# An exploratory study of species richness and dynamics in the cultivable mycobiota of Palmaria palmata

Yoran Le Strat, Nicolas Ruiz, Paul Déléris, Thibaut Robiou Du Pont, Samuel

Bertrand, Stella Debaets, Gaëtan Burgaud, Justine Dumay

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Yoran Le Strat, Nicolas Ruiz, Paul Déléris, Thibaut Robiou Du Pont, Samuel Bertrand, et al. An exploratory study of species richness and dynamics in the cultivable mycobiota of Palmaria palmata. Fungal Ecology, 2025, 75, pp.101417. 10.1016/j.funeco.2025.101417. hal-05225331

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# An exploratory study of species richness and dynamics in the cultivable mycobiota of *Palmaria palmata*

Yoran Le Stratª, Nicolas Ruizª,*, D, Paul Délérisª, Thibaut Robiou du Pontª, Samuel Bertrandª,
Stella Debaetsᵇ, Gaëtan Burgaudᵇ, Justine Dumayª

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

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## ARTICLE INFO

Handling Editor: Prof. Allison K. Walker

**Keywords:**

Cultivable mycobiota

Fungal diversity

Marine fungi

*Palmaria palmata*

## ABSTRACT

This study presents the first exploratory analysis of the cultivable fungal communities associated with the red alga *Palmaria palmata* and their temporal dynamics over one year. The fungal isolation strategy employed two culture media - a non-specific Wickerham medium and a specific "*P. palmata*" medium - coupled with two incubation temperatures (12 °C and 27 °C), and a differentiation between epiphytic and endophytic fungi. This experimental design yielded 298 fungal isolates. Through morphological observations and molecular techniques, 142 fungal taxa were identified. The cultivable fungal diversity associated with *P. palmata* was dominated by members of the phylum Ascomycota, with the classes Dothideomycetes, Eurotiomycetes and Sordariomycetes, and the genera *Cladosporium*, *Penicillium* and *Aspergillus* emerging as the dominant groups. This fungal diversity exhibited significant seasonal variation, with culture conditions - including incubation temperature and culture medium composition - influencing the diversity and composition of the cultivated fungi obtained.

## 1. Introduction

*Palmaria palmata*, commonly known as dulse, sea lettuce flakes, creatnach, or söl, respectively in English, Irish, Scottish and Icelandic, is a red seaweed growing along the northern coasts of the Atlantic - from Portugal to Arctic - and Pacific Oceans (Pereira, 2015). The earliest recorded use of this macroalga for food consumption dates back approximately 1500 years (Indergaard, 1991), with traditional uses documented in Iceland, Ireland, and Brittany as early as the fifth century for both nutritional and medicinal purposes (Mouritsen et al., 2013).

Fungi have been detected in nearly every marine habitat yet explored, from coastal waters to the deep biosphere (Burgaud et al., 2022). Marine fungi were long classified as obligate, needing marine conditions to grow and sporulate, or facultative, which can grow in both terrestrial and marine environments (Kohlmeyer and Kohlmeyer, 1979). This distinction, while common, can underestimate marine fungal diversity by excluding some facultative fungi with clear marine adaptations. Pang et al. (2016) refined this definition, defining fungi that grow or sporulate in marine environments, form symbiotic relationships with marine organisms, or show genetic or metabolic adaptation to marine conditions, as marine, without regard for an obligate or facultative

nature. A recent update on the numbers of marine fungi has listed 1898 species affiliated to 9 different phyla (Calabon et al., 2023), a figure that, while relatively small, belies the significant ecological roles these fungi play. Although marine fungi are less studied compared to their terrestrial counterparts, they are abundant and play crucial roles in marine ecosystems, particularly in coastal areas and macroalgal habitats (Gnavi et al., 2017; Goecke and Imhoff, 2017; Ogaki et al., 2019; Stien, 2020). In recent years there has been a growing interest in the microbial communities associated with macroalgae, particularly in understanding the key roles these microbiota play in the algal growth. This has led to the concept of the algae and its microbiome as a single entity, known as the holobiont (Egan et al., 2013). While most studies have focused on bacterial communities (Selvarajan et al., 2019; Tournoeroche et al., 2020; Pei et al., 2021), the mycobiota associated with seaweeds remains largely unexplored.

Interest in marine fungi associated with macroalgae has increased over the last decade, both in terms of their diversity and ecological roles (Ogaki et al., 2019). Most studies have described the fungal communities associated with brown seaweeds using culture-based approaches, which, despite limitations, have generated less than 2 % of the total marine fungal diversity documented to date (Hong et al., 2015; Lee

<footnote>ª Nantes Université, Institut des Substances et Organismes de la Mer (ISOMER), UR 2160, F-44000, Nantes, France</footnote>

<footnote>ᵇ Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, F-29280, Plouzané, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nicolas.ruiz@univ-nantes.fr (N. Ruiz).</footnote>
et al., 2019; Wainwright et al., 2019; Tourneroche et al., 2020). The majority of macroalgae-derived fungi belong to the phylum Ascomycota (Gnavi et al., 2017; Goecke and Imhoff, 2017; Garzoli et al., 2018; Kamat et al., 2020), consistent with the broader dominance of ascomycetes in marine fungal diversity (Jones, 2011). Specific genera, such as *Penicillium*, *Aspergillus*, and *Cladosporium*, are particularly prevalent, likely due to their halotolerance, osmotolerance, cultivation efficiency, and high adaptive potential (Gnavi et al., 2017; Garzoli et al., 2018; Lee et al., 2019; Kamat et al., 2020). The ecological roles of endophytic fungi associated with macroalgae were defined as parasitic, saprophytic or mutualistic (Suryanarayanan et al., 2010; Burgaud et al., 2022; Cooper and Walker, 2022). Nevertheless, further investigation including in-depth approaches such as meta-omics is needed to delve deeper into the functional aspects of the algal mycobiome.

Several factors influence the fungal diversity associated with macroalgae, including salinity, geographical location, algal structure, and the synthesis of antifungal compounds (Suryanarayanan, 2012; Wainwright et al., 2019). Studies comparing fungi isolated from brown, red and green macroalgal lineages have failed to identify a clear trend that designates one macroalgal family as the primary reservoir of fungal biodiversity (Suryanarayanan et al., 2010; Suryanarayanan, 2012; Flewelling et al., 2013; Abdul Malik et al., 2020). Moreover, defining a fundamental fungal diversity even within a single macroalgal species remains challenging due to significant variability in diversity among different sampling sites (Tourneroche et al., 2020). This underscores the need for more investigations to better understand the cultivable myco-biota of seaweeds, with a focus on underexplored species.

To date, no comprehensive study has finely characterized the cultivable fungal community associated with the red macroalga *P. palmata*. Previous research has primarily focused on bacteria, with only sporadic mentions of fungal isolates (Liot et al., 1993; Moore et al., 2002; Flewelling et al., 2013). An important fungal isolation campaign was recently performed on *P. palmata*, but the identification of fungal isolates was limited to a morphological characterization (Kovalenko et al., 2023). Moreover, the composition of *P. palmata* changes with the seasons (in terms of polysaccharides and proteins), and that substrate composition affects microbial development (Dumay et al., 2014; Overy et al., 2019). Previous studies have already investigated the seasonal dynamics of bacteria associated with algae, particularly *P. palmata* (Martin et al., 2014; Brunet et al., 2023). In this context, the present study aims to provide the first exploratory analysis of the cultivable fungal community structure of the red alga *P. palmata* and its seasonal dynamics over one year, using both morphological and genetic tools for fungal identification. In addition, this work is part of a project involving the biotechnological use of the isolated fungal strains, and in particular their enzymatic potential in the extraction of R-phycoerythrin from *P. palmata* (Le Strat et al., 2023).

# 2. Material and methods

## 2.1. Seaweed sampling

*Palmaria palmata* sampling was performed in the west coast of France at Valentin beach in Batz-sur-Mer (47.276222, -2.495004), over a one-year period between 2019 and 2020. Sampling was conducted on eight dates, approximately twice per season, on the following dates: June 19, 2019 and July 18, 2019 (summer), September 16, 2019 and November 13, 2019 (autumn), January 09, 2020 and February 11, 2020 (winter), May 25, 2020 and June 22, 2020 (spring). During each sampling event, seawater temperature was measured and seawater samples were collected to wash the seaweeds in the laboratory. *Palmaria palmata* was collected from the shore at low tide and placed in buckets containing seawater sampled at the same time to maintain temperature during transport to the laboratory (approximately 1 hour). Thalli were then rinsed three times with tap water, wrung out and stored at 4 °C in plastic bags until processing occurred, within 48 hours.

## 2.2. Fungal isolation

Three distinct protocols were performed to specifically isolate (i) epiphytic, (ii) endophytic, and (iii) both epiphytic and endophytic fungi from *P. palmata* algal thalli under strict sterile and aseptic conditions to prevent contamination (working within a Microbiological Safety Cabinet, sterilizing glass instruments with electric bunsen burner, using autoclaved glassware, disinfecting hands between each isolation procedure). These protocols were adapted from established marine fungal isolation methods (Zuccaro et al., 2003; Zhang et al., 2009; Bovio et al., 2018). Controls were also performed with the absence of algal contact on nutrient plates exposed within the laminar flow hood during all isolation procedures. No fungal colonies were obtained on control plates, indicating the absence of aerial contamination during isolation.

For the isolation of epiphytic fungal isolates, the algal thalli were first washed twice with sterile artificial seawater (ASW), followed by agitation with glass beads in ASW to remove residual debris and non-specific microorganisms. The thalli were then rinsed twice with ASW and spread on solid culture media in 15 cm Petri dishes.

The isolation of endophytic fungal isolates involved an initial chemical disinfection of algal thalli in 70 % ethanol 30 s to eliminate epiphytic microorganisms. The thalli were then rinsed three times with ASW and ground using a T25 Ultra-Turrax instrument (IKA® Werke GmbH & Co. KG, Staufen, Germany) in ASW. The crushed material was subsequently plated on solid culture media in 15 cm Petri dishes.

For the isolation of both epiphytic and endophytic fungal isolates (referred in this study as "epi/endo"), the protocol started similarly to the epiphytic isolation protocol, with ASW washes, agitation with glass beads, and additional ASW washes. The thalli were then homogenized using the Ultra-Turrax in ASW, and the resulting material was spread on solid culture media in 15 cm Petri dishes.

Two different media were used to generate the fungal culture collection, the aspecific enriched Wickerham medium (Wickerham, 1946) and a specifically formulated "Palmaria palmata" medium designed to mimic *in situ* conditions. The Wickerham medium composition included glucose 10 g/L (Rectapur, VWR-Avantor, Rosny-sous-Bois, France), yeast extract 3 g/L, malt extract 3 g/L, agar 17 g/L (Condalab Madrid, Spain), peptone 5 g/L, (Biokar Diagnostics, Beauvais, France), sea salts 30 g/L (Reef Crystals, Aquarium Systems, Strasbourg, France), chloramphenicol 0.5 g/L (Merck Chimie, Fontenay sous Bois, France), and penicillin G sodium salt 0.2 g/L (Sigma-Aldrich, Saint-Quentin-Fallavier, France). The "Palmaria palmata" medium consisted of *P. palmata* lyophilized powder 15.6 g/L, agar 17 g/L, sea salts 30 g/L, chloramphenicol 0.5 g/L, and penicillin G 0.2 g/L. Both media were autoclaved before the addition of antibiotics. Incubations were conducted at two temperatures, 12 °C and 27 °C, with each protocol performed in triplicate.

Fungal colonies, identified as yeasts or filamentous fungi based on macro- and microscopic characteristics, were isolated under sterile conditions on DCA medium (dextrose 40 g/L, enzymatic digest of casein 10 g/L, agar 15 g/L, Condalab) at the same incubation temperature used for enrichment. Each isolate was subsequently subcultured, stored at 4 °C, and added to the laboratory fungal collection (from mms1678 to mms1998). The subcultured isolates were incubated at 12 °C or 27 °C and observed as described in subsequent procedures.

## 2.3. Fungal identification

Fungal identification was initially conducted based on macroscopic and microscopic morphological features, allowing identification at the genus level when conidiophores were observed (Supplementary data 1). Complementary identifications were performed using a molecular approach. DNA from all the fungal isolates was extracted using the FastDNA Spin Kit (MP Biomedicals, Illkirch, France) following manufacturer's recommendations. The DNA concentration was measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Illkirch,
France) and standardized to 7.5 ng/µL. DNA extracts were then stored at -20 °C.

A Mini/Micro-Satellite Primed PCR (MSP-PCR) protocol was used as a dereplication approach to cluster similar fungal isolates, as described by Rédou et al. (2015). Briefly, the M13 microsatellite primer (5'-GAGGGTGGCGGTTCT-3') was used for PCR, resulting in the amplification of DNA fragments of varying sizes, reflecting the degree of M13 sequence repetition in each fungal isolate. This amplification provided a unique genetic fingerprint for each fungal isolate. The PCR was performed in a 25 µL reaction mix, containing 1X of PCR buffer (Promega, Charbonnières-les-Bains, France), 2 mM of MgCl₂ (Promega), 0.8 µM of the M13 primer (Sigma-Aldrich), 0.4 mM of dNTP (Promega), 0.025 U/µL of Taq Polymerase (Promega), and 15 ng of fungal genomic DNA. The amplification protocol included an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 45 s at 93 °C, 60 s at 50 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 6 min. PCR products were analyzed *via* electrophoresis along with a negative control (DNA replaced with sterile distilled water) and a molecular size marker (1 kb). DNA bands representing M13 profiles were visualized under ultraviolet light using a Quantum ST4 system (Vilber, Marne-la-Vallée, France). M13 genetic fingerprints were analyzed using Bionumerics software version 6.6 (Applied Maths NV, Sint-Martens-Latem, Belgium). The Dice coefficient was used to group fingerprints by similarity, and clustering was performed using the UPGMA method. An arbitrary but stringent cut-off of 90 % sequence similarity was used to define distinct clusters (Rédou et al., 2015; Quemener et al., 2020). One representative isolate per cluster was selected for DNA sequencing.

The internal transcribed spacers (ITS) region was sequenced for selected isolates. The primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') were used for amplification. PCR reactions were performed on 10 ng of DNA in a 25 µL reaction mix containing 1X of PCR buffer, 2 mM of MgCl₂, 0.2 µM of both ITS4 and ITS5 primers (Thermo Fisher Scientifics), 0.2 mM of dNTP, and 0.025 U/µL of Taq Polymerase (Promega). The amplification involved an initial denaturation step at 95 °C for 5 min, followed by 29 cycles of 60 s at 95 °C, 60 s at 55 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 10 min. PCR products were sent to Eurofins Scientific (Ebersberg, Germany) for sequencing using ITS4 and ITS5 primers. For each fungal isolate, forward and reverse sequences were aligned using the *de novo* assembly feature of Geneious software (2021.2.2 version, http://www.geneious.com) to generate a consensus sequence. The consensus sequences were queried against the NCBI database using the Blast function (max target sequences: 100; expect threshold: 0.05; word size: 28; max matches in a query range: 0; match/mismatch scores: 1,-2; gap costs: linear; filter: low complexity region; mask: mask for lookup table only) for taxonomic assignment. Assignments were made when possible, and for several fungal isolates where ITS sequences provided only genus-level resolution, additional gene markers were sequenced (Table 1).

