# Geographic Genetic Divergence in Tychoplanktonic Taxa Dominating Diatom Communities in Marine Biofilms

Raphaëlle Barry-martinet, Thomas Pollet, Fabienne Fay, Alina Tunin-ley, Jean Turquet, Jean-françois Ghiglione, Cédric Garnier, Aurélie Portas, Gaëtan Burgaud, Frédéric Rimet, et al.

► To cite this version:

Raphaëlle Barry-martinet, Thomas Pollet, Fabienne Fay, Alina Tunin-ley, Jean Turquet, et al. Ge-
ographic Genetic Divergence in Tychoplanktonic Taxa Dominating Diatom Communities in Marine
Biofilms. Environmental DNA, 2025, 7 (3), pp.e70116. 10.1002/edn3.70116. hal-05108506

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ORIGINAL ARTICLE

OPEN ACCESS

# Geographic Genetic Divergence in Tychoplanktonic Taxa Dominating Diatom Communities in Marine Biofilms

Raphaëlle Barry-Martinet¹ | Thomas Pollet¹,² | Fabienne Fay³ | Alina Tunin-Ley⁴ | Jean Turquet⁴ | Jean-François Ghiglione⁵ |
Cédric Garnier⁶ | Aurélie Portas¹ | Gaëtan Burgaud⁷ | Frédéric Rimet⁸ | Jean-François Briand¹

<figure><img src="image_3.png" /></figure>

¹MAPIEM, Université de Toulon, Toulon, France | ²UMR ASTRE, CIRAD-INRAE, Montpellier, France | ³UMR LBCM, Université de Bretagne Sud, Lorient, France | ⁴CITEB, Reunion Island, France | ⁵CNRS, Sorbonne Université, UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique de Banyuls, Banyuls sur mer, France | ⁶Université de Toulon, M.I.O., Aix Marseille Université, Toulon, France | ⁷Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, Plouzané, France | ⁸INRAE, UMR CARRTEL, Université Savoie Mont Blanc, Thonon-Les-Bains, France

**Correspondence:** Jean-François Briand (briand@univ-tln.fr)

**Received:** 10 December 2024 | **Revised:** 22 April 2025 | **Accepted:** 24 April 2025

**Funding:** This work was supported by both the Direction Générale de l'Armement, French Government Defense procurement and technology agency (Program DRACONS) and Research French National Agency.

**Keywords:** biofilm | core microbiome | genetic variants | marine diatoms | molecular diversity | plastisphere

## ABSTRACT

Diatoms constitute the main photosynthetic group in marine biofilms throughout the world's oceans, in particular, on plastic debris, which has become a major problem in the marine environment. However, they remain largely unexplored in this context compared to prokaryotes. Here, we aim to understand the dynamics of diatom communities in the plastisphere at different levels: how taxa are selected from the planktonic community and how communities are structured over time and at large spatial scales. Biofilms were collected from PVC (polyvinyl chloride) panels immersed (i) for 1 year in two NW Mediterranean sites, a mesotrophic one (Toulon Bay) and an oligotrophic one (Banyuls Bay), and (ii) for 1 month in Toulon Bay and two other eutrophic sites (Lorient in South Brittany in the Atlantic Ocean and Reunion Island in the Indian Ocean). Plastispheres were analyzed using both microscopic and molecular approaches, focusing on the relationship between diatoms and other microorganisms in biofilms. Light microscopy revealed spatio-temporal differences in cell abundance and biovolume. Metabarcoding, targeting the rbcL gene for diversity and composition, revealed that the richness of diatom species was already maximal in the early stages of biofilm formation, and beta-diversity showed a clear temporal evolution in the Mediterranean Sea. Including prokaryotic and fungal communities, we described microorganism interactions within biofilms throughout the colonization process of the plastisphere. In addition, environmental parameters on a large geographical scale were shown to be stronger drivers in structuring diatom communities, considering both planktonic and biofilm lifestyles. A core biofilm community represented by a few abundant species was observed across sites. The occurrence of tychoplanktonic taxa emphasizes the specificity of diatoms among other microorganisms in biofilms. Finally, the complete absence of common genetic variants among Lorient, Reunion Island, and Toulon suggests that dispersal by marine currents over a large geographical scale has led to adaptation processes.

## 1 | Introduction

Diatoms are unicellular photosynthetic microeukaryotes, which are distributed across marine, freshwater, and terrestrial

ecological niches, contributing up to 20% of the global primary production, including roles in carbon fixation and nutrient cycling (Benoiston et al. 2017; Tréguer et al. 2018). Most information is based on their planktonic lifestyle (e.g., Malviya

<footnote>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Environmental DNA* published by John Wiley & Sons Ltd.</footnote>
et al. 2016). However, due to their ability to secrete extracellular polymeric substances that notably enable adhesion, as well as bacteria, they are a major component of biofilm communities in marine environments. These include natural and artificial substrates, such as microphytobenthos on sediments, rocks, seagrass, algae, various shells, ship hulls, aquaculture nets, marine renewable facilities, and (micro)plastics (Brandini et al. 2001; Patil and Anil 2005; Molino and Wetherbee 2008; Briand et al. 2017; Amaral-Zettler et al. 2020; Schlundt et al. 2020; Zhao et al. 2021). These primary autotrophic colonizers respond directly to chemical, physical, and biological changes. They have also been shown to be excellent biological indicators in freshwater systems (e.g., Morin et al. 2008; Coste et al. 2009; Tlili et al. 2010, 2011; Wachnicka et al. 2011), although little is known about them in the marine environment (Belando et al. 2017).

Despite their enormous diversity (e.g., Malviya et al. 2016) and non-trivial roles in oceanic and coastal marine environments, few studies have investigated the spatial and temporal dynamics of diatom communities in marine biofilms (Abed et al. 2019; Mitbavkar et al. 2012; Qian et al. 2022). When considering the plastisphere, i.e., microorganisms that form biofilms on plastics, although its scientific output has exploded in recent years, most studies have focused on prokaryotes (e.g., Amaral-Zettler et al. 2020). Only recently have fungal communities begun to be studied (Philippe et al. 2023, 2024; Sérvulo et al. 2023; Lacerda et al. 2020). Although diatoms are reported to be an important part of the plastisphere, e.g., as primo-colonizers (Amaral-Zettler et al. 2020) or involved in the carbon cycle (Conan et al. 2022), surprisingly, studies on their abundance or diversity are rare, probably due to the lack of taxonomic knowledge on marine taxa. If polyethylene (26.9%) and polypropylene (19.3%) are the most commonly collected polymers, polyvinyl chloride (PVC) accounts for 12.9% of global plastic production (Plastics Europe 2022). Probably underestimated (Fernández-González et al. 2022), PVC was among the six dominant polymers found in marine litter (Carney Almroth and Eggert 2019).

Thanks to continuous improvements in DNA-based methods, it is now possible to initiate conclusive studies on the factors that shape microbial communities in biofilms (Briand et al. 2022; Song et al. 2022; Yeh and Fuhrman 2022; Zhao et al. 2021). Global eukaryotic databases still have limited resolution for Bacillariophyceae, mainly limiting taxonomic assignment to the genus level. However, Rimet et al. (2015) developed a specific and open-access database (diat::syst) for freshwater diatoms and biomonitoring, providing a necessary and complementary tool to morphological analysis of diatom communities. This database, now known as Diat.barcode (Rimet et al. 2019), has significantly improved the understanding of diatom community dynamics in river and lake biofilms. Gradually, sequences of marine taxa have also been added to this database, leading to its successful application in marine environments (Rivera et al. 2018; Pérez-Burillo et al. 2022; Védie et al. 2022; Laroche et al. 2023), opening the door to more comprehensive studies of diatom community control in marine biofilms. Such a DNA-based approach appears to be particularly well suited to the study of large datasets covering large geographical areas.

