sm84n3-5035

INTRODUCTIONTop Toxic benthic dinoflagellates have been related to seafood poisoning in humans and negative impacts on some marine organisms (). Several toxic genera frequently co-exist in epiphytic microalgal assemblages: Gambierdiscus Adachi and Fukuyo, 1979, which produce the toxins responsible for ciguatera fish poisoning (, , ); Fukuyoa F. Gómez, D.X. Qiu, R.M. Lopes et Senjie Lin, 2015, which produce haemolytic substances and a maitotoxin-like compound (, , ); Ostreopsis Schmidt, 1901, associated with clupeotoxicity (), skin irritations and respiratory disorders (, , ); and some toxic species of Amphidinium Claparède et Lachmann, 1859, Coolia Meunier, 1919, and Prorocentrum Ehrenberg, 1834, which may cause human health issues (). In the last two decades, the geographical area of the study of potentially toxic benthic dinoflagellates has increased considerably (, , and references therein). However, observations on marine diversity are still lacking from low latitudes, which have hitherto been overlooked by the scientific community (). 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 (). Furthermore, the GMR is known worldwide for its unique biodiversity, influenced by currents, local upwellings and other oceanographic features, representing biodiversity hotspots (). 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 (), representing an ecological threat for coastal marine ecosystems and for human health that may result in negative economic and social impacts in the GMR (). 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 ). Ostreopsis 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 (). It has been demonstrated, in some cases by bioassay and in others by analytical techniques, that several species/genetic clades of the genus Ostreopsis produce palytoxin (PLTX)-like compounds: O cf. ovata (, ), O. siamensis Schmidt, 1901 (), O. mascarenensis Quod, 1994 (), Ostreopsis sp. 1 and Ostreopsis sp. 6 (, ), and O. fattorussoi Accoroni, Romagnoli et Totti, 2016. Ostreopsis lenticularis Fukuyo, 1981 (), O. heptagona Norris, Bomber et Balech, 1985 and Ostreopsis sp. 7 () have been reported as toxic by mouse bioassay. However, within the O. cf. ovata strains there is a high infraspecific variability concerning toxin production (), as has been reported in other dinoflagellates such as the Alexandrium tamarense species complex (). The taxonomic status of the genus Ostreopsis is presently in flux and requires extensive revision (). 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 (, ). 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 and O. 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 and O. marina Faust, 1999. All the species share a similar plate pattern, which complicates their identification based on morphology (). 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 species, and considerable infraspecific variability in cell diameter has been observed both in field samples and in cultures(, , ). 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 (, ), apart from an unidentified phylotype (proposed as Ostreopsis sp. 8 in ) reported from Reunion Island in the Indian Ocean (). 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 () on the basis of the morphological features of the original description of the cells from the same location (). Most recently, Ostreopsis mascarenensis has been reinvestigated by morphological and molecular phylogenetic methods using specimens collected from the type locality of the species by . 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. MATERIALS AND METHODSTop Sampling Sampling occurred at two southern islands in the GMR. One site was sampled on Santa Fé Island (0°48′16.36″S; 90°5′7.522″W) on 29 March 2017, and two sites were sampled on Santa Cruz Island: Tortuga Bay (0°45′58.43″S; 90°20′42.373″W) on 30 March 2017 and Venecia Bay (0°302′5.755″S; 90°30′56.646″W) on 6 April 2017 (Fig. 1). The surface water temperature was 28.25°C to 28.80°C, salinity was 34.24-34.68, pH was 7.78-7.84 and dissolved oxygen was 5.11-6.34 mL L^–1 (94.3%-100.6%). Macroalgae and scrapings on the surface of sessile benthic invertebrates, Tetraclita sp. (Crustacea: Cirripedia), were collected for analysis of the epibenthic dinoflagellates growing on them. Samples were taken by hand at 1 to 2 m depth and placed in a plastic bag immediately; the volume of the surrounding water was subsequently measured with a plastic graduated cylinder.

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Fig. 1. – Sampling sites: Santa Fé Island (0°48′16.36″S; 90°5′7.522″W), Tortuga Bay (0°45′58.43″S; 90°20′42.373″W) and Venecia Bay (0°302′5.755″S; 90°30′56.646″W) in the Galapagos Marine Reserve.

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 (), grown in a 24-well microplate containing f/10 medium () 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^–1under 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 >10⁴ 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:

