Ludovic Sawelew
email: ludo.sawelew@gmail.com

Christopher Nuccio

Colin Foord

Jean Lorquin

Yvan Perez

P Cf Toxica

Ca

Chondria Murata

Ca-Ii

Ostreocin-B O Siamensis

N D Terajima

Nd

Symbiodiniaceae diversity and characterization of palytoxin in various zoantharians (Anthozoa, Hexacorallia)

Keywords: Liquid chromatography, mass spectrometry imaging, Palythoa, palytoxin, phylogenetics, Symbiodiniaceae, Zoanthus

Anemone-like animals belonging to the order Zoantharia are common anthozoans widely distributed from shallow to deep tropical and subtropical waters. Some species are well-known because of their high toxicity due to the presence of palytoxin (PLTX) in their tissues. PLTX is a large polyhydroxylated compound and one of the most potent toxins known. Currently, the PLTX biosynthetic pathway in zoantharians and the role of the host or the putative symbiotic organism(s) involved in this pathway are entirely unknown. To better understand the presence of PLTX in some Zoantharia, twenty-nine zoantharian colonies were analysed in this study. All zoantharian samples and their endosymbiotic dinoflagellates (Symbiodiniaceae = Zooxanthellae) were identified using DNA barcoding and phylogenetic reconstructions. Quantification of PLTX and its analogues showed that the yields contained in Palythoa heliodiscus, Palythoa aff. clavata and one potentially undescribed species of Palythoa are among the highest ever found (up to > 2 mg/g of wet zoantharian). Mass spectrometry imaging was used for the first time on Palythoa samples and revealed that in situ distribution of PLTX is mainly located in ectodermal tissues such as the epidermis of the body wall and the pharynx. Moreover, high levels of PLTX have been detected in histological regions where few or no Symbiodiniaceae cells could be observed. Finally, issues such as host-specificity and environmental variables driving biogeographical patterns of hosted Symbiodiniaceae in zoantharian lineages were discussed in light of our phylogenetic results as well as the patterns of PLTX distribution. It was concluded that (1) the variability of Symbiodiniaceae diversity may be related to ecological divergence in Zoantharia, (2) All Palythoa species hosted Cladocopium Symbiodiniaceae (formerly clade C), (3) the sole presence of Cladocopium is not sufficient to explain the presence of high concentrations of PLTX and/or its analogues and (4) the ability to produce high levels of PLTX and/or its analogues highlighted in some Palythoa species could be a plesiomorphic character inherited from their last common ancestor and subsequently lost in several lineages.

Introduction

Zoantharians (Anthozoa, Hexacorallia, Zoantharia) are sessile and usually colonial anemone-like animals widely distributed from shallow to deep tropical and subtropical waters. The order is mainly represented by the genera Palythoa Lamouroux, 1816 and Zoanthus Lamarck, 1801 that are morphologically similar. The molecular approach revealed that these two genera exhibit a wide intraspecific morphological plasticity, suggesting that previous systematics of Palythoa and Zoanthus species based on morphological characters (mainly the oral disc colour of polyps and several forms of colony growth) included a large number of synonyms [START_REF] Burnett | Zoanthids (Anthozoa, Hexacorallia) from the Great Barrier Reef and Torres Strait, Australia: systematics, evolution and a key to species[END_REF][START_REF] Reimer | Preliminary molecular examination of zooxanthellate zoanthid (Hexacorallia, Zoantharia) and associated zooxanthellae (Symbiodinium spp.) diversity in Singapore[END_REF][START_REF] Reimer | Reconsidering Zoanthus spp. diversity: molecular evidence of conspecifity within four previously presumed species[END_REF]Reimer et al. , 2006a[START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF][START_REF] Low | The order Zoantharia Rafinesque, 1815 (Cnidaria, Anthozoa: Hexacorallia): supraspecific classification and nomenclature[END_REF]. Moreover, many of the remaining species exist in sibling pairs between the Atlantic and Indo-Pacific basins [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. It is therefore almost impossible to tell species pairs apart without collection location and genetic data.

Although little is known about what exactly induces changes in the phenotype of zoantharians, some authors claimed that morphological variations in Palythoa and Zoanthus species may be a response to water turbidity [START_REF] Costa | Morphological plasticity in the reef zoanthid Palythoa caribaeorum as an adaptive strategy[END_REF], depth [START_REF] Kamezaki | Different zooxanthellae types in populations of the zoanthid Zoanthus sansibaricus along depth gradients in Okinawa, Japan[END_REF] or, more generally, to light intensity [START_REF] Ong | Morphologically plastic responses to shading in the zoanthids Zoanthus sansibaricus and Palythoa tuberculosa[END_REF]. Morphological phenotypic plasticity would facilitate the colonization of a wide variety of habitats and help organisms adapt to changes in their environment [START_REF] Burnett | Zoanthids (Anthozoa, Hexacorallia) from the Great Barrier Reef and Torres Strait, Australia: systematics, evolution and a key to species[END_REF][START_REF] Costa | Morphological plasticity in the reef zoanthid Palythoa caribaeorum as an adaptive strategy[END_REF][START_REF] Ong | Morphologically plastic responses to shading in the zoanthids Zoanthus sansibaricus and Palythoa tuberculosa[END_REF]. Reimer et al. (2006b) observed that, due to volcanic ash incorporated into their tissues, samples of Palythoa mutuki (Haddon and Shackleton, 1891) and Palythoa tuberculosa (Esper, 1805) harvested in a volcanic area were darker in colour than samples harvested in non-volcanic areas. Moreover, [START_REF] Kamezaki | Different zooxanthellae types in populations of the zoanthid Zoanthus sansibaricus along depth gradients in Okinawa, Japan[END_REF] showed that specimens of Zoanthus sansibaricus Carlgren, 1900 sampled at different depth levels had different colours.

Host-tissue colour variations of zoantharians could be due to the synthesis of different GFP-like proteins in response to different environmental parameters as observed in another Anthozoa, Montastraea cavernosa (Linnaeus, 1767) [START_REF] Kelmanson | Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida)[END_REF]. The use of phylogenetic analyses, particularly those based on a combination of mitochondrial and nuclear markers [START_REF] Sinniger | Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia)[END_REF], are therefore more appropriate to identify zoantharian species and clarify the potentially numerous synonyms [START_REF] Reimer | Reconsidering Zoanthus spp. diversity: molecular evidence of conspecifity within four previously presumed species[END_REF](Reimer et al. , 2006a[START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF].

The genus Palythoa is known for comprising highly toxic species due to the presence of palytoxin (PLTX) in their tissues [START_REF] Moore | Palytoxin a new marine toxin from a Coelenterate[END_REF][START_REF] Béress | A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zoanthid) Palythoa caribaeorum[END_REF][START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF]). PLTX, one of the most toxic natural compounds ever discovered, is a non-protein marine toxin which consists of a long, partially unsaturated (with eight double bonds) aliphatic backbone with spaced cyclic ethers and 64 chiral centres [START_REF] Uemura | Studies on palytoxins[END_REF]. Initially isolated from a Palythoa species [START_REF] Moore | Palytoxin a new marine toxin from a Coelenterate[END_REF], it can also be found in numerous other marine organisms from the same ecological region [START_REF] Aratake | Distribution of palytoxin in coral reef organisms living in close proximity to an aggregation of Palythoa tuberculosa[END_REF]. Moreover, several analogues of PLTX were discovered in various microorganisms (see Table 1). To date, Palythoa heliodiscus (Ryland and Lancaster, 2003) is the largest PLTX producer known among zoantharians with the highest recorded yield (1 mg of PLTX /g wet Palythoa) and deoxy-PLTX (3.51 mg/g wet Palythoa [START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF]). However, in most cases the toxin yield in other Palythoa species is low. During the last decade, more attention was focused on PLTX because of (1) sanitary problems due to toxic Ostreopsis spp. outbreaks [START_REF] Del Favero | Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach[END_REF] or numerous cases of severe domestic poisoning from aquarium zoantharians [START_REF] Moshirfar | Aquarium coral keratoconjunctivitis[END_REF][START_REF] Nordt | Palytoxin poisoning after dermal contact with zoanthid coral[END_REF][START_REF] Snoeks | Family with fever after cleaning a sea aquarium[END_REF][START_REF] Hall | A case of palytoxin poisoning in a home aquarium enthusiast and his family[END_REF][START_REF] Ruiz | Dangerous reef aquaristics: palytoxin of a brown encrusting anemone causes toxic corneal reactions[END_REF][START_REF] Farooq | Corneal toxicity associated with aquarium coral palytoxin[END_REF][START_REF] Barbany | Toxicidad corneal por exposición a palitoxina [Toxic corneal reaction due to exposure to palytoxin[END_REF][START_REF] Gaudchau | Verätzung durch Krustenanemone [Chemical burns caused by crust anemone[END_REF][START_REF] Jalink | Wegsmeltende hoornvliezen na de verhuizing van een zeeaquarium [Corneal melting after moving a tropical aquarium[END_REF][START_REF] Schulz | Inhalation poisoning with palytoxin from aquarium coral: case description and safety advice[END_REF][START_REF] Chang | A case of long-term neurological and respiratory sequelae of inhalational exposure to palytoxin[END_REF][START_REF] Moshirfar | Palytoxin keratitis[END_REF][START_REF] Wonneberger | Corneal perforation due to palytoxin exposure of domestic zoanthid corals[END_REF], and (2) significant toxic effects on proliferation and survival of cancer cells [START_REF] Valverde | Characteristics of palytoxin induced cytotoxicity in neuroblastoma cells[END_REF][START_REF] Ledreux | Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)[END_REF][START_REF] Kerbrat | First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium[END_REF]. Consequently, there is a growing need for high-yield sources of PLTX to facilitate its functional characterization.