Here we aimed to specifically understand the dynamics of diatom communities in the plastisphere at several levels: (i) how taxa are selected from the planktonic community, (ii) how communities are structured over time, including their potential interactions with prokaryotes and fungi in the biofilm community, and (iii) how communities are structured at large spatial scales. The temporal dynamics were studied over 1 year in the northwestern Mediterranean Sea in the Bay of Toulon, a mesotrophic site. The diatom plastisphere was then compared with a second Mediterranean site in Banyuls sur Mer, considered to be oligotrophic, after 75 and 365 days. In addition, the effect of geographical location and seawater parameters on the diversity of 1-month diatom biofilm diversity was studied in Toulon (with high salinity and temperature) and in two eutrophic sites: one with lower salinity and temperature along the French Atlantic Coast, and the other in a tropical area on Reunion Island in the Indian Ocean. Finally, the specificity of the plastisphere was characterized alongside the planktonic ones (surrounding seawater), in order to compare the two lifestyles for these sites. The biofilms were studied using both light microscopy for cell abundance and metabarcoding targeting the *rbcL* gene for diversity and composition. By considering prokaryotic and fungal communities from the same biofilm samples, interactions between microorganisms within biofilms have also been discussed.

## 2 | Materials and Methods

### 2.1 | Sampling Strategy and Environmental Variables

Diatom communities were studied at four sampling sites (Figure S1) selected for their contrasting environmental conditions: Toulon Bay (43°06'25" N;5°55'41" E) and Banyuls sur Mer (47°27'13" N;03°32'360" E) in the Mediterranean Sea (considered as mesotrophic and oligotrophic sites, respectively), Lorient (47°43'8.0178" N;3°22'7.2048" W) on the south coast of Brittany in the Atlantic Ocean, and Le Port, Reunion Island (20°56'03" S;55°17'0.6" E) in the Indian Ocean (both considered as eutrophic sites, with lower temperature and salinity for Lorient, and in a tropical area for Reunion Island).

Prior to immersion, 5 × 5 cm PVC panels (AbaqPlast) were sandblasted (roughness increased the contact surface area and reduced the biofilm release). The panels were immersed at a depth of 1 to 2 m, with a metal frame fixed to the dock (in Toulon Bay, Lorient, and Reunion Island) or suspended under the SOLA oceanographic buoy (at Banyuls sur Mer). Immersion started on June 18 at 2015, in Toulon and Banyuls sur Mer for 1 to 367 days. Sampling took place at Toulon after 1, 4, 8, 12, 20, 28, 75, and 367 days. In Banyuls sur Mer, sampling was performed only twice, after 75 and 365 days (for details, see (Briand et al. 2022)). One year later (in 2016), new panels were immersed during another immersion campaign as described above, this time for 30 days, starting on July 15 in Toulon and Lorient, and on December 1 in Reunion Island (i.e., all immersions started in summer). Two liters of seawater were sampled each week during the 30 days of immersion (i.e., four samples) for plankton DNA extraction (1 liter) and chemical analysis (1 liter).
For each sampling point, biofilms were scraped as previously described (Védie et al. 2022) directly at the sampling site with sterile scalpel blades from six panels—three panels for microscopic analysis and three panels for metabarcoding. Water temperature and salinity were measured at a depth of 1–2 m using a Hydrolab DS5X probe (Hatch Hydromet, United States). Dissolved organic carbon (DOC) and total nitrogen (TN) were analyzed using a TOC-VCSH analyzer (Shimadzu) (Oursel et al. 2014). Nutrients ($NO_3^-$, $PO_4^{3-}$, Si(OH)$_4$) were measured using standard colorimetric methods (Coclet et al. 2018). Data from Banyuls sur Mer were obtained directly from the French coastal monitoring network SOMLIT (Service d'Observation en Milieu Littoral, https://www.SOMLIT.fr/). Trace metal concentrations (Cd, Cu, Pb, Zn) were measured by voltammetry using a fully automated Metrohm/Ecochemie system (Cindrić et al. 2015).

## 2.2 | Determination of Diatom Abundance by Microscopy

Collected biofilms were fixed in 10mL of 0.3% Lugol's sterile ASW solution (with a sea salt concentration of 36gL⁻¹) and stored at 4°C in the dark until analysis. Cell counts were performed under an inverted light microscope (×1000 magnification, minimum 30 fields or 100 cells per sample). Biovolumes were estimated from cell sizes (Hillebrand et al. 1999). Sampling times with missing values were excluded from statistical analysis.

## 2.3 | DNA Extraction, Amplification, and Sequencing

Scraped biofilms were immediately immersed in liquid nitrogen. In parallel, seawater samples were filtered through 0.2-µm filters. Scraped biofilms and filters were stored at -80°C until DNA extraction using the Power Biofilm Kit (Qiagen). PCR amplification of diatom communities was performed using the Ribulose Bisphosphate Carboxylase Large subunit (rbcL) gene, a genetic marker commonly used for diatom metabarcoding (e.g., Bailet et al. 2020; Vasselon et al. 2018). Equimolar mixtures of forward primers (Diat_rbcL_708F_1 (AGGTGAAGTAAAAGGTTCWTACTTAAA), Diat_rbcL_708F_2 (AGGTGAAGTTAAAGGTTCWTAYTTAAA) and Diat_rbcL_708F_3 (AGGTGAAACTAAAGGTTCWTACTTAAA)) and reverse primers (R3_1 (CCTTCTAATTTACCWACWACTG) and R3_2 (CCTTCTAATTTA CCWACAACAG)) were used as described by Vasselon et al. (2017). The PCR program was as follows: 95°C-15', 95°C-45", 55°C-45", 75°C-45", 75°C-5'. Steps 2-5 were performed in 40 cycles. Amplicons were then paired-end sequenced (2×300 bp, Illumina platform at Eurofins Genomics (Konstanz, Germany)).

## 2.4 | Sequencing Data Processing

The DADA2 pipeline adapted for diatom metabarcoding data was applied as described by Tapolczai et al. (2019) (available on

Github: https://github.com/fkeck/DADA2_diatoms_pipe
The data are available in the NCBI BioProject database (accession number PRJNA1166302).

The global ASV table was taxonomically assigned using the FROGS pipeline with the *rbcL* Rsyst Diat.barcode 10.1 database (Rimet et al. 2019) and the RDP assignment. Multi-affiliations were manually selected for the most abundant ASVs (> 1000 reads for the entire table), prioritizing species previously recorded in marine habitats. The final taxonomic table was filtered based on several criteria: sequences with at least 95% blast identity were retained (as for the chosen multi-affiliations), while taxonomic ranks, from species to classes, with an RDP value <0.6 were considered uncertain and labeled as “unknown” (e.g., “unknown genus”, “unknown species,” etc.). Species listed as “sp.” in the Diat.barcode database (e.g., “*Stricosus* sp.”) were linked to their original environmental sequence numbers to maintain traceability and to distinguish them from uncertain assignments (due to an RDP value <0.6). ASVs classified as uncertain Bacillariophyta (RDP value ≠ 1) were discarded. The corresponding biom table and phylogenetic tree were exported from FROGS for subsequent statistical analyses. Reads were normalized to the minimum number of reads among the entire dataset (17,563 reads). The 365-day sampling point at Banyuls sur Mer was removed as it did not meet the rarefaction threshold.

