Supporting Information Data for Bittner et al. 2026 including environmental data of Neuse River Estuary, North Carolina (USA), and dissolved and particulate B-vitamin measurements.

<b>Data S1. </b>Physical, chemical and biological characteristics of NRE across stations and sampling dates. Abundances of the four small phytoplankton morphotypes were detected with flow cytometry <i>Synechococcus</i>-like phycoerythrin (PE)-rich (PE-SYN), <i>Synechococcus</i>-like phycocyanin (PC)-rich cells (PC-SYN), picoeukaryotic phytoplankton (PEUK) larger eukaryotic phytoplankton cells (LEUK).<b>Data S2. </b>Compound specific LC-MS parameters for dissolved (<b>A</b>) and particulate samples 0.2-3.0 µm (<b>B</b>) and 3-90 µm size-fractions (<b>C</b>). Mass over charge (m/z) values for the transitions used for quantification of the compound. The limit of detection (LOD) is calculated as three times the variation of the quality control sample. Limit of quantification (LOQ) is calculated as ten times the variation of the quality control sample. LOD and LOQ are given in fmol. Response of OH-pseudocobalamin and Me-pseudocobalamin assumed to be the same as for OH-cobalamin and Me-cobalamin, respectively. Asterisk (*) indicates values between LOD and LOQ were verified by the batch per batch method, otherwise only values above LOQ were quantified.<b>Data S3</b>. Concentrations of B-vitamins and vitamers present in the dissolved (<b>A</b>), small particulate (0.2-3 µm) size-fraction (<b>B</b>), and large (3-90 µm) particulate size-fraction (<b>C</b>) in the near-surface waters of the Neuse River Estuary across time points and stations sampled. Number of biological replicates for each sample for which data is available is provided in the column labelled n, as some replicates were lost during sample processing or measurement (see Methods). Not all metabolites were detected in all biological replicates as, therefore the number of biological replicates, on which the measurement is based, is provided for each metabolite. If the metabolite was detected in minimum three replicates the standard deviation (sd) is listed. LOD: measurements were below limit of detection. LOQ: measurements were below limit of quantification. Compounds specific LODs and LOQs are provided per dataset and matrix grouping in Supporting Information <b>Data S2</b>.<b>ABSTRACT</b>As B-vitamins are organic cofactors required by prokaryotic and eukaryotic planktonic cells, their availability impacts aquatic microbial communities and associated biogeochemistry. Contrary to inorganic nutrients, measurements of B-vitamins from brackish systems are scarce and relationships between B-vitamins and plankton composition in estuaries are unclear, limiting our understanding of estuary biology in general as well as how B-vitamins are distributed and dispersed in marine systems. Here, we quantify multiple B-vitamins and their vitamers in particulate and dissolved phases, and characterize microbial community composition, across fresh to polyhaline zones of the Neuse River Estuary (NRE), North Carolina, USA. We uncover elevated concentrations of B-vitamins within the mid-estuary, Chl <i>a</i> maximum along with a unique suite of dissolved B-vitamin associated with sporadic surges in pico and microplankton populations. The dynamics of both dissolved and particulate B-vitamin concentrations in space and time were striking - from subpicomolar to high picomolar levels observed and strong short-term (weeks) variability. We find notable autochtonous B-vitamin production in the estuary, but we expect the ability of the system to deliver these micronutrients to the ocean will depend on flushing as well as changes in microbial community. We identify vitamin B1, B12, psB12 (pseudocobalamin), and B3 as key explanatory variables for change in prokaryotic and eukaryotic NRE plankton, providing new evidence of B-vitamin influence upon estuarine plankton community composition. Our work reveals new complexities in B-vitamin production and consumption within zones of estuaries while underscoring these micronutrients as key drivers of microbial plankton composition.<br>