So far, the PLTX biosynthetic pathway and the respective roles of putative symbiotic organism(s) involved are entirely unknown [START_REF] Aratake | Distribution of palytoxin in coral reef organisms living in close proximity to an aggregation of Palythoa tuberculosa[END_REF]. The leading hypothesis, based on structural similarities observed between PLTX and zooxanthellatoxins [START_REF] Nakamura | Isolation of zooxanthellatoxins, novel vasoconstrictive substances from the zooxanthella Symbiodinium sp[END_REF][START_REF] Drainville-Higgins | Isolation of marine metabolites from Symbiodinium species of dinoflagellates[END_REF][START_REF] Onodera | Zooxanthellamide B, a novel large polyhydroxy metabolite from a marine dinoflagellate of Symbiodinium sp[END_REF]), proposed that endosymbiotic dinoflagellates (Symbiodiniaceae = zooxanthellae) are responsible for PLTX synthesis in Palythoa. Several species of free-living dinoflagellates are also able to produce PLTX and some analogues [START_REF] Ukena | Structure elucidation of ostreocin D, a palytoxin analog isolated from the dinoflagellate Ostreopsis siamensis[END_REF][START_REF] Lenoir | First evidence of palytoxin analogues from an Ostreopsis mascarenensis (Dinophyceae) benthic bloom in southwestern Indian Ocean[END_REF][START_REF] Ciminiello | Putative palytoxin and its new analogue, ovatoxin-a, in Ostreopsis ovata collected along the Ligurian coasts during the 2006 toxic outbreak[END_REF][START_REF] Rossi | New palytoxin-like molecules in Mediterranean Ostreopsis cf. ovata (dinoflagellates) and in Palythoa tuberculosa detected by liquid chromatography-electrospray ionization time-of-flight mass spectrometry[END_REF]). However, it is noteworthy that dinoflagellates are not the only organisms to produce PLTX or analogues. For example, PLTX and 42-hydroxy-PLTX are also produced by a marine Cyanobacteria belonging to the genus Trichodesmium [START_REF] Kerbrat | First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium[END_REF][START_REF] Frolova | An enzyme linked immunosorbent assay for detecting palytoxin-producing bacteria[END_REF] detected PLTX-like compounds in Gram-negative Aeromonas sp. and Vibrio sp. bacteria using anti-PLTX antibodies. Similarly, bacteria isolated from Palythoa caribaeorum (Duchassaing and Michelotti 1860) were found to display a PLTX-like haemolytic activity [START_REF] Seemann | Detection of hemolytic bacteria from Palythoa caribaeorum (Cnidaria, Zoantharia) using a novel palytoxin-screening assay[END_REF] confirming that several prokaryotic organisms can produce at least one PLTX type. It is, therefore, possible to assume that the Symbiodiniaceae and symbiotic prokaryotes might collaborate to produce PLTX within their zoantharian host.

In an attempt to discover new valuable sources of PLTX and learn more about its biosynthetic pathway and storage into zoantharians, twenty-nine colonies were analysed. This study aims to compare the diversity of Symbiodiniaceae among closely related Palythoa and Zoanthus species and determine whether one or several Symbiodiniaceae could be related to high levels of PLTX in zoantharians. Our integrative results based on molecular analyses, HPLC, mass spectrometry and MALDI-IMS strongly suggest that the "holobiont" (host plus its microbiote) plays a crucial role in the synthesis and storage of PLTX in Zoantharia, rather than Symbiodiniaceae alone.

Materials and methods

Biological material. Twenty-eight zoantharian colonies visually consistent with Palythoa or Zoanthus species and one colony morphologically similar (Z20) were used in the present study: eighteen specimens from Florida Miami (n=13) and the Reunion Island (n =5) have been collected by the teams of Coral Morphologic and Coral Biome respectively. The remaining ten colonies have been obtained from suppliers of the aquarium industry located in Indonesia (Table 2). Representative morphotypes of all samples are illustrated in supplementary information (Fig. A). Colonies were placed at Coral Biome's facility in the same recirculating aquaculture system connected to one biological filter fed by artificial seawater (Instant Ocean salts, from Seachem, Madison, USA). The physical and chemical real-time monitored parameters were: temperature, 25 -26°C; density, 1023 -1025 kg.m -3 ; KH, 7 -8°; pH, 8.2 -8.4; Ca, 400 -450 mg.l -1 ; Mg, 1200 -1300 mg.l -1 ; nitrates < 5 mg.l -1 ; phosphates < 0.1 mg.l -1 . Specimens were exposed to a daytime photoperiod of 12 hr with an irradiance of 70 mol.quanta.m -2 .s -1 and fed twice a week with a powder of lyophilized copepods (Calanus finmarchicus) (Dupla, Gelsdorf, Germany) at a concentration of 700 -900 µg/l. DNA extraction, PCR amplification and sequencing. To amplify both zoantharian and Symbiodiniaceae genes, tissue samples consisting of a complete oral disk (small polyps) or tentacles joined by a small piece of the oral disc (large polyps) were placed in 80% (v/v) alcohol. Total genomic DNA was extracted using the DNAeasy Kit (Qiagen, Valencia, CA).

Two commonly used DNA barcode markers (e.g. Reimer et al. 2102[START_REF] Reimer | Molecular analyses of shallow-water zooxanthellate zoanthids (Cnidaria: Hexacorallia) from Taiwan and their Symbiodinium spp[END_REF][START_REF] Reimer | Ascension Island shallow-water Zoantharia(Hexacorallia: Cnidaria) and theirzooxanthellae (Symbiodinium)[END_REF][START_REF] Reimer | Zooxanthellate zoantharians (Anthozoa: Hexacorallia: Zoantharia: Brachycnemina) in the northern Red Sea[END_REF]Reimer et al. , 2020) ) were amplified to identify the zoantharian species : the mitochondrial cytochrome oxidase subunit I (COI) and the entire length of the nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA: internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2). COI sequences were amplified with the primers HCO2198 (5' -TAA ACT TCA GGG TGA CCA AAA AAT CA -3') and LCO1490 (5' -GGT CAA CAA ATC ATA AAG ATA TTG G -3'). PCR amplifications were performed as follows: an initial denaturing step at 94°C for 2 min followed by 5 cycles of 15 s denaturation at 92°C, 45 s annealing at 48°C followed by a gradual temperature increase up to 72°C during 1 min, 1 min 30 extension at 72°C, followed by 30 cycles of 15 s denaturation at 92°C, 45 s annealing at 52°C, 45 s extension at 72°C, followed by 7 min at 72°C. ITS-rDNA sequences were amplified using the primers Zoanf-ITS (5' -CTT GAT CAT TTA GAG GGA GT -3') and Zoanr-ITS (5' -CGG AGA TTT CAA ATT TGA GCT -3'). The PCR program was carried out as follows: an initial denaturing step at 94°C for 3 min, 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 50°C, 2 min extension at 72°C followed by 10 min at 72°C.

To identify the Symbiodiniaceae genera associated with the zoantharian samples, the ITS2-rDNA sequences

(5.8S ribosomal RNA, partial sequence; internal transcribed spacer 2, complete sequence; 28S ribosomal RNA gene, partial sequence) were amplified using the specific primers ITS2-F1 (5' -GAA TTG CAG AAC TCC most used marker for analysing the diversity of Symbiodiniaceae [START_REF] Shi | Untangling ITS2 genotypes of algal symbionts in zooxanthellate corals[END_REF].

