Journal of Eukaryotic Microbiology ISSN 1066-5234 SHORT COMMUNICATION A Revised Taxonomy of Diplonemids Including the Eupelagonemidae n. fam. and a Type Species, Eupelagonema oceanica n. gen. & sp. Noriko Okamotoa , Ryan M.R. Gawryluka,1, Javier del Campoa,Jurgen€ F.H. Strasserta, Julius Lukesb, Thomas A. Richardsc, Alexandra Z. Wordend, Alyson E. Santoroe & Patrick J. Keelinga a Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Boulevard, British Columbia, Canada b Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Faculty of Sciences, University of South Bohemia, Branisovsk a 31, 370 05 Cesk e Budejovice (Budweis), Czech Republic c Biosciences, University of Exeter, Geoffrey Pope Building, Exeter EX4 4QD, United Kingdom d Monterey Bay Aquarium Research Institute, Moss Landing, California 95039, USA e Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 93106, USA Keywords ABSTRACT Deep-sea pelagic diplonemids; euglenozoa; heterotrophic flagellate; kinetoplastids; Recent surveys of marine microbial diversity have identified a previously unrec- marine diplonemids; single-cell amplified ognized lineage of diplonemid protists as being among the most diverse het- genome. erotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally Correspondence known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. N. Okamoto and P.J. Keeling, Department Recently, we documented morphology and molecular sequences from several of Botany, University of British Columbia, DSPDs, one of which is particularly widespread and abundant in environmental 3529-6270 University Boulevard, Boulevard, sequence data. To simplify the communication of future work on this impor- British Columbia, Canada tant group, here we formally propose to erect the family Eupelagonemidae to Telephone number: +1 (604) 822-2845; encompass this clade, as well as a formal genus and species description for Fax number: +1 (604) 822-6089; the apparently most abundant phylotype, Eupelagonema oceanica, for which e-mails: [email protected]; morphological information and single-cell amplified genome data are currently [email protected] available. Received: 9 November 2017; revised 16 June 2018; accepted July 12, 2018. doi:10.1111/jeu.12679 HETEROTROPHIC flagellates remain one of the most Diplonemids are one example of such heterotrophic pro- poorly studied fractions of microbial diversity, consistently tists with tremendous diversity that had slipped under our and substantially less well-studied than their parasitic or radar. Diplonemids are group of heterotrophic flagellates photosynthetic cousins. They tend to be hard to culture in that are sister to the kinetoplastids within the Euglenozoa. the laboratory and often possess relatively few morpholog- They have been known for a little over a century, but until ical characters that would allow easy identification. As cul- recently, only three genera of diplonemids had been for- turing and morphological identification were the twin mally described, namely, Diplonema Griessmann (1913), pillars of traditional protistology up to the late twentieth Rhynchopus Skuja (1948), and Hemistasia Griessmann century, the challenges in both areas have left the diver- (1913) (Cavalier-Smith 2016; Yabuki and Tame 2015). A sity of heterotrophic flagellates “a neglected majority” fourth proposed genus, Isonema, is generally considered a (Caron et al. 2017). One result of this is that molecular junior synonym of Diplonema (Triemer and Ott 1990). In surveys of protist diversity have revealed a great deal of 2018, three new diplonemid genera were reported previously unsuspected diversity in clades most likely to (Lacrimia, Sulcionema, and Flectonema) (Tashyreva et al. be heterotrophic flagellates (de Vargas et al. 2015). 2018a), and novel morphological and behavioral features, © 2018 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 0, 1–6 1 Revised Taxonomy of Diplonemids Including Eupelagonemidae Okamoto et al. as well as endosymbionts, were described for the genus “unofficial” names or acronyms for the same lineage. To Diplonema (Tashyreva et al. 2018b), demonstrating the circumvent this confusion as early as possible, we here under-explored diversity of this group. formally erect the family Eupelagonemidae n. fam. to These genera were mostly collected from marine envi- encompass the abundant and diverse “DSPD I” lineage ronments: Diplonema and Rhynchopus are primarily ben- based on their phylogenetic coherence and distribution in thic, while Hemistasia is found among marine plankton in nature. We also formally describe the Cell 37 phylotype as coastal waters (Cavalier-Smith 2016; Elbrachter€ et al. the type species Eupelagonema