**Figure S2:** phylogenetic relationships among *Eunicella* species. The phylogenetic reconstruction has been performed with mitochondrial MutS sequences obtained from Genbank, with a search focused on *Eunicella* species. Sequences from the *Complexum* and *Swiftia* genera have been used as outgroups on the basis of a Blast search with the MutS sequence of *E. cavolini*, and according to the current systematics of octocorals (McFadden et al., 2022). The sequences have been edited with ugene (Okonechnikov et al., 2012). The phylogenetic reconstructions have been performed with the Maximum-Likelihood (ML) approach of IQ-TREE 2.1.1 (Nguyen et al., 2015). We used the ModelFinder option (Kalyaanamoorthy et al., 2017), and robustness was evaluated with 1000 ultrafast bootstraps (Hoang et al., 2018). The tree has been visualized with FigTree 1.4.4 (Rambaut, 2006) and was rooted with *Swiftia simplex* as outgroup. The numbers to the left of the nodes indicate the percentages of bootstraps. The Genbank accession numbers are listed in table S1.

References:

Hoang, D. T., Chernomor, O., Von Haeseler, A., Minh, B. Q., & Vinh, L. S. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. *Molecular Biology and Evolution*, *35*(2), 518-522.

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. *Nature Methods*, *14*(6), 587-589.
https://doi.org/10.1038/nmeth.4285

McFadden, C. S., van Ofwegen, L. P., & Quattrini, A. M. (2022). Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. *Bulletin of the Society of Systematic Biologists*, *1*(3).

Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Molecular Biology and Evolution*, *32*(1), 268-274.
https://doi.org/10.1093/molbev/msu300

Okonechnikov, K., Golosova, O., Fursov, M., & Ugene Team. (2012). Unipro UGENE: a unified bioinformatics toolkit. *Bioinformatics*, *28*(8), 1166-1167.

Rambaut, A. (2006). FigTREE v1.4. University of Edinburgh. http://tree.bio.ed.ac.uk/software/figtree/
**Figure S2:** phylogenetic relationships among *Eunicella* species. The phylogenetic reconstruction has been performed with mitochondrial MutS sequences obtained from Genbank, with a search focused on *Eunicella* species. Sequences from the *Complexum* and *Swiftia* genera have been used as outgroups on the basis of a Blast search with the MutS sequence of *E. cavolini*, and according to the current systematics of octocorals (McFadden et al., 2022). The sequences have been edited with ugene (Okonechnikov et al., 2012). The phylogenetic reconstructions have been performed with the Maximum-Likelihood (ML) approach of IQ-TREE 2.1.1 (Nguyen et al., 2015). We used the ModelFinder option (Kalyaanamoorthy et al., 2017), and robustness was evaluated with 1000 ultrafast bootstraps (Hoang et al., 2018). The tree has been visualized with FigTree 1.4.4 (Rambaut, 2006) and was rooted with *Swiftia simplex* as outgroup. The numbers to the left of the nodes indicate the percentages of bootstraps. The Genbank accession numbers are listed in table S1.

References:

Hoang, D. T., Chernomor, O., Von Haeseler, A., Minh, B. Q., & Vinh, L. S. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. *Molecular Biology and Evolution*, *35*(2), 518-522.

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. *Nature Methods*, *14*(6), 587-589.
https://doi.org/10.1038/nmeth.4285

McFadden, C. S., van Ofwegen, L. P., & Quattrini, A. M. (2022). Revisionary systematics of Octocorallia (Cnidaria: Anthozoa) guided by phylogenomics. *Bulletin of the Society of Systematic Biologists*, *1*(3).

Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Molecular Biology and Evolution*, *32*(1), 268-274.
https://doi.org/10.1093/molbev/msu300

Okonechnikov, K., Golosova, O., Fursov, M., & Ugene Team. (2012). Unipro UGENE: a unified bioinformatics toolkit. *Bioinformatics*, *28*(8), 1166-1167.