PCR amplifications were performed under stringent conditions to specifically target the Symbiodiniaceae genes rather than those of zoantharians: an initial denaturing step at 94°C for 3 min followed by 12 cycles of 45 s denaturation at 94°C, 45 s annealing at 58°C and an incremental decrease of 0.5°C every cycle, 1 min extension at 72°C, followed by 20 cycles of 45 s denaturation at 94°C, 45 s annealing at 52°C and 1 min extension at 72°C followed by 7 min at 72°C. After amplification, all PCR fragments were visualized by denaturing gradient gel electrophoresis and were directly sequenced (without a cloning step) in both directions using the amplicon primers with an ABI 96-capillary 3730XL sequencer at Eurofins Genomics (Ebersberg, Germany).

Phylogenetic analyses. Three data sets were used for molecular analyses: dataset 1 for zoantharian COI sequences, dataset 2 for zoantharian ITS-rDNA sequences and dataset 3 for Symbiodiniaceae ITS2-rDNA sequences. All sequences obtained in this study were first checked using NCBI BLAST, then aligned with orthologous sequences available in public databases using CLUSTALW implemented in BioEdit v7.1.9 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The program MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]) was used to carry out a multiple alignment of ITS2-rDNA sequences (dataset 3). For each dataset, the program MODELTEST v3.0b4 [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF] was used to determine the best model of DNA evolution using the Bayesian information criterion (BIC). Molecular analyses were conducted through neighbour-joining (NJ) and maximum likelihood (ML) methods using MEGA v7.0 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF]. Bayesian inference (BI) was performed with MrBayes v3.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Topological robustness was determined using 100 non-parametric bootstrap replicates for NJ and ML analyses. For BI, Markov Chain Monte Carlo searches were done with four chains for 1,000,000 generations, with a random starting tree, default priors and Markov chains (with default heating values) sampled every 1,000 generations. It must be noted that previous studies have shown that Zoanthus sansibaricus collected along the Japanese coasts [START_REF] Reimer | Molecular evidence suggesting interspecific hybridization in Zoanthus spp. (Anthozoa: Hexacorallia)[END_REF][START_REF] Aguilar | Molecular phylogenetic hypotheses of Zoanthus species (Anthozoa: Hexacorallia) using RNA secondary structure of the internal transcribed spacer 2 (ITS2)[END_REF] and in the Red Sea (Reimer et al. 2017a) possesses two distinct types of ITS-rDNA sequences: a "normal" type that is closely related to Zoanthus gigantus ITS-rDNA sequences and a "distant" type, which is more divergent. According to the specimen considered, three combinations of alleles were identified: "normal" only, "distant" only or both [START_REF] Reimer | Molecular evidence suggesting interspecific hybridization in Zoanthus spp. (Anthozoa: Hexacorallia)[END_REF][START_REF] Aguilar | Molecular phylogenetic hypotheses of Zoanthus species (Anthozoa: Hexacorallia) using RNA secondary structure of the internal transcribed spacer 2 (ITS2)[END_REF].

MALDI-IMS analyses and localization of Symbiodiniaceae cells in Palythoa.

Polyps were collected from cultured colonies, harvested with care and then quickly frozen in a container of isopentane plunged in liquid nitrogen. After 5 min, dry polyps were stored at -80°C overnight. Tissue sections were cut using a Leica CM 1900 UV Microsystems cryostat (Leica Microsystems SAS) with a microtome chamber and a specimen holder chilled at -20°C. Sections of 18 μm were cut in crosswise directions at three levels of the polyp body.

Lengthwise sections were also made in some specimens. Sections were thaw mounted onto Indium Tin Oxide coated microscopic slides (Bruker Daltonics) adapted for MALDI-IMS, and onto Superfrost plus slides (Thermo Scientific) for epifluorescence imaging. Both types of target slides were dried in a desiccator for 45 min. Polyp sections were scanned before matrix deposition with a histology slide scanner (Opticlab H850 scanner, Plustek).

Then, 2,5-dihydroxybenzoic (DHB) acid (Bruker, Daltonics) 30 mg/ml in 50/50 (v/v) MeOH/H2O containing 0.1% (v/v) trifluoroacetic acid (TFA) was used as a matrix and applied on sections using an automatic matrix sprayer (TM-Sprayer, HTX Technologies). MALDI calibration was carried out manually with Peptide Calibration Standard 2 (Bruker Daltonics). MALDI-IMS data acquisition was performed on an Ultraflextreme mass spectrometer using the FlexControl 3.3 software (Bruker Daltonics) in positive reflectron mode and FlexImaging 3.0. The measurement regions were manually defined and surimposed with the histological images. The spatial resolution was set at 30 μm with a laser diameter of 20 μm, and 300 laser shots were accumulated for each spot. The laser power was optimized at the start directly on tissue and then fixed for the overall MALDI-IMS experiment. The images were opened with SCILS Lab v2.5 in RAW data with a baseline subtraction to maintain the resolution of the average mass spectrum. Due to the high intensity of the PLTX signal, root means square normalization was chosen for better visualization of the distribution. Endogenous autofluorescence of chlorophyll and peridinin pigments of the Symbiodiniaceae cell was revealed under an inverted microscope (Nikon Eclipse TE2000-U) using a filter cube B-2E/C corresponding to a medium band blue excitation. Images were acquired with NIS-Elements BR v2.30 imaging software.

PLTX extraction and purification. About one gram of entire zoantharian polyps was gently detached from the colony with a scalpel, chopped into several pieces and placed in 20 ml of 80% (v/v) MeOH in milliQ H2O.

MeOH was evaporated using a rotavapor and the aqueous phase extracted several times with dichloromethane to remove carotenoid and chlorophyll pigments. The aqueous phase containing PLTX was evaporated and deposited onto a two-centimetre diameter glass column filled with 10 cm 3 of C18 reversed-phase powder (Lichroprep RP18, from MERCK, France). The column was washed with acidified H2O (0.2% (v/v) formic acid), then with 50% (v/v) MeOH in acidified H2O. PLTX was finally eluted with 75% (v/v) MeOH in acidified H2O, and a dried pale-yellow solid of purified PLTX was obtained after N2 flow evaporation. The toxin was solubilised in Dimethyl sulfoxide (DMSO) and quantified using high-performance liquid chromatography (HPLC, see below). PLTX was then stored at concentrations comprised between 25 and 100 µg/µl at 4°C in darkness.

HPLC analyses and PLTX quantifications. Solvents were of HPLC grade from Biosolve (Dieuze, France). To control the purity of the PLTX fraction and quantify the toxin, 2-5 µg of PLTX in DMSO were injected and analysed by reverse-phase (RP) HPLC with a Waters equipment composed of a 1525 binary pump, a 2996 diode array detector, a 7725i Rheodyne injector fitted with a 20-µl loop, and a temperature control system. Files were acquired with the Empower software. Separations were carried out on a Cortecs RP-C18 column (4.6 × 75 mm, 2.7 µm) protected with a guard cartridge. Elution was performed at 30°C, at a flow rate of 0.8 ml/min with two linear gradients using solvent A (acetonitrile containing 0.2% (v/v) acetic acid) and solvent B (H2O containing 0.2% (v/v) acetic acid): 5% to 100% A for 10 min then 100% to 5% A for 2 min. PLTX was visualized at 263 nm and total spectra were analysed from 200 to 700 nm with the Empower software. PLTX peak areas were measured and interpolated within a calibration curve established with commercial PLTX (from Wako Pure Chemical Industries, Japan) dissolved in DMSO. The linearity of the calibration curve was indicated by a correlation coefficient (R 2 ) of 0.9948. PLTX concentration in each zoantharian sample was determined by averaging the results obtained from 3 extractions. When PLTX was not detected by HPLC in purified coral extracts, the calculated detection limit was 9 ng/mL at  265 nm and 5 ng/mL at  235 nm, the absence of the characteristic ions (bis-charged and molecular ions) in mass spectrometry (LC-QToF) systematically confirmed the absence of the toxin in these corals.

Mass spectrometry. Identification of mass spectra was done on an accurate mass spectrometer Agilent 6530 Q-TOF-MS (Agilent Technologies, USA). Chromatographic separation was performed on an RP-C18 Cortecs column (4.6 x 75 mm, 2.7 μm, Waters, UK) protected with a guard cartridge.The effluent of the HPLC mobile phase was split and guided into the electrospray ionization (ESI) source. Parameter conditions were performed as follows: capillary voltage, 3000 V; nebulizer pressure, 45 psi; nozzle voltage, 500 V; flow rate of drying gas, 7 l/min; temperature of sheath gas, 300°C; flow rate of sheath gas, 10 l/min; skimmer voltage, 65 V; OCT1 RF Vpp, 750 V; fragmentor voltage, 175 V. Elution was performed at 30°C at a flow rate of 0.4 ml/min with three linear gradients using solvent A (acetonitrile containing 0.2% (v/v) acetic acid) and solvent B (H2O containing 0.2% (v/v) acetic acid): 20% to 60% A for 35 min, 60% to 100% A for 5 min and 100% to 20% A for 5 min. The calibration was performed with a solution of CsI 1 mg/ml and used in the 100 -3200 mass range with a precision of +/-3 ppm. The data of the selected compounds were obtained by regulating diverse collision energy between 45 and 100 eV.

