Morphology and Ribosomal Amplicons: Complementary Methods in the Analysis of Waquoit Bay Phytoplankton Diversity and Abundance

Microscopy and ribosomal amplicon sequencing are methods commonly used to examine microbial plankton communities. Each has strengths and limitations. Microscopy provides reliable estimates of abundance and biomass, but it is time consuming and not useful for small planktonic species or those lacking defined morphologies. DNA sequencing can be used to analyze a large number of samples and provides a wider range of species identification, but abundances are only relative estimates and the recovery of species amplicons is affected by primer biases and database information. Here we utilize both methods to obtain a more complete assessment of the phytoplankton community and its change over the spring and summer 2019 in Waquoit Bay, MA. The proportional abundance of species identified using microscopy was compared to the proportional abundance of sequences identified through DNA analysis using Illumina sequencing. To identify active mixotrophic species (phytoplankton that consume particles) in our samples, ingestion experiments were conducted using bromodeoxyuridine (BrdU) labeled bacteria as prey. The DNA analysis allowed us to identify species that were too small or morphologically indistinct to be identified by microscopy. For example, amplicon sequencing clearly showed the presence of Microglena uva-maris, with a bloom on July 5^th, that was not identified through microscopy. In contrast, species of euglenids and cryptophytes that dominated some of the microscopy samples were not recovered in the sequence dataset. We were also able to combine the identification of mixotrophic species using BrdU with microscopy-based abundances to estimate the portion of the phototrophic community with mixotrophic potential. The use of complementary methods to expand identification of phytoplankton and mixotrophic species is currently necessary for a more complete understanding of phototrophic diversity.