s u r f a c e = π r \sqrt{h^{2} + r^{2}}

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)Fritz L., Triemer R.E. 1985. A rapid simple technique utilizing Calcofluor White M2R for the visualization of dinoflagellate thecal plates. J. Phycol. 21: 662-664. 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)Andree K.B., Fernandez-Tejedor M., Elandaloussi L.M., et al. 2011. Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea. Appl. Environ. Microb. 77: 1651-1659.. 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)Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983., 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 1992Tamura K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Mol. Biol. Evol. 9: 678-687.), conducted in MEGA X (Kumar et al. 2018Kumar S., Stecher G., Li M., et al. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547-1549.). 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)Riobó P., Paz B., Franco J.M., et al. 2008. Proposal for a simple and sensitive haemolytic assay for palytoxin: toxicological dynamics, kinetics, ouabain inhibition and thermal stability. Harmful Algae 7: 415-429. 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_2·2H₂O) and 1 mM boric acid (H₃BO₃) to a final concentration of 1.5 10⁶ 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)Ciminiello P., Dell’Aversano C., Dello Iacovo E., et al. 2015. Liquid chromatography-high-resolution mass spectrometry for palytoxins in mussels. Anal. Bioanal. Chem. 407: 1463-1473.. 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. 2014García-Altares M., Tartaglione L., Dell’Aversano C., et al. 2014. The novel OVTX-g and isobaric PLTX (so far referred to as putative PLTX) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MSn. Anal. Bioanal. Chem. 407: 1191-1204.), 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)García-Altares M., Tartaglione L., Dell’Aversano C., et al. 2014. The novel OVTX-g and isobaric PLTX (so far referred to as putative PLTX) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MSn. Anal. Bioanal. Chem. 407: 1191-1204., which was 6 ngmL^–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. 2011Ciminiello P., Dell’Aversano C., Dello Iacovo E., et al. 2011. LC-MS of palytoxin and its analogues: State of the art and future perspectives. Toxicon 57: 376-389.).

RESULTSTop

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).

Table 1. – Cell abundance estimation in cells g^–1 fw of macroalgae and in cells cm² for invertebrates (Tetraclita sp.); percentage of dominance is indicating in bold.

Sampling Site	Date	Substrate	Ostreopsis spp.						Coolia spp.		Amphidinium spp.		Prorocentrum spp.		P. lima		Gambierdiscus spp.
Sampling Site	Date	Substrate	Small-cell morphotype		Intermediate-cell morphotype		Large-cell morphotype		Coolia spp.		Amphidinium spp.		Prorocentrum spp.		P. lima		Gambierdiscus spp.
Santa Fé	29/03/2017	Tetraclita sp.	2924	97	0		0		0		15	1	5	<1	55	2	0
Santa Fé	29/03/2017		11590	98	0		0		101	1	55	<1	0		25	<1	0
Tortuga Bay	30/03/2017	Gracilaria sp.	5233	49	0		0		341	3	1809	17	1923	18	1339	13	0
			1601	58	0		0		45	2	19	1	370	13	708	26	0
			3857	35	0		0		136	1	4344	39	1656	15	1071	10	0
		Caulerpa sp.	1950	12	150	1	0		1500	9	2100	13	4650	29	5700	36	0
			72	5	0		0		0	0	0	0	648	46	612	44	72	5
			126	31	0		0		86	21	75	18	80	20	37	9	0
		Chlorophyta	46	6	0		0		11	1	5	1	409	52	312	40	0
		Dictyopteris sp.	30171	97	461	1	230	1	0		0		0		230	1	0
			9995	97	50	<1	165	2	0		33	<1	0		66	1	17	<1
			33405	82	0		4995	12	624	2	1561	4	0		0		0
		Dictyota sp.	3037	92	0		0		0		269	8	0		0		0
Venecia Bay	06/04/2017	Rhodophyta	882	10	0		0		2058	23	2940	33	2058	23	294	3	588	7
		Padina sp.	214	36	0		0		91	15	118	20	85	14	91	15	0
		Padina sp.	221	9	15	1	0		0		88	3	1253	48	1017	39	0
		Caulerpa sp.	298	19	0		0		256	16	511	32	256	16	256	16	0
		Pterocladia sp.	3313	23	95	1	0		3313	23	1798	13	5300	37	189	1	284	2

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).

Field morphology of Ostreopsis

A total of 369 Ostreopsis cells were measured from the epiphytic dinoflagellate samples obtained from macroalgae in Tortuga Bay, Santa Cruz Island. Three different morphotypes based on DV and W diameters were distinguished (Fig. 2). The small-cell morphotype corresponded to cells with a tear-drop shaped small size (mean±standard deviation; DV=54.48±7.12 µm; W=38.01±5.11 µm; DV/W=1.43). The group with the largest size had a more broadly oval shape, the large-cell morphotype (DV=98.13±7.59 µm; W=77.74±7.35 µm; DV/W=1.26), with a maximum of DV of 109.86 µm and W of 96.75 µm. An intermediate-cell morphotype was observed, having an elongated conical shape compared with the large-cell morphotype, and was larger than the small-cell morphotype (DV=80.14±1.59 µm; W=61.27±4.24 µm; DV/W=1.31) (Fig. 2).

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Fig. 2. – Dorsoventral (DV) and width (W) diameters of Ostreopsis cells from field samples, n=369.

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 together 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.

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Fig. 3. – Evolutionary relationships of Ostreopsis spp. 5.8S rDNA and ITS regions. Bootstrap values (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The tree with the highest log likelihood (-3268.95) is shown and is drawn to scale, with branch lengths measured in the number of substitutions per site. The percentage of trees in which the associated taxa clustered together is shown next to the branches.