Results

Molecular phylogenetic analyses. Whatever the dataset considered, the BI and NJ trees presented similar topologies to the ML bootstrapped trees. Thus, their statistical values were reported onto the ML topology.

Analyses were carried out with sequences from Parazoanthidae and Hydrozoanthidae used as outgroups.

COI and ITS-rDNA sequences of zoantharians. Both COI and ITS-rDNA markers were generated for 25 of 29 specimens (Table 2). For the remaining four specimens only COI sequences were successfully generated. All failed sequences were attempted at least three times. The resulting alignment of COI sequences had a length of 402 bp (dataset 1), while ITS-rDNA had aligned lengths of 760 bp (dataset 2). All sequences have been deposited in GenBank with accession numbers MW717422 to MW717450 for COI and MW219226 to MW219250 for ITS-rDNA. Distances were calculated using a Kimura's 2-parameter model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]) with either a discrete gamma distribution (K2+Γ, lnL = -957.423, dataset 1) or a proportion of invariable sites (K2+I, lnL = -1234.887, dataset 2). BI were performed using an HKY model [START_REF] Hasegawa | Dating of the human-ape splitting by a molecular clock of mitochondrial DNA[END_REF] with stationary state frequencies fixed to equal to get a Kimura's 2-parameter model (prset statefreqpr = fixed (equal)) either with a gamma-distributed rate (dataset 1) or a proportion of invariant sites (dataset 2).

The mid-rooted phylogenetic trees based on zoantharian COI sequences had very similar topologies whatever the method considered (Fig. 1). Sequences from all Palythoa plus Zoanthus species (suborder Brachycnemina) constituted a strongly supported monophyletic group (97/99/1). Within this clade, the Zoanthus monophyly (Zoanthidae) was similarly strongly supported whatever the method considered (98/99/1) while the Palythoa monophyly (Sphenopidae) was moderately supported (71/65/0.71). Among the Palythoa species, sequences from specimens Z05, Z06, Z26, Z27 and Z29 were identical to P. mutuki (KF499742, Taiwan), P. aff. clavata (KJ794177, Ascension Island) and P. sp. 'sakurajimensis' (KF499720, Taiwan). These sequences constituted a basal polytomy that included a sequence of P. aff. clavata (JX119166, Florida The monophyly of the genus Palythoa (Sphenopidae) was strongly confirmed (97/84/0.93) in phylogenetic reconstructions based on ITS-rDNA sequences (Fig. 2). Whatever the method considered, the sibling species pairs of Palythoa from Caribbean or Indo-Pacific basins were fully recovered with high support values: (1)

P. caribaeorum / tuberculosa (100/100/1) along with a first subclade composed of the sequences from Z06 and Z29 specimens (100/100/1) the latter being sister group to a second sublade composed of P. aff. mutuki and P.

grandiflora (99/96/0.58), (2) P. aff. clavata / sp. 'sakurajimensis' (100/100/1) including the sequences from Z05, Z26 and Z27 specimens, (3) a clade strongly supported (99/99/1) composed of two sister groups: P. variabilis / heliodiscus and the sequences from Z03, Z04 and Z07 on the one hand (99/64/1), and P. grandis plus Z02 on the other (100/100/1). Moreover, the ITS-rDNA analyses confirmed the sister group relationships of Z20 and Terrazoanthus sp. (82/84/1). While the validity of the Caribbean / Indo-Pacific sibling species pairs Z. pulchellus / kuroshio (100/100/1), Z. sociatus / sansibaricus (100/100/1) and Z. solanderi / gigantus (100/100/1) was confirmed, the Zoanthidae appeared paraphyletic in the ITS-rDNA trees because of the presence of an additional allele (the 'distant' allele) previously amplified in Z. sansibaricus and several related specimens (Z15, Z17, Z23 -Z25). No 'normal' allele could be identified in the Z. sansibaricus specimens analysed in this study, a result which confirms that the 'distant' allele is much more common (for an explanation on the origin of the "distant"

and "normal" ITS-rDNA alleles see Aguilar and Reimer 2010). ITS2-rDNA sequences of Symbiodiniaceae. In this study, we used the taxonomic nomenclature given by LaJeunesse et al. ( 2018) who revised the Symbiodiniaceae systematics and divided the former genus Symbiodinium into seven new genera: Symbiodinium, Breviolum, Cladocopium, Durusdinium, Effrenium, Fugacium and Gerakladium (formerly described as clades A, B, C, D, E, F, and G, respectively). Symbiodiniaceae ITS2-rDNA sequences were successfully generated from all zoantharian specimens (Table 2).

After the sequencing step, the ITS2-rDNA sequence displayed clear chromatograms in both forward and reverse directions with no 'double-peaks' or mixed signals. Therefore, there was no need for a cloning step to investigate the intragenomic variability. Although several Symbiodiniaceae taxa may be hosted by a single zoantharian colony, a unique one is commonly found to be predominant. When distinct Symbiodiniaceae are hosted by the same coral, they usually belong to distinct genera (see Hume et al. 2019 and references therein). Therefore, the sequences obtained in this study were interpreted as representing the sole or dominant Symbiodiniaceae genus in each sample.

The sequences isolated from all the 29 samples (Table 2), together with GenBank sequences, built up a total dataset of 47 sequences. The new Symbiodiniaceae sequences have been deposited in GenBank with accession numbers MW077616 to MW077644. The complete alignment displayed 277 positions (dataset 3). Distances were calculated using a Kimura's 2-parameter model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]) with a proportion of invariable sites (K2+I lnL = -1956.876). Phylogenetic analyses of ITS2-rDNA sequences, which were rooted on the midpoint, yielded well-resolved trees in which the main genera of Symbiodiniaceae previously described in cnidarians received high support values whatever the method considered (Fig. 3). Symbiodiniaceae belonging to the genus Cladocopium (formerly the generalist clade C) were identified in all Palythoa (Z01 -Z07, Z26 and Z27) as well as in the Terrazoanthus (Z20) and nine Zoanthus specimens (Z14 -Z17, Z21, Z22, Z24, Z25 and Z29). The sequence amplified from specimen Z12 constituted a well-supported group with the sequence EU333714

(90/78/0.61) within the genus Durusdinium (formerly clade D). ITS2-rDNA sequences belonging to the genus Symbiodinium (formerly clade A) were divided into three well-supported subclades: the sequences Z08 -Z11 and Z13 (100/77/0.88); the sequences Z18, Z19, Z28 along with the GenBank sequences AF427465 and HM130506 (100/99/0.93); the sequences Z23 and AB190280 (91/70/0.76). The concentrations of PLTX in samples, expressed in milligrams of toxin per gram of wet zoantharian, were determined by averaging 3 extractions. PLTX was detected in 6 Palythoa samples (Z03, Z04, Z05, Z07, Z26, Z27, Table 2). No PLTX was detected in P. caribaeorum (Z01), P. grandis (Z02), P. mutuki (Z06 and Z29),

Terrazoanthus sp. (Z20) and Zoanthus samples (Z11 and Z17) despite at least two attempts with final DMSO solutions highly concentrated to increase the HPLC detection threshold. The highest PLTX concentrations were measured in Palythoa sp. Z05 (2.22 ± 0.041 mg/g wet), P. heliodiscus (Z07, 1.618 ± 0.434 mg/g wet) and P. aff. clavata (Z26, 1.077 ± 0.072 mg/g wet; Z27, 1.265 ± 0.181 mg/g wet). Lower PLTX concentrations were detected in two others P. heliodiscus samples (Z03, 0.053 ± 0.003 mg/g wet; Z04, 0.200 ± 0.034 mg/g wet).

Three highly toxic samples were analysed using mass spectrometry (Fig. 4).

