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

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 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 Olkasia as its sister group, forming the new taxon Olkaspira. Ploeotids are paraphyletic, although Ploeotiidae (represented by Ploeotia spp.), Lentomonas, and Keelungia 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 Rapaza, and Anisonemia is a well-supported monophyletic group containing Anisonemidae (Anisonema and Dinema spp.), 'Heteronema II' (represented by H. vittatum), and a clade of Neometanema plus Aphagea. Among 'peranemid' phagotrophs, Chasmostoma branches with included Urceolus, and Peranema with the undescribed 'Jenningsia 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 Entosiphon 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.

Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages.

The Ciliophora is one of the most studied protist lineages because of its important ecological role in the microbial loop. While there is an abundance of molecular data for many ciliate groups, it is commonly limited to the 18S ribosomal RNA locus. There is a paucity of data when it comes to availability of protein-coding genes especially for taxa that do not belong to the class Oligohymenophorea. To address this gap, we have sequenced EST libraries for 11 ciliate species. A supermatrix was constructed for phylogenomic analysis based on 158 genes and 42,158 characters and included 16 ciliates, four dinoflagellates and nine apicomplexans. This is the first multigene-based analysis focusing on the phylum Ciliophora. Our analyses reveal two robust superclades within the Intramacronucleata; one composed of the classes Spirotrichea, Armophorea and Litostomatea (SAL) and another with Colpodea and Oligohymenophorea. Furthermore, we provide corroborative evidence for removing the ambiguous taxon Protocruzia from the class Spirotrichea and placing it as incertae sedis in the phylum Ciliophora.