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

Abstract

BackgroundThe class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea.ResultsWe cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S–28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I – III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II + III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms.ConclusionsThis altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1224-z) contains supplementary material, which is available to authorized users.

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

“…The use of both genes was proposed to more robustly derive the origin of environmental sequences, particularly in the case of fast‐evolving taxa, but this was based on Sanger sequencing of clone libraries (Marande, López‐García, & Moreira, ). Near full‐length 18S amplicons and even longer fragments including parts of the 28S gene have also recently been sequenced with PacBio for group‐specific investigations, demonstrating that long‐read high‐throughput sequencing is a promising complement to Illumina for investigating the environmental diversity of eukaryotes (Heeger et al, ; Orr et al, ). Here, we extended the approach to ~4,500 bp of the rDNA operon across the whole phylogenetic diversity of eukaryotes.…”

“…In the last few years, PacBio sequencing has started to be applied to metabarcoding studies, primarily on prokaryotic 16S rDNA (Mosher et al, 2014;Schloss, Jenior, Koumpouras, Westcott, & Highlander, 2016;Wagner et al, 2016) and most recently on larger amplicons also including the 23S rDNA (Martijn et al, 2019). For eukaryotes, the 18S rDNA was nearly fully sequenced for targeted microbial groups (Orr et al, 2018), whilst longer regions also spanning the ITS and the large subunit (LSU) 28S gene were used to analyse fungal diversity (Heeger et al, 2018;Tedersoo & Anslan, 2019;Tedersoo, Tooming-Klunderud, & Anslan, 2018). These studies showed that in spite of the high error rates of PacBio, when applying a corrective process based on multiple sequence passes (Circular Consensus Sequences [CCS]) together with rigorous quality filtering, long-amplicon sequencing is emerging as a robust approach for studying environmental diversity.…”

Perspectives from Ten Years of Protist Studies by High‐Throughput Metabarcoding

“…Using PacBio, near‐full‐length 18S rRNA gene sequences were obtained from environmental samples for the enigmatic protistan group of Diphyllatea (Orr et al. ). A few other studies mainly targeting fungal diversity were able to sequence longer fragments including also the ITS and 28S rRNA gene (Heeger et al.…”

Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

“…The use of both genes was proposed to more robustly derive the origin of environmental sequences, particularly in the case of fast-evolving taxa, but this was based on Sanger sequencing of clone libraries (Marande, López-García, & Moreira, 2009). Near full-length 18S amplicons and even longer fragments including parts of the 28S have also recently been sequenced with PacBio for group-specific investigations, demonstrating that long-read high-throughput sequencing is a promising alternative to Illumina for investigating the environmental diversity of eukaryotes (Heeger et al, 2018;Orr et al, 2018). Here, we extended the approach to ~4500bp of the rDNA operon across the whole phylogenetic diversity of eukaryotes.…”

“…In the last two years, PacBio sequencing has started to be applied to metabarcoding studies, primarily on prokaryotic 16S rDNA (Mosher et al, 2014;Schloss, Jenior, Koumpouras, Westcott, & Highlander, 2016;Wagner et al, 2016) and most recently on larger amplicons also including the 23S rDNA (Martijn et al, 2017). For eukaryotes, the 18S rDNA was nearly fully sequenced for targeted microbial groups (Orr et al, 2018), whilst longer regions also spanning the ITS and the 28S gene were used to analyze fungal diversity (Heeger et al, 2018;Tedersoo, Tooming-Klunderud, & Anslan, 2018). These studies showed that in spite of the high error rates of PacBio, when applying a corrective process based on multiple sequence passes (Circular Consensus Sequences -CCS) together with rigorous quality filtering, long-amplicon sequencing is emerging as a robust approach for studying environmental diversity.…”