Distribution of PLTX and Symbiodiniaceae cells. Sagittal and cross-sections were analysed by MALDI-IMS

to localize PLTX only in tissues of Palythoa sp. Z05 (representative of three polyps). Indeed, our access to the biological material was sometimes limited, in particular for P. aff. clavata (Z26 and Z27). Likewise, during handling, polyps closely related to P. heliodiscus (Z03, Z04 and Z07) decreased in size because of strong body contraction and secreted large amount of mucus what makes them poor candidates to carry out histological sectioning. Thus, we only selected Z05 specimen for MALDI-IMS because they were the biggest ones, with a solid body and producing less amount of mucus. It also exhibited the highest PLTX content allowing to expect a strong signal. PLTX was easily identified by MALDI-IMS and displayed a non-homogeneous distribution in polyp tissues (Fig. 5). The ectodermal tissues, e.g. the epidermis of the body wall and the pharynx, exhibited the highest concentrations (Fig. 5A,B). PLTX was also found in some tissues of endodermal origin but with concentrations usually lower than in the ectodermal ones (Fig. 5A-F). Amongst the endodermal tissues, the outer side of the endodermal fold close to the epidermis and the inner layer around the pharynx contained the highest PLTX concentrations (Fig. 5A,B). A very small amount of PLTX was detected in the gastrodermis of the enteron and septa (Fig. 5C-F). High concentrations of PLTX were also detected in the mucus-like secretion surrounding the polyps, especially well visible in the sagittal sections executed along the apical-basal axis of the body (data not shown). No PLTX was observed in the tentacles whatever the tissue considered (Fig. 5A,B).

Sections were also analysed by epifluorescence microscopy to localize the endogenous autofluorescence due to chlorophyll and peridinin pigments of the Symbiodiniaceae cells (Fig. 5G-I). A strong signal was detected in the epidermis of both the body wall and the tentacles, the endodermal fold (outer layer of the gastrodermis below the epidermis) at the level of the mouth opening and the inner layer of the gastrodermis constituting the enteron wall.

The gastrodermis constituting the septa walls located in the median part of the body also contained numerous Symbiodiniaceae cells (Fig. 5H), the number of which significantly decreased towards the apex (Fig. 5G) and the base (Fig. 5I).

Discussion

Zoantharia species identification. The unambiguous identification of a zoantharian species is a difficult task due to the lack of reliable diagnostic characters and high levels of intraspecific morphological variation mainly marked in their oral disk colour pattern and the form of the colony [START_REF] Burnett | Zoanthids (Anthozoa, Hexacorallia) from the Great Barrier Reef and Torres Strait, Australia: systematics, evolution and a key to species[END_REF][START_REF] Reimer | Reconsidering Zoanthus spp. diversity: molecular evidence of conspecifity within four previously presumed species[END_REF][START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. Six cases of intraspecific morphological plasticity were seen among the Palythoa and Zoanthus colonies used in this study: (1) P. aff. clavata Z26 and Z27;

(2) P. mutuki Z06 and Z29; (3) P. heliodiscus Z03, Z04 and Z07; (4) Z. kuroshio Z14 and Z21; (5) Z. sociatus / sansibaricus Z15, Z17, Z23 -Z25 and Z28; (6) Z. gigantus / solanderi Z08 -Z13. In all cases, these zoantharians had either identical or very similar COI and ITS-rDNA sequences whereas colour phenotypes varied considerably (see supplementary informations, Fig. A). In addition to this, a previous phylogenetic study based on mitochondrial and nuclear sequences indicated that Z. vietnamensis and Z. kuroshio might be conspecific (Reimer et al. 2006a).

Both our COI and ITS-rDNA tree patterns confirmed the previous topology of Zoanthus and Palythoa species, which displayed complexes of sibling species pairs between the Caribbean and Indo-Pacific regions [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. Indeed, in our results, the Palythoa species are clustered into three complexes: (1) P. caribaeorum / tuberculosa, (2) P. aff. clavata / sp. 'sakurajimensis', and (3) P. variabilis / heliodiscus. Zoanthus species were also divided into three complexes: (1) Z. pulchellus / kuroshio, (2) Z. sociatus / sansibaricus, and (3) Z. solanderi / gigantus. These sibling species pairs result from the closure of the Isthmus of Panama 3 million years ago [START_REF] Holcombe | Paleocurrents in the eastern Caribbean: geologic evidence and implications[END_REF][START_REF] O'dea | Formation of the Isthmus of Panama[END_REF] and they consist of genetically identical or closely similar zoantharians that were previously named differently depending on their external morphology and location (Caribbean-Atlantic or Indo-Pacific regions) [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. P. grandis, known for its endemism in the Caribbean Sea, is the only sample that does not fit into any of the complexes listed above and does not have any recorded sister species in the Indo-Pacific basin. These findings are congruent with those of [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF].

Finally, our molecular analyses showed that Palythoa sp. Z05 is a putatively undescribed Indo-Pacific species. Its closest relative is P. aff. clavata found in Florida waters [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. The morphology of Palythoa sp. Z05 does not match conclusively with any Indo-Pacific described species but displays striking similarities with a specimen from the Cape Verde Islands named Palythoa sp. 265 [START_REF] Reimer | Zoanthids of the Cape Verde Islands and their symbionts: previously unexamined diversity in the Northeastern Atlantic[END_REF]) which is also genetically very close to P. aff. clavata [START_REF] Reimer | Species diversity of shallow water zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida[END_REF]. Later, [START_REF] Reimer | Ascension Island shallow-water Zoantharia(Hexacorallia: Cnidaria) and theirzooxanthellae (Symbiodinium)[END_REF] identified new specimens from Ascension Island belonging to the clavata complex. Both COI and ITS-rDNA markers have

shown that the undescribed Indo-Pacific species P. sp. 'sakurajimensis' is close to Palythoa sp. Z05. However, this last result is not congruent with morphological data, emphasizing once more that, in zoantharians, genetic similarities do not necessarily reflect morphological ones. In general, species boundaries are primarily based on morphology and it is usually assumed that morphological variations reflect reproductive isolation along with genetic differentiation. However, genetic studies and fertilization trials producing viable larvae suggest that morphological and genetic distinctions do not always correlate in corals (for a review see [START_REF] Miller | No clear genetic distinction between morphological species within the coral genus Platygyra[END_REF]. Hybridization on coral reefs is common and widespread (for a review see [START_REF] Richards | Hybridisation on coral reefs and the conservation of evolutionary novelty[END_REF]. So far, even if it is reasonable to consider Palythoa sp. Z05 as an undescribed species based on its morphology and location of collection, it is not possible to assess to which extent hybridization with colonies of P. sp. 'sakurajimensis' occurs naturally on the reef.

Symbiodiniaceae diversity.

According to previous studies, different Zoantharia species may have different patterns of association with Symbiodiniaceae [START_REF] Reimer | Molecular analyses of shallow-water zooxanthellate zoanthids (Cnidaria: Hexacorallia) from Taiwan and their Symbiodinium spp[END_REF]. In Zoanthus samples, our results highlighted a correlation between the geographical origin of the colony and the Symbiodiniaceae taxon. Indo-Pacific species predominantly hosted Cladocopium (Z. kuroshio, 3/3; Z. sansibaricus, 4/5) and Durusdinium (Z. gigantus, 1/1) whereas most species from the Caribbean-Atlantic basin hosted Symbiodinium (Z. solanderi, 5/5; Z. aff.

pulchellus, 2/3; Z. sociatus, 1/1). These findings are consistent with previous studies that identified Symbiodiniaceae belonging to the genus Cladocopium in Indo-Pacific Zoanthus species sampled in Japanese coastal waters [START_REF] Reimer | Preliminary molecular examination of zooxanthellate zoanthid (Hexacorallia, Zoantharia) and associated zooxanthellae (Symbiodinium spp.) diversity in Singapore[END_REF][START_REF] Reimer | Molecular analyses of shallow-water zooxanthellate zoanthids (Cnidaria: Hexacorallia) from Taiwan and their Symbiodinium spp[END_REF]) while the genus Symbiodinium was predominant in colonies collected in coastal waters off Cape Verde and Brazil [START_REF] Reimer | Zoanthids of the Cape Verde Islands and their symbionts: previously unexamined diversity in the Northeastern Atlantic[END_REF][START_REF] Fontenele Rabelo | Symbiodinium diversity associated with zoanthids (Cnidaria: Hexacorallia) in Northeastern Brazil[END_REF][START_REF] López | Diversity of zoantharian species and their symbionts from the Macaronesian and Cape Verde ecoregions demonstrates their widespread distribution in the Atlantic Ocean[END_REF]. However, all Zoanthus specimens, including Z. pulchellus, Z. aff. pulchellus and Z. solanderi collected in the Canary Islands hosted Cladocopium [START_REF] López | Diversity of zoantharian species and their symbionts from the Macaronesian and Cape Verde ecoregions demonstrates their widespread distribution in the Atlantic Ocean[END_REF]. Cladocopium is considered as an Indo-Pacific generalist, known from multiple hosts and environment (Reimer et al., 2006c).

