Ostreopsis cf. ovata and Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) in the Galapagos Marine Reserve ; Ostreopsis cf. ovata y Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) en la Reserva Marina de Galápagos

1 Escuela de Gestión Ambiental, Pontificia Universidad Católica del Ecuador, Sede Esmeraldas (PUCESE), Calle Espejo y subida a Santa Cruz, Casilla 08-01-0065, Esmeraldas, Ecuador. (OC) (Corresponding author) E-mail: olgacarnicer@gmail.com. ORCID-iD: https://orcid.org/0000-0003-0821-5949 2 Laboratorio de Botánica Marina y Planctología, Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana (ICIMAP-UV), Calle Mar Mediterraneo 314, Costa Verde, C.P. 94294, Boca del Río, Veracruz, Mexico. (YBO) E-mail: yuriokolodkov@yahoo.com. ORCID-iD: https://orcid.org/0000-0003-3421-3429 3 Leibniz Institute for Natural Product Research and Infection Biology, Adolf-Reichwein-Straße 23, 07745, Jena, Germany. (MG-A) E-mail: maria.garciaaltares.perez@gmail.com. ORCID-iD: https://orcid.org/0000-0003-4255-1487 4 Charles Darwin Research Station, Charles Darwin Foundation, Santa Cruz, Galapagos, Ecuador. (IK) E-mail: inti.keith@fcdarwin.org.ec. ORCID-iD: https://orcid.org/0000-0001-9313-833X 5 Institut de Recerca i Tecnologia Agroalimentària (IRTA), Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain. (KBA) E-mail: karl.andree@irta.cat. ORCID-iD: https://orcid.org/0000-0001-6564-0015 (JD) E-mail: jorge.diogene@irta.cat. ORCID-iD: https://orcid.org/0000-0002-6567-6891 (MF-T) E-mail: margarita.fernandez@irta.cat. ORCID-iD: https://orcid.org/0000-0002-2875-1135