The PCR reaction was performed on 40 ng, 10 ng and 15 ng of genomic DNA for actin, EF1a and β-tubulin and 18S sequencing, respectively. The PCR reaction for actin sequencing was performed in 25 µL reaction mix, containing 1X of PCR buffer, 2 mM of MgCl₂, 0.5 µM of both ACT-512F and ACT-783R primers (Thermo Fisher Scientifics),

0.2 mM of dNTP, 0.05 U/µL of Taq Polymerase, and 40 ng of genomic DNA. The amplification consisted in an initial denaturation step at 95 °C for 8 min, followed by 29 cycles of 15 s at 95 °C, 20 s at 58 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 5 min. The PCR reaction for EF1a sequencing was performed in 30 µL reaction mix, containing 1X of PCR buffer, 1.5 mM of MgCl₂, 0.3 µM of EF1-f and EF1-r primers (Thermo Fisher Scientifics), 0.2 mM of dNTP, 0.025 U/µL of Taq Polymerase, and 10 ng of genomic DNA. The amplification consisted in an initial denaturation step at 95 °C for 5 min, followed by 29 cycles of 60 s at 95 °C, 90 s at 62 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 5 min. The PCR reaction for β-tubulin sequencing was performed in 25 µL mix, containing 1X of PCR buffer, 1.5 mM of MgCl₂, 0.2 µM of both Bt2a and Bt2b primers (Thermo Fisher Scientifics), 0.2 mM of dNTP, 0.025 U/µL of Taq Polymerase, and 10 ng of genomic DNA. The amplification consisted in an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 60 s at 94 °C, 60 s at 61 °C, and 60 s at 72 °C, with a final elongation step at 72 °C for 5 min. The PCR reaction for 18S sequencing was performed in 25 µL mix, containing 1X of PCR buffer, 1.5 mM of MgCl₂, 0.2 µM of both nu-ssu-1196 and nu-ssu-0814 primers (Thermo Fisher Scientifics), 0.2 mM of dNTP, 0.025 U/µL of Taq Polymerase, and 15 ng of genomic DNA. The amplification consisted in an initial denaturation step at 95 °C for 5 min, followed by 29 cycles of 60 s at 95 °C, 60 s at 55 °C, and 90 s at 72 °C, with a final elongation step at 72 °C for 7 min. PCR products were sent to Eurofins Scientific (Ebersberg, Germany) for sequencing using the respective primers. Forward and reverse sequences were aligned using Geneious software to generate consensus sequences. For each molecular marker, consensus sequences were aligned using MUSCLE software (https://www.ebi.ac.uk/Tools/msa/mu%20scle/) and manually checked to identify and cluster identical sequences into taxa. Fungal identification was based on the best BLAST hit with query coverage and identity higher than 99 % or, when lower, the highest possible match. Fungal classification was cross-referenced with NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), Mycobank (http://www.mycobank.org) and Index Fungorum (http://www.indexfungorum.org) databases. All sequences were deposited in GenBank, and the accession numbers for each isolate are available in Supplementary data 2.

## 2.4. Statistical analysis

The impact of four studied variables - ecological habitat, incubation medium, incubation temperature, and season - on fungal diversity was assessed using the Pearson's Chi-squared test. This evaluation was performed at three taxonomic levels: phylum, class and genus (or the most accurate taxonomical assignation). Specific chi-squared independence tests were performed using RStudio, taking into consideration the number of isolates and the number of taxa at different taxonomic levels (phylum, class and genus). Following the chi-squared independence tests, when p-values were lower than 0.05, a post hoc analysis was conducted to determine which combinations contributed most to the observed association. Standardized residuals were calculated for each cell in the contingency table, and a Bonferroni correction was applied to control for multiple comparisons.

Rarefaction curves were generated using RStudio with the *vegan*

<table><caption>**Table 1**
Supplementary molecular markers used for fungal identification.</caption><thead><tr><th>Markers</th><th>Primer's names</th><th>Sequences</th><th>Cladosporium</th><th>Aspergillus</th><th>Penicillium</th><th>Verticillium</th></tr></thead><tbody><tr><td rowspan="2">Act</td><td>ACT-512F</td><td>5'-ATGTGCAAGGCCGGTTTCGC-3'</td><td rowspan="2">X</td><td rowspan="2">X</td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td>ACT-783R</td><td>5'-TACGAGTCCTTCTGGCCCAT-3'</td></tr><tr><td rowspan="2">EF1a</td><td>EF1-f</td><td>5'-ATGGGTAGGAGGACAAGACTCA-3'</td><td rowspan="2">X</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2">X</td></tr><tr><td>EF1-r</td><td>5'-TGGAGATACCAGCCTCGAAC-3'</td></tr><tr><td rowspan="2">β-tub</td><td>Bt2a</td><td>5'-GGTAACCAAATCGGTGCTGCTTTC-3'</td><td rowspan="2"></td><td rowspan="2">X</td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td>Bt2b</td><td>5'-ACCCTCAGTGTAGTGACCCTTGGC-3'</td></tr><tr><td rowspan="2">18S</td><td>nu-ssu-1196</td><td>5'-TCTGGACCTGGTGAG-3'</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2">X</td></tr><tr><td>nu-ssu-0814</td><td>5'-TTAGCATGGAATAATRRAATAGGA-3'</td></tr></tbody></table>
package (version 2.6-4) and the *rarecurve* function, aiming to evaluate
how much of the total fungal community was sampled in this study.

**3. Results and discussion**

3.1. General structure of P. palmata fungal community (phylum, class and genus levels)

A total of 298 fungal isolates, representing 142 different taxa, were obtained. Among these, 268 isolates (90 %) belonged to the phylum Ascomycota, encompassing 115 taxa and representing 80 % of the total diversity. The remaining 30 isolates (10 %) belonged to the phylum Basidiomycota, with 27 taxa representing 20 % of the total diversity (Supplementary data 3). The predominance of Ascomycota has been widely documented in previous studies focusing on various marine habitats (Jones, 2011; Goecke and Imhoff, 2017). Algae-inhabiting fungi follow a similar trend, with most being ascomycetes (Jones, 2011; Agusman and Danging, 2017; Abdul Malik et al., 2020; Ahmed et al., 2021). Fungal isolates were sometimes assigned only to the genus level or higher taxonomic ranks (family, order, class, phylum). This low taxonomic resolution was due to a lack of comparable sequences in databases or difficulties in distinguishing between closely related taxa, which led to the presence of several arbitrary but distinct clades for some genera (as for example *Aspergillus* sp. I to VIII, *Cladosporium* sp. I to X, *Penicillium* sp. I to XXII, etc.). Nevertheless, the aim of the present study was to provide an overview of the *P. palmata* fungal community. The presence of fungal isolates not identified to the species level may indicate taxonomic novelty, depending strictly on the databases against which we compared our sequences. Indeed, the absence of a fungal sequence in a database does not exclude the possibility that the corresponding fungal species has already been described but may lack sequence data (Lücking et al., 2020). The global diversity of fungal

isolates at the class level is summarized in Fig. 1.

Differences in isolation frequency and species richness were observed among the fungal classes. Interestingly, Dothideomycetes isolates accounted for 39.2 % of total Ascomycota isolates (Fig. 1A), but this percentage decreased to 26.1 % when considering the number of taxa (Fig. 1B). Comparatively, Eurotiomycetes within Ascomycota had an isolation frequency of 31.0 % and a specific richness of 35.7 %. Sordariomycetes were the third major class within Ascomycota, representing 25 % of the isolates and 28.7 % of the taxa. These three main Ascomycota classes have already been recorded as dominant or abundant in several marine environments, such as sediments, seawater, and corals (Xu et al., 2018; Li et al., 2019; Yang et al., 2020; Baltar et al., 2021; Marchese et al., 2021). They are also recognized as major classes among fungal isolates from marine plants and macroalgae (Panzer et al., 2015; Vallet et al., 2018; Jones et al., 2019; Kamat et al., 2020). These classes were dominant in the fungal communities associated with the green alga *Flabellia petiolata* (Gnavi et al., 2017) and the brown alga *Padina pavonica* (Garzoli et al., 2018).