According to [START_REF] Finney | The relative significance of host-habitat, depth, and geography on the ecology, endemism, and speciation of coral endosymbionts in the genus Symbiodinium[END_REF], who analysed the Symbiodiniaceae diversity of 45 genera of Cnidaria, symbiont specificity in the Caribbean is higher than that observed in the Indo-Pacific where Cladocopium dominates in many coral communities. This could be influenced by geographical isolation and habitat depth correlated with changes in ambient irradiance. Indeed, it has been shown that stress tolerant Symbiodinium and

Durusdinium perform well at high irradiance and high temperature respectively and can increase bleaching resistance of the host [START_REF] Lajeunesse | Outbreak and persistence of opportunistic symbiotic dinoflagellates during the 2005 Caribbean mass coral 'bleaching' event[END_REF][START_REF] Wang | Determination of the thermal tolerance of Symbiodinium using the activation energy for inhibiting photosystem II activity[END_REF][START_REF] Silverstein | Change in algal symbiont communities after bleaching, not prior heat exposure, increases heat tolerance of reef corals[END_REF]. In our study, the predominance of Symbiodinium spp. in Z. solanderi, Z. sociatus and Z. aff. pulchellus from the Caribbean might suggest an adaptation to the environment in relation to abiotic stress tolerance. A similar strategy has been described in several Indo-Pacific zoantharian species collected in the Persian Gulf (Symbiodinium spp.

predominant) which was also interpreted as an adaptation to isolated regions characterised by an extreme physical environment [START_REF] Koupaei | Phylogeny of Symbiodinium populations in zoantharians of the northern Persian Gulf[END_REF].

All Palythoa species analysed in this study were associated with Symbiodiniaceae belonging to the generalist genus Cladocopium. This same genus has been observed in all samples of P. tuberculosa and P. mutuki harvested in Japanese waters (Reimer et al. 2006c;[START_REF] Mizuyama | Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats[END_REF] as well as in colonies of the Penghu Islands of Taiwan [START_REF] Reimer | Molecular analyses of shallow-water zooxanthellate zoanthids (Cnidaria: Hexacorallia) from Taiwan and their Symbiodinium spp[END_REF]. Similarly, all specimens of P. aff. clavata, P. grandiflora and P. caribaeorum examined from the Canary and Cape Verde Archipelagos hosted Cladocopium, regardless of location or depth [START_REF] López | Diversity of zoantharian species and their symbionts from the Macaronesian and Cape Verde ecoregions demonstrates their widespread distribution in the Atlantic Ocean[END_REF]. However, previous studies in Palythoa species revealed that their Symbiodiniaceae diversity is somewhat flexible being associated either with Cladocopium or Durusdinium, notably for Palythoa caesia Dana, 1846 from the Indian Ocean [START_REF] Burnett | Longitudinal variation in algal symbionts (zooxanthellae) from the Indian Ocean zoanthid Palythoa caesia[END_REF], P. tuberculosa and P. mutuki in Singapore waters [START_REF] Reimer | Preliminary molecular examination of zooxanthellate zoanthid (Hexacorallia, Zoantharia) and associated zooxanthellae (Symbiodinium spp.) diversity in Singapore[END_REF] and in the northern Persian Gulf [START_REF] Koupaei | Phylogeny of Symbiodinium populations in zoantharians of the northern Persian Gulf[END_REF]) and P. tuberculosa in the Red sea (Reimer et al. 2017a) and the South China Sea off the Vietnam coasts [START_REF] Sikorskaya | Lipidomes of phylogenetically different symbiotic dinoflagellates of corals[END_REF]. Our results, at least for the genus Zoanthus, support the idea that Symbiodiniaceae diversity is influenced by regional distribution and ecological specialisation, as previously suggested [START_REF] Reimer | Zooxanthellate zoantharians (Anthozoa: Hexacorallia: Zoantharia: Brachycnemina) in the northern Red Sea[END_REF][START_REF] Mizuyama | Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats[END_REF][START_REF] Wee | Symbiodiniaceae diversity of Palythoa tuberculosa in the central and southern Red Sea influenced by environmental factors[END_REF].

Interspecific and intraspecific toxicity variability in Zoantharia. One of the goals of this study was to use the evolutionary relationships revealed by molecular phylogenetic analyses, as well as the comparative analysis of PLTX contents, to predict zoantharian toxicity in a phylogenetic context and investigate the relation between toxicity and Symbiodiniaceae strains. To date, only one study of the relation between toxicity and phylogeny in Zoantharia has been conducted, with a reduced-scale taxonomic sampling [START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF]). To assess the potential exposure of PLTX to marine aquarium hobbyists, specimens were identified through genetic analysis of 16S and COI markers. They investigated 16 specimens and tested the toxicity for the first time. They found four toxic specimens closely related to P. heliodiscus containing PLTX and PLTX-like compounds (range 0.5-3.5 mg/g wet zoantharian). [START_REF] Hamade | Suspected palytoxin inhalation exposures associated with zoanthid corals in aquarium shops and homes-Alaska, 2012-2014[END_REF] reported 7.3 mg and 6.2 mg/g wet zoantharian of PLTX in two specimens consistent with P. heliodiscus. These authors did not identified another PLTX analogue in these highly toxic specimens.

In the present study, none of the Zoanthus and Terrazoanthus tested contained PLTX or PLTX-like compounds but we observed toxicity variations in Palythoa. Specimens belonging to the heliodiscus complex were highly toxic which is consistent with the earlier findings of [START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF]. Our results also demonstrated, for the first time, the presence of PLTX molecules in P. aff. clavata (Z26, Z27), an undescribed species from the Atlantic Ocean. Palythoa sp. Z05, another undescribed species from Indo-Pacific of the clavata complex, contains one of the highest amount of PLTX ever found in a zoantharian (2.22 ± 0.410 mg/g wet zoantharian).

The mean recorded value of 0.22% (w/w) PLTX is high compared to the values found in the literature (see Table 1) and is eight times more than the first value of 0.027% (w/w) recorded by [START_REF] Moore | Palytoxin a new marine toxin from a Coelenterate[END_REF] from Palythoa toxica (Walsh and Bowers, 1971). [START_REF] Fraga | First identification of palytoxin-like molecules in the Atlantic coral species Palythoa canariensis[END_REF] demonstrated the presence of PLTX, 42-OH-PLTX, and six minor PLTX-like molecules in Palythoa canariensis Haddon and Duerden, 1896, a species which has been considered a junior synonym to P. aff. clavata based on molecular data [START_REF] López | Diversity of zoantharian species and their symbionts from the Macaronesian and Cape Verde ecoregions demonstrates their widespread distribution in the Atlantic Ocean[END_REF]). The PLTX content in P. canariensis was estimated at 0.27 mg/g of lyophilized zoantharian using UPLC-IT-TOF-MS. In the sample of P. heliodiscus (Z07) we identified a mixture of bishomo-PLTX and PLTX. Although the concentration was high and corresponded to 0.16% (w/w), it was lower than the deoxy-PLTX concentration of 0.35% (w/w) measured in the same species by [START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF] who also found a maximum of 1.164 mg/g wet zoantharian of PLTX corresponding to 0.12% (w/w). This result is very close to the concentrations of PLTX measured in both the purple (Z27, 0.13%) and the green (Z26, 0.11%) morphotypes of P. aff. clavata.

The PLTX molecule identified in Palythoa sp. Z05, P. aff. clavata (Z26, Z27) and P. heliodiscus (Z07) has a molecular weight of 2679 Da corresponding to the PLTX previously found in P. tuberculosa, P. toxica, P. caribaeorum and P. heliodiscus [START_REF] Moore | Palytoxin a new marine toxin from a Coelenterate[END_REF][START_REF] Kimura | Purification of the toxin in a zoanthid Palythoa tuberculosa[END_REF][START_REF] Béress | A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zoanthid) Palythoa caribaeorum[END_REF][START_REF] Deeds | Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade[END_REF]. The bishomo-PLTX isolated from P. heliodiscus (Z07) has a molecular weight of 2706 Da and is identical to the toxin identified in P. tuberculosa [START_REF] Uemura | Studies on palytoxins[END_REF]. Finally, no PLTX or PLTX-like compounds were observed in P. mutuki, P. caribaeorum and P. grandis analysed in this study. Taken together, these results show a certain variability in both inter-and intraspecific toxicity in Palythoa. According to the most parsimonious scenario, phylogenetic patterns associated with comparative toxicity analysis suggest that the ability to produce and store a large amount of PLTX is likely a plesiomorphic character inherited from the last common ancestor of all Palythoa species that would have been lost independently in several lineages (Fig. 6). [START_REF] Béress | A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zoanthid) Palythoa caribaeorum[END_REF] were able to find 30 µg/g in wet Palythoa caribaeorum (Duchassaing and Michelotti, 1860). In contrast to an earlier report [START_REF] Gleibs | Studies on the origin and distribution of palytoxin in a Caribbean coral reef[END_REF] and according to our results and the study of Arakate et al. (2016), PLTX or PLTX-like compounds were only detected in Palythoa colonies but not in other zoantharians such as Zoanthus or Terrazoanthus. Scale bars = 1 cm.