In the last two decades, the geographical area of the study of potentially toxic benthic dinoflagellates has increased considerably (Hachani et al. 2018, Irola-Sansores et al. 2018, Durán-Riveroll et al. 2019 and references therein). However, observations on marine diversity are still lacking from low latitudes, which have hitherto been overlooked by the scientific community (Menegotto and Rangel 2018). Sampling efforts should thus be intensified in tropical areas, such as the Galapagos Marine Reserve (GMR), where epiphytic dinoflagellate occurrence has only been reported as preliminary results of the present study (Yépez Rendón et al. 2018). Furthermore, the GMR is known worldwide for its unique biodiversity, influenced by currents, local upwellings and other oceanographic features, representing biodiversity hotspots (Liu et al. 2014). There is little information about microalgae diversity in the Archipelago, and the risk of harmful algal blooms (HAB) in the area has not been assessed. A recent study on the southern islands of the GMR reported 18 harmful taxa (Carnicer et al. 2019), representing an ecological threat for coastal marine ecosystems and for human health that may result in negative economic and social impacts in the GMR (Kislik et al. 2017).
Ostreopsis is of particular interest because some species of this genus are known to negatively impact human health (causing fever, dyspnoea, bronchoconstriction, conjunctivitis and skin irritations) and to cause mortality in marine benthic organisms in temperate regions (reviewed in Accoroni and Totti 2016). Os-treopsis cf. ovata is the most widely distributed species of the genus; it has been studied in detail, mostly because of its recurrent blooms in the Mediterranean Sea, which pose a health risk to bathers (Vila et al. 2016).
The taxonomic status of the genus Ostreopsis is presently in flux and requires extensive revision (Berdalet et al. 2017). Eleven Ostreopsis species have been identified on the basis of morphological features, but the characteristics used to delineate those species have proven that unambiguous species identification based on morphology is difficult or even impossible. Instead, molecular characters, particularly the internal transcribed spacer (ITS) region and the D1-D3 large subunit (LSU) ribosomal genes, have proven to be more efficient and consistent for discriminating between dinoflagellate species (Litaker et al. 2007, Penna et al. 2014. For this reason, the two recently described species O. fattorussoi  and O. rhodesiae Verma, Hoppenrath et Murray, 2016 were defined on the basis of both molecular and morphological criteria. Morphologically, six species have a tear-drop cell shape: O. cf. siamensis, O. cf. ovata, O. heptagona, O. belizeana Faust, 1999, O. caribbeana Faust, 1999, O. fattorussoi andO. rhodesiae. The other four species of the genus are characterized by a broadly oval, lenticular-shaped cell: O. lenticularis, O. mascarenensis, O. labens Faust et Morton, 1995 andO. marina Faust, 1999. All the species share a similar plate pattern, which complicates their identification based on morphology (Penna et al. 2005). Only O. heptagona is easily distinguishable under a light microscope because the 2′′′′ plate narrows toward the centre of the hypotheca. Moreover, cell sizes overlap among spe-cies, and considerable infraspecific variability in cell diameter has been observed both in field samples and in cultures (Aligizaki and Nikolaidis 2006, David et al. 2013, Carnicer et al. 2016b).
In addition, ITS phylogenies based on sequencing the ITS region from numerous Ostreopsis isolates indicate the existence of an additional seven genetic clades (Ostreopsis spp. 1-7), designated numerically, pending formal taxonomic assignation (Sato et al. 2011, Tawong et al. 2014, apart from an unidentified phylotype (proposed as Ostreopsis sp. 8 in Tibiriçá et al. 2019) reported from Reunion Island in the Indian Ocean (Carnicer et al. 2015). Without genetic material from the originally described species location, it is impossible to determine whether the newly sequenced isolates belong to a previously described species. Fortunately, a recent study performed in French Polynesia has associated Ostreopsis sp. 5 with O. lenticularis (Chomérat et al. 2019) on the basis of the morphological features of the original description of the cells from the same location (Fukuyo 1981). Most recently, Ostreopsis mascarenensis has been reinvestigated by morphological and molecular phylogenetic methods using specimens collected from the type locality of the species by Chomérat et al. (2020).
New characterizations of Ostreopsis species from unexplored areas, including the study of morphology, phylogeny and toxin profiles, may be helpful in consolidating the original species described in the last century solely by morphology. In addition, reporting existing species will provide valuable data on their geographic distribution and support for current molecularly defined species. The present study aimed to identify the associated epibionthic dinoflagellate assemblage in the GMR and describe the morphology, molecular biology and toxicity of Ostreopsis strains found in the area.
Macroalgae and the surrounding water were transferred to a 500 mL plastic bottle, vigorously shaken for one minute and then filtered through a 300 µm mesh. For invertebrates, the surface was scraped off using a razor blade and resuspended in the surrounding water sample for filtration through a 300 µm mesh to collect the epibionthic microalgal community. The resulting water with suspended microalgae was fixed in 3% acid Lugol's solution for cell counting. Aliquots of the water samples from Tortuga Bay were kept unfixed for cell isolation. Macroalgae were placed in plastic bags and transported in coolers to the laboratory for weighing (Mettler Toledo SB32001 DeltaRange).

Cell isolation and culture conditions
Cells were isolated by the capillary method (Hoshaw and Rosowski 1973), grown in a 24-well microplate containing f/10 medium (Guillard 1975) for a week and then inoculated in 50 mL flat plastic flasks containing 30 mL f/10 medium. Cultures were transferred during the exponential phase to 500 mL non-treated, sterile polystyrene flat flasks (Thermo Scientific™ Nunc™) and grown at a constant temperature of 24°C. Salinity was adjusted to 36 by adding autoclaved Milli-Q water, and illumination was provided by fluorescent tubes with a photon irradiance of 100 mmol photons m -2 s -1 under a 12:12-h light:dark photoperiod. Cultures were acclimated to laboratory conditions for at least ten generations (three weeks). The exponential phase lasted for five days, and at the stationary phase (three weeks, density > 10 4 cells L -1 ) cells were collected on a 0.45 µm nylon filter (Whatman®, GE). Filters were stored at -20ºC until toxin extraction.

Cell counting and measurements
For cell counting, fixed field water samples were settled in 3 mL Utermöhl chambers for three hours before observation with an inverted Nikon Eclipse TE2000-S microscope. The entire bottom of the chamber was examined at 200x magnification to enumerate the larger organisms, and one/two transects at 200x or five/ten fields at 400x magnification were examined to count the small and more abundant organisms. Dinoflagellates were identified to genus except for Prorocentrum lima, O. cf. ovata and O. lenticularis. Epiphytic samples were expressed as cells per gram of fresh weight of macroalgae (cells g -1 fw) and as cells per cone surface area (cells cm -2 ) for conical shaped invertebrates, using the following equation: where r is base radius, and h is height.
Ostreopsis cells were measured from fixed water samples obtained from macroalgae in Tortuga Bay. In addition, Ostreopsis cells from laboratory cultures were measured during the exponential phase (5 days); dorsoventral (DV) and width (W) diameters were recorded using an image capture system (MCDI TM Analysis) with an Olympus DP70 camera connected to an inverted microscope (Nikon Eclipse 80i) at 400× magnification.

