopsis lenticularis from French Polynesia. Hence, the presence of this species in southwestern Indian Ocean is confirmed by molecular data. Since all the sequences obtained by [START_REF] Carnicer | Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): molecular, morphologic and toxicity characterization[END_REF] are closely related and belong to O. lenticularis, the record of O. marina (as a paratype) in Mayotte Island (south west Indian Ocean) by [START_REF] Faust | Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov., O. belizeanus sp. nov., and O. caribbeanus sp[END_REF] remains in question owing to the very similar size and morphology between these two species. Nevertheless, further molecular studies are necessary in the Indian Ocean since [START_REF] Hansen | Poentially harmful microalgae of the western Indian Ocean, Manual and guides[END_REF] also reported the presence of O. siamensis.

In the Caribbean, O. lenticularis has been reported on several occasions (e.g. [START_REF] Ballantine | Seasonal abundance of Gambierdiscus toxicus and Ostreopsis sp. in coastal waters of Southwest Puerto Rico[END_REF][START_REF] Carlson | Distribution and periodicity of toxic dinoflagellates in the Vrigin Islands[END_REF][START_REF] Faust | Observation of sand-dwelling toxic dinoflagellates (Dinophyceae) from widely differing sites, including two new species[END_REF][START_REF] Delgado | Epiphytic dinoflagellates associated with ciguatera in the northwestern coast of Cuba[END_REF][START_REF] Marchan-Álvarez | Dinoflagelados bentónicos nocivos, asociados con el sedimento arenoso en dos playas de la isla de Margarita, Venezuela[END_REF] but all the identifications were based on morphological observations only, which caused some confusions as emphasized in section 4.2. As seen in [START_REF] Tindall | Toxicity of Ostreopsis lenticularis from the British and United States Virgin Islands[END_REF][START_REF] Faust | Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae)[END_REF] and [START_REF] Faust | Identifying harmful marine dinoflagellates[END_REF] studies, several species very similar in shape and size did co-occur in this area, but specimens morphologically identical to O. lenticularis were reported, suggesting the likely presence of this species in the dinoflagellate assemblages.

From all these observations, it can be concluded that Ostreopsis lenticularis is widely distributed in the tropical areas of the world oceans but to date, molecular data supporting its identification are still lacking from the Caribbean Sea and the Atlantic Ocean.

Toxicity assessment

All 19 strains of Ostreopsis lenticularis tested for their toxicity using the CBA-N2a showed no toxic activities on neuroblastoma celles. Likewise, neither PlTX-like compounds nor OTX-1 and OTX-3 were detected in any of the 4 extracts analyzed by LC(-UV)-MS/MS, which is consistent with [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] who previously observed no toxic effect on mice in Ostreopsis sp. 5 (now identified as O. lenticularis) extracts. Contrastingly, these authors reported that Ostreopsis sp. 6 was toxic to mice and, more specifically, mentioned the detection of OST-D in strain s0587 extract [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF], although some strains genetically distinct (OU11, IR33) of this species did not produce this compound [START_REF] Suzuki | LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan[END_REF]. Overall, these findings highly support the idea that Ostreopsis lenticularis and Ostreopsis sp. 6 correspond to two different species.

