Morphological Identification and Single-Cell Genomics of Marine Diplonemids

Recent global surveys of marine biodiversity have revealed that a group of organisms known as “marine diplonemids” constitutes one of the most abundant and diverse planktonic lineages 1. Though discovered over a decade ago 2, 3, their potential importance was unrecognized, and our knowledge remains restricted to a single gene amplified from environmental DNA, the 18S rRNA gene (small subunit SSU). Here, we use single-cell genomics (SCG) and microscopy to characterize ten marine diplonemids, isolated from a range of depths in the eastern North Pacific Ocean. Phylogenetic analysis confirms that the isolates reflect the entire range of marine diplonemid diversity, and comparisons to environmental SSU surveys show that sequences from the isolates range from rare to superabundant, including the single most common marine diplonemid known. SCG generated a total of ∼915 Mbp of assembled sequence across all ten cells and ∼4,000 protein-coding genes with homologs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, distributed across categories expected for heterotrophic protists. Models of highly conserved genes indicate a high density of non-canonical introns, lacking conventional GT-AG splice sites. Mapping metagenomic datasets 4 to SCG assemblies reveals virtually no overlap, suggesting that nuclear genomic diversity is too great for representative SCG data to provide meaningful phylogenetic context to metagenomic datasets. This work provides an entry point to the future identification, isolation, and cultivation of these elusive yet ecologically important cells. The high density of nonconventional introns, however, also portends difficulty in generating accurate gene models and highlights the need for the establishment of stable cultures and transcriptomic analyses. © 2016 Elsevier Lt

Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α

BACKGROUND: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa-in particular determining the root-is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. PRINCIPAL FINDINGS: Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. CONCLUSIONS/SIGNIFICANCE: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids

A Revised Taxonomy of Diplonemids Including the Eupelagonemidae n. fam. and a Type Species, Eupelagonema oceanica n. gen. & sp.

Recent surveys of marine microbial diversity have identified a previously unrecognized lineage of diplonemid protists as being among the most diverse heterotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. Recently, we documented morphology and molecular sequences from several DSPDs, one of which is particularly widespread and abundant in environmental sequence data. To simplify the communication of future work on this important group, here we formally propose to erect the family Eupelagonemidae to encompass this clade, as well as a formal genus and species description for the apparently most abundant phylotype, Eupelagonema oceanica, for which morphological information and single-cell amplified genome data are currently available. © 2018 International Society of Protistologist

Data from: Morphological identification and single-cell genomics of marine diplonemids

Recent global surveys of marine biodiversity have revealed that a group of organisms known as “marine diplonemids” constitutes one of the most abundant and diverse planktonic lineages [1]. Though discovered over a decade ago [2 and 3], their potential importance was unrecognized, and our knowledge remains restricted to a single gene amplified from environmental DNA, the 18S rRNA gene (small subunit [SSU]). Here, we use single-cell genomics (SCG) and microscopy to characterize ten marine diplonemids, isolated from a range of depths in the eastern North Pacific Ocean. Phylogenetic analysis confirms that the isolates reflect the entire range of marine diplonemid diversity, and comparisons to environmental SSU surveys show that sequences from the isolates range from rare to superabundant, including the single most common marine diplonemid known. SCG generated a total of ∼915 Mbp of assembled sequence across all ten cells and ∼4,000 protein-coding genes with homologs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, distributed across categories expected for heterotrophic protists. Models of highly conserved genes indicate a high density of non-canonical introns, lacking conventional GT-AG splice sites. Mapping metagenomic datasets [4] to SCG assemblies reveals virtually no overlap, suggesting that nuclear genomic diversity is too great for representative SCG data to provide meaningful phylogenetic context to metagenomic datasets. This work provides an entry point to the future identification, isolation, and cultivation of these elusive yet ecologically important cells. The high density of nonconventional introns, however, also portends difficulty in generating accurate gene models and highlights the need for the establishment of stable cultures and transcriptomic analyses

Single cell genomics reveals plastid-lacking Picozoa are close relatives of red algae

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids