Epiphytic diatoms associated with marine macroalgae play vital ecological roles in nutrient cycling and primary production, yet their study remains limited due to the lack of standardized methodologies. This study focuses on diatom communities growing on Gelidium corneum, a key red alga in the Cantabrian coast (Spain). Samples were collected from two depths along the northern coast of Spain and processed using both morphological and molecular approaches. Morphological analysis involved diatom frustule preparation using hydrogen peroxide digestion, acid treatments, and permanent slide mounting, enabling identification through light microscopy. Molecular analysis employed DNA extraction and rbcL marker-based metabarcoding, allowing detailed taxonomic characterization. Results highlight the efficacy of combining morphological and molecular techniques to overcome the limitations of either approach individually. By standardizing procedures, we enhance the reproducibility and comparability of studies focused on diatom epiphytes. Our results highlight the ecological significance of diatom-macroalgal interactions and provide a framework for future investigations into these essential but underexplored communities.•A polyphasic method was developed for studying epiphytic diatoms on Gelidium corneum, combining morphological and molecular tools.•The approach overcomes challenges in diatom characterization, including intricate host morphology and cryptic species identification.•Standardized protocols enhance reproducibility and offer insights into diatom-macroalgal ecological interactions.

Department of Ecology Faculty of Science Charles University Viničná 7 Prague 2 CZ 12844 Czech Republic

Diatom Lab IMA La Serna St León Spain

Ecology Unit Department of Biodiversity and Environmental Management Faculty of Biological and Environmental Sciences University of León León Spain

IHCantabria Instituto de Hidráulica Ambiental de la Universidad de Cantabria Santander Spain

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