Rambaut, A. (2006). FigTREE v1.4. University of Edinburgh. http://tree.bio.ed.ac.uk/software/figtree/
<figure><img src="image_13.png" /></figure>

Eunicella_verrucosa_JQ397300

Eunicella_cavolinii_NC_035667

Eunicella_verrucosa_NC_073494

Eunicella_cavolini_JQ397292

Eunicella_cavolini_JQ397290

Eunicella_cavolini_JQ397291

Eunicella_verrucosa_JQ397305

Eunicella_singularis_KX051572

Eunicella_singularis_JQ397293

Eunicella_sp._JQ397311

Eunicella_verrucosa_JQ397306

Eunicella_sp._JQ397308

Eunicella_singularis_JQ397294

Eunicella_verrucosa_JQ397302

Eunicella_sp._JQ397307

Eunicella_singularis_KX051571

Eunicella_racemosa_KX051577

Eunicella_tricoronata_NC_062012

Eunicella_tricoronata_JX203795

Eunicella_albicans_NC_035666

Complexum_monodi_KP036906

Swiftia_pacifica_KX904973

Swiftia_simplex_KX905018
<figure><img src="image_14.png" /></figure>

Eunicella_verrucosa_JQ397300

Eunicella_cavolinii_NC_035667

Eunicella_verrucosa_NC_073494

Eunicella_cavolini_JQ397292

Eunicella_cavolini_JQ397290

Eunicella_cavolini_JQ397291

Eunicella_verrucosa_JQ397305

Eunicella_singularis_KX051572

Eunicella_singularis_JQ397293

Eunicella_sp._JQ397311

Eunicella_verrucosa_JQ397306

Eunicella_sp._JQ397308

Eunicella_singularis_JQ397294

Eunicella_verrucosa_JQ397302

Eunicella_sp._JQ397307

Eunicella_singularis_KX051571

Eunicella_racemosa_KX051577

Eunicella_tricoronata_NC_062012

Eunicella_tricoronata_JX203795

Eunicella_albicans_NC_035666

Complexum_monodi_KP036906

Swiftia_pacifica_KX904973

Swiftia_simplex_KX905018
**Figure S3:** distribution of the frequency of Symbiodiniaceae sequences in the individual transcriptomes according to the species based A) on the number of reads estimated with Salmon, and B) on the proportion of assembled sequences with the BLAT analyses.

**A)** Read counts with Salmon: Mean values per group: *E. cavolini*: 16508; hybrids: 10238; *E. singularis*: 26023; *E. verrucosa*: 4285. Kruskal-Wallis test of the differences among groups: chi-squared = 7.9467, df = 3, p-value = 0.047.

<figure><img src="image_14.png" /></figure>

**B)** Assembled sequences with BLAT: Mean values per group: *E. cavolini*: 0.0034; hybrids: 0.0029; *E. singularis*: 0.0219; *E. verrucosa*: 0.0028. Kruskal-Wallis test of the differences among groups: chi-squared = 14.352, df = 3, p-value = 0.002.

<figure><img src="image_15.png" /></figure>
**Figure S3:** distribution of the frequency of Symbiodiniaceae sequences in the individual transcriptomes according to the species based A) on the number of reads estimated with Salmon, and B) on the proportion of assembled sequences with the BLAT analyses.

**A)** Read counts with Salmon: Mean values per group: *E. cavolini*: 16508; hybrids: 10238; *E. singularis*: 26023; *E. verrucosa*: 4285. Kruskal-Wallis test of the differences among groups: chi-squared = 7.9467, df = 3, p-value = 0.047.

<figure><img src="image_15.png" /></figure>

**B)** Assembled sequences with BLAT: Mean values per group: *E. cavolini*: 0.0034; hybrids: 0.0029; *E. singularis*: 0.0219; *E. verrucosa*: 0.0028. Kruskal-Wallis test of the differences among groups: chi-squared = 14.352, df = 3, p-value = 0.002.

<figure><img src="image_16.png" /></figure>
**Figure S6:** distribution of F<sub>ST</sub> estimates over loci, for the pairwise comparisons among the three species, with the exclusion of potential hybrids.

