DOI: 10.5061/dryad.hqbzkh1pj
title: Data from: Phylogenomic position of eupelagonemids, abundant and diverse deep-ocean heterotrophs

description: Eupelagonemids, formerly known as Deep Sea Pelagic Diplonemids I (DSPD I),
 are among the most abundant and diverse heterotrophic protists in the deep
 ocean, but little else is known about their ecology, evolution, or biology
 in general. Originally recognized solely as a large clade of environmental
 ribosomal subunit RNA gene sequences (SSU rRNA), branching with a smaller
 sister group DSPD II, they were postulated to be diplonemids, a
 poorly-studied branch of Euglenozoa. Although new diplonemids have been
 cultivated and studied in depth in recent years, the lack of cultured
 eupelagonemids has limited data to a handful of light micrographs, partial
 SSU rRNA gene sequences, a small number of genes from single amplified
 genomes (SAGs), and only a single formal described
 species, Eupelagonema oceanica. To determine exactly where this
 clade goes in the tree of eukaryotes and begin to address the overall
 absence of biological information about this apparently ecologically
 important group, we conducted single-cell transcriptomics from two
 eupelagonemid cells. A SSU rRNA gene phylogeny shows these two cells
 represent distinct subclades within eupelagonemids, each different from E.
 oceanica. Phylogenomic analysis based on a 125-gene matrix contrasts with
 the findings based on ecological survey data, and shows eupelagonemids
 branch sister to the diplonemid subgroup Hemistasiidae.
description: Two single cells of eupelagonemids were isolated from seawater
 that was collected with a Niskin bottle from 300m depth at station KSC10
 (Lat. 51.6505, Lon. -127.9516; Calvert Island, British Columbia, Canada)
 on July 3rd 2022. The cells were manually isolated from the concentrated
 seawater with a microcapillary and imaged on a Leica DMIL-LED inverted
 microscope equipped with a Sony alpha7S III camera at 630X
 magnification. The isolated cells were rinsed three
 times in drops of clean seawater and dispensed into 2µl of Smart-seq3
 lysis buffer. cDNA was generated using Smart-seq3 with 24 PCR-cycles for
 cDNA amplification, libraries were prepared with Illumina DNA library prep
 and sequenced on an Illumina NextSeq 500 platform with 2x150bp paired-end
 reads. Raw reads were read-corrected with rcorrector
 version 1.0.5, adapter- and quality-trimmed with trimmomatic version 0.39
 using parameters ILLUMINACLIP: 2:30:10 LEADING:5 SLIDINGWINDOW:5:16
 MINLEN:60, with the following sequences trimmed: Transposase1 (5’
 CTGTCTCTTATACACATCTCCGAGCCCACGAGAC 3’), Transposase2RC (5’
 CTGTCTCTTATACACATCTGACGCTGCCGACGA 3’), SmartSeq3_TSO_N8 (5’
 AGAGACAGATTGCGCAATGNNNNNNNNGGG 3’), SmartSeq3_oligo-dT (5’
 ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 3’). The trimmed reads
 were then assembled with rnaSPAdes version 3.15.5 with default parameters.
 Protein-coding sequences were predicted with transdecoder version
 5.5.0. SSU rRNA gene sequences were extraced from
 assemblies Eupelagonemid 7 and Eupelagonemid 8 with barrnap version 0.9.
 Diplonemid SSU rDNA sequences were then aligned with 237 other diplonemid,
 kinetoplastid, and symbiontid sequences. This dataset was aligned with
 MAFFT E-INS-I version 7.481, trimmed with BMGE version 1.12, and a Maximum
 likelihood tree was estimated with RAxML-NG version 1.1.0 under the
 GTR+GAMMA model and 1,000 non-parametric bootstraps. To
 generate a multigene dataset, predicted proteomes of both cells were used
 as input into phylofisher version 1.2.6. We also added nine diplonemid and
 three kinetoplastid taxa (<em>Hemistasia phaeocysticola, Artemidia
 motanka, Namystinia karyoxenos, Lacrimia lanifica, Rhynchopus humris, R.
