DOI: 10.5061/dryad.w3r2280vv
title: Phylogenomics of novel ploeotid taxa contribute to the backbone of the euglenid tree

description: Euglenids are a diverse group of flagellates that inhabit most
 environments and exhibit many different nutritional modes. The most
 prominent euglenids are phototrophs, but phagotrophs constitute the
 majority of phylogenetic diversity of euglenids. They are pivotal to our
 understanding of euglenid evolution, yet we are only starting to
 understand relationships amongst phagotrophs, with the backbone of the
 tree being the most elusive. Ploeotids make up most of this backbone
 diversity—yet despite their morphological similarities, SSU rDNA analyses
 and multigene analyses show they are non-monophyletic. As more ploeotid
 diversity is sampled, known taxa have coalesced into some subgroups (e.g.
 Alistosa), but the relationships between these are not always supported
 and some taxa remain unsampled for multigene phylogenetics. Here, we used
 light microscopy and single-cell transcriptomics to characterize five
 ploeotid euglenids and place them into a multigene phylogenetic framework.
 Our analyses place Decastava in Alistosa;
 while Hemiolia branches with Liburna,
 establishing the novel clade Karavia. We describe Hemiolia limna,
 a freshwater-dwelling species in an otherwise marine clade. Intriguingly,
 two undescribed ploeotids are found to occupy pivotal positions in the
 tree: Chelandium granulatum nov. gen. nov. sp. branches
 as sister to Olkasia, and Gaulosia striata nov.
 gen. nov. sp. remains an orphan taxon.
description: Single cells of <em>Chelandium granulatum</em>,
 <em>Gaulosia striata</em>, <em>Hemiolia
 limna</em>, <em>Hemiolia trepidum</em>, and
 <em>Decastava</em> sp. were collected via manual single-cell
 isolation, imaged, and deposited in lysis buffer (see Picelli et al 2014).
 After 3-5 freeze-thaw cycles to lyse the cells, single-cell transcriptomes
 were generated with the SmartSeq2 protocol (Picelli et al 2014), and
 sequenced on Illumina NextSeq (2x150bp) or Illumina MiSeq
 (2x250bp). Raw reads were read-corrected with
 rcorrector (version 1.0.4), adapter- and quality-trimmed with trimmomatic
 (version 0.39), and assembled with rnaSPAdes (version 3.14.1). Coding
 regions were determined with Transdecoder (version 5.5.0).
 Additional assembly metrics shown in the multiQC report were
 generated with QUAST (version 5.2.0) and BUSCO (version 5.4.3).
 SSU-rDNA sequences from assemblies were extracted with barrnap
 (version 0.9), appended to an existing dataset (Lax & Simpson
 2020), aligned with MAFFT E-INS-I (version 7.475), and trimmed with trimAl
 (version 1.2rev59). A phylogeny was estimated with RAxML-NG under model
 GTR+GAMMA with 1000 non-parametric bootstraps (version 1.1.0), and MrBayes
 (version 3.2.7a on CIPRES webserver) under model GTR+GAMMA with 4 parallel
 chains under default heating parameters, with 50,000,000 generations each.
 Sampling was done every 10,000 trees, with the first 25% discarded as
 burn-in. The phylogenomic dataset was based on a
 20-gene dataset (Lax et al 2021), and homologs were extracted as described
 previously (Lax et al 2021). All 19 final genes had several rounds of
 single-gene tree checking to exclude paraloguous and contaminant
 sequences. After concatenation of all 19 protein alignments, several
 analyses were run on this base dataset:
 1) CAT_62S19F.fasta.UFB-C60.treefile: IQ-TREE (version 2.2.0)
 under LG+C60+F+G and 1,000 Ultrafast bootstraps
 2) CAT_62S19F.fasta.PMSF-C60.treefile: IQ-TREE (version 2.2.0)
 under LG+C60+F+G+PMSF and 500 non-parametric bootstraps
 3) CAT_60S19F.fasta.NoRogue-UFB-C60.treefile: IQ-TREE (version
 2.2.0) under LG+C60+F+G and 1,000 Ultrafast bootstraps, with
 'rogue' taxa <em>Dinema litorale</em> UB26 and SAG
 D1 removed
 4) Phylobayes_CAT-GTR_7500-10-30000_bpcomp.con.tre:
 PhyloBayes-MPI (version 1.8) under CAT+GTR with 4 cold chains, running for
 30,000 cycles each with a burn-in of 25%, sampled every 10 trees
 5) FSR (fast-site removal) analysis: Removed fast-evolving sites
 in 5% increments, with each resulting alignment having a phylogeny
 estimated in IQ-TREE (version 2.2.0) under LG+C20+F+G and 1,000 Ultrafast
 bootstraps
description: Alignment-files (.fasta, .bmge, .linsi, .new) can be opened with
 any text editor (e.g. SublimeText) or alignment viewer like AliView or
 SeaView. Assemblies (.fasta and .pep) files can be
 opened with any text editor like SublimeText.
 Tree-files in NEWICK-format (ending in .tre) can be opened with a
 treeviewer like FigTree, Archaeopteryx, or iTOL EMBL.
 The multiQC-report (ending in .html) can be opened with any
 browser. Images in JPEG-format can be opened with any
 image viewer.