DOI: 10.5061/dryad.34tmpg4qb
title: Molecular phylogenetics of sessile Dolium sedentarium, a petalomonad euglenid

description: The euglenids are a species-rich group of flagellates with varying modes
 of nutrition that can be found in diverse habitats. Phagotrophic members
 of this group gave rise to phototrophs and hold the key to understanding
 the evolution of euglenids as a whole, including the evolution of complex
 morphological characters like the euglenid pellicle. Yet to understand the
 evolution of these characters, a comprehensive sampling of molecular data
 is needed to correlate morphological and molecular data and to estimate a
 basic phylogenetic backbone of the group. While the availability of SSU
 rDNA and, more recently, multigene data from phagotrophic euglenids has
 improved, several ‘orphan’ taxa remain without any molecular data
 whatsoever. Dolium sedentarium is one such taxon: It is a rarely-observed
 phagotrophic euglenid that inhabits tropical benthic environments and is
 one of the few known sessile euglenids. Based on morphological characters,
 it has been thought of as part of the earliest branch of euglenids, the
 Petalomonadida. We report the first molecular sequencing data
 for Dolium using single-cell transcriptomics. Both SSU
 rDNA and multigene phylogenies confirm it as a solitary branch within
 Petalomonadida.
description: A single cell of <em>Dolium sedentarium</em> was
 collected from Spaanse Water mangrove in Curaçao in April 2022 (12.070621,
 -68.860269) via manual isolation with a micropipette, imaged on a Leica
 DLIM inverted microscope at 630x and a Sony a7RIII camera, and deposited
 in lysis buffer (see Picelli et al 2014). After 4 freeze-thaw cycles for
 lysis, a single-cell transcriptome was generated with the SmartSeq2
 protocol (Picelli et al 2014), and sequenced on an Illumina MiSeq
 (2x250bp), multiplexed with other libraries. Raw reads
 were read-corrected with <em>rcorrector</em> (version 1.0.4),
 adapter- and quality-trimmed with <em>trimmomatic</em>
 (version 0.39), and assembled with <em>rnaSPAdes</em> (version
 3.15.1). Coding regions were determined with
 <em>Transdecoder</em> (version 5.5.0).
 Additional assembly metrics shown in the multiQC report were
 generated with <em>QUAST</em> (version 5.0.2) and
 <em>BUSCO</em> (version 5.4.3). SSU-rDNA
 sequences from the assembly were extracted with
 <em>barrnap</em> (version 0.9), and those sequences were
 blasted against GenBank's NCBI nr/nt to identify the euglenid SSU
 rDNA. This sequence was then appended to an existing dataset (Lax et al
 2023), aligned with <em>MAFFT</em> <em>E-INS-I
 </em>(version 7.481), and trimmed with <em>trimAl</em>
 (version 1.2rev59, -gt 0.5 -st 0.001). A phylogeny was estimated with
 RAxML-NG under model GTR+GAMMA with 1000 non-parametric bootstraps
 (version 1.1.0). The phylogenomic dataset was based on
 a 19-gene dataset (Lax et al 2023), and homologs were extracted as
 described previously (Lax et al 2023). All 19 genes had 2 rounds of
 single-gene tree checking to exclude paraloguous and contaminant
 sequences. After concatenation of all 19 protein alignments, 9 orthologs
 from <em>Dolium sedentarium</em> were retained. Two analyses
 were run on this base 19-gene, 63-taxon dataset: 1)
 CAT_63S19F_UFB.treefile: IQ-TREE (version 2.2.0) under LG+C60+F+G and
 1,000 Ultrafast bootstraps 2) CAT_63S19F_PMSF.treefile:
 IQ-TREE (version 2.2.0) under LG+C60+F+G+PMSF and 500 non-parametric
 bootstraps (using the tree from 1 as a guide-tree)
description: Alignment-files in .fasta format can be opened with any text
 editor (e.g. SublimeText) or alignment viewers 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 .treefile or .tre) can be
 opened with a tree viewer like FigTree, Archaeopteryx, or iTOL
 EMBL. The multiQC-report (ending in .html) can be
 opened with any browser.