The present results may appear contradictory with previous studies mentioning the toxicity in O. lenticularis strains from the Caribbean area [START_REF] Lassus | Toxic and harmful microalgae of the world ocean[END_REF]. These data should be considered cautiously since the identification of the toxic species was not clear. The first toxic effect on mice associated with O. cf. lenticularis was reported by [START_REF] Tosteson | Comparative toxicity of Gambierdiscus toxicus, Ostreopsis cf. lenticuiaris, and associated microflora[END_REF] but without any taxonomical assessment of the species. [START_REF] Ballantine | Population dynamics and toxicity of natural populations of benthic dinoflagellates in southwestern Puerto Rico[END_REF] mentioned higher toxicity levels in strains of 'O. lenticularis' from the Caribbean as compared to those from Tahiti localities where this species was found in abundance but not obviously toxic according to [START_REF] Bagnis | The dynamics of three toxic benthic dinoflagellates and the toxicity of ciguateric surgeonfish in French Polynesia[END_REF]. [START_REF] Ballantine | Population dynamics and toxicity of natural populations of benthic dinoflagellates in southwestern Puerto Rico[END_REF] explained this apparent discrepancy by the use of different extraction methods but, surprisingly, did not question the taxonomic identification of the species. [START_REF] Tindall | Toxicity of Ostreopsis lenticularis from the British and United States Virgin Islands[END_REF] further performed a taxonomic investigation on the toxic clones from the Caribbean and observed that (i) the cells size range was compatible with both O. siamensis and O. lenticularis, and (ii) cells had an undulating cingulum and one type of thecal pores. Although these features should have definitely excluded O. lenticularis, these authors kept using the name O. lenticularis under the pretext that O. siamensis had never been reported from the Caribbean [START_REF] Tindall | Toxicity of Ostreopsis lenticularis from the British and United States Virgin Islands[END_REF], thus perpetuating the confusion. It was yet rather clear that another species than O. lenticularis was the toxic species producing the water soluble ostreotoxins (OTXs) inhibiting the acetylcholine response [START_REF] Tindall | Toxicity of Ostreopsis lenticularis from the British and United States Virgin Islands[END_REF]. Several subsequent studies investigated the mode of action of the toxic compounds and effects of bacteria on the toxicity of Ostreopsis strains from the Caribbean designated doubtfully as 'O. lenticularis' [START_REF] Mercado | An extraction procedure modification changes the toxicity, chromatographic profile and pharmacologic action of Ostreopsis lenticularis extracts[END_REF][START_REF] Mercado | Differences in the toxicity and biological activity of Ostreopsis lenticularis observed using different extraction procedures[END_REF][START_REF] Meunier | Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis[END_REF][START_REF] Pérez-Guzmán | Bacteria associated with toxic clonal cultures of the dinoflagellate Ostreopsis lenticularis[END_REF], but in their review, [START_REF] Parsons | Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology[END_REF] highlighted the fact that these reports of toxic strains of O. lenticularis in the Caribbean were subject to caution, and purposely used quotations marks.

From the present study, it appears more likely that the toxic strains formerly assigned to the species O. lenticularis were, in fact, misidentified due to morphological confusions and the lack of molecular data to confirm such identifications. Further studies using molecular techniques to characterize these toxic strains are needed to clarify this point. So far, in light of the present findings, there is still no true evidence for the existence of toxic strains in the species O. lenticularis, which raises the question of the relevance of keeping this species in the IOC-UNESCO taxonomic reference list of harmful micro-algae [START_REF] Akselman | other Gonyaulacales[END_REF].

Lectotype designation

When describing O. lenticularis from Tahiti Island, [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] did not designate a holotype associated with the name, but provided five LM pictures and two interpretation drawings.

From the analysis conducted on several culture strains and field specimens from the same area, it can be concluded that the observations made by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] were perfectly accurate and morphological features clearly separate this species from all other known species. In addition, this study also proves unambiguously that the species found in French Polynesia (O. lenticularis) is genetically and morphologically distinct from that found in the Gulf of Thailand, especially at the TF site [START_REF] Tawong | Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand[END_REF], which corresponds to the type locality of O. siamensis. Hence the taxonomic ambiguity with O. siamensis should no longer persist and the absence of type for O. lenticularis can be easily solved by the designation of a lectotype from the original illustrations by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF]. The line drawing of the epitheca (Fig. 52) provides an unambiguous description of the major characters, including the presence of two types of pores, which appears as the most distinctive feature constant in all specimens studied from French Polynesia.

Ostreopsis lenticularis Y. Fukuyo LECTOTYPE (designated here): Fig. 52 in [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] Bulletin of the Japanese Society of Scientific Fisheries, 47(8): p. 978.

Conclusions

The present re-investigation of O. lenticularis in French Polynesia showed that this species is present in all the four archipelagos investigated, including Tahiti island and Gambier Archipelago, which confirms the report by [START_REF] Fukuyo | Taxonomical study on benthic dinoflagellates collected in coral reefs[END_REF] in the original description. The morphological features observed in this study were all in perfect agreement with the original description, and the presence of two kinds of thecal pores on the theca of O. lenticularis was seen in all specimens from cultures and field samples studied. Hence this character appears as reliable taxonomic feature which can distinguish O. lenticularis from other large-species such as O. siamensis, O. labens and O. marina.

