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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., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Author(s): Faktorova, Drahomira; Nisbet, R Ellen R; Fernandez Robledo, Jose A; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E; Ares, Manuel; Areste, Cristina; Balestreri, Cecilia; Barbrook, Adrian C; Beardslee, Patrick; Bender, Sara; Booth, David S; Bouget, Francois-Yves; Bowler, Chris; Breglia, Susana A; Brownlee, Colin; Burger, Gertraud; Cerutti, Heriberto; Cesaroni, Rachele; Chiurillo, Miguel A; Clemente, Thomas; Coles, Duncan B; Collier, Jackie L; Cooney, Elizabeth C; Coyne, Kathryn; Docampo, Roberto; Dupont, Christopher L; Edgcomb, Virginia; Einarsson, Elin; Elustondo, Pia A; Federici, Fernan; Freire-Beneitez, Veronica; Freyria, Nastasia J; Fukuda, Kodai; Garcia, Paulo A; Girguis, Peter R; Gomaa, Fatma; Gornik, Sebastian G; Guo, Jian; Hampl, Vladimir; Hanawa, Yutaka; Haro-Contreras, Esteban R; Hehenberger, Elisabeth; Highfield, Andrea; Hirakawa, Yoshihisa; Hopes, Amanda; Howe, Christopher J; Hu, Ian; Ibanez, Jorge; Irwin, Nicholas AT; Ishii, Yuu; Janowicz, Natalia Ewa; Jones, Adam C; Kachale, Ambar; Fujimura-Kamada, Konomi; Kaur, Binnypreet; Kaye, Jonathan Z; Kazana, Eleanna; Keeling, Patrick J; King, Nicole; Klobutcher, Lawrence A; Lander, Noelia; Lassadi, Imen; Li, Zhuhong; Lin, Senjie; Lozano, Jean-Claude; Luan, Fulei; Maruyama, Shinichiro; Matute, Tamara; Miceli, Cristina; Minagawa, Jun; Moosburner, Mark; Najle, Sebastian R; Nanjappa, Deepak; Nimmo, Isabel C; Noble, Luke; Novak Vanclova, Anna MG; Nowacki, Mariusz; Nunez, Isaac; Pain, Arnab; Piersanti, Angela; Pucciarelli, Sandra; Pyrih, Jan; Rest, Joshua S | Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Euglenids comprise a distinct clade of flagellates with diverse modes of nutrition, including phagotrophy, osmotrophy and phototrophy. Much of the previous research on euglenids has focused on phototrophic species because of their ecological abundance and significance as indicators for the health of aquatic ecosystems. Although largely understudied, phagotrophic species probably represent the majority of euglenid diversity. Phagotrophic euglenids tend to be either bacterivorous or eukaryovorous and use an elaborate feeding apparatus for capturing prey cells. We characterized the ultrastructure and molecular phylogenetic position of Heteronema scaphurum, a eukaryovorous euglenid collected in freshwater. This species was equipped with a distinct cytoproct through which waste products were eliminated in the form of faecal pellets; a cytoproct has not been reported in any other member of the Euglenida. Heteronema scaphurum also had a novel predatory mode of feeding. The euglenid ensnared and corralled several green algal prey cells (i.e. Chlamydomonas) with hook-like flagella covered in mucous before engulfing the bundle of prey cells whole. Molecular phylogenetic analyses inferred from small subunit rDNA sequences placed this species with other eukaryovorous euglenids, which was consistent with ultrastructural features associated with the feeding apparatus, flagellar apparatus, extrusomes, and pellicle.

Molecular phylogenies of euglenids are usually based on ribosomal RNA genes that do not resolve the branching order among the deeper lineages. We addressed deep euglenid phylogeny using the cytosolic form of the heat-shock protein 90 gene (hsp90), which has already been employed with some success in other groups of euglenozoans and eukaryotes in general. Hsp90 sequences were generated from three taxa of euglenids representing different degrees of ultrastructural complexity, namely Petalomonas cantuscygni and wild isolates of Entosiphon sulcatum, and Peranema trichophorum. The hsp90 gene sequence of P. trichophorum contained three short introns (ranging from 27 to 31 bp), two of which had non-canonical borders GG-GG and GG-TG and two 10-bp inverted repeats, suggesting a structure similar to that of the non-canonical introns described in Euglena gracilis. Phylogenetic analyses confirmed a closer relationship between kinetoplastids and diplonemids than to euglenids, and supported previous views regarding the branching order among primarily bacteriovorous, primarily eukaryovorous, and photosynthetic euglenids. The position of P. cantuscygni within Euglenozoa, as well as the relative support for the nodes including it were strongly dependent on outgroup selection. The results were most consistent when the jakobid Reclinomonas americana was used as the outgroup. The most robust phylogenies place P. cantuscygni as the most basal branch within the euglenid clade. However, the presence of a kinetoplast-like mitochondrial inclusion in P. cantuscygni deviates from the currently accepted apomorphy-based definition of the kinetoplastid clade and highlights the necessity of detailed studies addressing the molecular nature of the euglenid and diplonemid mitochondrial genome.

