# Inter- and intraspecific diversity in bacterial communities associated with two crustose coralline algae from the NW Mediterranean Sea

Elisabetta Manea, Lorenzo Bramanti, Laura Pezzolesi, Annalisa Falace, Sara

Kaleb, Lucia Bongiorni, Pierre E. Galand

► To cite this version:

Elisabetta Manea, Lorenzo Bramanti, Laura Pezzolesi, Annalisa Falace, Sara Kaleb, et al. Inter- and intraspecific diversity in bacterial communities associated with two crustose coralline algae from the NW Mediterranean Sea. 2024. hal-04441255v2

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1 **Inter- and intraspecific diversity in bacterial communities associated with two crustose coralline algae from the NW Mediterranean Sea**

Manea, E.¹*, Bramanti, L.¹, Pezzolesi²,³, L., Falace, A.⁴, Kaleb, S.⁴, Bongiorni, L.⁵, Galand, P.E.¹

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¹Laboratoire d’Ecogechimie des Environnements Benthiques, LECOB, Observatoire Océanologique de
Banyuls sur Mer Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université, Banyuls-
sur-Mer, France

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²Department of Biological, Geological and Environmental Sciences (BiGeA), University of Bologna, Via
Sant'Alberto 163, 48123 Ravenna, Italy

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³Interdepartmental Centre for Industrial Research in Renewable Resources, Environment, Sea and Energy
(CIRI-FRAME), University of Bologna, Via Sant'Alberto 163, 48123 Ravenna, Italy

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⁴Department of Life Sciences, University of Trieste, Via Licio Giorgieri 10, 34127 Trieste, Italy

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⁵Institute of Marine Sciences, National Research Council, CNR-ISMAR, Arsenale, Tesa 104, Castello
2737/F, 30122, Venice, Italy

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*Corresponding author: elisabetta.manea@obs-banyuls.fr

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**Abstract**

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Crustose coralline algae (CCAs) are key components and primary builders of coralligenous reefs,
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which are among the most biodiverse marine habitats in the Mediterranean Sea. Beyond their
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structural role, CCAs play a crucial role in promoting larval settlement in a variety of invertebrate
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species, including corals. In addition, CCAs form complex holobionts through association with
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symbiotic microorganisms that can modulate their ability to induce larval settlement and provide
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information on their health status. Despite their ecological significance and potential for
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biomonitoring, the microbial communities associated with Mediterranean CCA holobionts remain
25
largely understudied. Using electron microscopy and barcoding techniques, we identified
26 *Lithophyllum stictiforme* complex and *Macroblastum dendrospermum*, two widespread CCAs
27 from the Western Mediterranean Sea, living in sympatry with gorgonians. The 16S rRNA gene
28 amplicon sequencing revealed that these CCAs host distinct and diverse bacterial communities,
29 even among thalli of the same species, with closer thalli showing more similar bacterial
30 assemblages. Our findings suggest that the primary factors influencing the bacterial communities
31 include the host genotype, local environmental conditions at the individual thallus scale, and
32 possible horizontal transfer between adjacent thalli. The predominance of bacterial sequences
33 affiliated to the *Blastopirellula* genus and *Pir4_lineage* in phylum Planctomycetes across all CCA
34 individuals examined is noteworthy. These bacterial groups have been hypothesized to promote
35 coral larvae settlement in tropical regions, raising the possibility of a similar facilitative interaction
36 between CCAs and Mediterranean coral larvae.

37
38 **Keywords:** marine holobiont, microbiome, *Lithophyllum stictiforme*, *Macroblastum dendrospermum*, marine monitoring, coralligenous habitat
39
40

41 1. Introduction

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48
Crustose coralline algae (CCAs) are a group of rhodophytes characteristic of coastal and
mesophotic seascapes (Kahng et al., 2010; Castellan et al., 2022; Schubert et al., 2020). In the
Mediterranean, they are key structural components of coralligenous concretions, which, due to
their wide spatial and bathymetric extent (up to 2,700 km² in area and from 20 to 130 m depth;
Martin et al., 2014, Iborra et al., 2022), constitute one of the most representative and biodiverse
benthic habitat in the region (Ballesteros, 2006; Bracchi et al., 2022). Mediterranean CCAs also
form a highly biodiverse group, potentially harboring significant cryptic diversity at both species
and genus level (Pezzolesi et al., 2019; Rindi et al., 2019). Through their calcareous three-dimensional thalli, CCAs serve as ecosystem engineers fostering high species richness (Asnaghi et al., 2015; Pezzolesi et al., 2017; Piazzi et al., 2022). Like their tropical counterparts, Mediterranean CCAs are increasingly affected by climate change and pathogenic diseases (Martin & Gattuso, 2009; Short et al., 2015; Quéré et al., 2019; Cornwall et al., 2022; Vitelletti et al., 2023), with widespread documented cases of mortality (Hereu & Kersting, 2016). As a whole, this context underscores the urgent need for targeted research on CCAs to better assess coralligenous biodiversity and health status.

Although the taxonomy, biology, and distribution of CCAs have been studied for a limited number of species, one of the least explored aspects is their associated microbiome (Rindi et al., 2019). To date, only two studies have addressed this topic focusing on *Neogoniolithon brassica-florida* (Quéré et al., 2019; Gefen-Treves et al., 2021). These studies provided crucial insights into CCA-bacteria associations and emphasized the need for further investigations into the functioning of these holobionts. There is growing evidence that CCAs harbor species-specific microbiomes (Barott et al., 2011; Cavalcanti et al., 2014; Sneed et al., 2015), with the associated bacteria playing a key role in the morphological development of the algae and exhibiting antifouling properties against epibionts (Singh & Reddy, 2016; Gómez-Lemos & Diaz-Pulido, 2017; Singh et al., 2017). For instance, bacteria can facilitate the settlement of *Ulva* spores (Joint et al., 2002), produce bioactive compounds that promote algal morphogenesis (Matsuo et al., 2005), and even form biofilms on CCAs that inhibit colonization by epiphytes, thereby reducing competition for light (Gómez-Lemos & Diaz-Pulido, 2017). The microbial community associated with algae may also serve as an indicator of algal and habitat health status (Glasl et al., 2017; Quéré et al., 2019). For instance, Zozaya-Valdes and colleagues (2015) identified a pathogen affecting the alga *Delisea*
72 *pulchra*, providing valuable information for predicting the spread of disease. Studies on 73 Mediterranean CCA holobionts are crucial for establishing a foundation for future biomonitoring 74 and conservation measures.

75 CCAs are also known to facilitate the recruitment process of several invertebrate species 76 (Lau et al., 2005; O'Leary et al., 2017; Seabra et al., 2019), promoting the settlement and 77 metamorphosis of tropical coral larvae through chemical cues (Gómez-Lemos et al., 2018; Siboni 78 et al., 2020; Jorissen et al., 2021). In the Mediterranean, field observations and an experimental 79 study also showed gorgonian-CCA facilitative interactions (Toma et al., 2022; Zelli et al 2020). 80 Gorgonian species, which are priority for conservation, are highly threatened by climate change 81 and local impacts (Iborra et al., 2022; Tignat-Perrier et al., 2022; Chatzimentor et al., 2023). In 82 recent decades, mass mortalities related to marine heat waves have severely affected shallow 83 gorgonian populations (10-40 m, Garrabou et al., 2022; Bramanti et al., 2023). Efforts are currently 84 underway to reintroduce these species to their natural habitat (e.g., Bramanti et al., 2007; Linares 85 et al., 2008; Montseny et al., 2019; Casoli et al., 2022). Understanding how CCA holobionts 86 interact with gorgonians (e.g., fostering larval settlement) could provide valuable insights for 87 restoration strategies.

88 In this study, we analyzed the diversity of the bacterial communities associated with two 89 widespread CCAs from the Western Mediterranean, *Lithophyllum stictiforme* and *Macroblastum 90 dendrospermum*, both of which are either colonized by or in close proximity to gorgonians. To 91 ensure consistent sampling, algae were collected in a restricted geographical area (approximately 92 30 m²) to maximize the likelihood of obtaining thalli from the same species in similar 93 environmental conditions. Previous research, in fact, has shown that bacterial communities 94 associated with macroalgae, including CCAs, can vary considerably between individuals of the
same species due to diverse environmental conditions and exogenous factors (Staufenberger et al.

2008; Burke et al. 2011; Barott et al. 2011; Miranda et al. 2013; Gefen-Treves et al., 2021).

Given the high cryptic diversity of CCAs (Rindi et al., 2019), which often makes identification based on morphological characters alone difficult, we used a combination of morphological traits and molecular techniques to identify these CCA species. To investigate species-specific bacterial associations, we characterized the diversity of bacteria associated with CCAs at different taxonomic levels using DNA metabarcoding. We further investigated both the common and distinct components of bacteria diversity, as well as the differential enrichment of bacterial taxa in CCA holobionts. These analyses were performed both between individuals of the two algal species and among individuals within the same CCA taxonomic group.

## 2. Material and Methods

### 2.1 Samples collection and preparation

Thalli of CCAs (ca. 5 cm wide and 5-10 cm long) were collected in December 2022 at the Canadels site in Banyuls-sur-Mer, France (42°26.900' N, 3°10.347' E). The sampling area hosts populations of two octocoral species: the white gorgonian (*Eunicella singularis*) and the Mediterranean red coral (*Corallium rubrum*). The thalli of each CCA species were collected at three neighboring sites (*L. stictiforme*: ST25, ST27, ST7; *M. dendrospermum*: ST12, ST13, ST17), in three replicates per site, (n = 18). Samples were collected using hammer and chisel at 25 m depth by SCUBA diving. The sampling area covered approximately 30 m² and the distance between the samples and the octocoral colonies varied from 30 cm and 2 m.

In the laboratory, the epiphytes were carefully removed from the CCA surfaces with sterile forceps. After cleaning, each thallus was divided into fragments. One half was immediately frozen
118 at -80°C for DNA extraction, the other half was air-dried and stored in plastic bags with silica gel
119 for morpho-anatomical characterization and DNA extraction.

120
121 **2.2 Morpho-anatomical characterization of CCA**
122 For detailed morpho-anatomical characterization, all samples were examined with a scanning
123 electron microscope (SEM) according to Kaleb et al. (2018). Small fragments of thalli were
124 mounted on aluminum stubs with acrylic adhesive, coated with a thin layer of chromium (Quorum
125 Q150T ES coater), and analyzed with a SEM Zeiss Gemini300 at a working distance (WD)
126 between 8 and 11 mm.

