Flood-Driven Salinity Shifts Restructure Estuarine Pico-Sized Chlorophyte Assemblages at a Subtropical River-Sea Interface

Chlorophytes are important contributors to aquatic primary production and biomass, yet the ecological role of freshwater-associated pico-sized chlorophytes in estuarine succession remains poorly resolved. Here, we conducted a high-frequency, year-round survey from August 2021 to September 2022 in the Minjiang estuarine-coastal system using Illumina MiSeq V4 and PacBio full-length 18S rRNA gene sequencing to examine chlorophyte dynamics across seasonal and flood-driven hydrographic transitions. Marine Mamiellophyceae, particularly Micromonas, Bathycoccus, and Ostreococcus, dominated during autumn and winter but were rapidly displaced during the 2022 spring-summer flood pulse by freshwater-associated Trebouxiophyceae. This transition coincided with reduced salinity, elevated chlorophyll a, and dissolved oxygen, associated with strong freshwater intrusion. Both platforms detected freshwater trebouxiophytes differently during the flood, with MiSeq capturing Chloroidium and PacBio identifying Auxenochlorella as the dominant taxon. Notably, both genera represent freshwater-associated trebouxiophytes that are rarely reported in estuarine coastal systems. MiSeq provided broader temporal and genus-level coverage, whereas PacBio improved phylogenetic resolution of freshwater-associated lineages, demonstrating complementary ecological and taxonomic resolution between platforms. Along the salinity gradient, Picochlorum declined during peak freshening but recovered under euhaline conditions, whereas Tetraselmis peaked under mesohaline conditions, suggesting a transitional niche between freshwater- and marine-associated assemblages. Together, these results demonstrate that episodic flooding can rapidly restructure pico-sized chlorophyte communities through freshwater intrusion and reveal an underappreciated ecological role of freshwater chlorophytes in subtropical river-sea interface systems.