DOI: 10.5061/dryad.tht76hdx7
title: Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs

description: 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 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.
description: Single cells of phagotrophic euglenids were isolated into lysis
 buffer using hand-drawn glass pipettes and an inverted microscope.
 Subsequent cDNA generation followed Picelli et al. 2014 (SmartSeq2).
 Generated cDNA was then sequenced on either Illumina MiSeq (2x250bp PE) or
 HiSeq (2x150bp PE). Transcriptomes were assembled in a pipeline with
 rcorrector, trimmomatic, and rnaSPAdes. The phylogenomic analysis involved
 a pipeline published in Brown et al 2018 (also see Lax et al 2018), using
 20 phylogenomic marker genes that were widely covered by most of the taxa
 analysed. All phylogenetic trees were generated with IQ-tree using LG+C60
 or LG+C20 site-heterogeneous mixture models.
description: Please see included README.txt for information.