Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life

Abstract

The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL(-1) were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 μm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.

Fig. 1.

Environmental diversity and sample locations. Maximum likelihood phylogenetic tree of environmental 16S rDNA sequences obtained herein (bold) as well as OM270. (Right) Clone library sample sites (Table S2) and the number of sequences obtained are shown, including marine samples from the North Pacific (NP), Florida Straits (FS), and BATS, as well as UK coastal and freshwater samples. The scale bar indicates the inferred number of nucleotide substitutions per site. Bootstrap support values (≥50%) are from RaxML and Log-Det distance analyses, respectively. (Inset) Map shows the approximate positions of the sites sampled.

Fig. 2.

Maximum likelihood (ML) phylogenetic tree of plastid 16S-Ile tRNA-Ala tRNA-23S rDNA sequences rooted with select cyanobacteria. Sequences in bold were generated as part of this study, including newly obtained Pavlovophyceae sequences. The scale bar indicates the inferred number of nucleotide substitutions per site. Bootstrap support values (≥50%) are from ML and Log-Det distance analyses, respectively. (Inset) Bootstrap support for nodes labeled A, B, and C using 16S rDNA, 23S rDNA, and near-complete rDNA operon alignments (representative 16S and 23S rDNA tree topologies are shown in Fig. S1).

Fig. 3.

Rappemonad distributions in the Sargasso Sea in 2003. Seasonal transitions are shown for BATS, as revealed by qPCR assays for the 16S rRNA gene (dark red, gene copies mL⁻¹). In vivo chlorophyll fluorescence is shown in relative fluorescence units (green, r.f.u.). Note differences in x-axis scales.

Fig. 4.

Fluorescence micrographs of rappemonads in the North Pacific. The DAPI-stained nucleus (blue) was often slightly elongated with a tapering end. Two to four plastids appeared to be present per cell (green, tyramide signal amplification FISH-labeled). Cells shown in A1–A3 were detected using the RappeA probe, whereas cells in B1–B3 were detected with the RappeB probe. (Scale bar: 5 μm.)