## 2.5 | Bioinformatics and Statistical Analyses

All statistical analyses were performed using Microsoft Excel and R (version 4.1.1) with the phyloseq, vegan, microbiome, stats, agricolae, and pairwiseAdonis packages. Shannon and Chao1 indices were calculated at the species level. Non-metric multidimensional scaling (NMDS) was performed using the “ordinate” function of the phyloseq package, with the Bray-Curtis dissimilarity method, again at the species taxonomic level. Analysis of variance (ANOVA) and permutational multivariate analysis of variance (PERMANOVA) were performed on the distance matrix, considering site, lifestyle (in biofilm or planktonic), and immersion time. Interactions between diatom species from biofilms (co-occurrence network) were assessed using the CoNet plugin (v 1.1.1 beta) (Faust and Raes 2016). Data were processed using Pearson and Spearman correlations, with Bray-Curtis and Kullback-Leibler distances, and a mutual information score, applying a threshold of 150 edges. Randomization was calculated according to Brown's method, with the Bonferroni correction for multiple testing (*p* < 0.05).

Interactions between microorganisms in biofilms were processed using raw sequencing data obtained from the same DNA extracts from previous studies focusing on prokaryotic communities (Catão et al. 2021, 2019) (NCBI accession n°. PRJNA608237, BioSamples SAMN14167926 to 28 and under PRJNA608293, BioSamples SAMN14169847 to 52, and under PRJNA504753). A similar processing workflow in R (cutadapt, dada2) and ASV assignment to the 16S rDNA Silva 138.1 database was performed with FROGS. For the fungal communities, the ASV table provided by G. Burgaud (Philippe et al. 2023) was used. A multi-block analysis DIABLO from the MixOmics R package (v 6.26.0) was
<figure><img src="image_4.png" /><figcaption>**FIGURE 1** | Alpha-diversity of diatom communities. Graphical representation of (A) Richness, (Chao1) and (B) Specific diversity (Shannon) of diatoms in Toulon (circle), Banyuls sur Mer (star), Lorient (square) and La Reunion (triangle), with both biofilm (filled or star shapes) and planktonic (empty shapes) samples. Immersions in 2015 in the Mediterranean Sea are shown with green shades (for the kinetic in Toulon Bay) and gray (Banyuls sur Mer). One-month immersions in 2016 ("_N + 1" in the legend) correspond to the blue (Toulon), red (Lorient), and yellow (Reunion Island) shapes.</figcaption></figure>

also performed to obtain a network showing positive correlations between diatoms, prokaryotes, and fungal species. The networks were then imported and analyzed using Cytoscape software (v 3.9.1).

Distance-based Redundancy analysis (dbRDA) was performed on a reduced subsampled dataset (biofilms emerged 30 days in 2016 in Lorient, Toulon and La Reunion) against environmental variables using the Bray–Curtis distance matrix at the species taxonomic level. The “anova.cca” function was used to test the statistical significance of global analyses, as well as axes and variables. LEfSe analysis (Segata et al. 2011) with a linear discriminant analysis (LDA) set at 4 or 4.5 allowed us to identify specific taxa according to conditions, using lifestyles used as classes to identify relevant taxa for each site. Finally, ASVs and species were exported from R using the “core_members” function to generate Venn diagrams for each site or between sites, distinguishing exclusive and core (common to all samples) communities, with results visualized using the online program Venny 2.1 (Oliveros 2007).

# 3 | Results

## 3.1 | Sequencing Data

Processing of the sequencing data with DADA2 resulted in 6,311,870 reads (from an initial 7,112,973) yielding 3105 ASVs. Of these, 34 ASVs (84,118 reads), mainly assigned to the brown algae *Feldmannaia irregularis* and *Hecatonema terminale*, were unclassified, while 99 ASVs (151,474 reads) were identified outside the Bacillariophyta phylum. Our decision to filter Bacillariophyta with an RDP value ≠ 1 removed a further 520 ASVs (1,678,022 reads), including 425 ASVs with an “unknown class” (RDP < 0.6). Of these, six ASVs, representing 888,816 reads, were among the 10 most abundant ASVs in the entire set. After normalization, all further analyses were performed on the resampled ASV table containing 2295 ASVs (843,024 reads) in 48 samples.

## 3.2 | Temporal Dynamics in the Northwestern Mediterranean Sea

### 3.2.1 | Diatom Density and Surface Coverage in Biofilms

In Toulon Bay, diatoms were undetectable after 1 day of immersion, and the second sampling time after 4 days showed 68 cells/cm² ± 44 cells/cm² (Figure S2A). Diatom abundance increased steadily, showing a significant increase after 28 days (*p-value* = 0.001, 56,930 cells/cm²) and reaching a plateau after 365 days (*p-value* < 0.0001, 125,678 cells/cm²). Although no statistical differences in biovolumes were observed between sampling points from 1 to 365 days due to large standard deviations, a general increase in mean biovolume was observed (Figure S2B). Furthermore, diatom numbers and biovolumes remained stable from 2015 (Tln_T28) to 2016 (Tln_T30_N + 1), under similar conditions after 1 month of immersion in summer (*p-value* = 0.579 and *p-value* = 1000, respectively). Comparing the two Mediterranean sites, Banyuls sur Mer showed a decrease in biovolume after 1 year compared to 75 days of immersion (*p-value* < 0.0001), while their density remained unchanged (*p-value* = 1.000, Figure S2A).

### 3.2.2 | Alpha- and Beta-Diversity of Diatom Communities

Alpha-diversity was analyzed at the species level (Figure 1). Similar values were found for different immersion times in Toulon and Banyuls sur Mer, all in the Mediterranean Sea.

Beta-diversity was analyzed using NMDS ordination based on the Bray–Curtis dissimilarity (Figure 2). In Toulon, a significant temporal dissimilarity in diatom communities was observed between T1 and T365 in 2015 (*p-value* = 0.0001), and PERMANOVA showed no significant difference between diatom communities in biofilms immersed for 1 month in 2015 and 2016.
<figure><img src="image_5.png" /><figcaption>**FIGURE 2** | Beta-diversity of diatom communities. Non-metric Multidimensional Scaling (NMDS) on Bray-Curtis distances, between all samples. Biofilm communities (filled and star shapes) were evaluated in 2015 with a kinetic from 1 day to 365 days (shades from light to dark green) in Toulon and a complementary point in the Mediterranean Sea, in Banyuls sur Mer (gray stars) after 75 days. Both biofilm and planktonic (empty shapes) lifestyles were also analyzed on samples after 1 month of immersion in 2016 (“_N+1” in the legend) in three sites—Lorient (red), Reunion Island (yellow), and Toulon (blue).</figcaption></figure>