Morphological descriptions

Small-cell morphotype – Ostreopsis cf. ovata

Cells were oval-pointed and tear-drop shaped, tapering ventrally in apical/antapical view and anteroposteriorly compressed. Cells were measured from five different O. cf. ovata strains (1G, 3G, 5G, 10G and 11G), a total of 477 cells, DV=44.73±5.62 µm (max=57.92 µm; min=28.26 µm); W=32.32±5.35 µm (max=48.71 µm; min=18.57 µm); DV/W=1.39±0.12. Plate 1′ is large, elongated, subhexagonal, slightly shifted to the left side of the cell, about 3.5 to 4 times long as it is wide (Fig. 4A, C). Plate 2′ is as narrow as the latter, contacts plate 4″ (Fig. 4D), and plate 3′ is small and hexagonal. The Po plate is moderately long, slightly shorter than plate 2′ (Fig. 4D). Plate 2′′′′ is pentagonal, relatively short, about half the DV diameter, slightly shifted to the right side of the cell, with almost straight longitudinal sides parallel to each other, of the same width in its anterior and posterior parts, its contact with 4′′′ about 1.5-2 times longer than with 3′′′ (Fig. 4B). Thecal pores are of one type: 0.19-0.23 µm in diameter (Fig. 4D, E).

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Fig. 4. – Thecal morphology of Ostreopsis cf. ovata (strain 1G) viewed with scanning electron microscopy. A, apical (epithecal) view; B, antapical (hypothecal) view; C, anterior-dorsal-left-side view; D, the apical pore plate and adjacent epithecal plates in left-side view; E, a fragment of the 1 plate with irregularly scattered trichocyst pores; F, a fragment of the hypotheca and the sulcal area. Plate labels: 1′-3′, the apical plates; 1″-7″ the precingular plates; 1′′′-5′′′, the postcingular plates; 1′′′′ and 2′′′′, the antapical plates; Po, the apical pore plate; Vo, the ventral opening. The plates are named according to Hoppenrath et al. (2014). Scale bars: 20 μm in A-C, 5 μm in D-F.

Large-cell morphotype – Ostreopsis lenticularis

Cells were broadly oval-pointed and lenticular, tapering ventrally in apical/antapical view, anteroposteriorly compressed. Cells were measured from one O. lenticularis (=Ostreopsis sp. 5) strain (17G), a total of 61 cells; DV=88.49±7.22 µm (max=105.36 µm; min=70.94 µm); W=67.29±6.11 µm (max=82.69 µm; min=55.5 µm); DV/W=1.32 ±0.06. Plate 1′ large, elongated, subhexagonal, slightly shifted to the left side of the cell, more than twice as long as wide (Fig. 5A, C). Plate 3′ is small, hexagonal. Plate 2′′′′ is somewhat curved longitudinally with its convex side to the right, slightly wider in its posterior part; its contact with 4′′′ is frequently about 1.5 to 2 times longer than with 3′′′ or its contacting sides are about equal (Fig. 5B, D). Thecal pores are of two types, large (the trichocyst pores) and small (Fig. 5E). Large pores (min=0.20 µm; rarely) 0.28 to 0.35 µm, and small thecal pores (min=0.04 µm; rarely) 0.07 to 0.12 µm. The ventral opening (the ventral pore) is 2 µm in diameter (Fig. 5F).

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Fig. 5. – Thecal morphology of Ostreopsis lenticularis (strain 17G) (A and B, cells stained with Calcofluor White M2R viewed with epifluorescence microscopy; C-F, cells viewed with scanning electron microscopy). A and C, apical (epithecal) view; B and D, antapical (hypothecal) view; E, a fragment of a thecal plate, with the irregularly scattered trichocyst pores and small pores; F, a fragment of the hypotheca and the sulcal area. Plate labels: 1′-3′, the apical plates; 1″–7″, the precingular plates; 1′′′-5′′′, the postcingular plates; 1′′′′ and 2′′′′, the antapical plates; Po, the apical pore plate; Sa, the anterior sulcal plate; Sda, the right sulcal plate; stp, small thecal pores; Sp, the posterior sulcal plate; Ssa, the left sulcal plate; tp, the trichocyst pores; Vo, the ventral opening (also known as the ventral pore). The plates are named according to Hoppenrath et al. (2014). Scale bars: 20 μm in A and B; 50 μm in C; 30 μm in D; 5 μm in E; 10 μm in F.

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 IRTASMM-11-10), 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. 2011Ciminiello P., Dell’Aversano C., Dello Iacovo E., et al. 2011. LC-MS of palytoxin and its analogues: State of the art and future perspectives. Toxicon 57: 376-389.). Mass errors between theoretical and experimental accurate mass of the monoisotopic peak of [M+3H-H₂O]3+ions of PLTX and OVTXs (-a to -e and -g) were below 3 ppm.

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Fig. 6. – LC-HRMS analysis (total ion chromatograms and full scan MS) of PLTX standard (25 ng mL^–1 from Palythoa tuberculosa) and methanolic extracts of Ostreopsis cf. ovata strain IRTA-SMM-11-10 (toxin profile described in García-Altares et al. 2014García-Altares M., Tartaglione L., Dell’Aversano C., et al. 2014. The novel OVTX-g and isobaric PLTX (so far referred to as putative PLTX) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MSn. Anal. Bioanal. Chem. 407: 1191-1204.), used as a reference sample in the present study for the detection of PLTX-like compounds.