A) comparison between *E. cavolini* and *E. singularis*

Fst cavolini-singularis

<figure><img src="image_16.png" /></figure>

B) comparison between *E. cavolini* and *E. verrucosa*

Fst cavolini-verrucosa

<figure><img src="image_17.png" /></figure>
**Figure S6:** distribution of F<sub>ST</sub> estimates over loci, for the pairwise comparisons among the three species, with the exclusion of potential hybrids.

A) comparison between *E. cavolini* and *E. singularis*

Fst cavolini-singularis

<figure><img src="image_17.png" /></figure>

B) comparison between *E. cavolini* and *E. verrucosa*

Fst cavolini-verrucosa

<figure><img src="image_18.png" /></figure>
C) comparison between *E.singularis* and *E. verrucosa*

Fst singularis-verrucosa

<figure><img src="image_18.png" /></figure>
C) comparison between *E.singularis* and *E. verrucosa*

Fst singularis-verrucosa

<figure><img src="image_19.png" /></figure>
<figure><img src="image_19.png" /><figcaption>**Figure S5:** result of the cross-entropy analysis with the LEA R package</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S5:** result of the cross-entropy analysis with the LEA R package</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S6:** Principal Component Analysis based on the "polymorphic SNPs" dataset. The axis 1 corresponded to 33.2% of the variance, and axis 2 to 13% of the variance.</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Figure S6:** Principal Component Analysis based on the "polymorphic SNPs" dataset. The axis 1 corresponded to 33.2% of the variance, and axis 2 to 13% of the variance.</figcaption></figure>
**Symbiotic status does not preclude hybridisation in Mediterranean octocorals**

**Supplementary Material S2: 18S rDNA metabarcoding**

Didier Aurelle<fnref n="1" />,<fnref n="2" />*, Anne Haguenauer<fnref n="3" />, Marc Bally<fnref n="1" />,
Frédéric Zuberer<fnref n="4" />, Jean-Baptiste Ledoux<fnref n="5" />, Stéphane
Sartoretto<fnref n="6" />, Lamya Chaoui<fnref n="7" />, Hichem Kara<fnref n="7" />, Sarah
Samadi<fnref n="2" />, Pierre Pontarotti<fnref n="8" />,<fnref n="9" />,<fnref n="10" />

<figure><img src="image_21.png" /><figcaption>CC-BY 4.0 https://creativecommons.org/licenses/by/4.0/</figcaption></figure>

<footnote n="1">¹ Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France</footnote>

<footnote n="2">² Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, CP 26, 75005 Paris, France.</footnote>

<footnote n="3">³ CNRS - Délégation Provence et corse, Marseille, France</footnote>

<footnote n="4">⁴ Aix Marseille Univ, CNRS, IRD, INRAE, OSU Inst. PYTHEAS, Marseille, France</footnote>

<footnote n="5">⁵ CIIMAR/CIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Porto, Portugal.</footnote>

<footnote n="6">⁶ Ifremer, LITTORAL, 83500 La Seyne-sur-Mer, France</footnote>

<footnote n="7">⁷ Laboratoire Bioressources marines. Université d'Annaba Badji Mokhtar, Annaba - Algérie.</footnote>

<footnote n="8">⁸ Aix Marseille Univ, MEPHI, Marseille, France.</footnote>

<footnote n="9">⁹ IHU Méditerranée Infection, Marseille, France.</footnote>

<footnote n="10">¹⁰ CNRS SNC5039</footnote>

<footnote>*Corresponding author</footnote>

<footnote>Correspondence: didier.aurelle@univ-amu.fr</footnote>
**Symbiotic status does not preclude
hybridisation in Mediterranean octocorals**

**Supplementary Material S2: 18S rDNA
metabarcoding**

Didier Aurelle<fnref n="1" />,<fnref n="2" />*, Anne Haguenauer<fnref n="3" />, Marc Bally<fnref n="1" />,
Frédéric Zuberer<fnref n="4" />, Jean-Baptiste Ledoux<fnref n="5" />, Stéphane
Sartoretto<fnref n="6" />, Lamya Chaoui<fnref n="7" />, Hichem Kara<fnref n="7" />, Sarah
Samadi<fnref n="2" />, Pierre Pontarotti<fnref n="8" />,<fnref n="9" />,<fnref n="10" />