Considering the taxonomic composition of the diatom communities in the whole dataset (Figure 3), the proportion of unknown species (49 species out of 489 total species) ranged from 1% (in seawater from La Reunion) to 69% (in biofilm from La Reunion) of species depending on the sample. Of the 25 most abundant species, 14 have been previously observed in marine habitats, while 2 species were only known from freshwater (*Minutocellus polymorphus* and *Nitzschia gracilis*), and 8 species had a habitat not yet defined (Table S1). *Hyalosynedra al-shareefii*, *Microfissurata* sp. HF563527, *Entomoneis Paludosa*, *Licmophora paradoxa*, *Licmophora* sp. MG684325, *Neosynedra provincialis*, *Nitzschia gracilis*, *Nitzschia cf. longissima* were prevalent in Toulon in the early stages but decreased with time and became rare after 28 days. *Cylindrotheca closterium* followed a similar trend but remained abundant at Banyuls after 75 days. Conversely, *Psammodictyon constrictum*, *Papiliocellulus simplex*, and *Thalassiosira profunda* (which was also present in Banyuls) became abundant after 28 days and persisted. Interestingly, the latter was still present the following year but with reduced relative abundances. Taxa identified as less abundant at T28 in 2015, such as *Berkeleya fennica* or *Minutocellus* sp. FJ002097, became dominant in 2016, highlighting the variation in species dominance in mature biofilms at Toulon between consecutive years.

### 3.2.3 | Interactions Among Diatom Species in the Marine Environment

The diatom network (Figure 4) showed 103 positive interactions (edges or degrees) between 51 species (nodes) across the

entire dataset. A main cluster of 46 nodes (Figure 4A) was identified, together with two smaller clusters of three and two nodes (Figure 4B,C, respectively). Cluster A consisted of species mainly from Toulon and Banyuls sur Mer, which were mostly found in biofilms. An unknown *Catenulaceae*, *Licmophora paradoxa*, *Nitzschia gracilis*, *N. laevis* (associated with species found during the first month of immersion), *Nanofrustulum shiloi*, and *Pierrecomperia catenuloides* (mostly associated with to species observed after long-term immersion) had the highest number of putative interactions (edges> = 8), each co-occurring with 4–108 other genera. They can therefore be considered as keystones in the network. Some of these genera (e.g., *Nitszchia*, *Licmophora*, *Navicula*, *Halamphora*, *Diploneis*, *Serratifera*, *Gedaniella* and *Entomoneis*) seemed to be more represented and clustered in groups ranging from two to five species. Cluster B contained three species mostly from 2015 samples in Toulon and Banyuls sur Mer, belonging to the *Catenulaceae* (*Halamphora* species) and Fragilariaceae classes. Cluster C contained two co-occurring species (*Arcocellulus mammifer* and *Thalassiosira spinulifera*) mainly from Lorient or Banyuls sur Mer, belonging to the Mediophyceae.

### 3.2.4 | Effect of Geographical Location on Biofilm and Planktonic Communities of Diatoms After 1 Month

#### 3.2.4.1 | Diatom Density in Biofilms Depending on Sites.

PVC substrates immersed in Reunion Island (Reu_T30_N+1) tended to be more colonized (76,741 cells/cm²) and more covered (891 µm³/cm²) than in Toulon (17 µm³/cm²)
<figure><img src="image_6.png" /><figcaption>**FIGURE 3** | Taxonomic composition of diatom communities. Composition of diatom communities in both lifestyles—biofilm and planktonic (“_pk”)—and in the four sites (Toulon, Banyuls, Lorient and La Reunion). Each bar plot represents the composition of one replicate, with three replicates for biofilms and four replicates (one for each week) for planktonic samples. Each colored bar (including gray, which represents the proportion of unknown species) highlights one of the 25 most abundant species. “Other” represents the remaining species.</figcaption></figure>

(Figure S2), although statistical analyses could not be performed due to missing values for Reunion Island. After 30 days, significant differences were observed for the Chao1 (Figure 1A) and Shannon (Figure 1B) indices between Toulon and Lorient (*p*-value = 0.00012 and *p*-value = 0.00061, respectively) or Reunion

Island (*p*-value = 0.9 × 10⁻⁶ and *p*-value = 0.6 × 10⁻⁴, respectively). Notably, the only difference between planktonic and biofilm samples was observed in Toulon, with a lower richness (Chao1, *p*-value = 0.037) in planktonic communities, as observed in Lorient and in Reunion Island.
<figure><img src="image_7.png" /><figcaption>**FIGURE 4** | Interactions between diatom species in the marine environment. (A–C) Co-occurrence network analysis of diatom species associations from biofilms within the full set of sampling sites. The relative abundance of each species (nodes) is shown for the four sites and all the immersion times in each central pie chart: 2015 immersions in the Mediterranean Sea are represented by green shades (for the kinetic in Toulon) and gray (Banyuls sur Mer) and 1-month immersions in 2016 correspond to the blue (Toulon), red (Lorient), and yellow (La Reunion) quarters. The network shows statistically significant co-occurrence relationships (*p* < 0.05) obtained with a threshold set at 150 edges. The labels indicate the last known level of taxonomy. Keystone species (8 or more edges) are shown in bold.</figcaption></figure>

The diatom communities appeared to be structured by two main factors (Figure 2): immersion site (with Toulon differed from Lorient and Reunion (*p-value*=0.006), and oceanic sites differed from each other (*p-value*=0.012)), and lifestyle (biofilm vs. surrounding seawater, *p-value*=0.001). Regarding the taxonomic composition (Figure 3), biofilms from Reunion Island and Lorient were dominated by two species pairs, *Stricosus* sp. MK454983 and *Minutocellus polymorphus* in the Indian Ocean, *Navicula* sp. MH064105 and *Thalassiosira spinulifera* in the Atlantic Ocean. In addition, *Chaetoceros sporotuncatus* and *Minutocellus polymorphus* (reaching 74%) were prevalent in the planktonic mode in Reunion Island, while *Thalassiosira mediterranea* dominated in Lorient, *Chaetoceros* sp. Na11C3 in Toulon, and *Pseudo-nitzschia galaxiae* in both the Indian Ocean and the Mediterranean Sea.

### 3.2.4.2 | Core Community Between Sites and Life-styles.
Venn diagrams allowed us to detect common taxa (core community) among the three sites of Toulon, Lorient, and Reunion Island, for each lifestyle (Figure 5A,B), and for both lifestyles at each site (Figure 5C). The core community of planktonic samples (Figure 5A) contained fewer species or ASVs than the communities of the individual sites. Nevertheless, the 22 species involved (8%) accounted for 52% of the reads, indicating a core community composed of abundant taxa such as *Minutocellus polymorphus*, *Pseudo-nitzschia galaxiae*, or an unknown *Bacillariophyceae* (with 12%, 9%, and 9% of the reads, respectively). The number of common genetic variants (ASVs) was much lower (1% of ASVs for 14% of the reads), reflecting a greater site specificity in terms of genetic variants. Toulon and Lorient shared more species (11%, corresponding to 20% of the reads) and genetic variants (4%, corresponding to 16% of the reads) than Reunion.

Analyses of the biofilm communities (Figure 5B) showed a similar pattern. Although the core community also contained 22 species (9%), they represented a lower proportion of reads (36%) compared to those from planktonic samples collected from surrounding waters. Common species included *Nitzschia* sp. (17%), *Minutocellus polymorphus* (4%), and two unknown species (6% and 3% of the reads). Notably, the three biofilm communities did not share any ASV. For comparison, looking specifically at Mediterranean biofilms (Figure 5D), Toulon and Banyuls sur Mer ( ) shared 41% of the species and 16.5% of the ASVs (corresponding to 88% and 59% of the reads, respectively). Furthermore, similar analyses after 1 month (Table S2) of immersion at Toulon (Tln_T28 and Tln_T30_N+1), 1 year apart (2015 and 2016), revealed 42% of shared species and 26% of shared ASVs (corresponding to 97% and 86% of the reads, respectively).