The LC-HRMS conditions applied in this study were those of Ciminiello et al. (2015)Ciminiello P., Dell’Aversano C., Dello Iacovo E., et al. 2015. Liquid chromatography-high-resolution mass spectrometry for palytoxins in mussels. Anal. Bioanal. Chem. 407: 1463-1473., which have been used to report the detection of PLTXs in several studies (García-Altares et al. 2014García-Altares M., Tartaglione L., Dell’Aversano C., et al. 2014. The novel OVTX-g and isobaric PLTX (so far referred to as putative PLTX) from Ostreopsis cf. ovata (NW Mediterranean Sea): structural insights by LC-high resolution MSn. Anal. Bioanal. Chem. 407: 1191-1204., Tartaglione et al. 2016Tartaglione L., Mazzeo A., Dell’Aversano C., et al. 2016. Chemical, molecular, and eco-toxicological investigation of Ostreopsis sp. from Cyprus Island: structural insights into four new ovatoxins by LC-HRMS/MS. Anal. Bioanal. Chem. 408: 915-932., 2017Tartaglione L., Dello Iacovo E., Mazzeo A., et al. 2017. Variability in toxin profiles of the Mediterranean Ostreopsis cf. ovata and in structural features of the produced ovatoxins. Environ. Sci. Technol. 51: 13920-13928.). 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.

DISCUSSIONTop

This study is the first accurate report of O. cf. ovata and O. lenticularis in the GMR and confirms the presence of potentially toxic benthic dinoflagellate species. Since the early 20th century, species of the genera Gambierdiscus, Ostreopsis, Prorocentrum, Coolia and Amphidinium have been reported in tropical and subtropical regions such as the eastern (Vargas-Montero et al. 2012Vargas-Montero M., Morales A., Cortés J. 2012. Primer informe del género Gambierdiscus (Dinophyceae) y otros dinoflagelados bentónicos en el Parque Nacional Isla del Coco, Costa Rica, Pacífico Tropical Oriental. Rev. Biol. Trop. 60 (Suppl. 3): 187-199., Maciel-Baltazar 2015Maciel-Baltazar E. 2015. Dinoflagelados (Dinoflagellata) tóxicos de la costa de Chiapas, México, Pacífico centro oriental. Cuad. Invest. UNED 7: 39-48.) and western Pacific Ocean (Rhodes et al. 2017Rhodes L.L., Smith K.F., Verma A., et al. 2017. The dinoflagellate genera Gambierdiscus and Ostreopsis from subtropical Raoul Island and North Meyer Island, Kermadec Islands. New Zeal. J. Mar. Fresh. Res. 51: 490-504.), the Indian Ocean (Carnicer et al. 2015Carnicer O., Tunin-Ley A., Andree K.B., et al. 2015. Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): Molecular, morphologic and toxicity characterization. Cryptogamie Algol. 36: 101-119.), the west Atlantic Ocean (Mendes et al. 2017Mendes M.C.Q., Nunes J.M.C., Menezes M., et al. 2017. Toxin production, growth kinetics and molecular characterization of Ostreopsis cf. ovata isolated from Todos os Santos Bay, tropical southwestern Atlantic. Toxicon 138: 18-30.) and the Caribbean Sea (Irola-Sansores et al. 2018Irola-Sansores E.D., Delgado-Pech B., García-Mendoza E., et al. 2018. Population dynamics of benthic-epiphytic dinoflagellates on two macroalgae from coral reef systems of the northern Mexican Caribbean. Front. Mar. Sci. 5: 487., Boisnoir et al. 2019Boisnoir A., Pascala P.Y., Cordonniera S., et al. 2019. Spatio-temporal dynamics and biotic substrate preferences of benthic dinoflagellates in the Lesser Antilles, Caribbean Sea. Harmful Algae 81: 18-29.). 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. 2019Núñez-Vázquez E.J., Almazán-Becerril A., López-Cortés D.J., et al. 2019. Ciguatera in Mexico (1984-2013). Mar. Drugs 17: 13.). 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. 2010Litaker R.W., Vandersea M.W., Faust M.A., et al. 2010. Global distribution of ciguatera causing dinoflagellates in the genus Gambierdiscus. Toxicon 56: 711-730., Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91., Carnicer et al. 2016aCarnicer O., García-Altares M., Andree K.B., et al. 2016a. First evidence of Ostreopsis cf. ovata in the eastern tropical Pacific Ocean, Ecuadorian coast. Bot. Mar. 59: 267-274.).