Morphological identification
Cultured cells were fixed at the exponential phase with a stock formaldehyde solution (37%) to a final concentration of 4%, examined and photographed in a Hitachi S-3500N scanning electron microscope (SEM) at a working distance of 5 to 6 mm and a voltage of 5.0 kV after a preliminary wash in distilled water followed by dehydration in a series of ethanol solutions of increasing concentration (30, 50, 70, 90 and 100%), critical point drying with pin-type stubs and sputter coating with gold-palladium using a Quarum Q150RS (Quorum Technologies, Newhaven, East Sussex, U.K.). Some strains were analysed under the inverted microscope (Nikon Eclipse 80i) after staining with fluorescent Calcofluor White M2R, based on the Fritz and Triemer (1985) technique.

Molecular identification
For DNA analysis, 15 mL of culture were transferred to plastic Eppendorf vials and centrifuged for 10 min at 2500 rpm. Resulting pellets were stored at -20°C until DNA extraction, following Andree et al. (2011). Primers used for the polymerase chain reaction (PCR) were ITSA (5′ -GTA ACA AGG THT CCG TAG GT -3′) and ITSB (5′ -AKA TGC TTA ART TCA GCR GG -3′), previously described by Sato et al. (2011), and the Taq DNA polymerase was from Invitrogen. ITS and 5.8S ribosomal DNA (rDNA) regions were amplified in an Applied Biosytems 2720 Thermal cycler (initial 5 min heating step at 94°C, 30 cycles at 94°C for 1 min, at 55°C for 2 min, and at 72°C for 3 min, and a final extension at 72°C for 10 min. Resulting fragments of approximately 400-base pair (bp) rRNA were evaluated by electrophoresis in agarose gel (1.5% wt/vol) stained with GelRed™ (Biotium Inc., Hayward, CA, USA) and were sent to be sequenced (GENOSCREEN, Paris, France). Amplicons were read by direct sequencing using the same primers as those applied for the initial amplification. Each amplicon was sequenced bi-directionally to resolve any ambiguities in the electropherograms that might have been attributed to polymorphisms.
Sequences were aligned using the CLUSTAL W utility built into MEGA X, and small adjustments were subsequently made to correct the alignment where needed, using the more conserved 5.8S rDNA sequence as an anchor guide to align sequences from all taxa.
The evolutionary history was inferred using the maximum likelihood method and Tamura 3-parameter model+G (Tamura 1992), conducted in MEGA X (Kumar et al. 2018). The least complex phylogenetic model was chosen as that with the lowest BIC score as indicated in the model test utility built into MEGA X. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach. The final dataset analysed included 73 nucleotide sequences, each containing 291 positions.

Toxin extraction
Nylon filters from the 16 clonal Ostreopsis cultures described above were added to methanol:water (80:20) and sonicated (Vibra-Cell™ Ultrasonic Liquid Processor VCX 750) in pulse mode for 10 min while being cooled in an ice bath before centrifugation (600 × g for 10 min). The supernatant was filtered through 0.22 µm polytetrafluorothylene membrane syringe filters (Kinesis Ltd.). This procedure was repeated twice, and the final volume was adjusted to 10 mL.

Hemolytic assay
The hemolytic assay protocol given in Riobó et al. (2008) was followed. A calibration curve was made using PLTX standard (extracted from Palythoa tuberculosa) from Wako Chemicals GmbH, (Neuss, Germany) dissolved in methanol:water (1:1) to a concentration of 25 ng PLTX mL -1 . Toxin extracts and PLTX standard were evaporated and refilled with phosphate buffered saline solution (PBS) to eliminate methanol and water from the extraction. A calibration curve was performed with 12 concentrations of standard from 12.5 to 1250 pg PLTX mL -1 adjusted to an exponential regression. The working solution was prepared with washed sheep blood (OXOID), centrifuged (4000 × g, 10°C, 10 min) twice and diluted with PBS 0.01 M, pH 7.4 (Sigma), 0.1% bovine serum albumin (BSA), 1 mM calcium chloride (CaCl 2· 2H 2 O) and 1 mM boric acid (H 3 BO 3 ) to a final concentration of 1.5 10 6 cells mL -1 . The assay for PLTX specificity was verified by a blank assay with ouabain (1 mM final concentration). The assay was performed in two non-treated 96 well microplates, and samples were analysed in triplicate. After 22 h of incubation at 24°C, microplates were centrifuged (416 × g, 10 min), and 200 µL of the supernatant was transferred to another microplate for absorbance reading by a KC4 microplate reader from BioTec Instruments, Inc. (Winooski, VT, USA) at 405 nm absorbance.