### 3.2.4.3 | Effects of Environmental Parameters on Diatom Community Structure and Composition.
Reunion Island had the highest temperatures and phosphate concentrations, while Lorient had the highest nitrate concentrations (Table S1). Salinity was lower at Lorient compared to Toulon, with intermediate values in Reunion Island. Toulon had the lowest silicate concentrations, which were significantly different from Reunion Island and Lorient. Bray–Curtis distance-based redundancy analysis (dbRDA, Figure S3) including both biofilm and planktonic samples, significantly (*p-value*=0.001) explained 44% (dbRDA1) and 43% (dbRDA2) of the variance accounted for by the constrained variables (43%). Statistical analysis showed that temperature (anova.cca, *p-value*=0.001), salinity (*p*=0.001), and nitrates (*p-value*=0.05) influenced the distribution of diatom communities in both planktonic and biofilm
<figure><img src="image_8.png" /><figcaption>**FIGURE 5** | Core community between sites and lifestyles. Venn diagrams (A,B) and table (C) showing the percentage of exclusive and shared diatom species, or ASVs, after 30 days between Toulon (blue), Lorient (red) and Reunion Island (La Reunion), according to each lifestyle (in the surrounding water (A) or biofilms (B)), or between lifestyles within each site (C). An additional Venn diagram (D) shows exclusive and shared diatom species, or ASVs, between biofilms from two different Mediterranean sites, after the same period of immersion (75 days): Toulon (blue) and Banyuls sur Mer (gray). The percentages of species are in bold and the percentages of ASVs are in italics. The corresponding percentages of reads are written in brackets.</figcaption></figure>

lifestyles. Phosphates and silicates were significantly correlated with temperature ($r=0.89$) and salinity ($r=-0.89$), respectively.

LEfSe analysis (Figure 6) identified the Cymatosirales, together with the family and genus of *Minutocellus polymorphus*, as discriminant taxa in Reunion Island, with no distinction between lifestyles. This was also the case for Bacillariales and Bacillariaceae. *Nitszchia* spp. and *Pseudo-nitzschia* spp. (and especially the species *Pseudo-nitzschia galaxiae*) were specific for biofilms (Figure 6A) and planktonic lifestyles (Figure 6B), respectively. *Pseudo-nitzschia galaxiae* was also found as a discriminant species in Toulon Bay biofilms, together with *Cylindrotheca closterium* and *Psammodictyon panduriforme*. Thalassiophysales, Pleurosigmataceae, and Catenulaceae (together with *Halamphora* spp., and an unknown species of *Halamphora*) were also specific taxa of Toulon biofilms. Fragilariophyceae appeared specific to Toulon biofilms as well (except for the genus *Tabularia*, which was discriminant in Lorient), with the genus *Hyalosira* and the species *Hyalosira al-shareefii*, or with *Licmophorales* represented by two species (*Licmophora* sp. MG684325 and *L. paradoxa*). *Diploneis* sp. MH064083 was discriminant in biofilms from Reunion Island. This was also the case for Toxariales, Paraliales, Paraliaceae, and *Paralia* spp. Mediophyceae and Coscinodiscophyceae were two discriminant classes in Lorient biofilms, as were the family Stephanodiscaceae (except for the genus *Cyclotella*, which

was specific to Toulon), the genus *Extubocellulus*, and *E. spinifer* species. In addition, two *Chaetoceros* species (and their respective family and order) were discriminant species in the planktonic community the surrounding waters of Toulon. Apart from an unknown Coscinodiscophyceae species, the surrounding waters of Lorient were characterized by Thalassiosirales, Thalassiosiraceae, Thalassiosira, and in particular *T. mediteranea* and *T. spinulifera*, which were also discriminant in the biofilms.

**3.2.4.4 | Interactions Among Prokaryotes, Fungi, and Diatoms in Marine Biofilms.** Within biofilms, we also focused on co-occurrences between diatoms, fungi, and bacterial taxa represented in a MixOmics network (Figure 7A). This network contained 1076 edges and 94 nodes (Table S5), including 61 bacteria (nodes 1–61), 22 fungi (nodes 62–83), and 11 diatoms (nodes 84–94), distributed in two clusters. Bacteria had fewer edges (1–31) than fungi (1–68) and diatoms (1–52) (Figure 7B). Co-occurrences between bacteria and fungi or bacteria and diatoms were more frequent than co-occurrences between fungi and diatoms (Figure 7C). However, node 84 (an unknown diatom species from the order Achnanthales) did not co-occur with any bacteria but with a *Coniothyrium* fungus (node 62). Some nodes had a minimal number of edges, such as bacteria from nodes 1 to 17 (see Table S5), which co-occurred with only one to four fungi (nodes 77, 81, 82, and 83). The diatom *Extubocellulus*
<figure><img src="image_9.png" /><figcaption>**FIGURE 6** | Effect of environmental parameters on diatom community structure and composition. Cladograms after LEfSe analysis of diatom communities to determine discriminant taxa in samples recovered after 1 month of immersion in 2016, from Lorient (red), Toulon (blue) and La Reunion (yellow and black), with respect to biofilm (A) or planktonic (B) lifestyle (LDA scores >4 and 4.5, respectively). Non-specific information is shown in gray.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**FIGURE 7** | Interactions between bacteria, fungi, and diatoms in marine biofilms. (A) Network analysis of associations between prokaryotic, diatom and fungal species in biofilms. The relative abundance of each species (nodes) is shown for the four sites and all the immersion times in each central chart pie: 2015 immersions in the Mediterranean Sea are represented with shades from light to dark green (for the kinetic in Toulon) and gray (Banyuls sur Mer) and 1-month immersions in 2016 correspond to the blue (Toulon), red (Lorient) and yellow (La Reunion) quarters. The surrounding ring are either orange (for bacterial species), green (for diatom species) or purple (for fungal species) depending on the community. The network shows statistically significant and discriminant co-occurrences with a cutoff of 0.70. Labels indicate the number of each node attributed from the least to the most connected node of each community. (B) Box plot showing the number of edges for each node. Statistical groups are represented by the letters "a" and "b" (p < 0.05). (C) Bar plot representing the number of edges whether they represent co-occurrences with diatom, fungal, or bacterial taxa. Statistical differences are indicated by "*" (p < 0.0001).</figcaption></figure>

spinifer (node 85) was related to five bacteria (nodes 58–61) belonging to the genera *Methyloceanibacter*, *Flagellimonas*, *Pelagibius*, and *Ahrensia*. Two species of diatoms, *Chaetoceros socialis* and a *Thalassiosirales* species (nodes 86–88), mainly present in Lorient, were only associated with bacterial

species. This was also the case for fungi from nodes 63 to 76 (see Table S5). In the large and central cluster of bacteria (nodes 18–61), co-occurrences were distributed with fungi (5–21 edges) and diatoms (1–10 edges). Finally, the nodes 89–93 and node 91 (*Chaetoceros diversus*, *C. danicus*, *Skeletonema costatum*,
*Minutocellus polymorphus*, *Rhizosolenia imbricate*, and *R. fallax*) also co-occurred with fungi from nodes 81 to 83 (*Nectriopsis* sp., *Parathyridaria rosae* and *Buckleyzyma aurantiaca*) and nodes 77 to 80 (*Exophiala xenobiotica*, *Lycoperdon pretense*, unknown species of the class *Agaricomycetes*, and *Flavoplaca* sp.).

4 | Discussion

4.1 | Diatom Richness Is Nearly Established in the Early Stages of Biofilm Formation

Microbial adhesion and colonization of plastic substrates in the marine environment occurs rapidly, typically within minutes to hours (Latva et al. 2022). Our experiment started in summer, corresponding to maximum irradiance and temperature, when the maximum diversity of diatom species is generally observed (Munda 2005). The colonization trend of diatoms in Toulon Bay is consistent with previous studies (Molino and Wetherbee 2008; Molino et al. 2009; Patil and Anil 2005). The evolution of diatom density determined by microscopy (Figure S2) indicated that initial colonization rates for diatoms are lower than for bacteria. The maximum richness and diversity observed by metabarcoding within the first few days (Figure 1) also confirmed that after an initial settlement phase, most diatoms on surfaces proliferate primarily by asexual reproduction of existing biofilm cells, rather than direct recruitment from planktonic cells, resulting in "monospecific" cloning patches (Cooksey et al. 1984; Mitbavkar et al. 2012). Mitbavkar and Anil (2008) also found that diatom recruitment and invasive reproduction increased cell numbers on surfaces. This process likely explains the lack of variation in both richness and diversity of such r-strategists over time. This is also consistent with recent studies using long-term deployments (up to 52 weeks) where diversity was highest after 1 week of immersion (Amaral-Zettler et al. 2020; Laroche et al. 2023). However, this pattern, however, contrasts with observations for prokaryotes and fungi in the same samples. Prokaryotic communities in Toulon displayed rapid temporal dynamics with a rapid increase in diversity during the first few days. For fungi, the increase in richness and diversity seems to come later (based on ITS, Philippe et al. 2023). As no clear trend was observed for diatom alpha-diversity, this indicates a distinct dynamic. However, the specificity of biofilm communities associated with geographical locations, including the similarity of Mediterranean sites and their higher diversity (H'), seems to be shared by all microbial components studied and linked to environmental parameters.

Beta-diversity analysis (Figure 2) revealed a gradual but nota-
ble evolution of diatom communities over 1 year, with biofilm
assemblages generally clustering, particularly during the first
20 days. Laroche et al. (2023) recently confirmed that exposure
time significantly affects diatom community structure on con-
ventional plastics, with significant differences observed after
12 weeks of immersion. Furthermore, our experiment was con-
ducted in a nearly closed, highly contaminated area (Misson
et al. 2016; Pollet et al. 2018), which may explain the slow re-
cruitment of new species (Mitbavkar and Anil 2008; Breznak
et al. 1984) and minimal changes in biofilm composition due
to selection pressure. However, the diatom communities after
365 days of immersion appeared highly different, with an

increase in the number of abundant taxa. This shift may be due
to the presence of multicellular colonizers competing for dia-
toms for space or providing additional substrates through epibi-
osis. In addition, recent work supports our findings, suggesting
that the cell size distribution shifts towards smaller cells with
longer exposure times, as observed with conventional polymers
over periods extending up to 12 weeks (Zhao et al. 2021).

The colonization dynamics of prokaryotes are known to be influenced by the characteristics of the substrate (natural or artificial), mostly during the first hours to days (e.g., Pinto et al. 2019). Among polymers, only the biodegradable ones will select different bacterial communities compared to conventional ones (Dussud et al. 2018; Derippe et al. 2024; Odobel et al. 2021; Jacquin et al. 2021), as well as for fungi (Kettner et al. 2019; Philippe et al. 2023). Although the effect of substrates on diatoms has not been extensively studied, existing evidence suggests that conventional polymers do not significantly affect the composition of mature diatom biofilm communities. This is consistent with observations from both Toulon and a Swedish site where diatom communities on different conventional polymers showed similar characteristics (Védie et al. 2022).

The analyses carried out in Toulon over two consecutive years and in the bay of Banyuls sur Mer, which experiences stronger hydrodynamic effects, gave us the opportunity to document the relatively little studied diatom biofilm communities in the Mediterranean Sea. Considering the cell size observed with microscopy, larger diatom species seemed to be favored on sandy substrates with strong hydrodynamic conditions (Andriana et al. 2021), which may explain why diatoms in Toulon were generally smaller compared to those in Banyuls sur Mer. In any case, these results should be interpreted with caution considering the limit of the numeration of individual cells from diversified and complex biofilms, resulting in high variation between replicates. Nevertheless, the Banyuls sur Mer biofilm communities analyzed by metabarcoding were more similar to those in Toulon, regardless of the immersion time, than to those in Lorient or in Reunion Island, both at the species (Figure 2) and ASV (data not shown) level. Mediterranean samples (Toulon and Banyuls sur Mer) shared almost 40% of the abundant species, representing 88% of the reads. This suggests that environmental parameters common to Mediterranean regions (Table S4), mainly temperature and salinity, are more important drivers than nutrients or contaminants such as trace metals. In particular, biofilms sampled after 1 month of immersion at the same site in the Toulon Bay in 2015 and 2016 with similar seawater parameters at the beginning of summer showed very similar communities with 26% of ASVs and 86% of the reads shared between the 2 years, suggesting a relative seasonal stability of the communities.

The co-occurrence network captured well the temporal variability of these and other keystone diatom species. *Licmophora paradoxa* and other species of the same genus are known for their weak attachment strength, which explains their absence from the most turbulent areas (Woods and Fletcher 1991; Hunsucker et al. 2014). This trait may explain why *L. paradoxa* became a dominant (Figure 3) and discriminant (Figure 6) species in the Bay of Toulon, which is protected from strong hydrodynamics by a dam. Moreover, their dominance during the first month of immersion indicates
that they are r-strategists. Not surprisingly, *L. paradoxa* and *Nitzschia gracilis* (among the most abundant species in Toulon Bay) stood out in the co-occurrence network (Figure 4) with the highest number of interactions with other diatom species mainly occurring during the early stages of our study. In contrast, *Pierrecomperia catenuloides*, which was present in all Mediterranean biofilms, was not abundant but emerged as a keystone species with potential interactions with several other diatom species in mature biofilms. This seems quite surprising for a centric diatom in biofilms where mostly pennates have been reported. Unfortunately, this taxon which is phylogenetically close to *Extubocellulus* spp. (Sabbe et al. 2010; Dąbek et al. 2017, 2019), has hardly been described so far. The abundance of *Cylindrotheca closterium* in all Mediterranean samples was expected. As an important cosmopolitan benthic diatom species (Djaoudi et al. 2022), it thrives in high salinity conditions (Rao and Ragothaman 1988; Chiu et al. 2005) and has already been observed as a dominant pioneer microalga in French sites (Briand et al. 2012). Interestingly, its dominance after 75 days of immersion in Banyuls sur Mer, whereas it became rare after the same immersion time in Toulon, may indicate that nutrient limitation in Banyuls sur Mer slowed down the temporal dynamics of diatom colonization. Furthermore, *Berkeleya fennica* and *Neosynedra provincialis*, known for their metal and nutrient tolerance, may reduce the colonization success of more sensitive species (Belando et al. 2017). Their higher abundance in Toulon compared to Banyuls sur Mer may highlight their preference for Cu and Zn concentrations, which differed notably between these two sites (Table S4).

A progressive shift in the abundance of species was observed in Toulon Bay, starting after 20 days of immersion. Predation, interactions between plastisphere microorganisms, recruitment of new taxa during the time of the immersion, completion and diversification of functional traits within the biofilms are inevitably involved in these temporal dynamics (Tobias-Hünefeldt et al. 2021; Bech et al. 2024). However, environmental parameters may also offer some explanation as temperature increased from 23.4°C (T0) to 25.8°C (T20), along with Cu and Zn (2-fold increase and 2.6-fold decrease, respectively) between T0 and T28, while other parameters remained relatively stable. This phenomenon is complex, and several variables not investigated here (e.g., the influence of light (Doose and Hubas 2024)) may also play a role. *Psammodictyon constrictum* became remarkably dominant (reaching up to 48%) after 1 year of immersion. This widely distributed species has recently been detected in biofilms from sea turtles and floating debris in the Persian Gulf (Azari et al. 2020) and described as a dominant species in red tides along the Egyptian coast (El-Hadary et al. 2022). It would be worthwhile to study the possible reappearance of these species in relation to biotic and abiotic factors. The increase in relative abundance of the centric tychoplanktonic diatoms *Papilliocellus simplex*, *Thalassiosira* spp., and *Minutocellus* sp. could be due to blooms that displace the benthic diatom community and temporarily alter the pennate/centric ratio, as previously shown for *Melosira nummuloides* by Patil and Anil (2005). Furthermore, Pollet et al. (2018) showed that after 75 days of immersion, the bacterial community in biofilms showed 65% of dissimilarity compared to those in the first month of immersion. Thus, the variable abundance of bacterial taxa within biofilms should be

considered as a factor that may influence the composition of diatom communities (Tang et al. 2011). Cruz and Neuer (2022) showed that the bacterial community influenced the aggregation capacity and consequently the settlement of *Minutocellus polymorphus*, one of the planktonic discriminant taxa in Reunion Island.

## 4.2 | Environmental Parameters Shape Diatom Communities in Marine Biofilms More Than Lifestyle

All three sites, although exposed to harbor activities, differed in terms of temperature, salinity, nutrients, and trace metal contamination, which largely explain the diatom dissimilarities in both biofilm and planktonic communities (dbRDA, Figure S3). Our results are consistent with findings that biofilm diatom communities differ significantly from surrounding seawater communities (Chiu et al. 2005; Patil and Anil 2005), as do bacteria or fungi. Thus, our data are in agreement with Patil and Anil (2005), showing that physico-chemical factors of the surrounding environment influence both biofilm diatom and planktonic communities. Furthermore, it was observed that site remained the primary clustering factor even in the case of biofilms on biocidal antifouling coatings (Zargiel et al. 2011; Briand et al. 2017). Here, environmental parameters accounted for up to 40% of the variation in community clustering between biofilm and planktonic samples recovered in 2016 (Figure S3). Recent studies have highlighted the structuring role of seawater parameters on biofilm diatom communities using metabarcoding, a finding consistent with previous microscopy-based observations of marine plastispheres (Belando et al. 2017; Azari et al. 2020; Munda 2005; Mitbavkar and Anil 2008; Chiu et al. 2005; Nodine and Gaiser 2014; Hillebrand and Sommer 2000).

For prokaryotes and fungi, biofilm communities showed higher diversity than planktonic communities (Yeh and Fuhrman 2022; Catão et al. 2021; Philippe et al. 2023). However, diatom diversity did not show similar differences in our samples. To date, studies on the alpha-diversity of diatom communities from biofilms and surrounding waters are scarce and have shown contrasting results depending on the site (Patil and Anil 2005; Laroche et al. 2023). The lower richness and diversity observed in biofilms from both oceanic sites (Lorient and Reunion Island) compared to Mediterranean biofilms echo recent results on fungal communities (Philippe et al. 2023). This trend can be attributed to nutrient loading (Desrosiers et al. 2013), which is the only common measured factor between the sites, although Lorient has higher nitrate concentrations and Reunion is characterized by higher phosphate levels. Hydrodynamics is also a notable factor structuring diatom assemblages in biofilms (Wachnicka et al. 2011; Hunsucker et al. 2014; Nodine and Gaiser 2014), with diatom species possessing stronger attachment capabilities (morphological and adhesive properties) being favored (Zargiel and Swain 2014). Although lower diatom biomass is typically observed on sheltered shores compared to moderately exposed ones, regardless of site and season (Thompson et al. 2005), the generally sheltered nature of our sites (all within harbors, except for Banyuls sur Mer) suggests that hydrodynamics may not be a primary driver of community composition when
comparing abundances in Toulon Bay, Lorient, and Reunion. As there is still a gap in the literature regarding lifestyle-specific diversity comparisons for marine diatoms, it was not possible to investigate this parameter further.

Prokaryotes and fungi exhibited distinctly different clear dissimilar communities when, as mentioned above, the diatom lifestyle appeared to be less specific as evidenced by the definition of tychoplanktonic taxa. The tychoplanktonic lifestyle (Kuhn and Waser 1981), corresponding to taxa able to switch from planktonic to biofilm and vice versa, was dominant at all sites with about a third of the species but only a quarter of the ASVs, representing about 80% of the reads. Our analysis revealed that some planktonic biomarkers from centric genera (*Chaetoceros*, *Thalassiosira*, *Minutocellus*, *Skeletonema*) were represented by specific species in biofilm communities (e.g., *Minutocellus polymorphus* in Reunion Island, *Thalassiosira spinulifera* in Lorient). Conversely, few diatoms were specific to the planktonic lifestyle (2% of species and 10% of reads in Toulon), suggesting that the biofilm lifestyle may be more common for diatoms, at least for certain life stages Notably, while bacteria mostly exhibit a specific lifestyle (Catão et al. 2021; Zhang et al. 2019), fungi tend to adapt to both lifestyles (Philippe et al. 2023).

Diatom communities are characterized by few dominant taxa complemented by a high number of rare taxa, a pattern also observed in planktonic diatom communities worldwide (Malviya et al. 2016). Rare species exhibited a specific lifestyle and a higher genetic diversity. As these comparisons were limited to summer (T30 samples), further studies are needed to understand seasonal variability in diatom biofilm diversity.

Among the dominant (Figure 3) and site-specific (Figure 6) species, *Navicula* and *Nitzschia* (motile diatoms) are known to be high competitors under nutrient-enriched conditions, which can explain why the genus *Nitzschia* was found as a biomarker in Reunion Island and why the community was largely dominated by *Navicula* sp. in Lorient. However, this selection may vary between species (Belando et al. 2017) as we were able to observe several species of *Nitzschia* among the abundant ones in Toulon. Similarly, *Tabularia* spp. are highly nutrient tolerant (Hillebrand and Sommer 2000), which is consistent with the specificity of this genus in Lorient (Figure 6). *Thalassiosira* spp. are centric and cosmopolitan marine diatoms (195 species referenced), already described in both the Mediterranean Sea and the French Atlantic coasts (Percopo et al. 2011). This seems to be in line with the fact that, depending on the species, *Thalassiosira* has been found either as abundant in Mediterranean biofilms and waters (in Toulon and Banyuls) or as a biomarker of Lorient. Finally, *Pseudo-nitzschia*, which includes at least 12 toxic species potentially producing domoic acid (Trainer et al. 2012), has been reported to be widespread in the Mediterranean Sea, with a high abundance of cells attached to plastic matrices (Casabianca et al. 2019). In our study, *P. galaxiae* appeared as a major species in the surrounding waters of Toulon and Reunion and, interestingly, serves as a biomarker either in both biofilms in Toulon and planktonic species in Reunion.

