Harmonized coexistence of intragenomic variations in diatom Skeletonema strains

Metabarcoding analysis has been demonstrated to be an effective technology for monitoring diversity and dynamics of phytoplankton including Skeletonema species. Although molecular diversity uncovered in metabarcoding projects has generally been interpreted as sum of interspecies diversity and intraspecies diversity, accumulating evidence suggests that it also harbors unprecedentedly high levels of intra-genomic variations (IGVs). As up to thousands of amplicon sequence variants (ASVs) identified in a typical metabarcoding project can be annotated to be Skeletonema species, we hypothesize that substantial portions of these ASVs are contributed by IGVs. Here, the nature of IGVs in Skeletonema species was quantitatively analyzed by carrying out single-strain metabarcoding analysis of 18S rDNA V4 in 49 strains belonging to seven Skeletonema species. Results showed that each Skeletonema strain harbored a high level of IGVs as expected. While many Skeletonema strains each contained one dominant ASV and a substantial number of ASVs displaying much lower relative abundance, other Skeletonema strains each contained multiple ASVs with comparable or nearly equally abundances. Thus the co-existence of multiple dominant ASVs in a single cell indicated a tug-of-war of these variants in evolution, which may eventually result in harmonized coexistence of multiple dominant ASVs. A total of nine dominant ASVs and 652 non-dominant ASVs were found in 49 strains of seven Skeletonema species, indicating rich interspecies and intraspecies variations, and complex evolution of IGVs in genus of Skeletonema. The results confirmed that the extensive degree of IGVs was the main contributor to the high molecular diversity revealed by metabarcoding analysis. This study highlights the importance of quantitative characterization of IGVs in Skeletonema species for accurate interpretation of species diversity in metabarcoding analysis.