Draining 31 states and roughly 3 million km 2 , the Mississippi River (MSR) and its tributaries constitute an essential resource to millions of people for clean drinking water, transportation, agriculture, and industry. Since the turn of the 20 th century, MSR water quality has continually rated poorly due to human activity.Acting as first responders, microorganisms can mitigate, exacerbate, and/or serve as predictors for water quality, yet we know little about their community structure or ecology at the whole river scale for large rivers. We collected both biological (16S and 18S rRNA gene amplicons) and physicochemical data from 38 MSR sites over nearly 3000 km from Minnesota to the Gulf of Mexico. Our results revealed a microbial community composed of similar taxa to other rivers but with unique trends in the relative abundance patterns among phyla, operational taxonomic units (OTUs), and the core microbiome. Furthermore, we observed a separation in microbial communities that mirrored the transition from an 8 th to 10 th Strahler order river at the Missouri River confluence, marking a different start to the lower MSR than the historical distinction at the Ohio River confluence in Cairo, IL. Within MSR microbial assemblages, we identified subgroups of OTUs from the phyla Acidobacteria, Bacteroidetes, Oomycetes, and Heterokonts that were associated with, and predictive of, the important eutrophication nutrients nitrate and phosphate. This study offers the most comprehensive view of MSR microbiota to date, provides important groundwork for higher resolution microbial studies of river perturbation, and identifies potential microbial indicators of river health related to eutrophication.

Draining 31 states and roughly 3 million km2, the Mississippi River (MSR) and its tributaries constitute an essential resource to millions of people for clean drinking water, transportation, agriculture, and industry. Since the turn of the 20th century, MSR water quality has continually rated poorly due to human activity. Acting as first responders, microorganisms can mitigate, exacerbate, and/or serve as predictors for water quality, yet we know little about their community structure or ecology at the whole river scale for large rivers. We collected both biological (16S and 18S rRNA gene amplicons) and physicochemical data from 38 MSR sites over nearly 3000 km from Minnesota to the Gulf of Mexico. Our results revealed a microbial community composed of similar taxa to other rivers but with unique trends in the relative abundance patterns among phyla, OTUs, and the core microbiome. Furthermore, we observed a separation in microbial communities that mirrored the transition from an 8th to 10th Strahler order river at the Missouri River confluence, marking a different start to the lower MSR than the historical distinction at the Ohio River confluence in Cairo, IL. Within MSR microbial assemblages we identified subgroups of OTUs from the phyla Acidobacteria, Bacteroidetes, Oomycetes, and Heterokonts that were associated with, and predictive of, the important eutrophication nutrients nitrate and phosphate. This study offers the most comprehensive view of MSR microbiota to date, provides important groundwork for higher resolution microbial studies of river perturbation, and identifies potential microbial indicators of river health related to eutrophication.

The environmental health of aquatic ecosystems is critical to society, yet traditional assessments of water quality have limited utility for some bodies of water such as large rivers. Sequencing of environmental DNA (eDNA) has the potential to complement if not replace traditional sampling of biotic assemblages for the purposes of reconstructing aquatic assemblages and, by proxy, assessing water quality. Despite this potential, there has been little testing of the ability of eDNA to reconstruct assemblages and their absolute and relative utility to infer water quality metrics. Here, we reconstruct phytoplankton communities by amplifying and sequencing DNA from a portion of the 23S rRNA region from filtered water samples along a 2900-km transect in the Mississippi River. Across the entire length, diatoms dominated the assemblage (72.6%) followed by cryptophytes (8.7%) and cyanobacteria (7.0%). There were no general trends in the abundances of these major taxa along the length of the river, but individual taxon abundance peaked in different regions. For example, the abundance of taxa genetically similar to Melosira tropica peaked at approximately 60% of all reads 2750 km upstream from the Gulf of Mexico, while taxa similar to Skeletonema marinoi began to increase below the confluence with the Missouri River until it reached approximately 30% of the reads at the Gulf of Mexico. There were four main clusters of samples based on phytoplankton abundance, two above the confluence with the Missouri and two below. Phytoplankton abundance was a poor predictor of NH4+ concentrations in the water, but predicted 61% and 80% of the variation in observed NO3-and PO43-concentrations, respectively. Phytoplankton richness increased with increasing distance along the river, but was best explained by phosphate concentrations and water clarity. Along the Mississippi transect, there was similar structure to phytoplankton and bacterial assemblages, indicating that the two sets of organisms are responding to similar environmental factors. In all, the research here demonstrates the potential utility of metabarcoding for reconstructing aquatic assemblages, which might aid in conducting water quality assessments.

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