Consequently, misidentifications of O. lenticularis were probably a consequence of mistaken interpretations of this feature by some authors but all data appear to be congruent. From a genetic point of view, the presence of a unique genotype in all the sites studied allows to associate unambiguously the species name with molecular data, which was not previously possible in absence of information from the type locality. Hence, the present paper provides reference sequences for further molecular identifications of O. lenticularis. Regarding the toxicity of this species, all the analyses conducted in the study showed that no toxic effect was observed using CBA-N2a assay, and no PlTX-like compounds could be detected from LC-MS/MS analyses, which is congruent with previous data obtained by [START_REF] Sato | Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan[END_REF] and [START_REF] Suzuki | LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan[END_REF]. Consequently, the previous reports of toxicity by this species need to be re-evaluated. In particular, the toxic strains should be characterized by molecular methods in order to ensure their correct identification since data in the literature are confused and no unambiguous evidence of a toxicity by O. lenticularis has been provided yet.

Authors contributions

NC and MC designed and supervised the study, drafted the paper and coordinated its revisions.

Moreover, MC also coordinated and contributed to the field samplings while NC also performed the microscopy observations and phylogenetic analyses. GB conducted the molecular analysis and

  3.1.1 Culture THT16-4 from Tahiti IslandSpecimens were broadly oval in apical and antapical views(Figs 2A,. The cells were biconvex, and flattened, with the cingulum straight in lateral view (Figs4C-D). They were60.5-89.3 µm (mean 80.3 µm; s.d. 7.5 µm, n = 30) deep (dorso-ventral length) and 56.1-73.4 µm (mean 65.8 µm; s.d. 5.4 µm, n = 30) wide. The DV/W ratio was 1.08-1.36 (mean 1.22; s.d. 0.08, n = 27). The thecal plate pattern was APC 3′ 7′ ′ 6c 4?s 5′ ′ ′ 2′ ′′ ′, and thecal plates were clearly visible both with light epifluorescence microscopy and SEM (Figs 3A-B, 4A-D, 5A-C). The apical pore complex (APC) consisted in a narrow, elongated and slightly curved Po plate bearing a slit and two rows of pores (Figs 5A-B). It was located parallel to the left mid-lateral to dorsal cell margin. The Po plate was 16.2-19.3 µm (mean 17.4 µm; s.d. 0.8 µm, n = 15) long. The first apical plate (1′) was elongated, located mostly on the left side of the cell (Fig. 4A). On its dorsal part, it is slightly protruding over the APC (Figs 4A, 5A). The second apical plate (2′) was narrow and elongated, and located below the APC, extending dorsally the Po plate, and reaching about the mid-position of the 3′ plate (Figs 3C,

  , 6A-B). They were biconvex and flattened (Figs 6C-D), 73.0-94.4 µm in dorso-ventral length (mean 81.2 µm, s.d. 5.7 µm, n = 22) and 58.0-78.2 µm in width (mean 67.5 µm, s.d. 6.1 µm, n = 22). The DV/W ratio was 1.08-1.32 (mean 1.21; s.d. 0.01, n = 22).

(

  2001) contradict with Fukuyo's original description of O. lenticularis and suggest a misidentification. Hence, the identity of the specimens clustering within Ostreopsis sp. 6 should be re-evaluated as, morphologically speaking, this clade corresponds to a large and lenticular species with thecal pores of one size only, such as O. siamensis (sensu Fukuyo 1981), O. labens or O.

  siamensis' clade likely correspond to another separate taxon, provided a proper description is proposed. Based on the present study conducted in the type locality of O. lenticularis, molecular comparisons show that specimens from the Gulf of Thailand and French Polynesia are clearly distinct, supporting Fukuyo's interpretation of O. siamensis and the description of O. lenticularis as a separate species. Similar investigations in the type localities of O. labens and O. marina are absolutely necessary to support that they are genetically distinct, since they are very difficult to separate from O. siamensis from a morphological point of view.

  did not observe O. lenticularis but O. siamensis in the Ryukyu Islands whereas Sato et al. (2011) found mixed populations of O. lenticularis (as Ostreopsis sp. 5) and Ostreopsis sp. 6. in Okinawa and Iriomote Islands, in the southern subtropical part. At the scale of the Pacific, O. lenticularis has a wide distribution, from the temperate Japan to the tropical Pacific while Ostreopsis sp. 6 is absent from French Polynesia

  sequencing. AD contributed to editing the paper. KH and AU contributed to the field samplings, and to establishing and maintaining Ostreopsis clonal cultures. JV contributed to the CBA-N2a analysis, while HTD contributed to the CBA-N2a analysis, and to drafting and editing the paper. CG contributed to the field samplings and to editing the paper. MR contributed to the design of the study and to editing the paper. FH, DR and ZA developed the three LC-(UV)-MS/MS methods and participated to the writing of the manuscript. FH also performed extraction and chemical analyses.