The four major fungal classes within Basidiomycota were Agar-
icomycetes, Exobasidiomycetes, Microbotryomycetes, and Trem-
ellomycetes, representing 36.7 %, 23.3 %, 20 %, and 10 % of
Basidiomycota isolates, and 37 %, 25.9 %, 14.8 %, and 11.1 % of
Basidiomycota taxa, respectively (Fig. 1C and D). A comparative anal-
ysis with the literature is challenging as this phylum was represented by
only one or two isolates per taxon in this study. However, the relative
dominance of Agaricomycetes among Basidiomycota has been previ-
ously observed in marine environments, particularly in Arctic sediments
(Zhang et al., 2015).

Interestingly, the global trends between isolation frequency and
species richness at the class taxonomic level, for both Ascomycota or
Basidiomycota phyla, appear comparable, indicating a relatively high
diversity. However, the quantitative distribution of Dothideomycetes is

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Fungal class diversity from *Palmaria palmata*. Quantitative distribution of the 268 Ascomycota strains (90 % of total strains) (A). Qualitative distribution of the 115 Ascomycota taxa (80 % of total taxa) (B). Quantitative distribution of the 30 Basidiomycota strains (10 % of total strains) (C). Qualitative distribution of the 27 Basidiomycota taxa (20 % of total taxa) (D).</figcaption></figure>
higher than its qualitative distribution, as this class is composed of few taxa relative to the number of isolates.

At the genus level, an important diversity of fungi has already been described in marine environments. Commonly found genera include *Acremonium*, *Alternaria*, *Arthrinium*, *Aspergillus*, *Cladosporium*, *Fusarium*, *Geomyces*, *Gliocladium*, *Microsphaeriopsis*, *Paecilomyces*, *Penicillium*, *Phoma*, *Phomopsis*, *Trichoderma*, and *Ulocladium* (Sallenave-Namont et al., 2000; Bugni and Ireland, 2004; Matallah-Boutiba et al., 2012; Abdul Malik et al., 2020). In the present study, the cultivable fungal diversity was characterized by four predominant genera in the phylum Ascomycota, i.e. *Cladosporium*, *Penicillium*, *Aspergillus*, and *Parengyodontium*, with 28.9 %, 20.5 %, 7.0 %, and 6.0 % of total fungal isolates, respectively (Fig. 2). Several taxa of these major genera were over-represented by a large number of isolates, as observed for *Cladosporium* sp. I, II, and III (Supplementary data 3). Other genera were represented by less than 5 % of the global amount of fungal isolates such as *Acremonium* (3.7 %), *Calcarisporium* (2.3 %), *Sporobolomyces* (2.0 %), *Lecanicillium* (1.7 %), and *Stachybotrys* (1.7 %) (data not shown), but together they represented 37.6 % of the diversity, highlighting the non-trivial occurrence of low-abundant taxa associated with *P. palmata*. The high representation of several genera in terms of isolate numbers does not necessarily correlate with their representation in terms of taxon numbers (Fig. 2). For instance, the *Cladosporium* genus represented 28.9 % of total isolates but only 9.2 % of total taxa. Similarly, the genus *Parengyodontium* was represented by 18 isolates (6.0 % of total isolates) but only one taxon (*Parengyodontium album*). The genera *Penicillium* and *Aspergillus* had similar representations when considering both isolate and taxon numbers.

The predominance and ubiquity of the genera *Aspergillus*, *Cladosporium*, and *Penicillium* have been well-documented in marine environments, including deep sediments, corals, and seagrasses (Subrmaniyan et al., 2018; Xu et al., 2018; Jones et al., 2019; Yang et al., 2020). These genera are also dominant in macroalgae, characterized by broad host specificity (Suryanarayanan, 2012; Abdel-Gawad et al., 2014; Abdul Malik et al., 2020; Kamat et al., 2020). For example, *Aspergillus* and *Cladosporium* dominated among 96 fungal algicolous isolates, and *Cladosporium* and *Penicillium* were dominant across seven macroalgae species (Furbino et al., 2018; Sahoo et al., 2021). However, caution must be taken with such conclusions. Indeed, studies performed on algicolous fungal communities have mostly focused on endophytic fungi, excluding epiphytic ones, which complicates the identification of clear trends (Abdul Malik et al., 2020). According to Kovalenko et al. (2023), the genera *Cladosporium* and *Penicillium* genera were particularly prominent in the cultivable fungal community associated with *P. palmata*, along with *Acremonium*.

Besides these ubiquitous taxa isolated from *P. palmata*, some species

have been isolated from this macroalga in other studies and not in the present one, ie *Alternaria tenuissima*, *Botrytis* sp., *Cadophora fastigiata*, *Chadefaudia marina*, *Didymella magnei*, *Fusarium oxysporum*, *Helicomyces* sp., *Mucor racemosus* or *Tolypocladium inflatum* (Kohlmeyer and Kohlmeyer, 1979; Flewelling et al., 2013; Kovalenko et al., 2023). An accumulation curve of global taxonomic richness was generated and the saturation point was not reached (Supplementary data 4). This may explain the absence of taxa previously mentioned and other taxa never isolated from *P. palmata* to date.

To the best of our knowledge, the following species seem to be reported for the first time in the marine environment: *Acremonium longisporum*, *Acrodontium luzulae*, *Acrodontium* sp., *Arachnomyces peruvianus*, *Aspergillus inflatus*, *Bullera alba*, *Calcarisporium cordycipiticola*, *Cladosporium pulvericola*, *Dioszegia hungarica*, *Exobasidium* sp., *Keissleriella* sp., *Leptobacillium leptobactrum*, *Metacordyceps chlamydosoria*, *Microstroma bacarum*, *Neosetophoma cerealis*, *Nothoacremonium* sp., *Penicillium angulare*, *Penicillium pancosmium*, *Penicillium spathulatum*, *Peniophora rufomarginata*, *Phlebia radiata*, and *Phlebiopsis ravenelii*. The remaining 56 taxa have already been described in marine environments (Supplementary data 3).

## 3.2. Impact of isolation and environmental parameters on the fungal community

Fungal isolation and cultivation from *P. palmata* were evaluated considering four variables: (i) location within the alga (epiphyte, endophyte and “epi/endo”), (ii) temperature (12 °C and 27 °C), (iii) culture medium (Wickerham or “*P. palmata*” medium), and (iv) season (from summer 2019 to spring 2020). The influence of these factors on the number of taxa is depicted in Fig. 3, representing the overall fungal community obtained.

