Seasonal dynamics of proteorhodopsin-gene diversity in the English Channel revealed by metabarcoding at the SOMLIT-Astan Time-Series
Proteorhodopsins (PRs) are light-driven proton pumps widely distributed among marine prokaryotes and represent a major functional trait associated with photoheterotrophic lifestyles. Although PR genes are known to be ubiquitous in surface oceans, their long-term temporal dynamics and seasonal structuring remain insufficiently characterized, particularly in temperate coastal ecosystems.  In this study, we investigated the seasonal and interannual dynamics of proteorhodopsin gene diversity in the western English Channel using a long-term coastal time series from the SOMLIT-Astan observatory. Seawater samples were collected from the 0.2–3 µm size fraction targeting free-living prokaryotic communities, and PR diversity was assessed by Illumina 2x250 pb paired-end amplicon sequencing of c.a. 330 pb fragment of the PR gene (PR-1aF / PR-1aR primers; Campbell et al. 2008). Sequencing was performed on one representative sample every two months between January 2009 and November 2010, providing a multi-seasonal view of PR community composition over two consecutive years.  Amplicon sequencing revealed a structured PR community dominated by a limited core assemblage of 62 proteorhodopsin operational taxonomic units (OTUs), accounting for approximately 96% of total PR sequence reads. These core PR types were mainly affiliated with Proteobacteria (a- and ?-Proteobacteria) and Firmicutes, indicating a phylogenetically constrained yet stable functional gene pool. Despite this core stability, PR community composition exhibited pronounced and reproducible seasonal patterns across the two-year period.  Distinct PR assemblages characterized winter and summer, while spring and autumn represented transitional phases marked by shifts in dominant PR types. Seasonal restructuring was evident both at the level of major taxonomic groups and individual OTUs. Firmicutes-related PR types dominated during spring and summer, whereas PR sequences affiliated with uncultured Euryarchaeota were more prominent during winter and fall. Additional seasonally restricted PR types affiliated with Bacteroidetes and Planctomycetes contributed to the temporal turnover of PR diversity.  Multivariate analyses integrating PR diversity with environmental descriptors indicated that seasonal changes in PR community structure were strongly associated with environmental forcing. Winter and fall PR assemblages were primarily related to elevated concentrations of inorganic nutrients, particularly nitrates and phosphates, whereas spring and summer communities were best explained by increasing temperature, day length, and chlorophyll-a concentration. These diversity patterns were consistent between years, highlighting a high degree of interannual reproducibility in the seasonal structuring of PR assemblages.  Overall, this study demonstrates that proteorhodopsin gene diversity in a temperate coastal ecosystem undergoes predictable seasonal reorganization despite a stable core community. The observed seasonal succession of PR-bearing clades suggests that PR-based photoheterotrophy encompasses multiple ecological strategies adapted to contrasting seasonal regimes. This work underscores the value of long-term, function-focused metabarcoding time series for resolving the temporal ecology of key microbial traits in coastal marine environments.