All toxic species investigated in this study were associated with Symbiodiniaceae belonging to the generalist genus Cladocopium which is the most species-rich, ecologically abundant, and broadly distributed genus within the Symbiodiniaceae [START_REF] Thornhill | Host-specialist lineages dominate the adaptive radiation of reef coral endosymbionts[END_REF][START_REF] Lajeunesse | Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts[END_REF]. Whilst ITS2 of the rRNA gene remains the most popular marker used to infer the Symbiodiniaceae systematics [START_REF] Shi | Untangling ITS2 genotypes of algal symbionts in zooxanthellate corals[END_REF], the species genetic delineation within Cladocopium is generally very difficult due to a very low interspecies variability in these sequences [START_REF] Thornhill | Host-specialist lineages dominate the adaptive radiation of reef coral endosymbionts[END_REF][START_REF] Reimer | Zooxanthellate zoantharians (Anthozoa: Hexacorallia: Zoantharia: Brachycnemina) in the northern Red Sea[END_REF][START_REF] Mizuyama | Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats[END_REF]. Considering ecological, geographic, reproductive, and genetic patterns, Cladocopium may consist of hundreds of species [START_REF] Thornhill | Host-specialist lineages dominate the adaptive radiation of reef coral endosymbionts[END_REF]). If so, there are likely distinct Cladocopium species present within toxic species of Palythoa, the identification of which may only be demonstrated with higher phylogenetic resolution markers (LaJeunesse and Thornhill 2011; [START_REF] Noda | A preliminary survey of zoantharian endosymbionts shows high genetic variation over small geographic scales on Okinawa-jima Island, Japan[END_REF][START_REF] Reimer | Zooxanthellate zoantharians (Anthozoa: Hexacorallia: Zoantharia: Brachycnemina) in the northern Red Sea[END_REF]). To further describe the relationships between the Cladocopium strains and toxicity variability of their Palythoa hosts, it will be necessary to carry out studies based on alternative markers such as, for example, the plastid mini-circle non-coding region [START_REF] Moore | Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium[END_REF][START_REF] Takishita | Comparison of phylogenies based on nuclearencoded SSU rDNA and plastid-encoded psbA in the symbiotic dinoflagellate genus Symbiodinium[END_REF][START_REF] Reimer | Zooxanthellate zoantharians (Anthozoa: Hexacorallia: Zoantharia: Brachycnemina) in the northern Red Sea[END_REF][START_REF] Mizuyama | Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats[END_REF]. Meta-barcoding using NGS-based analyses should be also carried out for a more detailed picture of intra-and interspecific ITS diversity (see [START_REF] Hume | SymPortal: a novel analytical framework and platform for coral algal symbiont nextgeneration sequencing ITS2 profiling[END_REF].

Biosynthesis and storage of PLTX in Palythoa species. Since its first isolation in 1971 by Moore and Scheuer, the origin of PLTX in Palythoa spp. is still a matter of debate. So far, the toxin synthetic pathway and the putative symbiotic organism(s) involved are entirely unknown. The main current hypothesis claims that the endosymbiotic Symbiodiniaceae is responsible for the presence of PLTX in Palythoa species [START_REF] Nakamura | Isolation of zooxanthellatoxins, novel vasoconstrictive substances from the zooxanthella Symbiodinium sp[END_REF][START_REF] Drainville-Higgins | Isolation of marine metabolites from Symbiodinium species of dinoflagellates[END_REF][START_REF] Onodera | Zooxanthellamide B, a novel large polyhydroxy metabolite from a marine dinoflagellate of Symbiodinium sp[END_REF]). However, the lack of correlation between chlorophyll a and PLTX contents contradicted its role in toxin synthesis [START_REF] Gleibs | Studies on the origin and distribution of palytoxin in a Caribbean coral reef[END_REF]. The implication of other potential producers, such as symbiotic prokaryotes, which can synthesize PLTX-like secondary metabolites may also be considered [START_REF] Nakamura | Isolation of zooxanthellatoxins, novel vasoconstrictive substances from the zooxanthella Symbiodinium sp[END_REF][START_REF] Frolova | An enzyme linked immunosorbent assay for detecting palytoxin-producing bacteria[END_REF][START_REF] Seemann | Detection of hemolytic bacteria from Palythoa caribaeorum (Cnidaria, Zoantharia) using a novel palytoxin-screening assay[END_REF][START_REF] Kerbrat | First evidence of palytoxin and 42-hydroxy-palytoxin in the marine cyanobacterium Trichodesmium[END_REF]. Interestingly, a PLTX-like haemolytic activity was identified in bacteria isolated from P. caribaeorum [START_REF] Seemann | Detection of hemolytic bacteria from Palythoa caribaeorum (Cnidaria, Zoantharia) using a novel palytoxin-screening assay[END_REF] showing that some symbiotic bacteria alone can produce at least one PLTX type. In this study, MALDI-IMS analysis carried out for the first time on histological cross-sections through a Palythoa specimen showed that high levels of PLTX were not exclusively colocalised with Symbiodiniaceae cells, notably in the epidermis of the body wall and the pharynx. It should be noted that we also carried out cultures of Symbiodiniaceae cells isolated from Palythoa sp. Z05 and P. heliodiscus, but we could not show that these isolated cells constituted the exclusive PLTX producers (data not shown). All this evidence certainly suggests that the sole implication of Cladocopium is unlikely and supports the involvement of others symbiotic microorganisms as well as the cnidarian host to produce and store PLTX. This could explain the toxicity differences observed between genetically close species of Palythoa hosting genetically close Symbiodiniaceae of the genus Cladocopium, as illustrated by our molecular results. Accordingly, this could mean that the holobiont itself holds a decisive role in providing a favourable environment where crucial organisms are united for the synthesis and storage of PLTX.

In conclusion, this study partially answers the longstanding question on the origin of PLTX in zoantharians.

Molecular phylogenies combined with chemical analyses showed that the ability to produce and store high levels of PLTX and/or its analogues in some Palythoa species could be inherited from their last common ancestor and subsequently lost in several lineages. All toxic and non-toxic Palythoa species hosted Cladocopium

Fig. 1 .

 1 Fig. 1. Mid-rooted maximum-likelihood phylogenetic tree based on zoantharian mitochondrial cytochrome oxidase subunit I (COI) sequences. Support values obtained using different reconstruction methods are indicated

Fig. 2 .

 2 Fig. 2. Mid-rooted maximum-likelihood phylogenetic tree based on zoantharian internal transcribed spacer of ribosomal DNA (ITS-rDNA) sequences. Support values obtained using different reconstruction approaches are indicated at branch nodes (maximum likelihood, ML; neighbour joining, NJ; and Bayesian inference, BI). Support values are displayed when ML > 80, NJ > 85 or BI > 0.85. Sequences newly obtained in this study are represented in bold. Sequences from previous studies are included with GenBank accession numbers and location when available.

Fig. 3 .

 3 Fig. 3. Maximum-likelihood tree of Symbiodiniaceae internal transcribed spacer of ribosomal DNA (ITS-2) sequences. Support values obtained using different reconstruction approaches are indicated at branch nodes (maximum likelihood, ML; neighbour joining, NJ; and Bayesian inference, BI). Support values are displayed when ML > 80, NJ > 85 or BI > 0.85. Sequences newly obtained in this study are represented in bold. Sequences from previous studies are included with GenBank accession numbers and location when available.

  Fig. B), which corresponds to the characteristic PLTX Amoiety (C16H27N2O5) and results from the cleavage between carbon 8 and 9. For P. heliodiscus (Z07), fragmentation of the bishomo-PLTX bi-and tri-charged molecular ions produced a fragment ion at m/z 355 (supplementary informations, Fig.B) corresponding to the bishomo-PLTX A-moiety (C18H31N2O5) and also resulting from the cleavage between carbon 8 and 9. These results confirmed the presence of both PLTX and bishomo-PLTX in P. heliodiscus (Z07) tissues.

Fig. 4 .

 4 Fig. 4. High-performance liquid chromatography profile at 263 nm (left column) and high-resolution mass spectrometric analysis (right column) of PLTX extracted from [A] Palythoa sp. Z05, [B] Palythoa heliodiscus (Z07) and [C] Palythoa aff. clavata (Z26). The inserted graph in the left column shows the UV spectra of the toxin at the HPLC 263 nm peak apex. All samples contained a 327 Da fragment and UV maxima at 233 and 263 nm, which are characteristic of PLTX and the majority of its analogues.