127
128 **2.3 CCA DNA extraction, amplification and sequencing**
129 The silica-dried CCA samples were pulverized using sterile mortars, and the DNA was extracted
130 following the modified protocol of the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Crawley,
131 UK) by Broom et al. (2008) and quantified with the use of Qubit dsDNA BR Assay Kit (Invitrogen,
132 Life Technologies). The psbA gene was PCR-amplified with the use of the forward and reverse
133 primers psbA-F 5'-ATGACTGCTACTTTAGAAAGACG-3' and psbA-R2 5'-
134 TCATGCATWACTTCCATACCTA-3' according to Pezzolesi et al. (2017, 2019), and the
135 QIAGEN Multiplex PCR Kit (Qiagen) according to the instructions. The amplification protocol
136 consisted of a HotStarTaq DNA polymerase activation step at 95°C for 15 min, followed by a 3-
137 step cycling with denaturation at 94°C for 30 sec, annealing at 57°C for 90 sec, extension at 72°C
138 for 90 sec for 35 cycles, and a final extension at 72°C for 10 min. The mitochondrial COI-5P
139 fragment was PCR-amplified in M. dendrospermum, to increase the number of reference
140 sequences available for the subsequent alignment, following Peña et al. (2015), with the use of the
141 forward and reverse primers GazF1 5'-TCAACAAATCATAAAGATATTGG-3' and GazR1 5'-
142 ACTTCTGGATGTCCAAAAAAYCA- 3' (Saunders, 2005). The amplification protocol consisted
143 of an initial denaturation at 95°C for 2 min followed by 40 cycles of 30 s denaturation at 94°C, 40
144 s annealing at 50°C and 40 s of extension at 72°C followed by an additional 5 min at 72°C. PCR
145 products were visualized in 1.5% agarose gels stained with Midori Green using Low DNA Mass
146 Ladder (Invitrogen, Carlsbad, CA, USA) as a reference, and PCR products with expected lengths
147 and yields were purified and sequenced by BMR Genomics (Padua, Italy). Specifically, PCR
148 products were treated with ExoSap (Applied Biosystems™, ThermoFischer Scientific, Walthman,
149 MA, USA), and sequencing was performed with BrilliantDye Terminator v1.1 Cycle Sequencing
150 Kit (Nimagen, The Netherlands) following the manufacturer's instruction on a AB3730xl DNA
151 Analyzer (Applied Biosystems™, ThermoFisher Scientific, Walthman, MA, USA).

152
153

154 The quality of the sequences was assessed by visual inspection of the electropherograms using the
155 Chromas software (Version 2.6.6; Technelysium Pty LTD, South Brisbane, Australia). All
156 sequences were scored as high quality, without double or confounding peaks, and were all retained
157 for the alignment and phylogenetic analyses. Alignment was performed using ClustalW and
158 default settings in AliView software (Version 1.28; Larsson, 2014) and phylogeny constructed by
159 means of MEGA software (Version 11.0.13, www.megasoftware.net; Tamura et al., 2021). In
160 addition, NCBI database (www.ncbi.nlm.nih.gov) was searched for publicly available sequences
161 of *Lithophyllum stictiforme* complex and *Mesophyllum* genus (as *Mesophyllum macroblastum* was
162 only recently recognized as *Macroblastum dendrospermum*, Athanasiadis & Ballantine, 2024),
163 and some representatives were selected to be included in the alignments. The alignment was
assembled after a series of preliminary phylogenetic analyses aimed at correct selection of ingroup
and outgroup taxa. Therefore, psbA sequences of *Lithophyllum dentatum* (Kützing) Foslie, *L. hibernicum* Foslie, and *L. bathyporum* Athanasiadis & D.L.Ballantine, and psbA and COI-5P
sequences of the Melobesioideae *Phymatolithon calcareum* (Pallas) W.H. Adey & McKibbin and
*Lithothamnion corallioides* (P.L. Crouan & H.M. Crouan) P.L. Crouan & H.M. Crouan were
included as outgroups in the respective data sets, according to previous studies (Pezzolesi et al,
2019; Peña et al., 2015). The sequences' alignment of *L. stictiforme* psbA sequences was 825 bp
long, while those of *M. dendrospermum* ranged from 598 (S17R1), 684 (S17R2), 725 (S12R1) to
851 base pairs (bp). The COI-5P of *M. dendrospermum* alignment resulted in 608 bp. Neighbor-
Joining (NJ) distance analyses were performed on all data sets using Maximum Composite
Likelihood model in MEGA software, with nodal support assessed by 1,000 bootstrap (BR)
resamplings. Phylogenetic relationships were inferred using Maximum Likelihood (ML) analyses
in MEGA software, under a generalized time-reversible with gamma+invariant sites heterogeneity
model (GTR+G+I), and a generalized time-reversible gamma distributed (GTR+G) alignments,
for the COI-5P and psbA alignments, respectively. The bootstrap consisted of 1000 replicates with
complete deletion option, i.e. eliminating the positions containing gaps and missing data (Saitou
& Nei, 1987; Nei & Kumar, 2000; Pezzolesi et al., 2019).

Genetic distances in *Lithophyllum stictiforme* and *Macroblastum dendrospermum* were computed
within and between clades using the Kimura distance (Kimura-2 parameter, Kimura, 1980) from
the alignments in MEGA (v.11.0.13).

## 2.5 Bacteria DNA extraction and sequencing
186 Frozen CCA samples were pulverized using sterile mortars. Total genomic DNA was extracted
187 from 18 CCA samples using Power Soil DNA Isolation kit according to the manufacturer's
188 instructions (QIAGEN, Hilden, Germany). After DNA quantification with the use of DeNovix
189 DS11 Spectrophotometer - Fluorometer (DeNovix Inc., Delaware), analyses of the prokaryotic
190 16S rDNA sequences were conducted through amplicons generation with the use of the forward
191 and reverse primers 341F 5'-CCTACGGGNGGCWGCAG-3' and 785R 5'-
192 GACTACHVGGGTATCTAATCC-3' amplifying the bacterial V3-V4 region (Klindworth et al.,
193 2013). Sequencing was performed on the Illumina MiSeq V3 platform at the Integrated
194 Microbiome Resource (IMR, imr.bio, Dalhousie University, Halifax, Nova Scotia, Canada) to
195 obtain 2x300 paired-end sequences. Complete details of the protocols applied for library
196 preparation and sequencing procedures can be found in Comeau and Kwawukume (2023).
197

198
2.6 Bacteria sequence analyses and statistics
199
Bacteria sequences were processed with the DADA2 R package, version 1.16 (Callahan et al.,
200 2016; https://benjjneb.github.io/dada2/) in R 4.2.3 (R Core Team, 2023). After inspection of
201 quality control profiles, the filtering parameters were set as follow: truncLen=c(280,220),
202 trimLeft=c(17,21), maxN=0, maxEE=c(2,5), truncQ=2, rm.phix=TRUE, compress=TRUE,
203 multithread=TRUE. The sequences were then dereplicated, denoised by removing sample
204 inference and chimeras, and merged. Representative amplicon sequence variants (ASVs) were
205 classified against the SILVA version 138.1 database (McLaren & Callahan, 2021). The dataset
206 was cleaned from chloroplast, Archaea, unknown ASVs at the phylum level and singletons with
207 the R package phyloseq (McMurdie & Holmes, 2013). After cleaning, a total of 503,450 reads and
208 2599 ASVs were retained.
Rarefaction curves were produced by rarefying the dataset to 9310 sequences through the use of vegan package in R (Oksanen et al., 2013), and all samples were retained for analyses. The curves reached a plateau for all samples indicating that the CCA-associated bacteria diversity was well covered (Supp. Fig. 1).

To estimate CCA-bacteria communities alpha diversity, the phyloseq package was used to calculate the Shannon Index with the *diversity* function. Significant differences among alpha diversity values at both inter and intra-CCA species levels were tested. The Shapiro Wilk test was applied to test for normality of data. When data were normal, the One-Way ANOVA with *aov* function followed by the Tukey test for pairwise comparison of samples were used. In case of non-normal data, the Kruskal-Wallis test followed by the Dunn test (dunn.test package) with the Benjamini-Hochberg method were performed. A multidimensional scaling ordination (MDS), based on Bray-Curtis distance matrix computed from Hellinger ASV transformed data (i.e., square root of relative abundance), was constructed with the vegan package to visualize similarities in bacteria community composition both between CCA species and within samples of the same CCA.

A permutational multivariate analysis of variance (PERMANOVA) was used to test for significant differences among bacteria communities associated with thalli of different and same CCA species on Bray Curtis distance matrix of Hellinger transformed ASV data using 999 permutations under reduced model with the vegan package by applying the *adonis2* function. Intraspecific differences of CCA bacteria communities were further analysed by the *pairwise.adonis2* function for pairwise comparison of samples considering the sampling sites as source of variance. Statistically significant differences were considered with *p*-value ≤ 0.05.

To identify the core microbiomes, i.e. ASVs shared between individual CCA-bacteria communities within and between CCA species, as well as the core ASVs unique to each CCA
species and sampling site, i.e. shared only at CCA species level, the microbiome package (Lahti et al., 2017) and the *core_members* function (Salonen et al., 2012) with detection = 0.01 (with relative abundance ≥1%) and prevalence = 0.75 (present in at least 75% of samples) were used.

The results were represented through Venn diagrams produced with the use of the ggvenn package.

To gain more details on the potential functional traits of the most contributing bacteria taxa among both the shared and unique ASVs associated to the two CCAs and characteristic of each species, the five most abundant ASVs were selected for further taxonomic analysis through a manual BLAST search against the NCBI nucleotide library (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

A differential abundance analysis was carried out to compare ASV data from the two CCA species. The analysis was done by applying DESeq2 analyses (Love et al., 2014) using phyloseq package in R v.4.4.1 (R Core Team, 2024) with *phyloseq_to_deseq2* function followed by *DESeq*. Significance was determined using a Wald test with a Benjamini and Hochberg adjusted *p*-value. An adjusted *p*-value ≤0.01 was used as the minimal cut-off, and analysis results were shown by selecting the ASVs with a significance ≤0.001. The 20 ASVs with the most significant different abundance were further represented with a heatmap showing also the hierarchical clustering of samples produced with microViz package and *comp_heatmap* function on clr transformed data (Barnett et al., 2021). Based on CCA phylogeny results (see section 3.2), the differential abundance analysis was carried out on ASVs associated with *L. stictiforme* thalli belonging to two different clades to deepen the comparison at intraspecific level. Species clade was adopted as comparison factor and ASVs meeting the minimal cut-off of an adjusted *p*-value ≤0.01 were considered.