<figure><img src="image_22.png" /><figcaption>CC-BY 4.0 https://creativecommons.org/licenses/by/4.0/</figcaption></figure>

<footnote n="1">¹ Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO, Marseille, France</footnote>

<footnote n="2">² Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, CP 26, 75005 Paris, France.</footnote>

<footnote n="3">³ CNRS - Délégation Provence et corse, Marseille, France</footnote>

<footnote n="4">⁴ Aix Marseille Univ, CNRS, IRD, INRAE, OSU Inst. PYTHEAS, Marseille, France</footnote>

<footnote n="5">⁵ CIIMAR/CIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Porto, Portugal.</footnote>

<footnote n="6">⁶ Ifremer, LITTORAL, 83500 La Seyne-sur-Mer, France</footnote>

<footnote n="7">⁷ Laboratoire Bioressources marines. Université d'Annaba Badji Mokhtar, Annaba - Algérie.</footnote>

<footnote n="8">⁸ Aix Marseille Univ, MEPHI, Marseille, France.</footnote>

<footnote n="9">⁹ IHU Méditerranée Infection, Marseille, France.</footnote>

<footnote n="10">¹⁰ CNRS SNC5039</footnote>

<footnote>*Corresponding author</footnote>

<footnote>Correspondence: didier.aurelle@univ-amu.fr</footnote>
**Objectives of the study**

This section describes a preliminary sequencing test carried out to analyse
the microeukaryotic community associated with gorgonians of the genus
*Eunicella*.

Given that gorgonian host DNA accounts for the vast majority of DNA extracted from colonies, the detection of microeukaryotic diversity is challenging as their less abundant sequences are severely disadvantaged by PCR, which favours amplification of dominant matrices, here the ribosomal DNA (rDNA) of the host. To circumvent this problem, we tested a strategy that relies on the use of a blocking primer complementary to the gorgonian rDNA sequence to reduce the proportion of host amplicons. This approach has been reported, for example, in previous studies on coral-associated protists (Clerissi et al., 2018) and krill stomach contents (Vestheim & Jarman, 2008).

**Methods**

PCR amplification and metabarcoding of 18S rDNA

Using the 18S rDNA gorgonian sequences available in GenBank and one
*Eunicella cavolini* sequence determined in the laboratory, we confirmed that
the 18SV4 blocking primer of Clerissi et al. (2018) (5'-
TCTTGATTAATGAAAACATTCTTGGC-3' modified with a C3 spacer at the 3' end)
initially designed for scleractinian corals was also complementary to the
octocorallia sequences.

We therefore tested the efficiency of amplification of microeukaryotes on DNA samples obtained from one colony of *E. singularis* and two colonies of *E. cavolini* sampled in Marseille Bay, using the blocking primer 18SV4 in combination with the primer pair 18SV4-F (5'-CCAGCASCYGCGGTAATTCC-3') and 18SV4-R (5'-ACTTTCGTTCTTGATYRA-3') (Stoek et al., 2010) targeting a fragment of approximately 420 base pairs in the V4 variable region of the 18S rRNA gene.

Gorgonian DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen)
and PCR reactions were performed according to the conditions of Clerissi et
al. (2018), except that different concentration ratios between the blocking
primer and the 18SV4 primers (1.5:1, 3:1, 5:1 and 10:1) were tested to
optimise the proportion of microeukaryotic amplicons.

Sequencing of the final library of pooled amplicons was performed at the
Génome Québec Centre of Expertise and Services (Montréal, Canada) on
Illumina MiSeq platform using 2 x 250 bp v2 chemistry and following the
manufacturer's guidelines.

**Analysis of sequencing data**
**Objectives of the study**

This section describes a preliminary sequencing test carried out to analyse
the microeukaryotic community associated with gorgonians of the genus
*Eunicella*.