Fig. 5 .

 5 Fig. 5. Cross-sections of Palythoa sp. Z05. Sections at the mouth and tentacles (A), actinopharynx (C) and basal peduncle (E) levels, demonstrating the location and relative concentrations of palytoxin (PLTX) by MALDIimaging mass spectrometry. Areas delimited by white rectangles are enlarged (B, D, F) and compared with images of the same histological regions exhibiting endogenous autofluorescence due to photosynthetic pigments of Symbiodiniaceae cells (G-I). Abbreviations: En, enteron; Ep, epidermis; Ph, pharynx; Se, septa; Te, tentacles. Colour scale, highest PLTX concentrations (100%) -no PLTX (0%). Scale bars = 3 mm (A, C, E), 0.5 mm (B, D, F, G-I).

Fig. 6 .

 6 Fig. 6. Palytoxin (PLTX) contents in the genera Palythoa, Terrazoanthus and Zoanthus determined in the present and previous studies. Red: high PLTX concentration (> 300 µg/g wet). Orange: weak PLTX concentration. Green: no PLTX detected. Black: not determined. CAP: common ancestor of all Palythoa species.Palythoa tuberculosa(Esper, 1805) has not been used in the present study but[START_REF] Hirata | Several compounds from Palythoa tuberculosa (Coelenterata)[END_REF] measured low concentration of PLTX (10 µg/g wet Palythoa) in this species. Similarly,[START_REF] Béress | A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zoanthid) Palythoa caribaeorum[END_REF] were able to find

Table 1 .

 1 Known molecules of the palytoxin family identified in Zoantharia, Cyanobacteria, algae and dinoflagellates.

	Compound	Organism	MW	Concentration	Reference

Table 2 .

 2 Sampling

	Z01	Caribbean (Florida, Miami)	-	+	+	Palythoa caribaeorum	Cladocopium	Not detected
	Z02	Caribbean (Florida, Miami)	+	+	+	P. grandis	Cladocopium	Not detected
	Z03	Indo-Pacific	+	+	+	P. heliodiscus	Cladocopium	0.053 ± 0.003
	Z04	Indo-Pacific	+	+	+	P. heliodiscus	Cladocopium	0.200 ± 0.034
	Z05	Indo-Pacific	+	+	+	P. sp. Z05	Cladocopium	2.22 ± 0.041
	Z06	Indo-Pacific	+	+	+	P. mutuki	Cladocopium	Not detected
	Z07	Indo-Pacific	+	+	+	P. heliodiscus	Cladocopium	1.618 ± 0.434
	Z08	Caribbean (Florida, Miami)	+	+	+	Zoanthus solanderi	Symbiodinium	/
	Z09	Caribbean (Florida, Miami)	+	+	+	Z. solanderi	Symbiodinium	/
	Z10	Caribbean (Florida, Miami)	+	+	+	Z. solanderi	Symbiodinium	/
	Z11	Caribbean (Florida, Miami)	+	+	+	Z. solanderi	Symbiodinium	Not detected
	Z12	Indo-Pacific	+	+	+	Z. gigantus	Durusdinium	/
	Z13	Caribbean (Florida, Miami)	+	+	+	Z. solanderi	Symbiodinium	/
	Z14	Indo-Pacific	+	+	+	Z. kuroshio	Cladocopium	/
	Z15	Indo-Pacific	+	+	+	Z. sansibaricus	Cladocopium	/
	Z16	Caribbean (Florida, Miami)	+	+	+	Z. pulchellus	Cladocopium	/
	Z17	Indo-Pacific	+	+	+	Z. sansibaricus	Cladocopium	Not detected
	Z18	Caribbean (Florida, Miami)	-	+	+	Z. pulchellus	Symbiodinium	/
	Z19	Caribbean (Florida, Miami)	-	+	+	Z. pulchellus	Symbiodinium	/
	Z20	Unknown	+	+	+	Terrazoanthus sp.	Cladocopium	Not detected
	Z21	Indian Ocean (Reunion island)	+	+	+	Z. kuroshio	Cladocopium	/
	Z22	Indian Ocean (Reunion island)	-	+	+	Z. kuroshio	Cladocopium	/
	Z23	Indian Ocean (Reunion island)	+	+	+	Z. sansibaricus	Symbiodinium	/
	Z24	Indian Ocean (Reunion island)	+	+	+	Z. sansibaricus	Cladocopium	/
	Z25	Indian Ocean (Reunion island)	+	+	+	Z. sansibaricus	Cladocopium	/
	Z26	Caribbean (Florida, Miami)	+	+	+	P. aff. clavata	Cladocopium	1.077 ± 0.072
	Z27	Caribbean (Florida, Miami)	+	+	+	P. aff. clavata	Cladocopium	1.265 ± 0.181
	Z28	Caribbean (Florida, Miami)	+	+	+	Z. sociatus	Symbiodinium	/
	Z29	Indo-Pacific	+	+	+	P. mutuki	Cladocopium	Not detected
	COI: mitochondrial cytochrome oxidase subunit I; ITS2-rDNA: internal transcribed spacer 2 of ribosomal DNA;
	ITS-rDNA: complete nuclear internal transcribed spacer region of ribosomal DNA ; /: not investigated in this
	study. +: sequence generated, -: not acquired.

location of zoantharian colonies used in this study, sequences generated and species identification of Zoantharia and their endosymbiotic Symbiodiniaceae and palytoxin content.

  916.1529 [M+2H+K] 3+ , 1365.7382 [M+H+Na] 2+ and 2707.4625 [M+H] + corresponding to a molecular mass of 2706 Da. This ion profile is typical to the bishomo-PLTX. Moreover, besides these ions, small peaks were also seen at m/z 1351.7413 and 906.8186 corresponding to the bi-[M+H+Na] 2+ and tri-charged [M+2H+K] 3+ molecular ions of a 2679 Da PLTX (data not shown). Molecular ion fragmentation tests showed that collision-induced dissociation (CID) gave the best results using collision energies of 45 eV for bi-and tri-charged molecular ions and 100 eV for the mono-charged molecular ions. In the three Palythoa samples, fragmentation of the PLTX molecular ions produced a fragment ion at m/z 327 [M + H] + in the full MS spectra (supplementary informations,

	The toxin extracted from Palythoa
	sp. Z05 and P. aff. clavata (Z26) displayed a clear ion profile corresponding to PLTX with prominent tri-charged
	[M+2H+K] 3+ (m/z 906.8158 and 906.8161, respectively), bi-charged [M+H+Na] 2+ (m/z 1351.7391 and
	1351.7387, respectively) and mono-charged [M+H]

+ (m/z 2680.4823 and 2680.4748, respectively)

molecular ions corresponding to a molecular mass of 2679 Da. The chromatogram of Z26 showed a small peak eluting before PLTX. Its composition was not determined, and it could correspond to impurities that were not removed during extraction or to PLTX degradation products. The toxin fraction extracted from P. heliodiscus (Z07) displayed prominent ions at m/z

Acknowledgements

We are deeply grateful to James Reimer for helpful discussions on the systematics and phylogeny of Zoantharia during this study. The authors also wish to thank Daniel Papillon who provided English corrections. Sampling of zoantharians by diving in the Caribbean was made possible by a commercial MLD collection license from the US Government (State of Florida SPL #932658 with MLD endorsement #1117).

Symbiodiniaceae (formerly the generalist Symbiodinium clade C). Analysis of PLTX distribution in Palythoa tissues showed that high levels of toxin do not colocalised with Cladocopium cells. This suggests that the sole implication of Symbiodiniaceae cells is unlikely and support the involvement of the holobiont (host plus its microbiote) in the synthesis and storage of PLTX. So far, transcriptomic analyses on the coral holobiont have essentially focused on environmental factors implicated in bleaching events including temperatures, acidification, nutrient stress, and disease [START_REF] Gierz | Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes[END_REF]. To identify the microorganism(s) involved in the PLTX synthesis in Palythoa, future integrative approaches incorporating transcriptomic and metabolomics should focus on the ability to produce PLTX in various types of holobionts and under different environmental conditions.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LS and YP carried out the molecular experiments and phylogenetic analyses. LS and JL did the PLTX purification, HPLC and mass spectrometry experiments. LS, CN and JL did the MALDI-IMS analyses. CF collected specimens of Zoanthus and Palythoa species in Florida and maintained the clonal populations of Palythoa aff. clavata and P. heliodiscus. JL, YP and LS drafted the manuscript. JL and YP conceived and supervised the study. All authors read, amended and approved the final manuscript.