## 3. Results

254
255 **3.1 CCA morpho-anatomy**

256 Based on the morpho-anatomical analyses, all *Lythophyllum stictiforme* individuals were
257 confirmed to belong to *L. stictiforme* complex (Supp. Fig. 2A-G) with no substantial differences
258 in diagnostic features observed across the three sampling sites. The thalli were encrusting with a
259 free laminar margin and the surface was either flat or slightly undulating. In all individuals, the
260 epithallus was abraded (Suppl. Fig. 2H), occasionally revealing the underlying cell layer.

261 The *M. dendrospermum* individuals displayed encrusting to warty thalli (Supp. Fig. 3A)
262 and exhibited the key diagnostic traits for this species (Supp. Fig. 3A-H). In surface view, large
263 areas of shedding epithallial cells were observed in all samples (Fig. 2B), and the epithallial cells
264 were flared.
265

265
266
**3.2 CCA phylogeny**

267 The molecular phylogenetic analysis based on the psbA marker confirmed that all 9 individuals of
268 *Lithophyllum stictiforme* belonged to the complex, previously described as formed by numerous
269 clades (Pezzolesi et al., 2019). Clade 3B comprised 6 samples from this study, belonging to two
270 of the three collection sites, together with some reference sequences from Italy and two from
271 France, including also one (isolate LLG3464) from the sampling area of the present study (S27
272 and S7; Fig. 1). Replicates collected at site S25 all belonged to a distinct haplogroup defined as
273 C3 (de Jode et al., 2019). It contained closely similar reference sequences (99% similarity) from
274 samples previously collected from the same study area (Banyuls-sur-Mer). The Kimura distances
275 within clades C3 and 3B were 0.0000, while the highest distance within clades was found for
276 clades 2 and 5 having a distance of 0.0042 and 0.0051, respectively. The genetic distance between
277 clades C3 and 3B was 0.0180. The mean distance to the reference group (outgroup) was 0.0591
278 for clade C3 and 0.0550 for clade 3B (Table S1).

279 As for *Macroblastum dendrospermum* thalli, 8 out of the 9 psbA sequences generated in
280 this study clustered with a Mediterranean *Mesophyllum macroblastum* specimen (voucher
281 VPF00506, now *Macroblastum dendrospermum*; Athanasiadis & Ballantine, 2024), collected in
282 the Alboran Sea (Spain), and with the Uncultured Corallinales clone LBC0055, collected at less
283 than 10 km distance from the area of collection (42.497219 N, 3.135617 E; Bittner et al., 2010;
284 Fig. 2). The last sequence (S12R2) was closely related to the main cluster. The Kimura distance
285 within this main group was 0.0018, while the mean distance with S12R2 or the outgroup was
286 0.0192 and 0.1095, respectively. The mean distance between the 8 specimens and the Uncultured
287 Corallinales from France (GQ917716) was 0.0009, while with *M. macroblastum* from Spain
288 (KJ638230) was 0.0028. For the COI-5P marker, only 5 sequences were generated as all
289 amplification attempts for station 13 samples were unsuccessful. The phylogenetic tree obtained
290 from the ML analysis of the COI-5P alignment was congruent with the psbA tree (Supp. Fig. 4)
291 and grouped our sequences within a big clade of Mediterranean *M. macroblastum* with high
292 support (99%), well separated from the other *Mesophyllum* species (*M. alternans*, *M. expansum*,
293 *M. lichenoides*, *M. erubescens*, and *M. sphaericum*) (Supp. Fig. 4). The genetic distance within
294 this group was 0.0125, while with the reference group (outgroup) was 0.2044.
295

296

3.3 Bacterial community diversity associated to CCAs

297 The average Shannon values ranged between 4.7±0.1 and 5.5±0.2, and between 5.7±0.3
298 and 6±0.1 in *L. stictiforme* and *M. dendrospermum* samples, respectively (Fig. 3a). The lowest
299 values were recorded for *L. stictiforme* taken at site ST27. In *M. dendrospermum* sampling sites
300 ST13 and ST17 values were significantly higher than in ST27 (one of *L. stictiforme* sampling site, 301 *p*-value < 0.05*, Fig. 3a, Table S2). In ST13 the value was also significantly higher than in *L. stictiforme* sampling site ST25 (*p*-value < 0.05*, Fig. 3a, Table S2). Bacteria diversity values at 302 intraspecific level differed only between individuals belonging to *L. stictiforme* complex, 303 specifically between ST7 and ST25, and ST7 and ST27 (*p*-value < 0.05* and *p*-value < 0.01**, 304 respectively; Fig. 3a, Table S3). The observed ASV numbers varied between 302 and 898 in the 305 low diversity site ST25 (*L. stictiforme*; on average 335-600 ASVs among sampling sites), and one 306 sample at site ST13 (*M. dendrospermum*; on average 626-740 ASVs among sampling sites), 307 respectively (Fig. 3b). 308

309 In the MDS ordination of microbial community composition, *L. stictiforme* samples 310 clustered separately from *M. dendrospermum* ones (Fig. 4). Among *L. stictiforme* samples, those 311 from ST27 and ST7 were closer to each other than those from ST25. *M. dendrospermum* samples 312 from sites ST12 and ST17 clustered together more closely than those collected in site ST13. 313

313 The comparison of CCA-associated bacteria communities, both between samples of the 314 two CCA species and between samples of the same CCA species collected in different sites, 315 showed significant differences (*p*-value = 0.001***, between CCA species; *p*-value = 0.005**, 316 between *L. stictiforme* sampling sites; *p*-value = 0.002**, between *M. dendrospermum* sampling 317 sites; Table S4). Intraspecific pairwise comparison did not capture significant differences between 318 any sample pair, but higher F values could indicate a larger difference between *L. stictiforme* 319 bacteria communities in ST7 and ST25, and *M. dendrospermum* bacteria communities in ST12 320 and ST13, and ST17 and ST13 (Table S6). 321

322

322 **3.4 CCA bacterial community composition**
The taxonomy of CCA-associated bacteria was first analyzed at the class and order levels considering the five most contributing taxa as relative abundance across the dataset. At the class level, the bacterial communities of both CCA species were dominated by Alphaproteobacteria, which accounted for 40% and 46% of the sequences in *L. stictiforme* and *M. dendrospermum*, respectively (Supp. Fig. 5A, C). They were followed by Gammaproteobacteria (18% and 10%), Acidimicrobiia (12% and 5%), Planctomycetacia (9% and 18%), and Bacteroidia (5% and 7%). At the order level, the most abundant group in *L. stictiforme* was Rhizobiales (15%), while in *M. dendrospermum* it was Pirellulales (18%) (Supp. Fig. 5B, D). These two orders were shared among the two CCAs. Order Rhodobacterales was also shared, while Cellvibrionales and Microtrichales (8% and 12%), and Flavobacterales and Rhodovibrionales (6% and 7%) were abundant only in *L. stictiforme* and *M. dendrospermum* bacteria communities, respectively.

At the family level, among the 20 most contributing families, Pirellulaceae (phylum Planctomycetes) were abundant in all CCA bacteria assemblages, contributing on average 4.5-15% to *L. stictiforme* bacteria communities in ST25 and ST7, and 13-23.4% to *M. dendrospermum* bacteria communities in ST13 and ST12, respectively (Fig. 5a, b). Shared bacteria families were also Microtrichaceae (phylum Actinobacteria) and Rhodobacteraceae (class Alphaproteobacteria), more abundant in *L. stictiforme* (average percentage contribution 7.4-15% in ST7 and ST25 samples, and 5.8-11.4% in ST25 and ST7 samples, respectively), and Kiloniellaceae (class Alphaproteobacteria) and Flavobacteriaceae (class Bacteroida), more abundant in *M. dendrospermum*, with average contribution of 3.1-12.5% in ST12 and ST13 and 4.1-6.9% in ST13, respectively.

Despite these similarities, differences were observed at the family level both between- and within-CCA species. Spongiibacteraceae (class Gammaproteobacteria) and Stappiaceae (class
346 Alphaproteobacteria) were mainly found in *L. stictiforme*. Within this species Spongiibacteraceae 347 were abundant in ST27 and Stappiaceae in ST25, representing on average 23.2% and 6.8% of the 348 bacterial community, respectively (Fig. 5a).

349 At the genus level, among the 10 most contributing genera only three were shared between 350 the two CCA species: undefined Geminicoccaceae, *Pir4_lineage* and *Blastopirellula* (Supp. Fig. 351 6). All the other genera were uniquely associated with one of the two CCAs. In particular, 352 undefined Microtrichaceae (phylum Actinobacteria) and BD1-7_clade were abundant in *L. 353 stictiforme*, while an unknown genus belonging to Alphaproteobacteriaceae and *Bythopirellula* 354 highly contributed to *M. dendrospermum* microbiome (Supp. Fig. 6).

355
356

**3.5 Core shared and unique and differently abundant bacteria**

357 A total of 65 ASVs (2.5% of the ASVs) constituted the core microbiome (ASVs present in at least 358 75% of samples) shared between *L. stictiforme* and *M. dendrospermum* (Fig. 6a). A total of 86 359 (3.3%) and 154 (5.9%) ASVs were uniquely associated to the core microbiome of *L. stictiforme* 360 and *M. dendrospermum*, respectively. When comparing sampling sites, in *L. stictiforme*, 23 ASVs 361 (0.5% of the total ASVs of *L. stictiforme* bacteria) were present as core taxa at all sites, with sites 362 ST7 and ST27 further sharing the highest number of ASVs (20) (Fig. 7b). For individuals collected 363 in the same site, 4.2 to 7.5% of core ASVs were shared among samples of ST27 and among 364 samples of ST25, respectively (Fig. 6b). In *M. dendrospermum*, 84 core ASVs (1.4% of the total 365 *M. dendrospermum* bacteria ASVs) were found in all sites, and ST12 and ST17 further shared the 366 most core ASVs (40). For *M. dendrospermum* samples collected in the same site, the percentage 367 of core ASVs ranged between 11.3-15.4% among samples of ST12 and ST13, respectively (Fig. 368 6c).
Among the core ASVs, ASVs 13 and 24 were identified as belonging to *Filomicrobium* 369 species, ASV 39 was classified as *Ruegeria atlantica*, ASV 48 was ascribed to 370 *Sva0996_marine_group*, and ASV 14 belonged to the family Rhizobiaceae (Table 1). Among the 371 core ASVs unique to each CCA species, ASVs of the genera *Pir4_lineage* and *Blastopirellula* 372 were present in all CCA bacteria assemblages. 373

A total of 152 ASVs were differently abundant between CCA species. Among them, 89 374 were the most significantly different, meeting an adjusted *p*-value ≤0.001***, and belonged 375 mainly to the families Pirellulaceae, Rhodobacteraceae, Flavobacteriaceae and Rubritaleaceae 376 (Fig. 7a). Among these, the 20 most differently abundant showed distinct distributional patterns 377 between CCA species that clustered separately (Fig. 7b), and felt within the list of non-shared 378 ASVs between the two algal species (Table 1). The complete list of differently abundant ASVs 379 between CCA species is reported in Table S7. 380

Between *L. stictiforme* clades 3B and C3, 17 ASVs were significantly different in 381 abundance, among which ASV876 Desulfobulbaceae, ASV686 Microtrichaceae, ASV47 and 382 ASV212 Geminicocaceae presented the highest log2 fold change values, and ASV20 383 Prochloraceae, ASV9 undefined Gammaproteobacteria and ASV1 Spongiibacteraceae presented 384 the lowest ones (Fig. 8a). Distinct distributional patterns of ASVs between clades are shown in 385 Fig. 8b. Samples belonging to the two clades clustered separately, as well as samples of clade 3B 386 when comparing ST7 and ST27 sampling sites. Detailed results are reported in Table S8. 387

4. Discussion 390
The results from both morpho-anatomical and molecular taxonomy confirmed the assignment of the analyzed CCA thalli to *Lithophyllum stictiforme* and *Macroblastum dendrospermum*. Our study shows that individuals belonging to diverse haplotypes of the *L. stictiforme* complex can coexist within a restricted area. Indeed, molecular taxonomy results revealed that two of the three sampling sites hosted individuals of clade 3B, while the CCAs from ST25 belonged to haplotype C3. The latter haplotype was previously and uniquely identified in the same sampling area (Pezzolesi et al., 2019; de Jode et al., 2019), indicating a possible Banyuls endemism.

The bacterial assemblages associated with the CCA samples showed similar or higher diversity compared to CCA microbiomes from other marine regions (e.g., Barott et al., 2011; Hester et al., 2016 and Yang et al., 2021). Mediterranean CCAs may therefore represent a reservoir of bacterial diversity, emphasizing the need for further research on the role of microbial communities in coralligenous habitat functioning.

In both CCA species, Proteobacteria dominated the bacterial communities, especially the Alpha and Gammaproteobacteria classes, followed by Planctomycetacia, Acidimicrobiia and Bacteroidia. These groups have been frequently detected in most of the investigated CCAs (Barott et al., 2011; Sneed et al., 2015; Brodie et al., 2016; Cavalcanti et al., 2018; Gefen-Treves et al., 2021). At the order level, Rhodobacterales, Rhizobiales and Rhodovibrionales (all Alphaproteobacteria) were among the dominant taxa, which is consistent with previous studies (Cavalcanti et al., 2014; Hester et al., 2016; Quéré et al., 2019). This shows that Mediterranean CCAs host bacterial communities that are taxonomically similar to those of other CCAs worldwide, at least at the class and order levels. In our study, the dominant bacterial families were also the most shared among the CCAs. Microtrichaceae and Rhodobacteriaceae, which were abundant in *L. stictiforme* individuals, were also well represented in *M. dendrospermum*, while
Kiloniellaceae and Flavobacteriaceae, which predominate in *M. dendrospermum*, were also abundant in *L. stictiforme* bacterial communities. Nonetheless, bacterial communities were found to differ between the two CCA species, with *M. dendrospermum* exhibiting higher bacterial alpha diversity based on the Shannon index. Studies from other regions have similarly reported species-specific microbiomes in other CCA species (e.g., see Barrott et al., 2011 - Caribbean; Sneed et al., 2015 - Belize; Brodie et al. 2016 - UK). At the family level, the bacterial taxa that contributed differentially to CCAs included Thiohalorhabdaceae and Illumatobacteraceae, which were exclusively associated with *L. stictiforme*, and Desulfobulbaceae and NB1-j, which occurred only in *M. dendrospermum*. These families were also unevenly distributed among host individuals. Indeed, bacterial communities differed not only between CCA species, but also between individuals of the same species, even if they were collected within a small geographical area (ca. 30 m²). CCA intraspecific variability of bacterial communities has previously been linked to geographical distance, depth gradient, seasonality, and anthropogenic influences (Gefen-Treves et al., 2021). Host genotype can also influence microbial diversity, as shown by studies on seaweeds (Wood et al., 2022; Vadillo Gonzalez et al., 2023). However, environmental conditions often play a more important role than genotype in shaping holobiont microbial communities, driving adaptive responses in the microbiome (Pearman et al., 2024; Wood et al., 2022; Baldassarre et al., 2023; Dubé et al., 2021).

Remarkably, we found two highly predominant bacteria families exclusively present in *L. stictiforme*, namely Spongiibacteriaceae and Stappiaceae. Spongiibacteriaceae occurred almost exclusively in clade 3B of ST27, while Stappiaceae was predominant only in clade C3. Spongiibacteriaceae are Gammaproteobacteria potentially able to use proteorhodopsin to exploit light as an additional energy source (Holert et al., 2018). This group was found in association with
the CCA *Neogoniolithon trichotomum* in the Gulf of California, at high temperature and silicate concentration (Valdespino-Castillo et al., 2021), and in Mediterranean sponges as steroid-degrading bacteria (Spring et al., 2015). In this sense, steroids may act as immune defense boosters under thermal stress and they have been associated to anti-inflammatory, antioxidant and antifouling effects in corals (Kong et al., 2012; Dembitsky, 2023; Ochsenkühn et al., 2023). They can also be produced by the tropical CCA *Hydrolithon boergesenii*, potentially acting as triggers for coral larval settlement (Quinlan et al., 2023). Stappiaceae have been found associated with benthic organisms (e.g., sponges, corals, anemones). They have genetic traits that hint to a possible role in sulfur cycling and dimethylsulfoniopropionate (DMSP) production, a scavenge ROS compound with antioxidant potential, and in increased abundance in response to thermal stress (Couceiro et al., 2021; Heric et al., 2023). The reason behind the selective abundance of these two families in two of the three *L. stictiforme* sampling sites remains unclear. We can hypothesize that the effects of the individual genotype on the microbial communities' composition, combined with microenvironmental variations (e.g., light and nutrients availability, presence of localized stressors) within and around CCA tissues, (see for instance Valdespino-Castillo et al., 2021), cause the observed differences between individuals of the same species. Furthermore, different thalli may vary in age, possibly influencing the diversity of the microbiome that has been shown to increase with age (Bengtsson et al., 2012; Sneed et al., 2015). Differential predation pressure on CCA thalli may also induce the proliferation of protective, biofilm-forming bacteria (Matz et al., 2008).

At the ASV level, only 2.5% of the total ASVs were shared between CCA species, while those that were differentially abundant corresponded to the 5.9%. The number of shared ASVs between individuals of the same CCA species was also low compared to previous studies (Miranda
et al., 2013; Brodie et al., 2016; Valdespino-Castillo et al., 2021). In *L. stictiforme*, not only few ASVs were shared between taxonomic clades, but also between thalli of the same clade. Hierarchical clustering on differently abundant ASVs showed *L. stictiforme* samples clustering primarily in relation to the clade they belonged to, and subsequently according to the sampling site. Indeed, these ASVs differently enriched the bacterial communities associated with clade 3B thalli collected in different sampling sites. These results further indicated that both genetic and environmental factors drive the composition of bacterial associations with CCAs, not only at species but also at haplotype level, and that CCA holobionts can possess a high level of adaptation to environmental changes. The percentage of shared ASVs only increased (4.2-7.5%) when individuals collected from the same sampling sites were compared. These results suggest a third potential driver in holobiont composition: the physical proximity between hosts may promote the development of more similar bacterial assemblages. We can hypothesize that this proximity effect could be due to bacteria horizontal transfer (Hester et al., 2016). These hypotheses need to be further investigated by specifically targeting these bacteria.

Among the core bacteria, we found ASVs shared among individuals of the two species. These ASVs were similar to some earlier found in biofilms, but also in endolithic communities of marine outcrops or in sponges and seaweeds (see Table 1). Diverse ASVs of *Blastopirellula* and *Pir4_lineage* fell within the list of top five abundant ASVs of both CCA species. Planctomycetes are commonly associated with red algae being able to resist the action of several algal antimicrobial compounds, and to live in biofilms taking advantage of the polysaccharides commonly produced by the algae, especially sulphated heteropolysaccharides (agars and carrageenans) (Lage & Bondoso, 2014; Wegner et al. 2013; Bergstrom et al., 2023). In particular, *Blastopirellula* and the *Pir4_lineage* can exploit such compounds thanks to sulfatase enzymes (Bondoso et al., 2017).
Interestingly, sulfated glucosaminoglycan, together with other glucosamine and galactose residues, can be produced by certain CCAs and act as potential triggers for coral larvae settlement and metamorphosis (Morse & Morse, 1991). Although these ASVs were not shared between the two CCA species, they were predominantly shared among individuals either of *L. stictiforme* and *M. dendrospermum*. These findings suggest that the facilitative interaction between Mediterranean CCAs and gorgonians could be mediated by bacteria, deserving further investigation.

## 5. Conclusions

Our results showed that both *Litophyllum stictiforme* and *Macroblastum dendrospermum* host holobiont-specific bacterial communities, potentially serving as reservoirs of bacterial diversity. The composition of these microbial communities differed also among individual CCA thalli. We hypothesize three main drivers of such differences: i) microbiome associations linked to host genotype, ii) adaptation of bacterial communities to exogenous factors acting at the scale of individual thalli, and iii) potential host-to-host bacteria transfer between holobionts in close proximity. These hypotheses should be further tested by examining bacterial communities responses to stress conditions, both in field and through manipulative experiments, also exploring their possible seasonal and geographical variability. In the two CCA species studied, we identified predominant bacterial genera affiliated with taxa known to potentially promote coral larval settlement in tropical environments. These findings offer promising insights for future research on the positive interaction between CCA holobionts and coral larvae, mediated by bacteria, in the Mediterranean Sea.

