Using quantitative polymerase chain reaction to assess phytoplankton and indicate eutrophication in freshwater rivers: A multiyear nationwide study across the United States

Phytoplankton are essential primary producers in fresh surface water that are critical to the health of ecosystems. However, phytoplankton overgrowth due to eutrophication threatens ecological, economic, and public health. Therefore, assessing phytoplankton is fundamental for understanding the productivity, health, and trophic status of freshwater ecosystems. Light microscopy and chlorophyll a assessment are common approaches for studying phytoplankton. They are easy to use, cost-effective, and reliable but have significant limitations. Microscopy has a low throughput and is time-consuming and labor-intensive. Chlorophyll a assessment does not reveal phytoplankton community composition and structure. For comparison, quantitative polymerase chain reaction (qPCR) is widely applied in quantifying microorganisms, offering multiple advantages, including high throughput, sensitivity, accuracy, and robustness. However, a research gap remains regarding the feasibility of using qPCR to assess phytoplankton and indicate trophic status of freshwater bodies. We conducted a nationwide, multiyear study in the United States to compare the performance of qPCR, microscopy, and chlorophyll a assessment in assessing phytoplankton and trophic statuses of multiple freshwater rivers. From early summer to late fall in 2017, 2018, and 2019, we assessed phytoplankton, chlorophyll a, pheophytin a, and the overall Trophic Level Index (TLI_Overall) at the sampling sites in 12 large freshwater rivers in three regions (western, midcontinent, and eastern) across the United States. The seasonal summed abundance of four major phytoplankton taxa [Bacillariophyta (diatoms), Cyanobacteria (blue-green algae), Chlorophyta (green algae), and Dinoflagellates (Dinophyta)] ranged from 6.88 log₁₀(GCN·L^–1) (the Connecticut River, 2017) to 9.29 log₁₀(GCN·L^–1) (the Kansas River, 2019) (GCN: gene or genome copy number). qPCR- and microscopy-based phytoplankton abundance of eight phytoplankton taxa had a significant positive allometric or log-linear correlation (adjusted R² = 0.836, p-value < 0.001, n = 815). In addition, qPCR-based phytoplankton abundance had positive allometric or log-linear correlations with chlorophyll a (adjusted R² = 0.5437, p-value < 0.001, n = 164), pheophytin a (adjusted R² = 0.3378, p-value < 0.001, n = 164), and TLI_Overall (adjusted R² = 0.4789, p-value < 0.001, n = 164). Therefore, qPCR is a promising alternative to microscopy and chlorophyll a for studying phytoplankton and trophic status in freshwater rivers. Moreover, phytoplankton abundance had limited temporal variation within each sampling season and over the three sampling seasons in 2017, 2018, and 2019 but showed clear spatial variation. The midcontinent sites had significantly higher phytoplankton abundance, chlorophyll a concentrations, pheophytin a concentrations, and TLI_Overall values than those in the eastern and western rivers, reflecting the higher trophic statuses of the midcontinent rivers. This work also provides the thresholds of qPCR-based phytoplankton abundance for delineating trophic statuses in freshwater rivers. Overall, this work demonstrates that qPCR is a promising tool for studying phytoplankton and characterizing the trophic status of freshwater rivers.