Abstract
BackgroundMost microbial eukaryotes are uncultivated and thus poorly suited to standard genomic techniques. This is the case for Polykrikos lebouriae, a dinoflagellate with ultrastructurally aberrant plastids. It has been suggested that these plastids stem from a novel symbiosis with either a diatom or haptophyte, but this hypothesis has been difficult to test as P. lebouriae dwells in marine sand rife with potential genetic contaminants.ResultsWe applied spliced-leader targeted PCR (SLPCR) to obtain dinoflagellate-specific transcriptomes on single-cell isolates of P. lebouriae from marine sediments. Polykrikos lebouriae expressed nuclear-encoded photosynthetic genes that were characteristic of the peridinin-plastids of dinoflagellates, rather than those from a diatom of haptophyte. We confirmed these findings at the genomic level using multiple displacement amplification (MDA) to obtain a partial plastome of P. lebouriae.ConclusionFrom these data, we infer that P. lebouriae has retained the peridinin plastids ancestral for dinoflagellates as a whole, while its closest relatives have lost photosynthesis multiple times independently. We discuss these losses with reference to mixotrophy in polykrikoid dinoflagellates. Our findings demonstrate new levels of variation associated with the peridinin plastids of dinoflagellates and the usefulness of SLPCR approaches on single cell isolates. Unlike other transcriptomic methods, SLPCR has taxonomic specificity, and can in principle be adapted to different splice-leader bearing groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1636-8) contains supplementary material, which is available to authorized users.
Highlights
Most microbial eukaryotes are uncultivated and poorly suited to standard genomic techniques
We employed this technique to amplify a partial plastid genome of Polykrikos lebouriae, and supplemented this with a dinoflagellate-specific transcriptomic approach, both to ensure that our plastid amplification did not stem from non-dinoflagellate environmental contaminants (i.e.; diatoms, haptophytes or other algae that share the same habitat as P. lebouriae), and to test whether the plastids are functionally integrated into the cell rather than being retained as kleptoplastids
The identification was confirmed by comparing DNA fragments of HSP90 and LSU rRNA genes from single cell sequence data to sequences obtained from previous isolates of Polykrikos lebouriae (LSU Small subunit ribosomal DNA (rDNA) sequences shared 96.8 % identity and HSP90 sequences shared 98.9 % identity)
Summary
Most microbial eukaryotes are uncultivated and poorly suited to standard genomic techniques. Polykrikoid dinoflagellates are a distinctive group of uncultivated eukaryotes, including heterotrophic, photosynthetic, and mixotrophic species They are recognized by their large, multinucleated cells or “pseudocolonies,” and include species that regulate harmful algal blooms by grazing on toxic dinoflagellates [7, 8]. Multiple displacement amplification is a powerful tool for whole-genome amplification from small amounts of template DNA, but it is nonspecific and prone to contamination [13] We employed this technique to amplify a partial plastid genome of Polykrikos lebouriae, and supplemented this with a dinoflagellate-specific transcriptomic approach, both to ensure that our plastid amplification did not stem from non-dinoflagellate environmental contaminants (i.e.; diatoms, haptophytes or other algae that share the same habitat as P. lebouriae), and to test whether the plastids are functionally integrated into the cell (ie; if the nucleus expresses plastidtargeted genes) rather than being retained as kleptoplastids. Previous researchers have established the effectiveness of SLPCR for amplifying dinoflagellate transcripts from a large volume of wild-caught plankton [14] or coral tissue [15], and this is the first study to apply SLPCR at the scale of single cells (Additional file 1)
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