## Declaration of interests
The authors declare no competing financial or non-financial interests.

507

## 508 Acknowledgments

EM was supported by the support of the European Union programme Marie Sklodowska-Curie
509
Actions through the project RESTORE [grant number 101062275] and the European Union's
510
Horizon 2020 research and innovation programme, ASSEMBLE Plus project [grant number
511
730984]. LB was supported by Fondation Prince Albert II (Project ROMERO). PEG
512
acknowledges the support of Agence Nationale de la Recherche (ANR) through the CoralMates
513
project (ANR-18-CE02-0009).
514

515

516 Supplementary Information

The link to the electronic supplementary material can be found online. Crustose coralline algae
517
(CCAs) and bacterial sequences are accessible at the European Nucleotide Archive (ENA;
518
https://www.ebi.ac.uk/ena/browser/home) under the project accession numbers PRJEB83080
519
and PRJEB82879, respectively.
520

521

522 References

- Acosta-González A, Rosselló-Móra R, Marqués S (2013) Characterization of the anaerobic
523
microbial community in oil-polluted subtidal sediments: aromatic biodegradation potential after
524
the Prestige oil spill. Environmental microbiology 15(1):77-92. doi.org/10.1111/j.1462-
525
2920.2012.02782.x
526
527 - Athanasiadis A, Ballantine DL (2024) Anatomy and Classification of the Mesophyllaceae (Corallinales, Rhodophyta), Based on Phylogenetic Principles. Smithsonian Institution Scholarly Press. Book. doi.org/10.5479/si.25267183

530 - Asnaghi V, Thrush SF, Hewitt JE, Mangialajo L, Cattaneo-Vietti R, Chiantore M (2015) Colonisation processes and the role of coralline algae in rocky shore community dynamics. Journal of Sea Research 95:132-138. doi.org/10.1016/j.seares.2014.07.012

533 - Ballesteros E (2006) Mediterranean coralligenous assemblages: A synthesis of present knowledge. Oceanography and Marine Biology: An Annual Review 44:123-195.

535 - Barnett DJ, Arts IC, Penders J (2021). microViz: an R package for microbiome data visualization and statistics. Journal of Open Source Software, 6(63):3201. DOI: 10.21105/joss.03201

537 - Barott KL, Rodriguez-Brito B, Janouškovec J, Marhaver KL, Smith JE, et al (2011) Microbial diversity associated with four functional groups of benthic reef algae and the reef-building coral *Montastraea annularis*. Environmental Microbiology 13(5):1192-1204. doi.org/10.1111/j.1462-2920.2010.02419.x

541 - Bengtsson MM, Øvreås L (2010) Planctomycetes dominate biofilms on surfaces of the kelp Laminaria hyperborea. BMC Microbiology 10:1-12. doi.org/10.1186/1471-2180-10-261

543 - Bengtsson MM, Sjotun K, Lanzen A, Ovreas L (2012) Bacterial diversity in relation to secondary production and succession on surfaces of the kelp *Laminaria hyperborea*. ISME Journal 6:2188-2198. doi.org/10.1038/ismej.2012.67
546 - Bergstrom E, Lahnstein J, Collins H, Page TM, Bulone V, Diaz-Pulido G (2023) Cell wall
547 organic matrix composition and biomineralization across reef-building coralline algae under
548 global change. Journal of Phycology 59(1):111-125. DOI: 10.1111/jpy.13290

549 - Bittner L, Halary S, Payri C, Cruaud C, de Reviers B, et al (2010) Some considerations for
550 analyzing biodiversity using integrative metagenomics and gene networks. Biol. Direct 5(1):1-17.
551 doi.org/10.1186/1745-6150-5-47

552 - Bondoso J, Godoy-Vitorino F, Balague V, Gasol JM, Harder J, Lage OM (2017) Epiphytic
553 Planctomycetes communities associated with three main groups of macroalgae. FEMS
554 Microbiology Ecology 93(3):fiw255. doi.org/10.1093/femsec/fiw255

555 - Bracchi VA, Bazzicalupo P, Fallati L, Varzi AG, Savini A et al (2022) The main builders of
556 Mediterranean coralligenous: 2D and 3D quantitative approaches for its identification. Frontiers
557 in Earth Science 10:910522. doi.org/10.3389/feart.2022.910522

558 - Bramanti L, Rossi S, Tsounis G, Gili JM, Santangelo G (2007) Settlement and early survival of
559 red coral on artificial substrates in different geographic areas: some clues for demography and
600 restoration. Hydrobiologia 580:219-224. doi.org/10.1007/s10750-006-0452-1

561 - Bramanti L, Manea E, Giordano B, Estaque T, Bianchimani O, et al (2023) The deep vault: a
temporary refuge for temperate gorgonian forests facing marine heat waves. Mediterranean Marine
562 Science 24(3):601-609. doi: 10.12681/mms.35564

564 - Brodie J, Williamson C, Barker GL, Walker RH, Briscoe A, Yallop M (2016) Characterising the
565 microbiome of *Corallina officinalis*, a dominant calcified intertidal red alga. FEMS Microbiology
566 Ecology 92(8):fiw110. doi.org/10.1093/femsec/fiw110
567 - Broom JES, Hart DR, Farr TJ, Nelson WA, Neill KF, et al (2008) Utility of psbA and nSSU for phylogenetic reconstruction in the Corallinales based on New Zealand taxa. Molecular Phylogenetics and Evolution 46:958-73. doi.org/10.1016/j.ympev.2007.12.016

570 - Burke C, Steinberg P, Rusch D, Kjelleberg S, Thomas T (2011) Bacterial community assembly based on functional genes rather than species. Proceedings of the National Academy of Sciences 108(34):14288-14293. doi.org/10.1073/pnas.110159110

573 - Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nature Methods 13:581-583. doi: 10.1038/nmeth.3869

576 - Casoli E, Ventura D, Mancini G, Cardone S, Farina F, et al (2022) Rehabilitation of Mediterranean animal forests using gorgonians from fisheries by-catch. Restoration Ecology 30(1):e13465. doi.org/10.1111/rec.13465

579 - Castellan G, Angeletti L, Montagna P, Taviani M (2022) Drawing the borders of the mesophotic zone of the Mediterranean Sea using satellite data. Scientific Reports 12:5585. doi.org/10.1038/s41598-022-09413-4

582 - Cavalcanti GS, Gregoracci GB, Dos Santos EO, Silveira CB, Meirelles PM, et al (2014) Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean. ISME Journal 8(1):52-62. doi.org/10.1038/ismej.2013.133

585 - Cavalcanti GS, Shukla P, Morris M, Ribeiro B, Foley M, Doane MP, Thompson CC, Edwards MS, Dinsdale EA, Thompson FL (2018) Rhodoliths holobionts in a changing ocean: host-microbes interactions mediate coralline algae resilience under ocean acidification. BMC Genomics 19:701. doi.org/10.1186/s12864-018-5064-4
589 - Chatzimentor A, Doxa A, Katsanevakis S, Mazaris AD (2023) Are Mediterranean marine
590 threatened species at high risk by climate change?. Global Change Biology 29(7):1809-1821.
doi.org/10.1111/gcb.16577

592 - Choi DH, Noh JH, Yu OH, Kang YS (2010) Bacterial diversity in biofilms formed on condenser
593 tube surfaces in a nuclear power plant. Biofouling 26(8):953-959.
doi.org/10.1080/08927014.2010.533267

595 - Comeau, AM, Kwawukume, A (2023). Preparing multiplexed 16S/18S/ITS amplicons for the
596 Illumina MiSeq. Protocols.io. dx.doi.org/10.17504/protocols.io.4r3l277k3g1y/v1

597 - Cornwall CE, Harvey BP, Comeau S, Cornwall DL, Hall-Spencer JM, et al (2022)
598 Understanding coralline algal responses to ocean acidification: Meta-analysis and synthesis.
599 Global Change Biology 28(2):362-374. doi.org/10.1111/gcb.15899

600 - Couceiro JF, Keller-Costa T, Marques M, Kyrpides NC, Woyke T, et al (2021) The *Roseibium
601 album* (*Labrenzia alba*) genome possesses multiple symbiosis factors possibly underpinning host-
602 microbe relationships in the marine benthos. Microbiology Resource Announcements 10(34):10-
603 1128. doi.org/10.1128/mra.00320-21

604 - Couradeau E, Roush D, Guida BS, Garcia-Pichel F (2017) Diversity and mineral substrate
605 preference in endolithic microbial communities from marine intertidal outcrops (Isla de Mona,
606 Puerto Rico). Biogeosciences 14(2):311-324. doi.org/10.5194/bg-14-311-2017

607 - Dang H, Li T, Chen M, Huang G (2008) Cross-ocean distribution of Rhodobacterales bacteria as
608 primary surface colonizers in temperate coastal marine waters. Applied and Environmental
609 Microbiology 74(1):52-60. doi.org/10.1128/AEM.01400-07
610 - Dat TTH, Cuc NTK, Cuong PV, Smidt H, Sipkema D (2021) Diversity and antimicrobial activity 611 of Vietnamese sponge-associated bacteria. Marine drugs 19(7):353. doi.org/10.3390/md19070353

612 - Dembitsky VM (2023) Biological Activity and Structural Diversity of Steroids Containing 613 Aromatic Rings, Phosphate Groups, or Halogen Atoms. Molecules 28(14):5549. 614 doi.org/10.3390/molecules28145549

615 - De Jode A, David R, Haguenauer A, Cahill AE, Erga Z et al (2019) From seascape ecology to 616 population genomics and back. Spatial and ecological differentiation among cryptic species of the 617 red algae *Lithophyllum stictiforme/L. cabiochiae*, main bioconstructors of coralligenous habitats. 618 Molecular Phylogenetics and Evolution 137:104-113. doi.org/10.1016/j.ympev.2019.04.005

619 - Dubé CE, Ziegler M, Mercière A, Boissin E, Planes S, Bourmaud CF, Voolstra CR (2021). 620 Naturally occurring fire coral clones demonstrate a genetic and environmental basis of microbiome 621 composition. Nature Communications 12(1), 6402. doi.org/10.1038/s41467-021-26543-x