Figure legends Fig. 1 .

 legends1 Figure legendsFig. 1. Map of French Polynesia (South Pacific Ocean), showing the location of the different archipelagos. The islands from where samples of Ostreopsis lenticularis were collected are encircled by thick lines.

Fig. 2 .

 2 Fig. 2. Light and epifluorescence micrographs of cells from strain THT16-4 fromTahiti Island (Society Archipelago). (A) Live specimen; (B) epifluorescence image showing the nucleus stained by SYBR green. Scale bars: 10 µm.

Fig. 3 .

 3 Fig. 3. Epifluorescence micrographs of different strains and field specimens of Ostreopsis lenticularis. (A-C) strain THT16-4 from Tahiti Island (Society Archipelago): (A) apical view; (B) antapical view; (C) detail of the apical pore and adjacent plates, note the presence of two kind of thecal pores (some smaller pores shown by arrowheads). (D-G) strain MGR17-1 from Mangareva Island (Gambier Archipelago): (D) apical view; (E) antapical view; (F) detail of the apical pore and adjacent plate; (G) detail of the thecal surface with two kinds of thecal pores. (H-K) strain RVV-RF8 from Raivavae Island (Australes Archipelago): (H) apical view; (I) antapical view; (J) detail of the apical pore and adjacent plates; (K) detail of the thecal surface with two kinds of thecal pores. (L-M) strain TIO6 from Nuku Hiva Island (Marquesas Archipelago): (L) apical view; (M) antapical view. (N-O) field specimens from Anaho Bay (Nuku Hiva Island): (N) apical view; (O) antapical view. Scale bars: 20 µm in A, B, D, E, H, I, L-O, 10 µm in F, J, 5 µm in C, K, and 2 µm in G.

Fig. 4 .

 4 Fig. 4. Scanning electron micrographs of cells from strain THT16-4 (Tahiti Island, Society Archipelago). (A) apical view; (B) antapical view; (C) ventral view; (D) left lateral view showing the straight cingulum and apical pore plate (Po). Scale bars: 10 µm.

Fig. 5 .

 5 Fig. 5. Scanning electron micrographs of cells from strain THT16-4 (Tahiti Island, Society Archipelago). (A) detail of apical plate series and apical pore; (B) detail of the Po plate and apical pore composed by a slit and 2 rows of pores; (C) ventral view of the sulcus, note the presence of a partially visible sulcal plate (arrowhead), a list on the Sda plate (asterisk) and the flagellar pore (fp); (D) detail of the thecal pores of two sizes, note that the internal structure is visible within larger pores. Scale bars: 2 µm in A, B and C, and 1 µm in D.

Fig. 6 .

 6 Fig. 6. Scanning electron micrographs of field specimens from Anaho Bay, Nuku Hiva Island (Marquesas Archipelago). (A) apical view; (B) antapical view; (C) ventral view; (D) ventro-antapical view showing the cell flattening; (E) detail of apical plates and apical pore (Po); (F) detail of sulcal plates, note that a plate is only partially visible (arrowhead) and the flagellar pore (fp); (G) detail of the surface of the theca with two kinds of pores. Scale bars: 10 µm in A-D, 2 µm in E, and 1 µm in F-G.

Fig. 7 .

 7 Fig. 7. Maximum Likelihood phylogenetic tree inferred from LSU D8-D10 sequences of various Ostreopsis strains. French Polynesian strains are indicated by bold face and a gray background. Coolia sp. is used as an outgroup. Black vertical bars show distinct Ostreopsis clade. For O. cf. ovata, three subclades are shown: Med/Pac for Mediterranean and Pacific subclade; SCS for the South China Sea subclade and Thai for the Thailand subclade. For O. lenticularis, the vertical gray bars show the geographic origin of the strains. Numbers at nodes represent bootstrap support values

Table 1 .

