Enigmatic Diphyllatea eukaryotes: Culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution

Abstract Diphyllatea is an ancient and enigmatic lineage of unicellular eukaryotes that possesses morphological features common to other deeply diverging eukaryotes, such as Amoebozoa and Excavata. In reconstruction of the evolutionary processes underlying diversification and morphological innovation among eukaryotes, Diphyllatea plays a key role together with other orphan lineages. Despite being of evolutionary significance, only three species of Diphyllatea have descripted morphology, with molecular data available from fewer. The lack of data means that the actual diversity of this key lineage of eukaryotes remains unresolved. We here present a first attempt to understand the species diversity and higher order structure of the Diphyllatea phylogeny. We have cultured several new strains, described these morphologically, and amplified their rRNA. We have sampled DNA from multiple globally distributed sites, using these as templates in a Diphyllatea-specific PCR. In contrast to recent diversity studies, which use short variable gene regions, we amplify nearly the whole 18S rRNA gene, and sequence using PacBio RS II technology, to provide enough information to resolve historically ancient speciation events. Phylogenetic inference of Diphyllatea rRNA reveals three deeply branching and distinct clades of Diphyllatea, here named Diphy I – III. Diphy I and II include the genera Diphylleia and Collodictyon . Notably, Diphy III is here shown as novel phylogenetic clade with all strains investigated having a congruent morphology to Collodictyon triciliatum (Diphy II). Altogether, Diphyllatea seems to constitute two morphotypes, a biflagellate (i.e. Diphy I) and a quadraflagellate (i.e. Diphy II and III) form, congruent with earlier descriptions of Diphylleia and Collodictyon. Further, our targeted environmental sequencing approach, which includes specific PCR primers, reveals a wider global distribution of Diphyllatea than earlier known. Altogether, the described protocol shows the usefulness of combining long amplicon high-throughput sequencing and lineage-specific PCR approach in surveys of enigmatic eukaryote lineages.