Given that gorgonian host DNA accounts for the vast majority of DNA extracted from colonies, the detection of microeukaryotic diversity is challenging as their less abundant sequences are severely disadvantaged by PCR, which favours amplification of dominant matrices, here the ribosomal DNA (rDNA) of the host. To circumvent this problem, we tested a strategy that relies on the use of a blocking primer complementary to the gorgonian rDNA sequence to reduce the proportion of host amplicons. This approach has been reported, for example, in previous studies on coral-associated protists (Clerissi et al., 2018) and krill stomach contents (Vestheim & Jarman, 2008).

**Methods**

PCR amplification and metabarcoding of 18S rDNA

Using the 18S rDNA gorgonian sequences available in GenBank and one
*Eunicella cavolini* sequence determined in the laboratory, we confirmed that
the 18SV4 blocking primer of Clerissi et al. (2018) (5'-
TCTTGATTAATGAAAACATTCTTGGC-3' modified with a C3 spacer at the 3' end)
initially designed for scleractinian corals was also complementary to the
octocorallia sequences.

We therefore tested the efficiency of amplification of microeukaryotes on DNA samples obtained from one colony of *E. singularis* and two colonies of *E. cavolini* sampled in Marseille Bay, using the blocking primer 18SV4 in combination with the primer pair 18SV4-F (5'-CCAGCASCYGCGGTAATTCC-3') and 18SV4-R (5'-ACTTTCGTTCTTGATYRA-3') (Stoek et al., 2010) targeting a fragment of approximately 420 base pairs in the V4 variable region of the 18S rRNA gene.

Gorgonian DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and PCR reactions were performed according to the conditions of Clerissi et al. (2018), except that different concentration ratios between the blocking primer and the 18SV4 primers (1.5:1, 3:1, 5:1 and 10:1) were tested to optimise the proportion of microeukaryotic amplicons.

Sequencing of the final library of pooled amplicons was performed at the
Génome Québec Centre of Expertise and Services (Montréal, Canada) on
Illumina MiSeq platform using 2 x 250 bp v2 chemistry and following the
manufacturer's guidelines.

**Analysis of sequencing data**
The FROGS pipeline v4.0 (Escudié et al., 2018) implemented in a Galaxy instance at GenoToul bioinformatics facility (Toulouse, France; https://bioinfo.genotoul.fr/) was used to align reads, remove chimera sequences, define Operational Taxonomic Units (OTUs), and to assign taxonomy based on the Silva 138.1 18S reference database (Quast et al., 2012).

For the phylogenetic analysis, the sequences were edited with ugene (Okonechnikov et al., 2012). The phylogenetic reconstructions were performed on a 376 bp alignment with the Maximum-Likelihood (ML) approach of IQ-TREE 2.1.1 (Nguyen et al., 2015). We used the ModelFinder option (Kalyaanamoorthy et al., 2017), and robustness was evaluated with 1000 ultrafast bootstraps (Hoang et al., 2018). The tree has been visualized with FigTree 1.4.4 (Rambaut, 2006) and rooted at mid-point.

## Results

### Identification of Symbiodiniaceae sequences in *E. singularis* and *E. cavolini*

Depending on the primer ratio used, the proportion of non-cnidarian 18S sequences reached up to 37.6% and 23.2% of the total sequences for *E. singularis* and *E. cavolini*, respectively. For both species, inhibition of host 18S rDNA gene amplification was most effective with the highest concentration of blocking primer.

In *E. singularis*, we identified 92 OTUs belonging to the family Symbiodiniaceae, in good agreement with the intracolonial diversity of zooxanthellae genotypes previously reported in this host species (Forcioli et al., 2011). Among these OTUs, a single OTU (OTU_7) was highly dominant and contributed up to 45.6% of the total Symbiodiniaceae abundance in the studied colony.

A small number of Symbiodiniaceae OTUs were detected in *E. cavolini* colonies (12 to 13 OTUs depending on the colony). Between the two colonies analysed, the proportion of Symbiodiniaceae sequences varied considerably, accounting for 0.21% to 2,3% of the non-cnidarian sequences when the blocking primer concentration was the highest. However, in both cases OTU_7 was the most abundant, representing up to 99% of all Symbiodiniaceae sequences. The sequence of this OTU_7 (381 bp in length) has been submitted to GenBank under reference SUB14400021.

