Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs

<b>Abstract</b><br/><p>Euglenids are a well-known group of single-celled eukaryotes, with phototrophic, osmotrophic and phagotrophic members. Phagotrophs represent most of the phylogenetic diversity of euglenids, and gave rise to the phototrophs and osmotrophs, but their evolutionary relationships are poorly understood. Symbiontids, in contrast, are anaerobes that are alternatively inferred to be derived euglenids, or a separate euglenozoan group. Most phylogenetic studies of euglenids have examined the SSU rDNA gene only, which is often highly divergent. Also, many phagotrophic euglenids (and symbiontids) are uncultured, restricting collection of other molecular data. We generated transcriptome data for 28 taxa, mostly using a single-cell approach, and conducted the first multigene phylogenetic analyses of euglenids to include phagotrophs and symbiontids. Euglenids are recovered as monophyletic, with symbiontids forming an independent branch within Euglenozoa. Spirocuta, the clade of flexible euglenids that contains both the phototrophs (Euglenophyceae) and osmotrophs (Aphagea), is robustly resolved, with the ploeotid <em>Olkasia</em> as its sister group, forming the new taxon Olkaspira. Ploeotids are paraphyletic, although Ploeotiidae (represented by <em>Ploeotia</em> spp.), <em>Lentomonas</em>, and <em>Keelungia</em> form a robust clade (new taxon Alistosa). Petalomonadida branches robustly as sister to other euglenids in outgroup-rooted analyses. Within Spirocuta, Euglenophyceae is a robust clade that includes <em>Rapaza</em>, and Anisonemia is a well-supported monophyletic group containing Anisonemidae (<em>Anisonema</em> and <em>Dinema</em> spp.), ‘<em>Heteronema</em> II’ (represented by <em>H. vittatum</em>), and a clade of <em>Neometanema</em> plus Aphagea. Among ‘peranemid’ phagotrophs, <em>Chasmostoma</em> branches with included <em>Urceolus</em>, and <em>Peranema</em> with the undescribed ‘<em>Jenningsia</em> II’, while other relationships are weakly supported and consequently the closest sister group to Euglenophyceae remains unresolved. Our results are inconsistent with recent inferences that <em>Entosiphon</em> is the evolutionarily pivotal sister either to other euglenids, or to Spirocuta. At least three transitions between posterior and anterior flagellar gliding occurred in euglenids, with the phylogenetic positions and directions of those transitions remaining ambiguous.</p>