Metabarcoding analysis has been widely applied to probe the diversity and dynamics of phytoplankton species responsible for the development of harmful algal blooms (HABs) that are common in the coastal regions of China and many other countries. Metabarcoding analysis has been proven to be effective in identifying HAB species with high resolution, being able to identify HAB species that could not be properly identified using morphology-based approaches because are too small in cell sizes, too similar in cell morphologies, and too fragile in sample fixation. However, how to accurately identify individual species from mountains of sequence results obtained in metabarcoding analyses remains a challenge.

Inaccurate interpretation of sequences obtained in metabarcoding analysis can lead to the overestimation of biodiversity of marine ecosystems. To address this problem, the common molecular marker 18S rDNA V4 of single cells (including the dinoflagellates Noctiluca scintillans and Tripos species) or single strains (including the diatoms Pseudo-nitzschia species and Skeletonema species, and the haptophyte Phaeocystis globosa) of a few representative HAB species were sequenced and analyzed to examine the nature of molecular diversity revealed in metabarcoding analyses.

Each cell was found to harbor a large number of variants with different copy numbers, with usually a single dominant variant and a many non-dominant variants, indicating previously neglected high intragenomic variations (IGVs). Different cells of a particular species usually share the dominant variant, but not always. For example, two different dominant variants were found in different cells of Pseudo-nitzschia pungens. Some variants were found to be the dominant variants of one species, but non-dominant variants in other species of the same genus. This research illustates the importance to understand the nature of molecular diversity and to properly distinguish species diversity and genetic diversity from intragenomic diversity.  A model was proposed to explain the origin and evolution of this molecular marker and the nature of molecular diversity. 

harmful algal bloom, metabarcoding, molecular diversity, genetic diversity, intragenomic diversity