### Phylogenetic analysis

The 18S rDNA OTU_7 shared between *E. cavolini* and *E. singularis* was used for a Blast search in GenBank. In the list of Blast hits, we retained a subset of sequences corresponding to different levels of identity and to different clades of Symbiodiniaceae for phylogenetic reconstruction. The phylogenetic inference on these data produced a tree (Fig. S2.1) that allowed sequences belonging to clades A, B, C and D to be distinguished (most previously defined clades were recovered with more than 90% bootstrap support). According to the tree topology, the putative Symbiodiniaceae species associated with OTU_7 is sister to a clade containing *Symbiodinium microadriaticum*, *S.*
The FROGS pipeline v4.0 (Escudié et al., 2018) implemented in a Galaxy instance at GenoToul bioinformatics facility (Toulouse, France; https://bioinfo.genotoul.fr/) was used to align reads, remove chimera sequences, define Operational Taxonomic Units (OTUs), and to assign taxonomy based on the Silva 138.1 18S reference database (Quast et al., 2012).

For the phylogenetic analysis, the sequences were edited with ugene (Okonechnikov et al., 2012). The phylogenetic reconstructions were performed on a 376 bp alignment with the Maximum-Likelihood (ML) approach of IQ-TREE 2.1.1 (Nguyen et al., 2015). We used the ModelFinder option (Kalyaanamoorthy et al., 2017), and robustness was evaluated with 1000 ultrafast bootstraps (Hoang et al., 2018). The tree has been visualized with FigTree 1.4.4 (Rambaut, 2006) and rooted at mid-point.

## Results

### Identification of Symbiodiniaceae sequences in *E. singularis* and *E. cavolini*

Depending on the primer ratio used, the proportion of non-cnidarian 18S sequences reached up to 37.6% and 23.2% of the total sequences for *E. singularis* and *E. cavolini*, respectively. For both species, inhibition of host 18S rDNA gene amplification was most effective with the highest concentration of blocking primer.

In *E. singularis*, we identified 92 OTUs belonging to the family Symbiodiniaceae, in good agreement with the intracolonial diversity of zooxanthellae genotypes previously reported in this host species (Forcioli et al., 2011). Among these OTUs, a single OTU (OTU_7) was highly dominant and contributed up to 45.6% of the total Symbiodiniaceae abundance in the studied colony.

A small number of Symbiodiniaceae OTUs were detected in *E. cavolini* colonies (12 to 13 OTUs depending on the colony). Between the two colonies analysed, the proportion of Symbiodiniaceae sequences varied considerably, accounting for 0.21% to 2,3% of the non-cnidarian sequences when the blocking primer concentration was the highest. However, in both cases OTU_7 was the most abundant, representing up to 99% of all Symbiodiniaceae sequences. The sequence of this OTU_7 (381 bp in length) has been submitted to GenBank under reference SUB14400021.