A limited biofilm core community, consisting of a few dominant species (9% with 36% of reads), showed no shared genetic variants between sites. This suggests that genetic variants from

the same species, transported by marine currents, were able to adapt to different local conditions. It also seems to indicate that a genetic variant from Lorient reaching the Mediterranean Sea may not thrive in this new habitat. The importance of intraspecific variation for adaptive success, which could also be related to the ecotype concept, has already been reported for planktonic diatoms (Godhe and Rynearson 2017), but this study provides the first evidence for marine biofilms. Such genetic divergence, mainly observed over large geographical distances, seems to be in line with our data showing intraspecific variability between oceans but high similarity between Mediterranean sites. Recent studies in French and Hungarian rivers (Pérez-Burillo et al. 2021; Tapolczai et al. 2021) based on ASVs have highlighted their utility in analyzing intraspecific and cryptic diversity among diatom species based on dispersal patterns and ecological preferences. Similar results have been shown for diatoms in alpine lakes from France and Georgia, with remarkable intraspecific differentiation (Rimet et al. 2023). Considering heterotrophic protists, a less clear trend has been observed, with some species showing restricted distribution patterns and others being widely distributed. It would be worthwhile to look at the functional potential of diatom communities to better unravel how diatom communities adapt to different environments (Pearson et al. 2015). To our knowledge, intraspecific genetic variability has not yet been explored for fungi. Finally, this adaptation process at the subspecies level remains difficult to test for prokaryotes as metabarcoding does not allow for a sufficient level of taxonomic assignment at the species level.

## 4.3 | Microbial Potential Interactions Between Prokaryotes and Microeukaryotes in Marine Biofilm Communities

The prokaryotic and fungal communities of the same samples used here for diatom diversity have already been published independently (Catão et al. 2021, 2019; Pollet et al. 2018; Philippe et al. 2023). This study advances our understanding of biofilm dynamics and interactions between the three main taxonomic groups using for the first time a co-occurrence approach with a MixOmics network. Although these interactions appear primordial, they remain poorly studied, particularly in marine biofilms.

In the network, most of the nodes were abundant in mature biofilms in Toulon, which could be related to the largest number of samples in Toulon and the increasing cell density over time. Prokaryotic nodes were mainly composed of low abundant but core biofilm taxa (Verrucomicrobiae and Planctomycetes, Catão et al. 2021), rare taxa in biofilms but involved in photosynthesis and nitrogen fixation (filamentous cyanobacteria and an ammonia-oxidizing Thaumarchaeota), a marine pathogen (*Vibrio splendidus*), and three abundant and diverse groups in biofilms (Flavobacteriaceae, Rhodobacteraceae, and Rhizobiales). Many of these taxa are known to be associated with diatoms in natural habitats or cultures (e.g., Amin et al. 2012; Abed et al. 2019). Recently, a Rhizobiales was identified as the first non-cyanobacterial nitrogen-fixing symbiont of a marine diatom belonging to the genus *Haslea* (Tschitschko et al. 2024). *Rhizosolenia* and *Chaetoceros* spp. constituted half of the diatom nodes, although, as centric diatoms, they represent low abundant taxa in biofilms. Interestingly, these genera
are involved in N₂-fixing symbioses with cyanobacteria (Foster et al. 2011), which may indicate that this lifestyle is also reflected in the network. We also noticed *Minutocellus polymorphus*, a diatom biomarker of La Reunion biofilms, but also abundant in mature biofilms in Toulon, highly associated with several bacteria and three fungi, yet little described in the marine environment, possibly including *Nectriopsis* sp., known as a mycoparasite and also a lichen parasite (Lawrey and Diederich 2003). The diatom biomarkers of Lorient (*Extubocellulus spinifer* and *Thalassiosira spinulifera*) show associations only with bacteria. Among the fungi, *Exophiala xenobiotica* co-occurred with several bacteria and the Coscinodiscophyceae diatom *Rhizosolenia fallax*. This fungus is an ubiquitous taxon frequently detected in soil and water contaminated by monoaromatic hydrocarbons and alkanes (De Hoog et al. 2006), with a closely related species, *E. jeanselmei*, described as a styrene degrader (Cox et al. 1997). Other fungi associated with a variety of bacteria, are known as parasites, such as *Ochroconis*, known to cause diseases of marine fish (Pang et al. 2016) or Rozellomycota, known as the basal fungal lineage, which includes species that are all parasites, whether of water molds and algae, crustaceans, or amoebae (Corsaro et al. 2020), epibionts (*Ochrocladosporium frigidarii*, isolated from brown algae (Crous et al. 2007)), or algal endophytes (*Acrostalagmus luteoalbus* (Cao et al. 2019)).

Interactions between microorganisms are critical to the functioning of ecosystems. For example, using isolated strains, Koedooder et al. (2019) suggested that interactions between bacteria and diatoms in mudflats are a key factor in the structuring of the benthic microbial community and its overall functioning, as reported for specific bacteria-diatom associations (Amin et al. 2012; Seymour et al. 2017; Filek et al. 2022). While our results are largely consistent with the literature, the complexity of natural, diverse communities makes it difficult to draw firm conclusions. Understanding the interactions and dynamics between prokaryotes, fungi, and diatoms in marine biofilms is still in its early stages.

## 5 | Conclusions

This study provided significant insights into diatom communities, as major players in marine biofilms living on plastics, by combining classical microscopic analysis with advanced molecular approaches. The results highlighted the specificity of diatoms within biofilms, with a strong influence of environmental parameters on the structuring of their communities, beyond lifestyle aspects. The presence of a core of dominant species on a large geographical scale, but with local genetic differentiation, underlines the importance of adaptive processes in the diversity of diatoms within marine biofilms. These findings contribute to a better understanding of the functioning and dynamics of such microbial ecosystems in coastal environments.

### Author Contributions

R.B.M. performed P.C.R., the bioinformatic and statistical analyses, and wrote the article. T.P. was responsible for experimental design and performed DNA extraction. C.G. was in charge of trace metal quantification as well as water quality analysis. F.F. was responsible for immersion and sample recovery in Lorient. A.T.L. and J.T. were responsible

for immersion and sample recovery in Reunion Island. A.P. assisted with the statistical analysis of the MixOmic network. G.B. supplied the fungi ASV table and reviewed the article. F.R. helped with the diatarcode use and reviewed the article. J.F.B. led the project and wrote the article.

### Acknowledgments

We thank Marine Maintenay for her help in biofilm samplings at Toulon. We also thank the scuba diving service of the Oceanographic Observatory of Banyuls sur Mer (OOB) for the immersion at the SOLA Buoy at Banyuls.

### Conflicts of Interest

The authors declare no conflicts of interest.

### Data Availability Statement

The datasets generated during the current study are available in the SRA (BioProject PRJNA1166302) using the MIxS package.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section.