This is the case for the eastern tropical Pacific (ETP), where there is little information on toxic benthic dinoflagellates (Durán-Riveroll et al. 2019Durán-Riveroll L.M., Cembella A.D., Okolodkov Y.B. 2019. A review on the biodiversity and biogeography of toxigenic benthic marine dinoflagellates of the coasts of Latin America. Front. Mar. Sci. 6: 148). 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. 2010Edgar G.J., Banks S.A., Brandt M., et al. 2010. El Niño, grazers and fisheries interact to greatly elevate extinction risk for Galapagos marine species. Global Change Biol. 16: 2876-2890.), 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. 2016Keith I., Dawson T.P., Collins K.J., et al. 2016. Marine invasive species: establishing pathways, their presence and potential threats in the Galapagos Marine Reserve. Pac. Conserv. Biol. 22: 377-385.). The studies from the ETP are limited to Colombia (Quintana-Manotas and Mercado-Gómez 2017Quintana-Manotas H., Mercado-Gómez J. 2017. Composición de dinoflagelados epífitos y forófitos en la Costa norte del golfo de Morrosquillo, Sucre, Colombia. Rev. Colombiana Cienc. Anim. 9: 129-140.), where Coolia sp., O. lenticularis, O. ovata, P. emarginatum and P. lima were recorded, and Costa Rica (Coco Island) (Vargas-Montero et al. 2012Vargas-Montero M., Morales A., Cortés J. 2012. Primer informe del género Gambierdiscus (Dinophyceae) y otros dinoflagelados bentónicos en el Parque Nacional Isla del Coco, Costa Rica, Pacífico Tropical Oriental. Rev. Biol. Trop. 60 (Suppl. 3): 187-199.), with the presence of Gambierdiscus spp., C. tropicalis, C. cf. areolota, P. concavum, P. compressum, Amphidinium carterae and O. siamensis. 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. 2016aCarnicer O., García-Altares M., Andree K.B., et al. 2016a. First evidence of Ostreopsis cf. ovata in the eastern tropical Pacific Ocean, Ecuadorian coast. Bot. Mar. 59: 267-274.). 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.).

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 2014Torres G., Andrade C. 2014. Dinámica del plancton marino costero con relación al flujo de mareas en Bahía Aeolián en septiembre 2006 (Baltra-Galápagos-Ecuador). OceanDocs: Repository of Ocean Publications.). They identified O. siamensis on Baltra Island (located north of Santa Cruz Island) from surface seawater samples collected in shallow areas in 2005. However, samples were only observed with a light microscope, so a misidentification may have occurred. For example, small tear-drop shaped cells such as O. cf. ovata, O. cf. siamensis, O. fattorussoi and O. rhodesiae are not distinguishable solely by light microscopy (Accoroni et al. 2016Accoroni S., Romagnoli T., Penna A., et al. 2016. Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea. J. Phycol. 52: 1064-1084., Verma et al. 2016)Verma A., Hoppenrath M., Harwood T., et al. 2016. Molecular phylogeny, morphology and toxigenicity of Ostreopsis cf. siamensis (Dinophyceae) from temperate south-east Australia. Phycol. Res. 64: 146-159., 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 Island 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 2016Accoroni S., Totti C. 2016. The toxic benthic dinoflagellates of the genus Ostreopsis in temperate areas: a review. Adv. Oceanogr. Limnol. 7: 1-15.). According to Hoppenrath et al. (2014)Hoppenrath M., Murray S.A., Chomérat N., et al. 2014. Marine benthic dinoflagellates - unveiling their worldwide biodiversity. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany, 276 pp., 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. 2014Penna A., Battocchi C., Capellacci S., et al. 2014. Mitochondrial, but not rDNA, genes fail to discriminate dinoflagellate species in the genus Ostreopsis. Harmful Algae 40: 40-50.). 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. 2013Ciminiello P., Dell’Aversano C., Dello Iacovo E., et al. 2013. Investigation of toxin profile of Mediterranean and Atlantic strains of Ostreopsis cf. siamensis (Dinophyceae) by liquid chromatography-high resolution mass spectrometry. Harmful Algae 23: 19-27.), with the exception of three strains from Japan reported as non-toxic by Suzuki et al. (2012)Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.. Clade ii) includes OVTX producing strains (Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91., Uchida et al. 2013Uchida H., Taira Y., Yasumoto T. 2013. 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. Rapid Commun. Mass Spectrom. 27: 1999-2008.), ostreol-A producers (a non-PLTX derivative compound) (Hwang et al. 2013Hwang B.S., Yoon E.Y., Kim H.S., et al. 2013. Ostreol A: a new cytotoxic compound isolated from the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island, Korea. Bioorg, Med. Chem. Lett. 23: 3023-3027.) and non-toxic strains (Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91., Carnicer et al. 2015Carnicer O., Tunin-Ley A., Andree K.B., et al. 2015. Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): Molecular, morphologic and toxicity characterization. Cryptogamie Algol. 36: 101-119.). In clade iii) some strains displayed toxicity in mouse bioassays (Tawong et al. 2014)Tawong W., Nishimura T., Sakanari H., et al. 2014. Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand. Harmful Algae 37: 160-171. and hemolytic assays (Penna et al. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841.), but the clade also includes non-toxic strains (Carnicer et al. 2016aCarnicer O., García-Altares M., Andree K.B., et al. 2016a. First evidence of Ostreopsis cf. ovata in the eastern tropical Pacific Ocean, Ecuadorian coast. Bot. Mar. 59: 267-274.). 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. 2016aCarnicer O., García-Altares M., Andree K.B., et al. 2016a. First evidence of Ostreopsis cf. ovata in the eastern tropical Pacific Ocean, Ecuadorian coast. Bot. Mar. 59: 267-274.), Belize (Penna et al. 2014Penna A., Battocchi C., Capellacci S., et al. 2014. Mitochondrial, but not rDNA, genes fail to discriminate dinoflagellate species in the genus Ostreopsis. Harmful Algae 40: 40-50.), Indonesia (Penna et al. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841.), Thailand (Tawong et al. 2014Tawong W., Nishimura T., Sakanari H., et al. 2014. Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand. Harmful Algae 37: 160-171.) and Malaysia (Leaw et al. 2001Leaw L.C., Teen L.P., Ahmad A., et al. 2001. Genetic diversity of Ostreopsis ovata (Dinophyceae) from Malaysia. Mar. Biotechnol. 3: 246-255.). 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 1963Muromtsev A.M. 1963. The principal hydrology features of the Pacific Ocean. Jerusalem Post Press, Jerusalem, Israel, 417 pp., Banks, 2002Banks S. 2002. Ambiente físico. In: Danulat E., Edgar G.J. (eds), Reserva Marina de Galápagos. Línea base de la biodiversidad. Fundación Charles Darwin/Servicio Parque Nacional Galápagos, Santa Cruz, Galápagos, Ecuador, pp., Hickman 2009Hickman C.P.Jr. 2009. Evolutionary responses of marine invertebrates to insular isolation in Galapagos. Galapagos Res. 66: 32-42.). 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. 2008Schaeffer B.A., Morrison J.M., Kamykowski D., et al. 2008. Phytoplankton biomass distribution and identification of productive habitats within the Galápagos Marine Reserve by MODIS, a surface acquisition system, and in-situ measurements. Remote Sens. Environ. 112: 3044-3054). 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. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841.). 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. 2012Nascimento S.M., Correa E.V., Menezes M., et al. 2012. Growth and toxin profile of Ostreopsis cf. ovata (Dinophyta) from Rio de Janeiro, Brazil. Harmful Algae 13: 1-9.) 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. 2016aCarnicer O., García-Altares M., Andree K.B., et al. 2016a. First evidence of Ostreopsis cf. ovata in the eastern tropical Pacific Ocean, Ecuadorian coast. Bot. Mar. 59: 267-274.).

At least one other species of Ostreopsis has been identified in this study. This species fell in Ostreopsis sp. 5 (Sato et al. 2011Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983.). Morphologically, it resembles the original description of O. lenticularis, but until recently (Chomérat et al. 2019Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111.) its known genetic clade assignment could not be used to unambiguously establish these isolates as O. lenticularis because it was described by Fukuyo (1981)Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Japan. Soc. Sci. Fish 47: 967-978. prior to routine molecular characterization. The absence of the undulation of the cingulum in side view was not verified, although, according to Fukuyo (1981)Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Japan. Soc. Sci. Fish 47: 967-978., 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)Hoppenrath M., Murray S.A., Chomérat N., et al. 2014. Marine benthic dinoflagellates - unveiling their worldwide biodiversity. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany, 276 pp., who suggested that the species under the name of O. lenticularis in Faust et al. (1996)Faust M.A., Morton S.L., Quod J.P. 1996. Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae). J. Phycol. 32: 1053-1065. 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)Faust M.A., Morton S.L., Quod J.P. 1996. Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae). J. Phycol. 32: 1053-1065. is described with the two types of pores consistent with the original O. lenticularis description. Cortés-Lara et al. (2005)Cortés-Lara M.C., Cortés-Altamirano R., Sierra-Beltrán A., et al. 2005. Ostreopsis siamensis (Dinophyceae) a new tychoplanktonic record from Isabel Island National Park, Pacific Mexico. Harmful Algae News. IOC Newsletter on toxic algae and algal blooms 28: 4-5. illustrated two pore size classes in O. siamensis from the Mexican Pacific and Penna et al. (2005)Penna A., Vila M., Fraga S., et al. 2005. Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences. J. Phycol. 41: 212-225. in O. ovata from the western Mediterranean. Aligizaki and Nikolaidis (2006)Aligizaki K., Nikolaidis G. 2006. The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece. Harmful Algae 5: 717-730. 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. 2014Hoppenrath M., Murray S.A., Chomérat N., et al. 2014. Marine benthic dinoflagellates - unveiling their worldwide biodiversity. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany, 276 pp.), is not given. The morphology of the sulcal plates in Ostreopsis spp. remains poorly examined. Similarly, in our study only close-ups 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)Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111. 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)Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111., 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)Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111., 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)Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Japan. Soc. Sci. Fish 47: 967-978. (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 1995Faust M.A., Morton S.L. 1995. Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae). J. Phycol. 31: 456-463.). 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).

Table 2. – Description of rounded shaped Ostreopsis cells regarding ITS phylogeny, location, toxicity, size, morphology and the number of types of pores; n.d., not determined. ^aFirst description of the species.