LC-HRMS toxin analysis
The liquid chromatography-high-resolution mass spectrometry (LC-HRMS) conditions were those of Ciminiello et al. (2015). The analyses were performed using a Q-Exactive Orbitrap mass spectrometer coupled to an Accela AS LC system (Thermo Fisher, San José, CA, USA). The organic solvents (LC-MS grade) and reagents used for LC-MS analysis were purchased from Sigma Aldrich. An Accucore C18 column (2.6 µm, 100×2.1 mm; Thermo Fisher) was eluted at 0.2 mL/min with water (eluent A) and acetonitrile (eluent B), both containing 0.1% formic acid. The gradient elution used was 26% to 29% B over 15 min, 29% to 99% B in 1 min, hold 3 min, 99% to 26% B in 0.5 min, and hold 10.5 min. The injection volume was 10 µl, and the oven temperature was 30°C. HR full MS experiments (positive ionization) were acquired in the range of m/z 700-2000. The following source settings were used: spray voltage = 3200 V; capillary temperature = 250ºC; sheath gas flow = 49; and auxiliary gas flow = 10 arbitrary units. Resolving power was set at 70000 (FWHM at m/z 400).
Palytoxin standard (from Palythoa tuberculosa) and strain IRTASMM-11-10 of Ostreopsis cf. ovata from the northwestern Mediterranean Sea (García-Altares et al. 2014), extracted as described above, were used as references to check for retention times and ionization behaviour of PLTX, isobaric PLTX and ovatoxin (OVTX)-a to -g. The instrumental limit of quantification was estimated to be comparable to that of the method reported in García-Altares et al. (2014), which was 6 ng mL -1 . The presence of both known and unknown PLTX-like compounds was investigated using their characteristic ionization profile of PLTXs, typically containing triply charged ions in the region m/z 830-950 and doubly charged ions in the region m/z 1250-1400 (Ciminiello et al. 2011).

Epiphytic dinoflagellate assemblage
Benthic dinoflagellate abundances were estimated in 18 samples: two samples of sessile benthic invertebrates and 16 of macroalgae. Invertebrate samples of Tetraclita sp. were collected at Santa Fé Island, and macroalgae were taken from the two other sampling sites at Santa Cruz Island; 11 samples were from Tortuga Bay and five were from Venecia Bay ( Table 1).
The "small cell morphotype" of Ostreopsis was the dominant species among the benthic dinoflagellate assemblage in 10 out of 18 samples, representing more than 90% of the dinoflagellate assemblage in five samples (Table 1). The maximum abundance of the "small cell morphotype" of Ostreopsis (33405 cells g -1 fw) was found on Dictyopteris sp. (Phaeophyceae: Dictyotales), where a brownish mucilage was easily observed. The "large cell morphotype" of Ostreopsis was only found on this macroalgal species (maximum abundance of 4995 cells g -1 fw). Prorocentrum spp. dominated in three samples, with a maximum abundance of 5300 cells g -1 fw on Pterocladia sp. (Florideophyceae: Gelidiales). Amphidinium spp. showed the highest abundance in three samples, with a maximum of 4344 cells g -1 fw on Gracilaria sp. (Florideophyceae: Gracilariales). Gambierdiscus spp. was present in four samples, two from each site, in low abundances (maximum of 588 cells g -1 fw) (Table 1).

Isolated strains
A total of 16 strains were isolated, 13 from the "small-cell morphotype" and three from the "large-cell morphotype" from field samples in Tortuga Bay. No isolates of the intermediate size were successfully established in culture.

Phylogenetic analysis
The PCR amplifications of 5.8S rDNA and ITS regions obtained from the 16 isolates were aligned to- gether with other sequences from GenBank. Thirteen strains that shared identical sequences corresponding to the "small-cell morphotype" clustered in the Atlantic/Indian/Pacific clade of O. cf ovata (GenBank accession number MH844087 for the strain 1G), and three strains with identical sequences corresponded to the "large-cell morphotype" in O. lenticularis (= Ostreopsis sp. 5) (GenBank accession number MH844088 for the strain 17G) (Fig. 3). The same tree topology was obtained using Bayesian inference (data not shown), with one caveat being that the Atlantic/Indian/Pacific clade was bifurcated into two clades, one group more distal to the Indian Pacific clade and one group containing the isolates from the Galapagos Islands in the more proximal clade.

Toxin profile
The 16 toxin extracts analysed (13 from O. cf. ovata and 3 from O. lenticularis) were below the limit of detection of the haemolytic assay (25 pg PLTX mL -1 ) and proved to be non-toxic. This result was supported by the absence of PLTX-like compounds, both known and unknown, in the analysis by LC-HRMS. Figure 6 shows the total ion chromatograms and full scan MS spectra of reference materials (PLTX standard and O. cf. ovata , showing the characteristic clusters of triply charged ions in the region m/z 830-950 and doubly charged ions in the region m/z 1250-1400 (Ciminiello et al. 2011). Mass errors between theoretical and experimental accurate mass of the monoisotopic peak of [M+3H-H 2 O]3+ions of PLTX and OVTXs (-a to -e and -g) were below 3 ppm.
The LC-HRMS conditions applied in this study were those of Ciminiello et al. (2015), which have been used to report the detection of PLTXs in several studies (García-Altares et al. 2014, Tartaglione et al. 2016. The instrumental limit of quantitation was estimated to be of the same order of magnitude as in other studies that reported the detection of PLTXs (6 ng PLTX mL -1 ). Moreover, chromatograms and mass spectra were manually explored to look for the characteristic ionization pattern of palytoxins to find potentially unknown analogues. It is therefore unlikely that the lack of toxicity was due to the insensitivity of the detection methods used.  Boisnoir et al. 2019). A recent study highlights the potential ecological and sanitary risks to Mexican coasts associated with the presence of Gambierdiscus, Ostreopsis and Prorocentrum, with special attention to the importance of an accurate genetic and toxic identification of these species of these genera (Núñez-Vázquez et al. 2019). The absence of these specific data prevents accurate determination of the potential impacts on marine ecosystems and human health because morphological features are not sufficient to describe a species, and toxin production is unevenly distributed among species and even among strains of the same species (Litaker et al. 2010, Suzuki et al. 2012, Carnicer et al. 2016a). This is the case for the eastern tropical Pacific (ETP), where there is little information on toxic benthic dinoflagellates (Durán-Riveroll et al. 2019). This area of the globe is of special concern because the marine ecosystem is sensitive to climate change and to El Niño-Southern Oscillation events (Edgar et al. 2010), affecting biodiversity due to changing temperatures and rainfall that, in turn, can influence the distribution of certain species that can adapt to new conditions (Keith et al. 2016 Unfortunately, none of these studies included nucleic acid sequencing or toxicity analysis, making the correct identification of species difficult. A third study was performed along the northern and central coasts of Ecuador (Esmeraldas and Manta provinces), where the Padina sp. epiphytic community was sampled in 2015 (Carnicer et al. 2016a). O. cf. ovata, Atlantic/Indian/ Pacific clade, non-toxic, P. lima and Coolia spp were present, but Gambierdiscus species were not observed (O. Carnicer, pers. comm.).