Three isolation protocols were employed, two selective (targeting either epiphytes or endophytes) and one non-selective (epi/endo). Both epiphytic and endophytic communities were represented in similar proportions, with 46 (32.4 %) epiphytic taxa and 51 (35.9 %) endophytic taxa. An additional 75 taxa (52.8 %) correspond to isolates obtained through non-selective isolation protocol and could thus not be attributed neither to epiphytic nor endophytic communities. Interestingly, the non-selective method yielded almost as many isolates as the combined selective methods. However, fast-growing fungi dominated in the non-selective cultures, thus inhibiting the growth of slower species. Future studies might address this issue by applying dilution-to-extinction techniques or incorporating growth-inhibiting compounds such as Rose Bengale to improve the detection and isolation of slow-growers (Out et al., 2016; Rédou et al., 2016; Delhaes et al., 2019). Application of these methodologies was not possible in our work,

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Main fungal genera from *Palmaria palmata*. Quantitative distribution of the main genera (represented by more than 5 % of total isolates) (A). Qualitative distribution of the main genera (represented by more than 5 % of total taxa) (B).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Distribution of the cultivable fungal community of *Palmaria palmata* in terms of taxon number according to the location within/on alga (A), culture medium (B), season (C) and incubation temperature (D).</figcaption></figure>

already dealing with an important isolation protocol. Moreover, it is
important to stay cautious with the fact that there is a great biological
variability from a biological sample to another, notably concerning
fungal cultivation and isolation, resulting in a low reproducibility of the
isolation procedure (Franco et al., 2000; Parsa et al., 2018; Tourneroche
et al., 2020). Accumulation curves of taxonomic richness under different
conditions (temperature, culture medium, season, and algal sampling
zone) have been generated and saturation point has not been reached for
any of the conditions tested (Supplementary data 4). This observation
suggests that, despite the diversification of the culturing conditions, the
total cultivable diversity remains here undersampled. This also indicates
that further exploration under even more varied or prolonged culturing
conditions could reveal additional taxa.

The number of fungal isolates from the Wickerham medium at 12 °C was nearly twice higher than the number of fungal isolates on "*P. palmata*" at 27 °C. The Wickerham medium, known for its nutrient richness, is commonly used for the isolation and growth of marine yeast and filamentous fungi (Kjer et al., 2010; Yue et al., 2015; Bovio et al., 2018; Fan et al., 2019). Moreover, since the research project to which this study belongs also includes a biotechnological aspect, involving the isolation of degradation enzymes (xylanolytic and cellulolytic), this culture medium appeared highly relevant (Le Strat et al., 2023). In the other hand, the presence of lyophilisate of *P. palmata* as carbon source in the "*P. palmata*" medium, may restrict the growth of less specialized fungi. Our findings thus support the notion that more fungal developments are observed on the non-selective Wickerham medium compared to the selective "*P. palmata*" one. Furthermore, the preference for 12 °C (~65 % of the collection) likely reflects the typical sea temperature at Batz-sur-Mer, where samples were collected, aligning with findings that marine fungal development tends to correspond to environmental conditions, thus strengthening the importance of mimicking at best *in situ* conditions (Overy et al., 2019; Pasqualetti et al., 2020).

Seasonality had a significant impact on fungal isolation rates. Spring yielded the highest number of isolates (44.3 %), while autumn had the lowest (10.7 %). Summer and winter yielded 18.5 % and 26.5 % of total isolates, respectively. The over-representation of spring isolates could be an artifact, as two separate sampling dates were conducted in that

season, including a June 2020 sampling that technically falls under
summer but was categorized as spring. Without any intention to estab-
lish early conclusion, the dynamic of fungal isolation frequency appears
to peak during spring and then to decrease from summer to autumn
before starting increasing from winter to peak again during spring.
Further investigation is needed to determine if this pattern is consistent
over time. Some investigations on the seasonality of the marine fungal
isolation frequency from the water column have demonstrated that
fungal isolation rates vary by taxa, geographical location, and are often
related to marine biomass blooms (Sen et al., 2022; Christmas et al.,
2023). Few studies focus specifically on algicolous fungi, but Abdel-
Gawad et al. (2014) reported a loss in fungal diversity and isolation
frequency with increasing temperatures, which is in accordance with the
current observations.

3.3. *Impacts of isolation and environmental parameters on the cultivable fungal diversity*

The relative distribution of each fungal taxon based on the four
studied parameters (ecological habitat, culture medium, incubation
temperature, and season) is illustrated in Fig. 4. In addition to their
overall impact on fungal isolation, the different parameters investigated
here may also correlate with the number of isolates obtained at different
taxonomic levels. Potential effects of environmental and isolation pa-
rameters are discussed in detail for the three main classes of Ascomy-
cota: Dothideomycetes, Eurotiomycetes, and Sordariomycetes. The
other Ascomycota classes and those within Basidiomycota lacked suffi-
cient representation for rigorous comparison. Environmental and
isolation parameters effects are also discussed at the genus level,
focusing on the three main genera Aspergillus, Cladosporium and Peni-
cillium. Because only one genus of Basidiomycota (Sporobolomyces) was
represented by more than two isolates, the taxonomic composition of
this phylum may not be reliable according to the studied environmental
and isolation parameters.
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** List of the cultivable fungi isolated from *Palmaria palmata*. The Bubble plots represent the classification of each taxa isolated (Division, Order, Class) and their relative abundance according to the different parameters assessed: ecological habitat (red), culture medium (orange), incubation temperature (yellow) and season (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

### 3.3.1. Influence of variables on fungal diversity

3.3.1.1. **Temperature.** The effect of incubation temperature on marine fungi has been studied from decades (Curran, 1980). In the present study, temperature significantly influenced culturing efficiency. Incubation at 12 °C led to the isolation of 100 taxa, represented by 193 isolates, while 27 °C incubation yielded 62 taxa, represented by 105 isolates (Fig. 3). Interestingly, the overlap between these two conditions is low with only 20 taxa (around 14 % of the diversity) but represented by 155 isolates. These overlapping taxa were mainly Ascomycota and mostly represented by genera such as *Cladosporium*, *Penicillium*, *Aspergillus*, *Acremonium*, *Calcarisporium*, *Parengyodontium*, and *Stachybotrys*. A chi-squared test conducted to assess the independence of variables did not reveal any dependence between temperatures and the established diversity at the phylum level in terms of isolate number. However, this test did indicate a significant dependence between temperature and diversity when focusing on the three primary classes Dothideomycetes, Eurotiomycetes and Sordariomycetes (p-value <0.001). Similarly, at the

genus level, with a specific focus on the three main genera - *Cladosporium*, *Penicillium*, and *Aspergillus* - the chi-squared test for independence of variables demonstrated a high dependence between temperature and diversity in terms of isolate number (p-value <0.001). Post hoc analyses revealed that *Cladosporium* and *Penicillium* showed significant differences in their occurrence at both temperature conditions (Bonferroni-adjusted p-values <0.05). In contrast, *Aspergillus* did not show any significant association with temperature, as the observed frequencies were similar to the expected values. This suggests that temperature has a significant impact on the occurrence of *Cladosporium* and *Penicillium*, with higher numbers of isolates at 12 °C, but not on *Aspergillus*.

Taxa belonging to Sordariomycetes seemed to be preferentially isolated at 27 °C while taxa belonging to Dothideomycetes and Eurotiomycetes seemed to be mainly isolated at 12 °C. Temperature seems to be a major parameter influencing the diversity of fungal communities (Booth and Kenkel, 1986; Cheung et al., 2018; Li et al., 2019). Interestingly, Dothideomycetes should be particularly adapted to low temperatures, compared with other classes such as Eurotiomycetes and
<figure><img src="image_8.png" /><figcaption>Fig. 4. (continued).</figcaption></figure>

Sordariomycetes (Li et al., 2019). Fungi belonging to Dothideomycetes, Eurotiomycetes and Sordariomycetes isolated from the green alga *Flabellia petiolata* were mostly isolated at 25 °C (compared with 15 °C). This preferential incubation temperature may be partially explained by the fact that this study on cultivable fungal community associated with *Flabellia petiolata* was performed in Mediterranean Sea (Gnavi et al., 2017), which may be warmer than Atlantic Ocean and more precisely Atlantic French coasts. Moreover, the low temperature preference of Dothideomycetes observed in the present study may be due to the high amount of *Cladosporium* isolates (representing 87 % of the Dothideomycetes), preferentially isolated at 12 °C. Indeed, from a study on fungi isolated from deep-sea sediments, *Cladosporium* isolates appeared particularly psychrophilic compared to other species (Wei et al., 2018).