oceanica n. gen. & sp. for 1996; Roy et al. 2007; Yabuki and Tame 2015). The newly the group, based on its unique molecular phylogenetic described genera are also marine, though their habitats position, in addition to 160.6 Mbp of genomic data com- are yet to be clarified (Tashyreva et al. 2018a). Some of prising 531 identifiable protein coding genes, as well as these diplonemids have been studied mostly due to their the morphological information currently available from this relationship to the more famous kinetoplastids, and uncultured taxon. because of their baroque mitochondrial genome architec- ture and posttranscriptional editing characteristics MATERIALS AND METHODS (Kiethega et al. 2013; Marande et al. 2005; Valach et al. 2016; Yabuki et al. 2016), as well as some curious meta- A single cell of Eupelagonema oceanica was collected bolic and molecular traits (Morales et al. 2016; Qian and from 100 m depth at 33°18.08 N, 29°24.03 on October 7, Keeling 2001). But overall, the group has not been exten- 2013 as previously described (Gawryluk et al. 2016). The sively described, with multiple undetermined phylotypes cell was photographed live using a Leica DM IL LED present in environmental molecular surveys (Tashyreva inverted microscope equipped with a Canon D5100 cam- et al. 2018a,b). era, then isolated into 10 ll of DNase-free water. The iso- Diplonemids were not found to be particularly common lated samples were immediately frozen at À80°C, and in early molecular surveys based on the 18S rRNA gene, kept frozen at or below À20°C. The same cell was subse- although one enigmatic sister group of other diplonemids, quently used for a single cell genomic survey, from which dubbed the ‘deep-sea pelagic diplonemids’ (Lara et al. the 18S rRNA genes and 160.6 Mbp of genomic sequence 2009; also referred to as DSPD I and DSPD II), was was obtained, as previously described (Gawryluk et al. observed (Lopez-Garc ıa et al. 2001, 2007; Scheckenbach 2016), and deposited in GenBank (18S RNA genes: et al. 2010). More recently, however, analyses based on #KY947154; genomic sequence: #SRX2014516). The 18S the Tara Oceans data (de Vargas et al. 2015) demon- gene sequence from E. oceanica was assembled from a strated that DSPDs are not restricted to the deep sea and single amplified genome (Gawryluk et al. 2016). Mapping are more common than previously thought (Flegontova 40 reads of 250 bp each over the contig, we detected et al. 2016; Lukes et al. 2015). They are present at various variable sites at 1.5% of alignment positions. Diplonemid depths ranging from surface water to deep oceans, with 18S rRNA genes were retrieved from NCBI and aligned the majority found in mesopelagic waters (200–1,000 m). with 18S rRNA genes from 10 diplonemid cells reported in They are also present in different geographic locations, Gawryluk et al. (2016) using MAFFT v.7.212 with the L- ranging from tropical to temperate to high latitude regions, INS-i iterative refinement method (Katoh and Standley as well as coastal to open ocean environments. In addition 2013). Alignments were trimmed automatically with trimAl to this wide distribution, they are one of the most abun- (Capella-Gutierrez et al. 2009), with -gt and -st equal to dant and diverse protist groups yet characterized based 0.3, and 0.001, respectively. Excessively short sequences only on 18S rRNA gene sequences (Flegontova et al. or sequences with long branches in a preliminary tree 2016; Lukes et al. 2015). were also removed. ML trees were reconstructed with Despite the diversity, ubiquity, and abundance of RAxML v.8.1.6 (Stamatakis 2014), under the GTR model DSPDs in amplicon data, direct information about the biol- of substitution rates, the gamma model of rate hetero- ogy of these organisms has been unavailable until a recent geneity, and an estimated proportion of invariable sites characterization of ten marine diplonemid cells that [GTRGAMMAI]. Bootstrap support values derived from included basic microscopy and single-cell amplified geno- 1000 replicates were mapped onto the highest likelihood mic (SAG) data (Gawryluk et al. 2016). Of the phylotypes ML tree generated from 100 independent heuristic characterized, “Cell 37” was found to be particularly highly searches. Since diplonemids are not ever known to be represented in Tara Oceans data; with more than photosynthetic and there is no evidence that any of the 6,000,000 mapped amplicon reads, this phylotype is more members of the DSPD lineage is photosynthetic, descrip- abundant in amplicon data than all ciliate phylotypes com- tions are according to the International Code of Zoological bined. Nomenclature. With the emerging