 euleeides, Diplonema japonicum, Paradiplonema papillatum, Sulcionema
 specki, Papus ankaliazontas, Apiculatamorpha spiralis</em>, SAG
 EU19). After checking each of the 240 single gene trees for contaminant,
 paralogous, or otherwise aberrant sequences, we recovered 28.1% of sites
 for cell Eupelagonemid 7 (54 genes), and 17.9% of sites for cell
 Eupelagonemid 8 (36 genes) out of a total of 77,659 sites (240
 genes). A final concatenated dataset of 125 genes from
 33 euglenozoan and outgroup taxa (all Discoba) with a total 32,780 sites
 was used to run a ML-phylogeny using IQ-TREE2 version 2.2.0 under the
 LG+C60+F+G model with 1,000 ultrafast bootstraps (UFB). We additionally
 ran the same dataset under a posterior mean site frequency model (PMSF)
 with 200 non-parametric bootstraps, using the previous LG+C60+F+G tree as
 a guide tree.
description: # Phylogenomic position of eupelagonemids, abundant and diverse deep-ocean
 heterotrophs
 [https://doi.org/10.5061/dryad.hqbzkh1pj](https://doi.org/10.5061/dryad.hqbzkh1pj) These are supplemental data for the publication "Phylogenomic position of eupelagonemids, abundant and diverse deep-ocean heterotrophs", in The ISME Journal [https://doi.org/10.1093/ismejo/wrae040](https://doi.org/10.1093/ismejo/wrae040). It contains the assembled single-cell transcriptomes of cells "Eupelagonemid 7" and "Eupelagonemid 8", two cells of eupelagonemids isolated from the deep pelagic ocean that were placed in a multigene phylogeny, resolving their position among diplonemids. This dataset also contains the SSU rRNA gene alignment and subsequent tree data, and all multigene data used to construct the final phylogenomic tree. ## Description of the data and file structure There are 4 main parts to this dataset: 1. Raw micrograph images of Eupelagonemid 7 and Eupelagonemid 8. In TIFF-format (can be opened with most image viewers, including ImageJ). 2. Assemblies of Eupelagonemid 7 and Eupelagonemid 8. In nucleotide format (ending in .fasta; generated with rnaSPAdes) and predicted peptides (ending in .pep; predicted with TransDecoder). Both in fasta-format. 3. SSU rRNA gene alignment of eupelagonemids and other euglenozoans. Unmasked (aligned with MAFFT) and trimmed alignment files (trimmed with BMGE; both in fasta-format), and corresponding treefile (RAxML-ng with 1,000 bootstraps under GTR+Gamma model; NEWICK-format). Treefiles are openable with FigTree or other tree-viewing software. 4. Multigene data. This includes: * a compressed PhyloFisher database folder (ending in .tar.gz; tarball format), which includes all single-gene alignments used (in fasta-format) and metadata information. This database can be opened with PhyloFisher: [https://github.com/TheBrownLab/PhyloFisher](https://github.com/TheBrownLab/PhyloFisher) * '28-gene-phylogeny': multigene concatenation alignment of 33 taxa and 28 genes (matrix_mod_33T28G.fas; in fasta-format) and corresponding treefiles (matrix_mod_33T28G.fas.UFB.treefile; in NEWICK-format) derived from phylogenetic analysis, generated with IQTree2 under the LG+C60+F+G model with 1,000 Ultrafast Bootraps (UFB). Treefiles are openable with FigTree or other tree-viewing software. * '125-gene-phylogeny': multigene concatenation alignment of 33 taxa and 125 genes (matrix_mod_33T125G.fas; in fasta-format) and corresponding treefiles (in NEWICK-format) derived from phylogenetic analysis, generated with IQTree2 under the LG+C60+F+G model with 1,000 Ultrafast Bootraps (file 'matrix_mod_33T125G.fas.UFB.treefile'), and 200 non-parametric bootstraps with Posterior Mean Site Frequency model (file 'matrix_mod_33T125G.fas.PMSF.treefile'). Treefiles are openable with FigTree or other tree-viewing software. ## Sharing/Access information Find the raw Illumina read data under NCBI BioProject accession PRJNA1041876, and the SSU rDNA sequences of cells Eupelagonemid 7 and Eupelagonemid 8 under NCBI accessions OR831206 and OR831207.