622 - Garrabou J, Gómez-Gras D, Medrano A, Cerrano C, Ponti M, et al (2022) Marine heatwaves 623 drive recurrent mass mortalities in the Mediterranean Sea. Global Change Biology 28(19):5708- 624 5725. doi.org/10.1111/gcb.16301

625 - Gefen-Treves S, Bartholomäus A, Horn F, Zaborowski AB, Tchernov D, et al (2021) The 626 microbiome associated with the reef builder *Neogoniolithon* sp. in the eastern 627 Mediterranean. Microorganisms 9 (7):1374. doi.org/10.3390/microorganisms9071374

628 - Glasl B, Webster NS, Bourne DG (2017) Microbial indicators as a diagnostic tool for assessing 629 water quality and climate stress in coral reef ecosystems. Marine Biology 164:1-18. 630 doi.org/10.1007/s00227-017-3097-x
631 - Gómez-Lemos LA, Diaz-Pulido G (2017) Crustose coralline algae and associated microbial
632 biofilms deter seaweed settlement on coral reefs. Coral Reefs 36:453-462.
633 doi.org/10.1007/s00338-017-1549-x

634 - Gómez-Lemos LA, Doropoulos C, Bayraktarov E, Diaz-Pulido G (2018) Coralline algal
635 metabolites induce settlement and mediate the inductive effect of epiphytic microbes on coral
636 larvae. Scientific Reports 8(1):17557. doi.org/10.1038/s41598-018-35206-9

637 - Goldsmith DB, Pratte ZA, Kellogg CA, Snader SE, Sharp KH (2019) Stability of temperate coral
638 Astrangia poculata microbiome is reflected across different sequencing methodologies. AIMS
639 Microbiology 5(1):62-76. doi: 10.3934/microbiol.2019.1.62

640 - Havemann SA, Foster JS (2008) Comparative characterization of the microbial diversities of an
641 artificial microbialite model and a natural stromatolite. Appl Environ Microbiol 74(23):7410-21.
642 doi:10.1128/AEM.01710-08

643 - Heric K, Maire J, Deore P, Perez-Gonzalez A, van Oppen MJ (2023) Inoculation with
644 Roseovarius increases thermal tolerance of the coral photosymbiont, *Breviolum
645 minutum*. Frontiers in Ecology and Evolution 11. doi.org/10.3389/fevo.2023.1079271

646 - Hereu B, Kersting DK (2016) Diseases of coralline algae in the Mediterranean Sea. Coral reefs
647 35(2):713-713. DOI 10.1007/s00338-016-1428-x

648 - Hester ER, Barott KL, Nulton J, Vermeij MJ, Rohwer FL (2016) Stable and sporadic symbiotic
649 communities of coral and algal holobionts. ISME Journal 10(5):1157-1169.
650 doi.org/10.1038/ismej.2015.190
651 - Holert J, Cardenas E, Bergstrand LH, Zaikova E, Hahn AS, et al (2018) Metagenomes reveal global distribution of bacterial steroid catabolism in natural, engineered, and host environments. mBio 9(1):10-1128. doi.org/10.1128/mbio.02345-17

654 - Iborra L, Leduc M, Fullgrabe L, Cuny P, Gobert S (2022) Temporal trends of two iconic Mediterranean gorgonians (*Paramuricea clavata* and *Eunicella cavolini*) in the climate change context. Journal of Sea Research 186:102241. doi.org/10.1016/j.seares.2022.102241

657 - Joint, I, Tait, K, Callow, ME, Callow, JA, Milton, D, Williams, P, Cámara, M (2002). Cell-to-cell communication across the prokaryote-eukaryote boundary. Science, 298(5596), 1207-1207. doi.org/10.1126/science.1077075

660 - Jorissen H, Galand PE, Bonnard I, Meiling S, Raviglione D, et al 2021. Coral larval settlement preferences linked to crustose coralline algae with distinct chemical and microbial signatures. Scientific Reports 11(1):14610. doi.org/10.1038/s41598-021-94096-6

663 - Kaleb S, Alongi G, Falace A (2018) Coralline algae preparation for scanning electron microscopy and optical microscopy. In: Charrier B, Wichard T, Reddy CRK (eds.) Protocols for Macroalgae Research. CRC Press, Taylor & Francis, Boca Raton, Florida. pp 413–29

666 - Kahng SE, Garcia-Sais JR, Spalding HL, Brokovich E, Wagner D, Weil E, Hinderstein L, Toonen RJ (2010) Community ecology of mesophotic coral reef ecosystems. Coral Reefs 29:255-275. doi.org/10.1007/s00338-010-0593-6

669 - Kimura M (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16 (2), 111-120. doi.org/10.1007/BF01731581.
672 Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, et al (2013) Evaluation of general 16S
673 ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity
674 studies. Nucleic Acids Research 41(1):e1-e1. doi.org/10.1093/nar/gks808

675 - Kong WW, Shao CL, Wang CY, Xu Y, Qian PY, et al (2012) Diterpenoids and steroids from
676 Gorgonian *Subergorgia mollis*. Chemistry of Natural Compounds 48:512-515.
677 doi:10.1007/s10600-012-0294-1

678 - Lage OM, Bondoso J (2014) Planctomycetes and macroalgae, a striking association. Frontiers in
679 microbioly 5:267. doi.org/10.3389/fmicb.2014.00267

680 - Lahti L, Shetty S., et al. (2017) Tools for microbiome analysis in R.
681 URL: http://microbiome.github.com/microbiome.

682 - Larsson A (2014) AliView: a fast and lightweight alignment viewer and editor for large
683 datasets. Bioinformatics 30(22):3276-3278. doi.org/10.1093/bioinformatics/btu531

684 - Lau SC, Thiyagarajan V, Cheung SC, Qian PY (2005) Roles of bacterial community composition
685 in biofilms as a mediator for larval settlement of three marine invertebrates. Aquatic Microbial
686 Ecology 38(1):41-51. doi:10.3354/ame038041

687 - Linares C, Coma R, Zabala M (2008) Restoration of threatened red gorgonian populations: an
688 experimental and modelling approach. Biological Conservation 141(2):427-437.
689 doi.org/10.1016/j.biocon.2007.10.012

690 - Love MI, Huber W, Anders S (2014). Moderated estimation of fold change and dispersion for
691 RNA-seq data with DESeq2. Genome biology 15:1-21. doi.org/10.1186/s13059-014-0550-8
692 - Manes CL, West N, Rapenne S, Lebaron P (2011) Dynamic bacterial communities on reverse-osmosis membranes in a full-scale desalination plant. Biofouling 27(1):47-58.
693
694 doi:10.1080/08927014.2010.536980.

695 - Martin S, Gattuso JP (2009) Response of Mediterranean coralline algae to ocean acidification
696 and elevated temperature. Global Change Biology 15(8):2089-2100. doi.org/10.1111/j.1365-6979.2009.01874.x

698 - Martin CS, Giannoulaki M, De Leo F, Scardi M, Salomidi M, et al (2014) Coralligenous and maërl habitats: predictive modelling to identify their spatial distributions across the Mediterranean
700 Sea. Scientific Reports 4(1):5073. doi.org/10.1038/srep05073

701 - Matsuo Y, Imagawa H, Nishizawa M, Shizuri Y (2005). Isolation of an algal morphogenesis
702 inducer from a marine bacterium. Science 307(5715):1598-1598.
703 doi.org/10.1126/science.1105486

704 - Matz C, Webb JS, Schupp PJ, Phang SY, Penesyan A, Egan S, et al. (2008) Marine biofilm
705 bacteria evade eukaryotic predation by targeted chemical defense. PloS One 3(7):e2744.
706 doi.org/10.1371/journal.pone.0002744

707 - McLaren MR, Callahan BJ (2021) Silva 138.1 prokaryotic SSU taxonomic training data
708 formatted for DADA2 [Data set]. Zenodo. doi.org/10.5281/zenodo.4587955

709 - McMurdie PJ, Holmes S (2013) phyloseq: an r package for reproducible interactive analysis and
710 graphics of microbiome census data. PLoS One 8:e61217. doi: 10.1371/journal.pone.0061217
711 - Miranda LN, Hutchison K, Grossman AR, Brawley SH (2013) Diversity and abundance of the
712 bacterial community of the red macroalga *Porphyra umbilicalis*: did bacterial farmers produce
713 macroalgae?. PLoS One 8(3):e58269. doi.org/10.1371/journal.pone.0058269

714 - Mohamed NM, Saito K, Tal Y, Hill RT (2010) Diversity of aerobic and anaerobic ammonia-
715 oxidizing bacteria in marine sponges. The ISME journal 4(1):38-48.
716 doi.org/10.1038/ismej.2009.84

717 - Montalvo NF, Hill RT (2011) Sponge-associated bacteria are strictly maintained in two closely
718 related but geographically distant sponge hosts. Applied and Environmental Microbiology,
719 77(20):7207-7216. doi.org/10.1128/AEM.05285-11

720 - Montseny M, Linares C, Viladrich N, Olariaga A, Carreras M, et al (2019) First attempts towards
721 the restoration of gorgonian populations on the Mediterranean continental shelf. Aquatic
722 Conservation: Marine and Freshwater Ecosystems 29(8):1278-1284. doi.org/10.1002/aqc.3118

723 - Morse DE, Morse AN (1991) Enzymatic characterization of the morphogen recognized by
724 *Agaricia humilis* (scleractinian coral) larvae. Biology Bulletin 181(1):104-122.
725 doi.org/10.2307/1542493

726 - Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New
727 York, 348 pp.

728 - Ochsenkühn M, Mohamed AR, Haydon TD, Coe LS, Abrego D, Amin SA (2023)
729 *Endozoicomonas* provides corals with steroid hormones during thermal stress. bioRxiv, 2023-09.