### Phylogenetic analysis

The 18S rDNA OTU_7 shared between *E. cavolini* and *E. singularis* was used for a Blast search in GenBank. In the list of Blast hits, we retained a subset of sequences corresponding to different levels of identity and to different clades of Symbiodiniaceae for phylogenetic reconstruction. The phylogenetic inference on these data produced a tree (Fig. S2.1) that allowed sequences belonging to clades A, B, C and D to be distinguished (most previously defined clades were recovered with more than 90% bootstrap support). According to the tree topology, the putative Symbiodiniaceae species associated with OTU_7 is sister to a clade containing *Symbiodinium microadriaticum*, *S.*
*pilosum* and other symbiotic *Symbiodinium* species belonging to Clade A (now corresponding to the *Philozoon* genus; Lajeunesse et al., 2022). The closest sequence to OTU_7 is from a dinoflagellate isolated from a South China Sea reef (accession MZ621018; to be released upon publication).
*pilosum* and other symbiotic *Symbiodinium* species belonging to Clade A (now corresponding to the *Philozoon* genus; Lajeunesse et al., 2022). The closest sequence to OTU_7 is from a dinoflagellate isolated from a South China Sea reef (accession MZ621018; to be released upon publication).
<figure><img src="image_22.png" /><figcaption>**Figure S2.1.** Maximum-likelihood phylogeny of Symbiodiniaceae based on the variable V4 region of the 18S rRNA gene, illustrating the relationship of OTU_7 sequence (in red) with Symbiodiniaceae spp. belonging to Clade A. The numbers to the left of the nodes indicate the percentages of bootstraps, for values superior to 75%. The first part of each sequence name corresponds to the accession number in GenBank.</figcaption></figure>
<figure><img src="image_23.png" /><figcaption>**Figure S2.1.** Maximum-likelihood phylogeny of Symbiodiniaceae based on the variable V4 region of the 18S rRNA gene, illustrating the relationship of OTU_7 sequence (in red) with Symbiodiniaceae spp. belonging to Clade A. The numbers to the left of the nodes indicate the percentages of bootstraps, for values superior to 75%. The first part of each sequence name corresponds to the accession number in GenBank.</figcaption></figure>
## References

Clerissi, C., Brunet, S., Vidal-Dupiol, J., Adjeroud, M., Lepage, P., Guillou, L., ... & Toulza, E. (2018). Protists within corals: the hidden diversity. *Frontiers in Microbiology, 9*, 2043. https://doi.org/10.3389/fmicb.2018.02043

Escudié, F., Auer, L., Bernard, M., Mariadassou, M., Cauquil, L., Vidal, K., ... & Pascal, G. (2018). FROGS: find, rapidly, OTUs with galaxy solution. *Bioinformatics*, 34(8), 1287-1294.
https://doi.org/10.1093/bioinformatics/btx791

Forcioli, D., Merle, P. L., Caligara, C., Ciosi, M., Muti, C., Francour, P., ... & Allemand, D. (2011). Symbiont diversity is not involved in depth acclimation in the Mediterranean sea whip Eunicella singularis. *Marine Ecology Progress Series*, 439, 57-71. https://doi.org/10.3354/meps09314

Hoang, D. T., Chernomor, O., Von Haeseler, A., Minh, B. Q., & Vinh, L. S. (2018). UFBoot2: Improving the ultrafast bootstrap approximation. *Molecular Biology and Evolution*, 35(2), 518-522.

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. *Nature Methods*, 14(6), 587-589.
https://doi.org/10.1038/nmeth.4285

LaJeunesse, T. C., Wiedenmann, J., Casado-Amezúa, P., D'ambra, I., Turnham, K. E., Nitschke, M. R., Oakley, C. A., Goffredo, S., Spano, C. A., & Cubillos, V. M. (2022). Revival of Philozoon Geddes for host-specialized dinoflagellates, 'zooxanthellae', in animals from coastal temperate zones of northern and southern hemispheres. *European Journal of Phycology*, 57(2), 166-180. https://doi.org/10.1080/09670262.2021.1914863

Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Molecular Biology and Evolution*, 32(1), 268-274.
https://doi.org/10.1093/molbev/msu300

Okonechnikov, K., Golosova, O., Fursov, M., & Ugene Team. (2012). Unipro UGENE: a unified bioinformatics toolkit. *Bioinformatics*, 28(8), 1166-1167. https://doi.org/10.1093/bioinformatics/bts091

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., ... & Glöckner, F. O. (2012). The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. *Nucleic acids research*, 41(D1), D590-D596. https://doi.org/10.1093/nar/gks1219

Rambaut, A. (2006). *FigTREE v1.4*. University of Edinburgh.
http://tree.bio.ed.ac.uk/software/figtree/

Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M. D., Breiner, H. W., & Richards, T. A. (2010). Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine
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Nguyen, L.-T., Schmidt, H. A., von Haeseler, A., & Minh, B. Q. (2015). IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Molecular Biology and Evolution*, **32**(1), 268-274.
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