Taxa with the highest number of isolates obtained through incubation at 12 °C were identified as *Cladosporium* sp. II, *Cladosporium* sp. III, *Penicillium* sp. I and *Cladosporium* sp. I (Fig. 4), with 35, 23, 15 and 12 strains isolated, respectively. Six isolates of *Penicillium brevicompactum* were obtained at 12 °C. All the five isolates of *Cladosporium ramotenellum* and the two isolates of taxa *Aspergillus* sp. II, *Aspergillus* sp. III, *Penicillium* sp. XVI, *Acrodontium luzulae* and *Helotiales* sp. I, were obtained with a 12 °C incubation. Many of these taxa belonged to Eurotiomycetes and Dothideomycetes, corresponding to the two major cultivable fungal classes associated with *P. palmata* (Fig. 1). All of these taxa belonged to ascomycetes, the main fungal phylum associated with *P. palmata*. Specifically, the *Trametes versicolor* taxon, with a putative preference to a 12 °C incubation temperature, belonged to basidiomycetes.

Among the taxa characterized by a more important number of isolates obtained with a 27 °C than a 12 °C temperature incubation, 11 and 5 strains belonged to *Parengyodontium album* and *Aspergillus* sp. VIII, respectively (Fig. 4). All the four strains of *Penicillium* sp. XIII were

isolated at a 27 °C incubation temperature. Six other taxa were characterized by a total of two or three isolates, all isolated at 27 °C, and were identified as *Aspergillus* sp. I, *Aspergillus* sp. V, *Penicillium* sp. VII, *Penicillium* sp. XX, *Calcarisporium* sp. I, and *Lecanicillium* sp. II. All taxa with a putative 27 °C incubation temperature preference belonged to Eurotiomycetes or Sordariomycetes classes (Ascomycota), but no Dothideomycetes was detected.

3.3.1.2. *Culture medium.* The composition of the culture medium plays a crucial role in the isolation of marine fungi, directly influencing the cultivable diversity observed (Overy et al., 2019). Algal-based media have already been employed for the growth of marine fungi (Gnavi et al., 2017; Garzoli et al., 2018; Fawzy et al., 2020; Landeta-Salgado et al., 2021), but this study represents the first instance of the macroalga *P. palmata* as a carbon source in an autoclaved culture medium for fungal isolation.

Culture medium composition has an important impact on culturing rate. The use of Wickerham medium yielded 108 taxa, represented by 183 isolates, while the use of the *P. palmata* medium produced 52 taxa, represented by 115 isolates (Fig. 3). Interestingly, the overlap between these two media is low with only 18 taxa, meaning around 13 % of the diversity, but represented by 153 isolates. These overlapping taxa are mainly ascomycetes and represented by genera such as *Cladosporium*, *Aspergillus*, *Penicillium*, *Acremonium*, *Parengyodontium*, and *Stachybotrys*. The conducted chi-squared tests for independence of variables showed dependence between the variable “culture medium” and the established diversity at the phylum level in terms of isolate number (p-value <0.001). Post hoc analyses revealed that *Ascomycota* and *Basidiomycota* showed significant differences in their occurrence across both culture media, with Bonferroni-adjusted p-values below 0.05, highlighting a
higher number of isolates on Wickerham medium compared to *P. palmata*. Chi-squared tests with a focus on the three main classes and the three main genera did not show any dependence between diversity and the culture medium. Comparative studies on marine fungal diversity obtained by using a non-selective medium in one hand, and a selective algal-based medium in the other hand are particularly limited (Gnavi et al., 2017; Garzoli et al., 2018). The fact that more fungal taxa were isolated by the use of a non-selective medium compared with a selective one has been observed, but no deeper comparisons could be made on the basis of the effect of culture media on fungal diversity.

Within the 35 isolates of *Cladosporium* sp. II and the 23 of *Cladosporium* sp. III, 21 and 14 were isolated from the Wickerham culture medium, respectively (Fig. 4). Moreover, among the 12 isolates of *Cladosporium* sp. I, seven were isolated from Wickerham culture medium. The eleven taxa of *Cladosporium* sp. IX, *Cladosporium* sp. X, Mycosphaerellales sp. II, *Microsphaerops arundinis*, *Aspergillus* sp. II, *Aspergillus* sp. V, Helotiales sp. I, *Calcarisporium* sp. I, *Calcarisporium* sp. II, *Emericellopsis* sp. and *Trametes versicolor* were characterized by a total of two or three isolates exclusively obtained on Wickerham medium. The three main classes Eurotiomycetes, Sordariomycetes and Dothideomycetes characterizing the fungal community of *P. palmata* were represented among the above-mentioned taxa.

Among the 15 isolates of *Penicillium* sp. I and the 7 isolates of *Acremonium* sp. I, nine and six strains were isolated from “*P. palmata*” culture medium, respectively. All the five isolates of *Cladosporium ramotenellum* were isolated from “*P. palmata*” culture medium. *Aspergillus* sp. II, *Penicillium* sp. VII, *Penicillium* sp. XVI and *Lecanicillium* sp. II, represented by two isolates, were exclusively isolated with “*P. palmata*” medium and belong to Ascomycota and either to Eurotiomycetes, Sordariomycetes or Dothideomycetes.

The three dominant Ascomycota classes Dothideomycetes, Sordariomycetes and Eurotiomycetes revealed in the present study seemed to be characterized by a slight preference for the non-selective Wickerham culture medium, which corresponds to the general observation on the global fungal community. Nevertheless, within these three fungal classes, several fungal taxa were recorded to be either mainly isolated from Wickerham or “*P. palmata*” media albeit no significant impact of the nature of the culture medium on the fungal diversity was detected (a significant influence was only detected at the phylum level with a *p*-value of 0.00069). In a previous study on fungi associated with *Flabellia petiolata*, similar numbers of isolates were obtained from both selective and non-selective media, in contrast with our study (Gnavi et al., 2017). This discrepancy may be explained by the nature of the macroalgal host, different environmental conditions, and slight differences in the isolation protocol (algal thalli sonication, homogenate filtration, incubation temperature, etc.). Results from Garzoli et al. (2018) study on fungal diversity from *Padina pavonica* are more consistent with the present findings. Regarding basidiomycetes, no taxa seemed to be preferentially isolated on the selective “*P. palmata*” medium. Indeed, a significant impact of the nature of culture medium on the diversity in terms of isolate number at the taxonomic level of phylum was established (*p*-value <0.05). It can then be hypothesized that basidiomycetes and particularly Agaricomycetes may not be well adapted to *P. palmata*.

### 3.3.1.3. Location in the algae (endophyte/epiphyte).
Algicolous fungi can be characterized either as epiphytes (colonizing the macroalgal surface) or endophytes (living within inner tissues). While endophytic fungal communities have received more attention than epiphytic ones, the present study employed three distinct protocols to isolate either epiphytes, endophytes, or a combination of both without selectivity (Abdel-Gawad et al., 2014; Abdul Malik et al., 2020). The location within the algae seems to have limited importance on the culturing rate. The endophytic-based approach led to the isolation of 51 taxa, represented by 85 isolates, while the epiphytic-based approach yielded 46 taxa represented by 84 isolates (Fig. 3). The non-targeted approach

(epi/endo) led to the isolation of 75 taxa represented by 129 isolates, clearly highlighting an improved isolation rate. However, the endophytic and epiphytic approaches generated 38 and 29 taxa that were not revealed by the non-targeted approach, respectively. This seems to underline the importance of the diversification of culture conditions to access a higher scale of fungal representatives. However, this can also be weighed a bit when considering chi-squared tests of independence since no dependence was visualized either at the phylum, class or genus levels, clearly strengthening the idea that in-depth analyses distinguishing endophyte and epiphyte did not affect culturing efficiency.