730 - Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, et al (2013) Vegan: Community
731 Ecology Package. R Package Version. 2.5-2.
732 - O'Leary JK, Barry JP, Gabrielson PW, Rogers-Bennett L, Potts DC, et al (2017) Calcifying algae maintain settlement cues to larval abalone following algal exposure to extreme ocean acidification. Scientific Reports 7(1):1-10. DOI:10.1038/s41598-017-05502-x

735 - Pearman WS, Duffy GA, Liu XP, Gemmell NJ, Morales SE, Fraser CI (2024). Macroalgal microbiome biogeography is shaped by environmental drivers rather than geographical distance. Annals of botany 133(1), 169-182. doi.org/10.1093/aob/mcad151

738 - Peña V, De Clerck O, Afonso-Carrillo J, Ballesteros E, Bárbara I, et al (2015) An integrative systematic approach to species diversity and distribution in the genus *Mesophyllum* (Corallinales, Rhodophyta) in Atlantic and Mediterranean Europe. European Journal of Phycology 50(1):20-36. DOI: 10.1080/09670262.2014.981294

742 - Pezzolesi L, Falace A, Kaleb S, Hernande-zKantun JJ, Cerrano C, Rindi F (2017) Genetic and morphological variation in an ecosystem engineer, *Lithophyllum byssoides* (Corallinales, Rhodophyta). Journal of Phycology 53(1):146-160. doi.org/10.1111/jpy.12837

745 - Pezzolesi L, Peña V, Le Gall L, Gabrielson PW, Kaleb S, et al (2019) Mediterranean *Lithophyllum stictiforme* (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats. Journal of Phycology 55(2):473-492. doi.org/10.1111/jpy.12837

749 - Piazzi L, Pinna F, Ceccherelli G (2022) Crustose coralline algae and biodiversity enhancement: The role of *Lithophyllum stictiforme* in structuring Mediterranean coralligenous reefs. Estuarine, Coastal and Shelf Science 278:108121. doi.org/10.1016/j.ecss.2022.108121
752 - Quéré G, Intertaglia L, Payri C, Galand PE (2019) Disease specific bacterial communities in a
753 Coralline Algae of the Northwestern Mediterranean Sea: A combined culture dependent and-
754 independent approach. Frontiers in Microbiology 10:1850. doi.org/10.3389/fmicb.2019.01850

755 - Quinlan ZA, Bennett MJ, Arts MG, Levenstein M, Flores D, et al (2023) Coral larval settlement
756 induction using tissue-associated and exuded coralline algae metabolites and the identification of
757 putative chemical cues. Proceedings of the Royal Society B 290(2009):20231476.
758 doi.org/10.1098/rspb.2023.1476

759 - R Core Team (2024). *R: A Language and Environment for Statistical Computing*. R Foundation
760 for Statistical Computing, Vienna, Austria. https://www.R-project.org/.

761 - Rindi F, Braga JC, Martin S, Peña V, Le Gall L, et al (2019) Coralline algae in a changing
762 Mediterranean Sea: how can we predict their future, if we do not know their present?. Frontiers in
763 Marine Science 6:723. doi.org/10.3389/fmars.2019.00723

764 - Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing
765 phylogenetic trees. Molecular Biology and Evolution 4:406-425.

766 - Salonen A, Salojärvi J, Lahti L, de Vos WM (2012) The adult intestinal core microbiota is
767 determined by analysis depth and health status. Clinical Microbiology and Infection 18:16-20.
768 doi.org/10.1111/j.1469-0691.2012.03855.x

769 - Saunders GW (2005). Applying DNA barcoding to red macroalgae: a preliminary appraisal holds
770 promise for future applications. Philosophical Transactions of the Royal Society B, 360:1879-
771 1888.
772 - Schubert N, Schoenrock KM, Aguirre J, Kamenos NA, Silva J, Horta PA, Hofmann, LC (2020)
Coralline algae: Globally distributed ecosystem engineers. Frontiers in Marine Science 7:352.
doi.org/10.3389/fmars.2020.00352

775 - Seabra M, Cruz T, Fernandes J, Silva T, Hawkins S (2019) Recruitment of the limpet *Patella ulyssiponensis* and its relationship with crustose coralline algae: Patterns of juvenile distribution and larval settlement. Journal of the Marine Biological Association of the United Kingdom 99(8):1787-1796. doi:10.1017/S0025315419000869

779 - Short J, Foster T, Falter J, Kendrick GA, McCulloch MT (2015) Crustose coralline algal growth, calcification and mortality following a marine heatwave in Western Australia. Continental Shelf Research 106:38-44. doi.org/10.1016/j.csr.2015.07.003

782 - Siboni N, Abrego D, Puill-Stephan E, King WL, Bourne DG, et al (2020) Crustose coralline algae that promote coral larval settlement harbor distinct surface bacterial communities. Coral Reefs 39:1703-1713. doi.org/10.1007/s00338-020-01997-5

785 - Singh RP, Reddy CRK (2016) Unraveling the functions of the macroalgal microbiome. Frontiers
786 in Microbiology 6:1488. doi.org/10.3389/fmicb.2015.01488

787 - Singh RP, Kothari R, Egan S (2017) Exploring the Complexity of Macroalgal-Bacterial Interactions Through Interkingdom Signalling System. pp 301-315. In: Kumar, M., Ralph, P. (Eds) Systems Biology of Marine Ecosystems. Springer, Cham. doi.org/10.1007/978-3-319-62094-7_15

791 - Sneed JM, Ritson-Williams R, Paul VJ (2015) Crustose coralline algal species host distinct
792 bacterial assemblages on their surfaces. ISME Journal 9(11):2527-2536.
doi.org/10.1038/ismej.2015.67
794 - Spring S, Scheuner C, Göker M, Klenk HP (2015) A taxonomic framework for emerging groups of ecologically important marine gammaproteobacteria based on the reconstruction of evolutionary relationships using genome-scale data. *Frontiers in Microbiology* 6:281. doi.org/10.3389/fmicb.2015.00281

798 - Staufenberger T, Thiel V, Wiese J, Imhoff JF (2008) Phylogenetic analysis of bacteria associated with *Laminaria saccharina*. *FEMS Microbiology Ecology* 64(1):65-77. doi.org/10.1111/j.1574-6941.2008.00445.x

801 - Tamura K, Stecher G, Kumar S (2021) MEGA 11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution. doi.org/10.1093/molbev/msab120.

803 - Tignat-Perrier R, van de Water JA, Guillemain D, Aurelle D, Allemand D, Ferrier-Pagès C (2022) The effect of thermal stress on the physiology and bacterial communities of two key Mediterranean gorgonians. *Applied and Environmental Microbiology* 88(6):e02340-21. doi.org/10.1128/aem.02340-21

807 - Toma M, Bo M, Cattaneo-Vietti R, Canese S, Canessa M, et al (2022) Basin-scale occurrence and distribution of mesophotic and upper bathyal red coral forests along the Italian coasts. *Mediterranean Marine Science* 23(3):484-498. doi: 10.12681/mms.28052

810 - Toupoint N, Mohit V, Linossier I, Bourgougnon N, Myrand B, Olivier F, Lovejoy C, Tremblay R (2012) Effect of biofilm age on settlement of *Mytilus edulis*. *Biofouling* 28(9):985-1001. doi:10.1080/08927014.2012.725202.

813 - Vadillo Gonzalez S, Vranken S, Coleman MA, Wernberg T, Steinberg PD, Marzinelli EM (2023). Host genotype and microbiome associations in co-occurring clonal and non-clonal kelp, *Ecklonia radiata*. *Molecular Ecology* 32(16), 4584-4598. DOI: 10.1111/mec.17056
816 - Valdespino-Castillo PM, Bautista-García A, Favoretto F, Merino-Ibarra M, Alcántara-Hernández 817 RJ, et al (2021) Interplay of microbial communities with mineral environments in coralline 818 algae. Science of the Total Environment 757:143877. doi.org/10.1016/j.scitotenv.2020.143877

819 - Vitelletti ML, Manea E, Bongiorni L, Ricchi A, Sangelantoni L, Bonaldo D (2023) Modelling 820 distribution and fate of coralligenous habitat in the Northern Adriatic Sea under a severe climate 821 change scenario. Frontiers in Marine Science 10:88. doi.org/10.3389/fmars.2023.1050293

822 - Vojvoda J, Lamy D, Sintes E, Garcia JA, Turk V, Herndl GJ (2014) Seasonal variation in marine-823 snow-associated and ambient-water prokaryotic communities in the northern Adriatic 824 Sea. Aquatic Microbial Ecology 73(3):211-224. doi.org/10.3354/ame01718

825 - Webster NS, Soo R, Cobb R, Negri AP (2011) Elevated seawater temperature causes a microbial 826 shift on crustose coralline algae with implications for the recruitment of coral larvae. The ISME 827 journal 5(4):759-770. doi.org/10.1038/ismej.2010.152

828 - Wegner CE, Richter-Heitmann T, Klindworth A, Klockow C, Richter M, et al (2013) Expression 829 of sulfatases in Rhodopirellula baltica and the diversity of sulfatases in the genus 830 Rhodopirellula. Marine Genomics 9:51-61. doi.org/10.1016/j.margen.2012.12.001

831 - Wood G, Steinberg PD, Campbell AH, Vergés A, Coleman MA, Marzinelli EM (2022). Host 832 genetics, phenotype and geography structure the microbiome of a foundational 833 seaweed. Molecular Ecology 31(7), 2189-2206. doi.org/10.1111/mec.16378

834 - Yang Z, Li Z (2012) Spatial distribution of prokaryotic symbionts andammoxidation, denitrifier 835 bacteria in marine spongeAstrosclera willeyana. Sci Rep. 2:528. doi: 10.1038/srep00528.
836 - Yang F, Mo J, Wei Z, Long L (2021) Calcified macroalgae and their bacterial community in relation to larval settlement and metamorphosis of reef-building coral *Pocillopora damicornis*. FEMS Microbiology Ecology 97(1):fiaa215. doi.org/10.1093/femsec/fiaa215

839 - Zelli E, Quéré G, Lago N, Di Franco G, Costantini F, et al (2020) Settlement dynamics and recruitment responses of Mediterranean gorgonians larvae to different crustose coralline algae species. Journal of Experimental Marine Biology and Ecology 530:151427. doi.org/10.1016/j.jembe.2020.151427

843 - Zozaya-Valdes, E, Egan, S, & Thomas, T (2015). A comprehensive analysis of the microbial communities of healthy and diseased marine macroalgae and the detection of known and potential bacterial pathogens. Frontiers in Microbiology, 6, 146. doi.org/10.3389/fmicb.2015.00146