Identification (morphological)	Genetic clade (ITS)	GENBANK	Location	Toxicity	Cell size (µm)	Morphology	Number of pore types	Study
O. lenticularis^a	n.d.		French Polynesia	n.d.	60-100 (DV); 45-85 (W) field sample	SEM	2	Fukuyo 1981Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Japan. Soc. Sci. Fish 47: 967-978.
O. lenticularis	n.d.		Mexican Pacific	n.d.	65-100 (DV); 50-80 (W) field sample	SEM	2	Gárate-Lizarraga et al. 2018 Gárate-Lizárraga I., González-Armas R., Okolodkov Y.B. 2018. Occurrence of Ostreopsis lenticularis (Dinophyceae: Gonyaulacales) from the Archipiélago de Revillagigedo, Mexican Pacific. Mar. Pollut. Bull. 128: 390-395.
O. lenticularis	n.d.		Colombian Caribbean	n.d.	102.1±7 (DV); 83.8±6.4 (W) field sample	SEM	2	Arbelaez et al. 2017Arbelaez N.M., Mancera Pineda J.E., Reguera B. 2017. Epiphytic dinoflagellates of Thalassia testudinum in two coastal systems of the Colombian Caribbean. Bol. Invest. Mar. Cost. - INVEMAR 46: 9-40.
O. lenticularis	n.d.		New Zealand	n.d.	70-95 (DV); 55-75 (W) field sample	SEM	2	Chang et al. 2000Chang F.H., Shimizu Y., Hay B., et al. 2000. Three recently recorded Ostreopsis spp. (Dinophyceae) in New Zealand: Temporal and regional distribution in the upper North Island from 1995 to 1997. New Zeal. J. Mar. Fresh. Res. 34: 29-39. (doubtful identification; see Chomérat et al. 2019Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111.)
O. lenticularis	O. sp. 5	AB674917/8/9	Japan	Non-toxic (LC)	n.d.	n.d.		Sato et al. 2011Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983., Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.
	O. sp. 5	JX065552	Hawaii (Pacific)	n.d.	n.d.	n.d.		Penna et al. 2014Penna A., Battocchi C., Capellacci S., et al. 2014. Mitochondrial, but not rDNA, genes fail to discriminate dinoflagellate species in the genus Ostreopsis. Harmful Algae 40: 40-50.
	O. sp. 5	KX129872	China Sea	n.d.	68-113.5 (DV); 56.5-97.3 (W) culture	SEM, Calcofluor	2-3	Zhang et al. 2018 Zhang H., Lu S., Li Y., et al. 2018. Morphology and molecular phylogeny of Ostreopsis cf. ovata and O. lenticularis (Dinophyceae) from Hainan Island, South China Sea. Phycol. Res. 66: 3-14.
	O. sp. 5	KM032221/2	Reunion Island (Indian Ocean)	Non-toxic (hemolytic)	103.9±5.1 (DV); 85.3±6.9 (W) field sample	Calcofluor	2	Carnicer et al. 2015Carnicer O., Tunin-Ley A., Andree K.B., et al. 2015. Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): Molecular, morphologic and toxicity characterization. Cryptogamie Algol. 36: 101-119.
	O. sp. 5	MH844088	Galapagos (Pacific)	Non-toxic (hemolytic; LC)	88.49±7.22 (70.94-105.36) (DV); 67.29±6.11 (W) (55.5-82.69) culture	SEM, Calcofluor	2	This study
	O. sp. 5	MH844088	Galapagos (Pacific)	Non-toxic (hemolytic; LC)	98.13±7.59 (86.29-109.86) (DV); 77.74±7.35 (68.24-96.75) (W) field sample	SEM, Calcofluor	2	This study
O. lenticularis	O. sp. 5	MK227240-48	French Polynesia (South Pacific Ocean)	Non-toxic (CBA-N2a, LC)	81.2±5.7 (DV); 67.5±6.1 (W) culture	SEM, Calcofluor	2	Chomérat et al. 2019Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111.
O. marina^a	n.d.		Caribbean and Indian Ocean	n.d.	83-111 (DV); 73-85 (W) field sample	SEM	1	Faust 1999Faust M.A. 1999. Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov., O. belizeanus sp. nov., and O. caribeanus sp. nov. Phycologia 38: 92-99.
O. labens^a	n.d.		Caribbean and Japan	n.d.	60-86 (DV); 70-80 (W) field sample	SEM	1	Faust and Morton 1995Faust M.A., Morton S.L. 1995. Morphology and ecology of the marine dinoflagellate Ostreopsis labens sp. nov. (Dinophyceae). J. Phycol. 31: 456-463.
O. lenticularis	n.d.		Japan, Southwest Indian Ocean, Caribbean	Toxic (mouse bioassay)	65-75 (DV); 57-63 (W) field sample	SEM	1	Faust et al. 1996Faust M.A., Morton S.L., Quod J.P. 1996. Further SEM study of marine dinoflagellates: the genus Ostreopsis (Dinophyceae). J. Phycol. 32: 1053-1065.
O. labens	O. sp. 6	FM244728	Malaysia	Toxic (hemolytic)	n.d.	n.d.		Penna et al. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841.
O. labens	O. sp. 6 (LSU)	upon request authors	Caribbean	Toxic (hemolytic; LC) epiphytic extract	60-85 (DV); 50-67 (W) culture	SEM	1	Moreira et al. 2012Moreira A., Rodríguez F., Riobó P., et al. 2012. Notes on Ostreopsis sp. from southern-central coast of Cuba. Cryptogamie Algol. 33: 217-224.
O. lenticularis	O. sp. 6	AF218465	Malaysia	n.d.	64-76 (DV); 52-65 (W) culture	n.d.		Leaw et al. 2001Leaw L.C., Teen L.P., Ahmad A., et al. 2001. Genetic diversity of Ostreopsis ovata (Dinophyceae) from Malaysia. Mar. Biotechnol. 3: 246-255. (doubtful identification; see Chomérat et al. 2019Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111.)
O. lenticularis	O. sp. 6	AB841255/4	Thailand	Toxic (mouse bioassay)	62.4±8.0 (DV) 48.2±5.5 (W) culture	Calcofluor	1	Tawong et al. 2014Tawong W., Nishimura T., Sakanari H., et al. 2014. Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand. Harmful Algae 37: 160-171.
O. lenticularis	O. sp. 6	JX065584	South China Sea (Vietnam)	n.d.	n.d.	n.d.		Penna et al. 2014Penna A., Battocchi C., Capellacci S., et al. 2014. Mitochondrial, but not rDNA, genes fail to discriminate dinoflagellate species in the genus Ostreopsis. Harmful Algae 40: 40-50.
O. lenticularis	O. sp. 6	AB674920/1/2	Japan	Toxic (LC), strain AB674922; non-toxic, strains AB674920/1	n.d.	n.d.		Sato et al. 2011Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983., Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.

