Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Dinoflagellates possess many cellular characteristics with unresolved evolutionary histories. These include nuclei with greatly expanded genomes and chromatin packaged using histone-like proteins and dinoflagellate-viral nucleoproteins instead of histones, highly reduced mitochondrial genomes with extensive RNA editing, a mix of photosynthetic and cryptic secondary plastids, and tertiary plastids. Resolving the evolutionary origin of these traits requires understanding their ancestral states and early intermediates. Several early-branching dinoflagellate lineages are good candidates for such reconstruction, however these cells tend to be delicate and environmentally sparse, complicating such analyses. Here, we employ transcriptome sequencing from manually isolated and microscopically documented cells to resolve the placement of two cells of one such genus, Abedinium, collected by Remotely Operated Vehicle (ROV) in deep waters off the coast of Monterey Bay, California, USA. One cell corresponds to the only described species, A. dasypus, while the second cell is distinct and formally described as Abedinium folium, sp. nov. Abedinium has classically been assigned to the early-branching dinoflagellate subgroup Noctilucales, which is weakly supported by phylogenetic analyses of small subunit ribosomal RNA (SSU rRNA), the single characterized gene from any member of the order. However, an analysis based on 221 proteins from the transcriptome places Abedinium as a distinct lineage, separate from and basal to Noctilucales and the rest of the core dinoflagellates. The transcriptome also contains evidence of a cryptic plastid functioning in the biosynthesis of isoprenoids, iron-sulfur clusters, and heme, a mitochondrial genome with all three expected protein-coding genes (cob, cox1, and cox3), and the presence of some but not all dinoflagellate-specific chromatin packaging proteins.

The calcium carbonate plates (coccoliths) surrounding most coccolithophorid cells are strikingly reminiscent of armor, and defense against predators has been hypothesized as a selective advantage provided by these mineral structures. Although microzooplankton are the main predators of small phytoplankton such as coccolithophores, few putative phytoplankton defenses have been tested against this group. In this study, predation by the heterotrophic dinoflagellate Amphidinium longum on three calcifying strains of Emiliania huxleyi was compared with predation on cells acid‐treated to remove coccoliths. As a control for acidification effects on other cell properties, we also compared predation on acid‐treated and untreated non‐calcifying (haploid) E. huxleyi strains. We found no systematic support for the defense hypothesis in this predator‐prey system. Acid‐treated cells experienced predation rates that were higher, the same as, and lower than rates on untreated cells, depending on E. huxleyi strain. Other cell properties showing no relationship to predation included size, dimethylsulfoniopropionate (DMSP) content, and DMSP release. For untreated cells, however, hydrogen peroxide (H2O2) concentration in the cell suspension was positively related to predation, suggesting that H2O2 acts as a signal promoting prey detection, or that H2O2 co‐varies with other (unmeasured) predation‐enhancing cell properties. A. longum uses two different prey capture and ingestion strategies, depending on prey type. This plasticity suggests co‐evolution of predation and defense strategies, facilitating a generalist feeding strategy compatible with the diversity of potential phytoplankton prey.

Dinoflagellates are a diverse protist group possessing many unique traits. These include (but are not limited to) expansive genomes packaged into permanently condensed chromosomes, photosynthetic or cryptic plastids acquired vertically or horizontally in serial endosymbioses, and a ruffle-like transverse flagellum attached along its length to the cell. When reconstructing character evolution, early branching lineages with unusual features that distinguish them from the rest of the group have proven useful for inferring ancestral states. The Noctilucales are one such lineage, possessing relaxed chromosomes in some life stages and a trailing, thread-like transverse flagellum. However, most of the cellular and molecular data for the entire group come from a single cultured species, Noctiluca scintillans, and because its phylogenetic position is unresolved it remains unclear if these traits are ancestral or derived. Here, we use single cell transcriptomics to characterize three diverse Noctilucales genera: Spatulodinium, Kofoidinium, and a new lineage, Fabadinium gen. nov. We also provide transcriptomes for undescribed species in Amphidinium and Abediniales, critical taxa for clarifying the phylogenetic position of Noctilucales. Phylogenomic analyses suggests that the Noctilucales are sister to Amphidinium rather than an independent branch outside the core dinoflagellates. This topology is consistent with observations of shared characteristics between some members of Noctilucales and Amphidinium and provides the most compelling evidence to date that the unusual traits within this group are derived rather than ancestral. We also confirm that Spatulodinium plastids are photosynthetic and of ancestral origin, and show that all non-photosynthetic Noctilucales retain plastid genes indicating a cryptic organelle.

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