Among the 142 taxa, 51 were characterized by at least one endophytic strain. Endophyte cultivable fungi were mainly represented by *Cladosporium* and *Penicillium* genera (Fig. 4). Among the total isolates of *Cladosporium* sp. II (35) and sp. III (23), nine strains were characterized as endophytes in both cases. Seven and six endophytic strains of *Penicillium* sp. I and *Cladosporium* sp. I were obtained, respectively.

Considering epiphytic communities associated with *P. palmata*, 46 of the 142 taxa were characterized by at least one epiphytic strain. Basidiomycetes and ascomycetes were both represented. Among the 35 isolates of *Cladosporium* sp. II and the 18 isolates of *Parengyodontium album*, eight strains were isolated as epiphytic strains in both cases and numerous epiphytes also belonged to *Aspergillus* and *Penicillium* genera (Supplementary data 3).

Finally, the isolation protocol without distinction between epiphytes and endophytes (epi/endo) allowed the isolation of the majority of the strains. Indeed, of the 298 isolates, 129 could not be characterized as epiphyte nor as endophyte, and among the 142 taxa, 75 counted at least one isolate without associated location. *Cladosporium* sp. II, *Parengyodontium album* and *Cladosporium* sp. III counted 18, 9 and 8 isolates without associated location, respectively. All five *Stachybotrys chartarum* isolates could not be defined as endophytic or epiphytic.

It was not possible to establish clear trends regarding the preferred ecological habitat of the observed taxa. For example, *Parengyodontium album* was characterized by eight epiphytic and one endophytic strains, but also by nine isolates belonging to the non-determined location. A previous study identified *Engyodontium album* (syn. *Parengyodontium album*) as a major macroalgal endophyte, but all isolates were obtained from a green alga and no strain was isolated from red algae (Sarasan et al., 2020). When referring to selective isolation procedures, epiphytes and endophytes are similarly distributed among taxonomic groups. It was notably the case regarding the classes Dothideomycetes, Eurotiomycetes and Sordariomycetes. According to the present study, *Cladosporium*, *Penicillium* and *Aspergillus* could be considered as major endophytic genera compared with other fungal genera but this over-representation was due to an absolute domination, also extended to epiphytes strains. It has already been observed that fungal strains of *Penicillium* and *Aspergillus* isolated from macroalgae were over-represented in both epiphytic and endophytic communities (Abdel-Gawad et al., 2014; Fan et al., 2019).

### 3.3.1.4. Seasonality.
Season is often related with temperature which is known to impact marine fungal diversity (Wang et al., 2021). Moreover, season influences macroalgal protein and carbohydrate composition, notably in *P. palmata* (Dumay et al., 2014; Laurens et al., 2020; Sasaki and Yoshikuni, 2022). As mentioned before, substrate composition also influences the development of fungi and the observed diversity in terms of isolate number, which may change according to variations in macroalgal host composition. In the present study, seasonality has a dramatic impact on culturing rate. A split between summer/autumn and winter/spring was clearly visible based on both number of taxa and isolate number (Fig. 3). Both sampling during winter and spring led to the isolation of 56 taxa. However, sampling during spring allowed the isolation of 132 isolates, higher than the 79 isolates obtained during the winter isolation campaign. Chi-squared tests for independence of variables confirmed these trends with *p*-values close to the threshold of
significance at the phylum level (p-value = 0.062) but clearly lower at the class level with a specific focus on the three main classes (p-values <0.05) and genus level targeting the 3 main genera (p-value <0.05). Sampling during summer and autumn led to the isolation of 31 and 23 taxa, represented by 55 and 32 isolates, respectively. Interestingly, around 85 % of the fungal taxa were obtained at a unique season (122 taxa), clearly highlighting the seasonal dynamics of this cultivable fungal community structure.

The season with the lowest number of isolates was autumn, with only 32 isolates among the 308 total strains. The taxa *Cladosporium* sp. IX and *Helotiales* sp. I were both characterized by a total of two strains isolated in autumn.

The isolates of *Aspergillus* sp. II, *Aspergillus* sp. III, *Penicillium* sp. V, *Penicillium* sp. XIII and *Stachybotrys chartarum* were principally obtained in summer compared with the three other seasons (Fig. 4). Regarding *Penicillium* sp. V and *Penicillium* sp. XIII, only one strain was not isolated during summer in both cases. All the five isolates of *Stachybotrys chartarum* corresponded to summer isolates. No Dothideomycetes taxon was preferentially isolated in summer.

Among the seven isolates of *Acremonium* sp. I, six were obtained in winter. *Microsphaeropsis arundinis*, *Penicillium* sp. VII, *Penicillium* sp. XVI, and *Trametes versicolor* were characterized by a total of two strains isolated in winter. These strains belong to the three major classes of *P. palmata* fungal communities, corresponding to Eurotiomycetes, Sordariomycetes and Dothideomycetes, with the exception of the Basidiomycota taxon *Trametes versicolor*. *Cladosporium* and most generally Dothideomycetes were recorded as mainly isolated from seawater in winter (Wang et al., 2021).

All 23 strains of *Cladosporium* sp. III were isolated in spring (Fig. 4). Among the 12 isolates of *Cladosporium* sp. I, the seven isolates of *Aspergillus* sp. VIII and the five isolates of *Cladosporium ramotenellum*, eight, five and four strains were obtained in spring, respectively. All the three isolates of *Calcarisporium* sp. I were characterized as spring isolates. *Cladosporium* sp. X, *Acrodontium luzulae*, *Calcarisporium* sp. II, *Lecanicillium* sp. II and *Lecanicillium* sp. III, were characterized by a total of two strains isolated in spring. The over-representation of spring isolates compared with other seasons was principally due to *Cladosporium* strains, which represented 42 % of the whole isolates obtained during spring. This genus represented 82 % of the Dothideomycetes in terms of number of isolates, with this class predominantly isolated in spring. This spring preference was also observed for both Eurotiomycetes and Sordariomycetes.

These results contrast with findings from Abdel-Gawad et al. (2014), where autumn and winter were associated with higher algicolous fungal diversity compared to spring and summer. The present observations and conclusions performed comparing fungal diversity across seasons must be interpreted cautiously even if a significant impact of the seasonality was established on the fungal diversity in terms of isolate number at both class and genus levels. Total isolate numbers obtained from each season were not similar, which constitute an interesting result but limit any rigorous comparison of diversity across season.

### 3.3.2. Impacts of environmental and isolation parameters on primary fungal taxa

Within the 142 total taxa, only 15 were characterized by more than two isolates and corresponded to *Cladosporium ramotenellum*, *Cladosporium* sp. I, *Cladosporium* sp. II, *Cladosporium* sp. III, *Aspergillus* sp. V, *Aspergillus* sp. VIII *Penicillium brevicompactum*, *Penicillium* sp. I, *Penicillium* sp. V, *Penicillium* sp. XIII, *Acremonium* sp. I, *Calcarisporium* sp. I, *Parengyodontium album*, *Stachybotrys chartarum* and *Sporobolomyces ruberrimus*.

The four main taxa of *Cladosporium* were *Cladosporium ramotenellum*, *Cladosporium* sp. I, *Cladosporium* sp. II and *Cladosporium* sp. III and were represented by 5, 12, 35 and 23 isolates, respectively. All of these taxa were characterized with a majority of isolates obtained with a 12 °C incubation temperature. It has already been reported that marine-

derived strains of *Cladosporium* were particularly adapted to low temperatures (Gonçalves et al., 2013; Del-Cid et al., 2014; Duarte et al., 2018). Regarding the culture medium, *Cladosporium* sp. II and *Cladosporium* sp. III were represented by a majority of strains isolated from Wickerham medium, whereas all the five isolates of *Cladosporium ramotenellum* were isolated from "*P. palmata*" medium. All these four taxa were mainly isolated in spring. Moreover, no particular link with isolation protocol could be established on the structuration of this fungal genus within *P. palmata*.