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848 **Figures and Table**

849
<figure><img src="image_1.png" /><figcaption>**Fig. 1** *Lithophyllum stictiforme* phylogenetic tree. Phylogenetic tree inferred from ML analysis of the psbA sequences of Mediterranean *Lithophyllum stictiforme* and other publicly available sequences for this complex. Bootstrap ML values > 60% are shown for each node. *L. dentatum*, *L. hibernicum* and *L. bathyporum* were used as outgroup. Clades 1 to 13 refer to the classification provided by Pezzolesi et al. (2019), while C3 corresponds to the haplotype identified by de Jode et al. (2019). Scale bar: 0.01 substitutions per site. Sequences generated in the present work are marked in bold</figcaption></figure>

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<figure><img src="image_2.png" /><figcaption>**Fig. 2** *Macroblastum dendrospermum* phylogenetic tree of the psbA sequences. Phylogenetic tree inferred from ML analysis of the psbA sequences of Mediterranean *Mesophyllum* and other publicly available sequences for this genus as *Mesophyllum macroblastum* was only recently recognized as *Macroblastum dendrospermum* (Athanasiadis and Ballantine, 2024). Bootstrap ML values > 60% are shown for each node. Members of the subfamily Melobesioideae were used as outgroup. Scale bar: 0.02 substitutions per site. Sequences generated in the present work are marked in bold</figcaption></figure>

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<figure><img src="image_3.png" /><figcaption>**Fig. 3** Alpha diversity of bacterial communities associated with the CCA *Lithophyllum stictiforme* and *Macroblastum dendrospermum*. **a.** Mean values of the Shannon index in prokaryotic communities associated with CCAs from the six sampling sites. Significant differences are reported coded by letter and "*" (p-value < 0.05). **b.** Bacterial community richness (as observed ASVs) reported in each CCA individual.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 4** Beta diversity of bacterial communities associated with CCA hosts. Multidimensional scaling ordination (MDS) based on Bray-Curtis distance matrix computed from Hellinger transformed data showing the distribution and dispersion of bacteria communities associated with the CCAs *Lithophyllum stictiforme* and *Macroblastum dendrospermum* sampled at different sites at the ASV level.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 5** CCA bacteria taxonomy at the family level. Relative abundance of the 20 most abundant bacteria families associated with the two CCA species: **a.** Individuals of *Lithophyllum stictiforme* and **b.** of *Macroblastum dendrospermum* presented by site.</figcaption></figure>
891

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Venn diagrams of shared core microbiomes. **a.** Core ASVs at 75% prevalence shared between bacteria communities associated with *Lithophyllum stictiforme* and *Macroblastum dendrospermum* samples. **b.** Core ASVs at 75% prevalence shared between bacteria communities associated with *Lithophyllum stictiforme* at the three sampling sites. **c.** Core ASVs at 75% prevalence shared between bacteria communities associated with *Macroblastum dendrospermum* at the three sampling sites.</figcaption></figure>

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<figure><img src="image_8.png" /></figure>

a

<figure><img src="image_9.png" /></figure>

b

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M. macroblastum

900

901 **Fig. 7.** Differently abundant ASVs between CCA species. **a.** Only ASVs presenting adjusted *p*-value

902 ≤0.001 are shown. The bacterial families to which the ASVs belong are reported in the legend. **b.** The 20

903 ASVs most significantly different in abundance, with a significance ≤0.001, are represented in the a

904 heatmap.

905
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Differently abundant ASVs between *L. stictiforme* clades. **a.** ASVs presenting adjusted *p*-value ≤0.01 are shown. The bacterial families to which the ASVs belong are reported in the legend. **b.** ASVs are represented in the heatmap.</figcaption></figure>
<table><caption>**Table 1** Five most abundant shared and unique bacteria ASVs. ASVs are shown per category: shared between all CCA, all *Lithophyllum stictiforme* specimens and all *Macroblastum dendrospermum* specimens, and unique to all *Lithophyllum stictiforme* and all *Macroblastum dendrospermum* specimens</caption><thead><tr><th></th><th>ASV</th><th>Order</th><th>Family</th><th>Species</th><th>Best match NCBI</th><th>Similarity</th><th>Origin</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">ASVs shared between the two CCA taxa bacteria communities</td><td>ASV 13</td><td>Rhizobiales</td><td>Hyphomicrobiaceae</td><td>Filomicrobium sp.</td><td>KF1854 66</td><td>100%</td><td>marine snow in North Adriatic Sea</td><td>Vojvoda et al., 2014</td></tr><tr><td>ASV 14</td><td>Rhizobiales</td><td>Rhizobiaceae</td><td>NA</td><td>JQ2360 06</td><td>100%</td><td>biofilms from water purification system</td><td>Manes et al., 2011</td></tr><tr><td>ASV 24</td><td>Rhizobiales</td><td>Hyphomicrobiaceae</td><td>Filomicrobium sp.</td><td>JX9840 50</td><td>100%</td><td>biofilms implicated in M. edulis settlement</td><td>Toupoint et al., 2012</td></tr><tr><td>ASV 39</td><td>Rhodobacterales</td><td>Rhodobacteraceae</td><td>Ruegeria atlantica</td><td>MN704 271</td><td>100%</td><td>sponges from the central coastal region of Vietnam</td><td>Dat et al., 2021</td></tr><tr><td>ASV4 8</td><td>Microtrichales</td><td>Microtrichaceae</td><td>Sva0996_marin e_group</td><td>JN1130 50</td><td>100%</td><td>marine sponge Astrosclera willeyana</td><td>Yang & Li, 2012</td></tr><tr><td rowspan="5">ASVs unique of L. stictiforme bacteria communities</td><td>ASV 19</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage sp.</td><td>EU9180 01</td><td>98,32%</td><td>biofilm on artificial microbialites and natural stromatolites</td><td>Havemann et al., 2008</td></tr><tr><td>ASV 22</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Blastopirellula sp.</td><td>KT9731 01</td><td>100%</td><td>endolithic communities from intertidal outcrops</td><td>Couradeau et al., 2017</td></tr><tr><td>ASV 30</td><td>Rhodobacterales</td><td>Rhodobacteraceae</td><td>Silicimonas sp.</td><td>MK175 845</td><td>99,75%</td><td>coral Astrangia poculata microbiome</td><td>Goldsmith et al., 2019</td></tr><tr><td>ASV 54</td><td>Thiohalorhabdales</td><td>Thiohalorhabdaceae</td><td>Granulosicoccu s sp.</td><td>KY4686 48</td><td>96,49%</td><td>oil-polluted subtidal sediments North Spain coast</td><td>Acosta-González et al., 2013</td></tr><tr><td>ASV 57</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage sp.</td><td>FJ65250 5</td><td>98,8%</td><td>sponge Mycale laxissima</td><td>Mohamed et al., 2010</td></tr><tr><td rowspan="5">ASVs unique of M. dendrospermum bacteria communities</td><td>ASV 7</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage</td><td>HM596 354</td><td>98,56%</td><td>biofilm on condenser tube surfaces in a nuclear power plant</td><td>Choi et al., 2010</td></tr><tr><td>ASV 34</td><td>Rhizobiales</td><td>Rhizobiaceae</td><td>NA</td><td>KT9731 29</td><td>97,76%</td><td>endolithic communities from marine intertidal outcrops</td><td>Couradeau et al., 2017</td></tr><tr><td>ASV 41</td><td>Rhodobacterales</td><td>Rhodobacteraceae</td><td>Roseovarius sp.</td><td>KT9738 98</td><td>99%</td><td>endolithic communities from marine outcrops</td><td>Couradeau et al., 2017</td></tr><tr><td>ASV 63</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Blastopirellula sp.</td><td>EF2157 70</td><td>98,01%</td><td>surface biofilm in biofilm formation process in the West Pacific coast</td><td>Dang et al., 2008</td></tr><tr><td>ASV 67</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage</td><td>FJ65250 5</td><td>98,08%</td><td>marine sponge Mycale laxissima</td><td>Mohamed et al., 2010</td></tr><tr><td rowspan="5">ASVs shared between L. stictiforme bacteria communities</td><td>ASV 13</td><td>Rhizobiales</td><td>Hyphomicrobiaceae</td><td>Filomicrobium sp.</td><td>KF1854 66</td><td>100%</td><td>marine snow in North Adriatic Sea</td><td>Vojvoda et al., 2014</td></tr><tr><td>ASV 15</td><td>Rhizobiales</td><td>Stappiaceae</td><td>Breoghania sp.</td><td>HM177 744</td><td>100%</td><td>CCA Neogoniolithon fosliei</td><td>Webster et al., 2011</td></tr><tr><td>ASV 19</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage sp.</td><td>EU9180 01</td><td>98,32%</td><td>biofilm on artificial microbialites and natural stromatolites</td><td>Havemann et al., 2008</td></tr><tr><td>ASV 22</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Blastopirellula sp.</td><td>KT9731 01</td><td>100%</td><td>endolithic communities intertidal outcrops</td><td>Couradeau et al., 2017</td></tr><tr><td>ASV 30</td><td>Rhodobacterales</td><td>Rhodobacteraceae</td><td>Silicimonas sp.</td><td>MK175 845</td><td>99,75%</td><td>coral Astrangia poculata microbiome</td><td>Goldsmith et al., 2019</td></tr><tr><td rowspan="5">ASVs shared between M. dendrospermum bacteria communities</td><td>ASV 6</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Bythopirellula sp.</td><td>JN5966 18</td><td>99,29%</td><td>tropical sponges, Xestospongia muta and Xestospongia testudinaria</td><td>Montalvo et al., 2011</td></tr><tr><td>ASV 7</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Pir4_lineage</td><td>HM596 354</td><td>98,56%</td><td>biofilm on condenser tube surfaces in nuclear power plant</td><td>Choi et al., 2010</td></tr><tr><td>ASV 13</td><td>Rhizobiales</td><td>Hyphomicrobiaceae</td><td>Filomicrobium sp.</td><td>KF1854 66</td><td>100%</td><td>marine snow in North Adriatic Sea</td><td>Vojvoda et al., 2014</td></tr><tr><td>ASV 14</td><td>Rhizobiales</td><td>Rhizobiaceae</td><td>NA</td><td>JQ2360 06</td><td>100%</td><td>biofilms from water purification system</td><td>Manes et al., 2011</td></tr><tr><td>ASV 23</td><td>Pirellulales</td><td>Pirellulaceae</td><td>Blastopirellula sp.</td><td>HM369 093</td><td>99,26%</td><td>biofilms on surfaces of the kelp Laminaria hyperborea</td><td>Bengtsson et al., 2010</td></tr></tbody></table>