The first ribosomal sequences for a strain from Malaysia identified as O. lenticularis were presented by Leaw et al. (2001)Leaw L.C., Teen L.P., Ahmad A., et al. 2001. Genetic diversity of Ostreopsis ovata (Dinophyceae) from Malaysia. Mar. Biotechnol. 3: 246-255., 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. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841.), and after the addition of three new sequences to the clade it was then called Ostreopsis sp. 6 (Sato et al. 2011Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983.). However, in a recent study conducted in the China Sea (Zhang et al. 2018Zhang H., Lu S., Li Y., et al. 2018. Morphology and molecular phylogeny of Ostreopsis cf. ovata and O. lenticularis (Dinophyceae) from Hainan Island, South China Sea. Phycol. Res. 66: 3-14.), O. lenticularis was clustered in another genetic clade, Ostreopsis sp. 5, together with the strains from Reunion Island (Carnicer et al. 2015Carnicer O., Tunin-Ley A., Andree K.B., et al. 2015. Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): Molecular, morphologic and toxicity characterization. Cryptogamie Algol. 36: 101-119.) and three strains isolated from Japan (Sato et al. 2011Sato S., Nishimura T., Uehara K., et al. 2011. Phylogeography of Ostreopsis along west Pacific coast, with special reference to a novel clade from Japan. PloS ONE 6: e27983.), 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)Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111., 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. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.), Reunion Island (Carnicer et al. 2015Carnicer O., Tunin-Ley A., Andree K.B., et al. 2015. Contribution to the genus Ostreopsis in Reunion Island (Indian Ocean): Molecular, morphologic and toxicity characterization. Cryptogamie Algol. 36: 101-119.) 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. 2010Penna A., Fraga S., Battocchi C., et al. 2010. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 37: 830-841., Tawong et al. 2014Tawong W., Nishimura T., Sakanari H., et al. 2014. Distribution and molecular phylogeny of the dinoflagellate genus Ostreopsis in Thailand. Harmful Algae 37: 160-171.), producers of ostreocin-d (Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.) and PLTX analogues (Moreira et al. 2012Moreira A., Rodríguez F., Riobó P., et al. 2012. Notes on Ostreopsis sp. from southern-central coast of Cuba. Cryptogamie Algol. 33: 217-224.), as well as non-toxic strains (Suzuki et al. 2012Suzuki T., Watanabe R., Uchida H., et al. 2012. LC-MS/MS analysis of novel ovatoxin isomers in several Ostreopsis strains collected in Japan. Harmful Algae 20: 81-91.) (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 GMR. The PCR amplifications of rDNA, 5.8S and ITS regions clustered the isolates obtained from 16 strains of the O. cf. ovata Atlantic/Indian/Pacific clade, and Ostreopsis sp. 5 (= O. lenticularis). The strains proved to be non-toxic according to the haemolytic assay and LC-HRMS. Morphological characters of Ostreopsis sp. 5 are similar to those of O. lenticularis according to the original description by Fukuyo (1981)Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Japan. Soc. Sci. Fish 47: 967-978. as well as by Chomérat el al. (2019)Chomérat N., Bilien G., Derrien A., et al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae 84: 95-111. 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.

ACKNOWLEDGEMENTSTop

The authors would like to thank Josselyn B. Yépez Rendón (Esmeraldas, Ecuador) for her help in field work, Àngels Tudó (Institut de Recerca i Tecnologia Agroalimentària, IRTA, Sant Carles de la Ràpita, Spain) for her assistance in data analysis, and Marcia M. Gowing (University of California at Santa Cruz, California, USA) for improving the English style. The two anonymous reviewers are thanked for their valuable comments. This work was funded by the Pontifical Catholic University of Ecuador - Sede Esmeraldas through the internal project “Characterization of the epibenthic and phytoplanktonic microalgae community in the Galapagos Islands”. IK thanks Danny Rueda and the Galapagos National Park for granting us authorization to carry out this investigation (research permit number: PC-15–19). Additionally, IK would like to thank Galapagos Conservancy, Lindblad Expedition/National Geographic Fund, Galapagos Conservation Trust, Paul M. Angell Foundation and Ecoventura for research funding provided for the CDF marine invasive species program. This publication is contribution number 2332 of the Charles Darwin Foundation for the Galapagos Islands.

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