A distinct subgroup of euglenozoans, referred to as the ‘Symbiontida,’ has been described from oxygen-depleted and sulfidic marine environments. By definition, all members of this group carry epibionts that are intimately associated with underlying mitochondrion-derived organelles beneath the surface of the hosts. We have used molecular phylogenetic and ultrastructural evidence to identify the rod-shaped epibionts of the two members of this group, Calkinsia aureus and B.bacati, hand-picked from the sediments of two separate oxygen-depleted, sulfidic environments. We identify their epibionts as closely related sulfur or sulfide-oxidizing members of the epsilon proteobacteria. The epsilon proteobacteria generally have a significant role in deep-sea habitats as primary colonizers, primary producers and/or in symbiotic associations. The epibionts likely fulfill a role in detoxifying the immediate surrounding environment for these two different hosts. The nearly identical rod-shaped epibionts on these two symbiontid hosts provides evidence for a co-evolutionary history between these two sets of partners. This hypothesis is supported by congruent tree topologies inferred from 18S and 16S rDNA from the hosts and bacterial epibionts, respectively. The eukaryotic hosts likely serve as a motile substrate that delivers the epibionts to the ideal locations with respect to the oxic/anoxic interface, whereby their growth rates can be maximized, perhaps also allowing the host to cultivate a food source. Because symbiontid isolates and additional small subunit rDNA gene sequences from this clade have now been recovered from many locations worldwide, the Symbiontida are likely more widespread and diverse than presently known.

Background Poorly understood but highly diverse microbial communities exist within anoxic and oxygen-depleted marine sediments. These communities often harbour single-celled eukaryotes that form symbiotic associations with different prokaryotes. During low tides in South-western British Columbia, Canada, vast areas of marine sand become exposed, forming tidal pools. Oxygen-depleted sediments within these pools are distinctively black at only 2-3 cm depth; these layers contain a rich variety of microorganisms, many of which are undescribed. We discovered and characterized a novel (uncultivated) lineage of heterotrophic euglenozoan within these environments using light microscopy, scanning and transmission electron microscopy, serial sectioning and ultrastructural reconstruction, and molecular phylogenetic analyses of small subunit rDNA sequences. Results Bihospites bacati n. gen. et sp. is a biflagellated microbial eukaryote that lives within low-oxygen intertidal sands and dies within a few hours of exposure to atmospheric oxygen. The cells are enveloped by two different prokaryotic episymbionts: (1) rod-shaped bacteria and (2) longitudinal strings of spherical bacteria, capable of ejecting an internal, tightly wound thread. Ultrastructural data showed that B. bacati possesses all of the euglenozoan synapomorphies. Moreover, phylogenetic analyses of SSU rDNA sequences demonstrated that B. bacati groups strongly with the Symbiontida: a newly established subclade within the Euglenozoa that includes Calkinsia aureus and other unidentified organisms living in low-oxygen sediments. B. bacati also possessed novel features, such as a compact C-shaped rod apparatus encircling the nucleus, a cytostomal funnel and a distinctive cell surface organization reminiscent of the pellicle strips in phagotrophic euglenids. Conclusions We characterized the ultrastructure and molecular phylogenetic position of B. bacati n. gen. et sp. Molecular phylogenetic analyses demonstrated that this species belongs to the Euglenozoa and currently branches as the earliest diverging member of the Symbiontida. This is concordant with ultrastructural features of B. bacati that are intermediate between C. aureus and phagotrophic euglenids, indicating that the most recent ancestor of the Symbiontida descended from phagotrophic euglenids. Additionally, the extrusive episymbionts in B. bacati are strikingly similar to so-called "epixenosomes", prokaryotes previously described in a ciliate species and identified as members of the Verrucomicrobia. These parallel symbioses increase the comparative context for understanding the origin(s) of extrusive organelles in eukaryotes and underscores how little we know about the symbiotic communities of marine benthic environments.

Euglenids comprise a group of single-celled eukaryotes with diverse modes of nutrition, including phagotrophy and photosynthesis. The level of morphological diversity present in this group provides an excellent system for demonstrating evolutionary transformations in morphological characters. This diversity also provides compelling evidence for major events in eukaryote evolution, such as the punctuated effects of secondary endosymbiosis and mutations in underlying developmental mechanisms. In this essay, we synthesize evidence for the origin, adaptive significance and diversification of the euglenid cytoskeleton, especially pellicle ultrastructure, pellicle surface patterns, pellicle strip number and the feeding apparatus. We also highlight holes in our knowledge that must be filled before we are able to confidently describe euglenid cell biology and infer the earliest stages in euglenid evolution. Nonetheless, by possessing combinations of characters resulting from adaptive change and morphostasis, euglenids have retained key pieces of evidence necessary for reconstructing the early evolution and diversification of eukaryotic life.