DISCUSSION
In the GMR, investigations have focused on planktonic species during several cruises undertaken by the Naval Oceanographic Institute of Ecuador (INOCAR). The first record of a benthic dinoflagellate was of the genus Ostreopsis, reported by the institution's journal (Torres and Andrade 2014 , Verma et al. 2016, and molecular techniques are mandatory for correct identification. In 2017, during the study period in the GMR, Ostreopsis cf. ovata and Ostreopsis cf. lenticularis (based on light microscopy observations) were reported from 2 to 10 miles from Santa Cruz Is-land and islands nearby. Their presence in the water column and the high epibionthic abundances suggest that there may be proliferations in some areas of the GMR that have not been reported. A brownish mucilage has been observed previously in the Archipelago (I. Keith, pers. comm.), but there is no confirmation of the species involved in those events. Further monitoring should be performed covering a larger area of the GMR to evaluate the presence of benthic HAB.
O. cf. ovata has been extensively studied, and there are many sequences from different regions around the world, because it is the most widely distributed species of the genus (Accoroni and Totti 2016). According to Hoppenrath et al. (2014), without a genetic characterization of O. ovata from the type locality, it is presently not possible to conclude which genotype corresponds to this species; therefore, most authors have reported O. cf. ovata. Phylogenetically, the O. cf. ovata species complex has been divided into three clades (Penna et al. 2014). These include the: i) Atlantic/Mediterranean/ Pacific, ii) Indian/Pacific, and iii) Atlantic/Indian/Pacific clades. In clade i) all strains produce PLTX-like compounds such as isobaric PLTX and OVTX analogues (e.g., Ciminiello et al. 2013), with the exception of three strains from Japan reported as non-toxic by Suzuki et al. (2012). Clade ii) includes OVTX producing strains (Suzuki et al. 2012, Uchida et al. 2013), ostreol-A producers (a non-PLTX derivative compound) (Hwang et al. 2013) and non-toxic strains (Suzuki et al. 2012, Carnicer et al. 2015. In clade iii) some strains displayed toxicity in mouse bioassays (Tawong et al. 2014) and hemolytic assays (Penna et al. 2010), but the clade also includes non-toxic strains (Carnicer et al. 2016a). The present study contributes additional physiological information on the characterization of the O. cf. ovata species complex by adding a strain from a geographical area not previously sampled. The O. cf. ovata strain from the GMR belongs to clade iii), as do the strains sequenced to date from the coasts of Ecuador (Carnicer et al. 2016a), Belize (Penna et al. 2014), Indonesia (Penna et al. 2010), Thailand (Tawong et al. 2014) and Malaysia (Leaw et al. 2001). As for the strains isolated from Ecuador and Belize, O. cf. ovata strains from the GMR are non-toxic.
The GMR is influenced by the convergence of three major currents that contribute to its unique environmental conditions favouring its high biodiversity (Muromtsev 1963, Banks, 2002, Hickman 2009). The South Equatorial Current flows westward and shows a marked seasonality. More intense cold-salty waters come during the dry season (June-November) with the Humboldt Current influenced by southern winds, while during the wet season (December-May) warmer waters come with the Panama Current. Eastward flowing, the Equatorial Undercurrent upwells in the western islands of the GMR, increasing primary production (Schaeffer et al. 2008). Thus, microalgal colonization from the western Pacific Ocean, as well as from Central America to the Archipelago, may have occurred. It is suspected that O. cf. ovata populations were separated by the Isthmus of Panama, and subsequent genetic differentiation took place (Penna et al. 2010). This hypothesis is supported by O. cf. ovata strains from the western Atlantic (Brazil), which are genetically clustered with the eastern Atlantic and Mediterranean strains (Nascimento et al. 2012) and produce OVTX analogues. However, strains from the Caribbean Sea are genetically clustered with the eastern Pacific and Galapagos strains. Further molecular studies need to be undertaken in the Caribbean Sea and along the eastern Pacific coast to validate the assumption of an introduction of cells through the Panama Chanel with ballast waters (Carnicer et al. 2016a).
At least one other species of Ostreopsis has been identified in this study. This species fell in Ostreopsis sp. 5 (Sato et al. 2011). Morphologically, it resembles the original description of O. lenticularis, but until recently (Chomérat et al. 2019) its known genetic clade assignment could not be used to unambiguously establish these isolates as O. lenticularis because it was described by Fukuyo (1981) prior to routine molecular characterization. The absence of the undulation of the cingulum in side view was not verified, although, according to Fukuyo (1981), it is a morphological feature that distinguishes O. lenticularis, which possesses additional minute thecal pores, from O. siamensis, which does not. This is in agreement with Hoppenrath et al. (2014), who suggested that the species under the name of O. lenticularis in Faust et al. (1996) with only one type of pore belongs to another species. In addition, the species illustrated under the name of O. siamensis in Faust et al. (1996: Figs 2-8) is described with the two types of pores consistent with the original O. lenticularis description. Cortés-Lara et al. (2005) illustrated two pore size classes in O. siamensis from the Mexican Pacific and Penna et al. (2005) in O. ovata from the western Mediterranean. Aligizaki and Nikolaidis (2006) also reported two types of pores in O. ovata and O. cf. siamensis, which makes delimitation of O. lenticularis even more complicated. The terms used in the literature in the description of the cell shape are vague and rather confusing, especially when the dorsoventral diameter/width (DV/W) ratio, which can be a useful feature for separating Ostreopsis spp. (Hoppenrath et al. 2014), is not given. The morphology of the sulcal plates in Ostreopsis spp. remains poorly examined. Similarly, in our study only closeups of the sulcal area viewed ventrally-antapically are presented (Figs 4F and 5F), revealing some details that we were unable to compare with the published data on the same plates.
The strains of O. lenticularis recently isolated by Chomérat et al. (2019) from the type locality (Tahiti Island) cluster with the sequences previously ascribed to Ostreopsis sp. 5. The morphological features of the O. lenticularis strains isolated by Chomérat et al. (2019), such as the presence of two types of thecal pores on the theca, are in agreement with the original description, and those authors suggest that this character be used to distinguish O. lenticularis from other large species. To confirm the findings obtained by Chomérat et al. (2019), all the known morphological, morphometrical, molecular and toxicity data for O. lenticularis and related species are assembled in Table 2 to determine how strongly the preponderance of data supports Ostreopsis sp. 5 compared with the closely related Ostreopsis sp. 6 being O. lenticularis. Comparing cell sizes, strains of Ostreopsis sp. 5 (references in Table 2) fit better with the original description of O. lenticularis in Fukuyo (1981) (60-100 (DV); 45-85 (W) µm), whereas Ostreopsis sp. 6 (references in Table 2) are, in general, smaller cells that do not exceed 85 µm in DV diameter and correspond better to the original description of O. labens: 60-86 µm (DV), 70-80 µm (W) (Faust and Morton 1995). The presence of the two types of thecal pores has been considered the main diagnostic feature of O. lenticularis, which differentiates it from other Ostreopsis spp. From the morphological descriptions available for Ostreopsis sp. 5, there are at least two different types of pores, whereas only one type of pore was observed in Ostreopsis sp. 6 ( Table 2).
The first ribosomal sequences for a strain from Malaysia identified as O. lenticularis were presented by Leaw et al. (2001), but the study lacks the necessary morphological description to confirm whether it corresponds to the original description of the species. In subsequent publications, O. labens was clustered in the same genetic clade (Penna et al. 2010), and after the addition of three new sequences to the clade it was then called Ostreopsis sp. 6 (Sato et al. 2011). However, in a recent study conducted in the China Sea (Zhang et al. 2018), O. lenticularis was clustered in another genetic clade, Ostreopsis sp. 5, together with the strains from Reunion Island (Carnicer et al. 2015) and three strains isolated from Japan (Sato et al. 2011), posing a new taxonomic question of whether Ostreopsis sp. 5 or Ostreopsis sp. 6 corresponds to O. lenticularis. This was resolved by Chomérat et al. (2019), who found that Ostreopsis sp. 5, a non-toxic species, is O. lenticularis, and Ostreopsis sp. 6 corresponds to a different species.
Another interesting pattern observed is related to toxin content. There is a homogeneity for Ostreopsis sp. 5 strains from Japan (Suzuki et al. 2012), Reunion Island (Carnicer et al. 2015) and the Galapagos (this study), which are non-toxic (Table 2). Within Ostreopsis sp. 6, there is higher variability in cell toxicity, including observations of toxic strains detected by hemolytic and mouse bioassays (Penna et al. 2010, Tawong et al. 2014, producers of ostreocin-d (Suzuki et al. 2012) and PLTX analogues (Moreira et al. 2012), as well as nontoxic strains (Suzuki et al. 2012) (Table 2).
In summary, the present study provides a description of epibionthic dinoflagellate assemblages from three sites of two southern islands in the GMR (Santa Cruz and Santa Fé) in March and April 2017. The potentially toxic genera of Amphidinium, Coolia, Gambierdiscus and Ostreopsis were found, the latter with abundances up to 38400 cells g -1 fw. The presence of these genera represents a potential threat to humans and to marine ecosystems. Thus, it is important to consider benthic dinoflagellate species in the surveillance of HAB in the GMR. This study also provides the first correct characterization of Ostreopsis strains based on molecular, morphological and toxicological data, corresponding to O. cf. ovata and O. lenticularis in the  Fukuyo (1981) as well as by Chomérat el al. (2019) regarding cell size and type of pores. Furthermore, in our study all the strains of Ostreopsis sp. 5 (=O. lenticularis) were non-toxic, revealing a possible discriminating character.