Both *Aspergillus* sp. V and *Aspergillus* sp. VIII were primarily isolated on Wickerham medium incubated at 27 °C. Five of the seven strains of *Aspergillus* sp. VIII were isolated in spring. A majority of *Aspergillus* strains could not be associated neither with epiphytic nor endophytic habitat, but for the ones that can be, a majority was epiphytic. Among the 21 strains of *Aspergillus*, 15 were isolated on Wickerham medium. Then, the isolation of *Aspergillus* isolates seemed not to be particularly related neither to the incubation temperature, nor to the season. Nevertheless, *Aspergillus* has previously been described as the main genus isolated in summer in seawater compared with spring and winter (Wang et al., 2021). This over-representation of *Aspergillus* isolates during summer, compared with other taxa, has also been observed in macroalgae, but this genus was similarly isolated during the three remaining seasons (Abdel-Gawad et al., 2014). Regarding the effect of the nature of the culture medium, *Aspergillus* isolates seemed to be preferentially isolated from non-selective Wickerham medium and characterized as more epiphytic than endophytic, even if the observed differences were not significant. This potential epiphytic strategy, along with a low affinity with the "*P. palmata*" selective medium may be related to a global strategy of superficially non-selective colonization of this genus. Interestingly, in Sarasan et al. (2020), *Aspergillus* isolates had not any host preference among the three macroalgal phyla, and were obtained from 7 to the 11 studied macroalgae. This ability to colonize a wide host variety has also been observed in mangrove, where *Aspergillus* has been described as the most speciose fungal genus (Devadatha et al., 2021). Furthermore, this genus has already been characterized in marine environments as ubiquist with various ecological strategies such as saprophytic, endophytic or pathogenic (Ramírez-Camejo et al., 2012; Burge et al., 2013; Wong Chin et al., 2021).

Several *Penicillium* taxa were characterized by opposite trends in response to environmental and isolation factors in the present study, but global trends can be mentioned. The four principal taxa of *Penicillium* were *Penicillium brevicompactum*, *Penicillium* sp. I, *Penicillium* sp. V and *Penicillium* sp. XIII and were represented by 7, 15, 5 and 4 isolates, respectively. All taxa represented at least by two isolates were isolated from "*P. palmata*" medium, suggesting here a host adaptation. Four on the seven strains of *Penicillium brevicompactum* were epiphytes, whereas three among the five strains of *Penicillium* sp. V were endophytic. A great majority of strains of both *Penicillium brevicompactum* and *Penicillium* sp. I were isolated with a 12 °C incubation temperature but all the four isolates of *Penicillium* sp. XIII were obtained with a 27 °C incubation temperature. It has already been reported that marine-derived *Penicillium* strains were particularly adapted to low temperatures (Gonçalves et al., 2013; Del-Cid et al., 2014; Duarte et al., 2018). The principal marine fungal genus found in association with mussels and marine sediments was *Penicillium* and isolates were mostly obtained with a 12 °C incubation temperature when compared with 27 °C (Matallah-Boutiba et al., 2012).

Both *Penicillium brevicompactum* and *Penicillium* sp. I taxa mainly obtained with a 12 °C incubation temperature were characterized with a majority of strains obtained in winter and spring. *Penicillium* sp. V and *Penicillium* sp. V XIII strains were mainly isolated in summer and principally isolated at 27 °C. Within the 30 remaining strains belonging to the *Penicillium* genus, 23 were obtained with a 12 °C incubation temperature and 14 were isolated in spring. Spring and winter preference of marine *Penicillium* strains has already been documented (De Moura
Sarquis and De Oliveira, 1996). Moreover, it has been previously observed in marine sediments that winter resulted in higher Penicillium diversity compared with summer (Park et al., 2019). Whether representatives of the Penicillium genus are physiologically adapted to low temperatures remains an open question and needs further investigation.

Acremonium sp. I was represented by seven isolates. Among them, six were obtained in winter and six were obtained on "P. palmata" medium. This observed selective adaptation of Acremonium for a macroalgal host has already been documented, with Acremonium fuci conidia germinating only in presence of Fucus serratus tissues (Zuccaro et al., 2003; Teixeira et al., 2019). The isolates of Acremonium sp. II, Acremonium sp. III and Acremonium sp. IV were also obtained in winter. The three strains of Calcarisporium sp. I were also isolated on Wickerham medium, incubated at 27 °C and obtained in spring. Even if Parengyodontium album was one of the most represented taxa in the present study, no clear impact of the different parameters of this study was observed. Parengyodontium album strains were more associated with the epiphytic habitat than endophytic one but it cannot be determined for 9 strains. However, Parengyodontium album isolates were obtained either in winter or in spring. Considering the five strains of Stachybotrys chartarum, they all were isolated in summer but without any other tendency. Distribution of the three Sporobolomyces ruberrimus isolates is not particularly discriminated considering the different conditions of isolation. Nevertheless, focusing on the four Sporobolomyces taxa, isolates were mostly obtained at 12 °C, from Wickerham medium and in summer.

Some genera were characterized with a high amount of underrepresented taxa, counting only one or two isolates. Thus, except genera previously mentioned, Ramularia, Acrodontium and Lecanicillium were characterized by more than two isolates. The three strains of Ramularia were isolated on Wickerham medium during spring. All strains of Acrodontium were isolated in spring with a 12 °C incubation temperature. The genus Lecanicillium was represented by 5 strains all isolated in spring, 4 of them at the 27 °C incubation temperature.

## 4. Conclusion

This study represents the first comprehensive description of the cultivable mycobiota of the red alga Palmaria palmata. A total of 298 fungal isolates were obtained, representing 142 taxa, predominantly ascomycetes, with fewer basidiomycetes. The three most common fungal classes were Dothideomycetes, Eurotiomycetes, and Sordariomycetes. The isolation design revealed that incubation at 12 °C on a non-selective Wickerham culture medium was particularly efficient for isolating a broad range of fungi, in comparison with an incubation at 27 °C on a selective "P. palmata" medium. Interestingly, Sordariomycetes were preferentially isolated at 27 °C, while Dothideomycetes and Eurotiomycetes were preferentially isolated at 12 °C. Most of the isolates were characterized as epiphytes and endophytes, without distinct communities according to the various isolation conditions used in this study. The influence of season on fungal diversity in terms of isolate number differed according to the considered taxa but was significant at both class and genus levels. The genera Cladosporium, Penicillium, and Aspergillus emerged as key players, reflecting their ubiquity in marine environments. The present study spanned one year and provides a baseline understanding of the cultivable fungal community associated with P. palmata, but further exploration of non-cultivable fungal diversity if necessary to assess the ecological relevance of these fungi.

### CRediT authorship contribution statement

Yoran Le Strat: Writing – original draft, Visualization, Methodology, Investigation, Conceptualization. Nicolas Ruiz: Writing – review & editing, Conceptualization. Paul Déléris: Writing – review & editing, Conceptualization. Thibaut Robiou du Pont: Investigation, Conceptualization. Samuel Bertrand: Visualization, Formal analysis. Stella

Deaba: Investigation. Gaëtan Burgaud: Writing – review & editing, Formal analysis. Justine Dumay: Writing – review & editing, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgement

This work was supported by a Ph.D grant for Yoran Le Strat from the French Ministry of Higher Education and Research. Authors gratefully acknowledge the LUBEM (Laboratoire Universitaire de Biodiversité et Ecologie Microbienne) where experiments related with the identification of fungal strains were performed.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.funeco.2025.101417.

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