Comparison of Metabarcoding and Shotgun Sequencing Confirms the Relevance of Chloroplastic <scp>rRNA</scp> Genes to Assess Community Structure of Lake Phytoplankton

Studying the taxonomic composition of phytoplankton has been revolutionised by the emergence of metabarcoding approaches which theoretically provides access to all phytoplankton diversity. However, metabarcoding has its limitations, including biases related to primers efficiency in covering all phytoplanktonic taxonomic groups, biases introduced during amplification and due to databases completion. To assess the importance of these biases, we compared the compositions of phytoplankton in two large peri-alpine lakes, using metabarcoding with primers targeting hypervariable regions of chloroplastic 16S and 23S rRNA, designed to cover the full taxonomic diversity of phytoplankton, and shotgun sequencing. To be able to directly compare the two methods, we extracted reads coming from the full sequences of these rRNA genes in shotgun sequencing data and used the same reference database for taxonomic assignation. The results show that the relative abundances of dominant groups of phytoplankton, including Cyanobacteria, Cryptophyta, Haptophyta, and Bacillariophyta, are consistent between the two approaches, validating the primers used for metabarcoding analysis. However, two phyla, Chlorophyta and non-diatom Ochrophyta showed greater divergence in their relative abundance, due to under-amplification or lack of amplification of certain taxonomic groups in metabarcoding. This is likely due to the high diversity of these groups, not covered yet by the reference databases, as well as a possible presence of introns in their choloroplastic ribosomal genes. These limitations are expected to be overcome with increasing reference database completion and the use of long-read metabarcoding. Overall, our study confirms the relevance of using chloroplastic primers for assessing the phytoplankton composition of lakes.