A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been recently reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and abundances in freshwater ecosystems remain unknown. Here, we performed 18S rRNA amplicon sequencing, shotgun metagenomics and CARD-FISH analyses in seven freshwater lakes in Japan, Czech Republic, and Switzerland in order to uncover the diversity and abundance of diplonemids in deep freshwater environments. We detected diplonemids in all lakes we examined. Phylogenetic analyses of the assembled long 18S rRNA sequences from deep-sequenced metagenomes showed that freshwater diplonemids form a distinct clade with a much lower diversity than their marine counterparts. The abundance estimations based on CARD-FISH quantification were similar to those observed from sequencing approaches and showed that in contrast to the marine counterparts, freshwater diplonemids display low abundances associated with a reduced diversity. The microscopy data showed that the freshwater diplonemids range in size between 5 and 10 μm and that they appear to contain intra-cytoplasmatic bacteria. The reduced diversity of the freshwater diplonemids clade, together with the group monophyly and its position inside a diversified marine group indicate a recent habitat transition from marine to freshwater environments. Overall, our study shows the widespread presence of diplonemids in freshwater ecosystems and suggests their presence through a recent habitat transition from marine environments.