 1 List of the strains of Ostreopsis lenticularis used in the study

	Strain	Archipelago	Island, locale	Origin coordinates	isolation date (month-y ear)	Toxicity analysis (CBA-N2a)	Toxin analysis (LC-MS/ MS)	ITS-5.8S rDNA sequences	LSU D8-D10 rDNA sequences
	ATH6	Society	Moorea, Atiha	17°35.416S 149°51.013W May-16	<LOD	-	MK227240	MK227180
	HPT6	Society	Moorea, Haapiti	17°35.459S 149°51.580W May-16	<LOD	-	MK227241	MK227181
	MRP23	Society	Moorea, Maharepa 17°28.427S 149°47.000W May-16	<LOD	-	MK227242	MK227182
	MRP25	Society	Moorea, Maharepa 17°28.427S 149°47.000W May-16	<LOD	-	MK227243	MK227183
	MRP26 MD03-03	Society Society	Moorea, Maharepa 17°28.427S 149°47.000W May-16 Tahiti, Papara 17°47.037S 149°28.088W Oct.-03	<LOD <LOD	-<LOD	MK227244 MK227245 †	MK227184 MK227185
	THT16-1	Society	Tahiti, To'ahotu	17°45.247S 149°20.115W Sept.-16	<LOD	<LOD	MK227246	MK227186
	THT16-2 THT16-4	Society Society	Tahiti, To'ahotu Tahiti, Vairao	17°45.247S 149°20.115W Sept.-16 17°48.628S 149°18.895W Sept.-16	<LOD <LOD	<LOD <LOD	MK227247 MK227248 †	MK227187 MK227188
	VRO17-1	Society	Tahiti, Vairao	17°48.642S 149°18.292W Aug.-17	-	-	-	MK227189
	RVV-RF8	Australes	Raivavae, Rocher de la femme	23°51.914S 147°41.093W May-08	<LOD	-	MK227230	MK227190
	MTR17-1	Australes	Tubuai, Mataura	23°20.375S 149°28.130W May-17	-	-	-	MK227191
	MTR17-2	Australes	Tubuai, Mataura	23°20.375S 149°28.130W May-17	-	-	-	MK227192
	MTR17-3	Australes	Tubuai, Mataura	23°20.375S 149°28.130W May-17	-	-	-	MK227193
	Tub8	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227194
	Tub9	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227195
	Tub10	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	<LOD	-	MK227231	MK227196
	Tub11	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227197
	Tub12	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227198
	Tub14	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227199
	Tub15	Australes	Tubuai, Mataura	23°20.588S 149°28.754W Oct.-15	-	-	-	MK227200
	Tub22	Australes	Tubuai, Mataura	23°20.670S 149°29.241W Oct.-15	-	-	-	MK227201
	Tub23	Australes	Tubuai, Mataura	23°20.670S 149°29.241W Oct.-15	-	-	-	MK227202
	Tub24	Australes	Tubuai, Mataura	23°20.670S 149°29.241W Oct.-15	-	-	-	MK227203
	Tub25	Australes	Tubuai, Mataura	23°20.670S 149°29.241W Oct.-15	-	-	-	MK227204
	Tub26	Australes	Tubuai, Mataura	23°20.670S 149°29.241W Oct.-15	-	-	-	MK227205

Acknowledgements

Tiriana Tchong and Tanguy Sergent are gratefully acknowledged for their technical assistance in the extraction steps and molecular analysis, respectively. NC wish to express his deep gratitude to Y. Fukuyo for sharing information on the original material from French Polynesia, and to J.

McNeill for kind advice on the way of dealing typification of O. lenticularis. This work is part of the TATOO project and was supported by funds from the Délégation à la Recherche de Polynésie Française (DREC-Pf). The Regional Council of Brittany, the General Council of Finistère, the urban community of Concarneau Cornouaille Agglomération and the European Regional Development Fund (ERDF) are also acknowledged for the funding of the Sigma 300 FE-SEM of the Concarneau Marine Biology Station.

from Maximum Likelihood (ML) and posterior probabilities from Bayesian Inference (BI).

Bootstraps values below 55 and posterior probabilities below 0.70 are not shown. Roman numerals (I-V) indicate the subclades as defined in Table 2. 3.

Supplementary material