DOI: 10.5061/dryad.1g1jwsv5v
title: Data from: Multiple plastid losses within photosynthetic stramenopiles revealed by comprehensive phylogenomics

description: Ochrophyta is a vast and morphologically diverse group of algae with
 complex plastids, including familiar taxa with fundamental ecological
 importance (diatoms or kelp), and a wealth of lesser-known and obscure
 organisms. The sheer diversity of ochrophytes poses a challenge for
 reconstructing their phylogeny, with major gaps in sampling and an
 unsettled placement of particular taxa yet to be tackled. We sequenced
 transcriptomes from 25 strategically selected representatives and used
 these data to build the most taxonomically comprehensive
 ochrophyte-centered phylogenomic supermatrix to date. We employed a
 combination of approaches to reconstruct and critically evaluate the
 relationships among ochrophytes. While generally congruent with previous
 analyses, the updated ochrophyte phylogenomic tree resolved the position
 of several taxa with previously uncertain placement, and supported a
 redefinition of the classes Picophagea and Synchromophyceae. Our results
 indicated that the heterotrophic plastid-lacking heliozoan Actinophrys sol
 is not a sister lineage of ochrophytes, as proposed recently, but rather
 phylogenetically nested among them, implying that it lacks a plastid due
 to loss. In addition, we found the heterotrophic ochrophyte Picophagus
 flagellatus to lack all hallmark plastid genes, yet to exhibit
 mitochondrial proteins that seem to be genetic footprints of a lost
 plastid organelle. We thus document, for the first time, plastid loss in
 two separate ochrophyte lineages. Furthermore, by exploring eDNA data we
 enrich the ochrophyte phylogenetic tree by identifying five novel
 uncultured class-level lineages. Altogether, our study provides a new
 framework for reconstructing trait evolution in ochrophytes and
 demonstrates that plastid loss is more common than previously thought.
description: For a majority of strains, cells grown in culture flasks were
 dislodged by scraping and collected by filtration over a 25mm 12 micron
 polycarbonate filter and flash frozen in liquid nitrogen. RNA was
 extracted using Machery Nagel plant RNA kit following manufacturers
 protocol except for cell lysis. This was carried out by bead beating for 5
 minutes using a mixture of 0.1 and 0.5mm zirconia/silica beads (BioSpec)
 in a BioSpec Mini-Beadbeater. Sequencing libraries were constructed using
 the New England BioLabs Next Ultra RNA prep kit and sequenced on the
 Illumina HiSeq (4000) in paired-end mode (2×150bp) at either University of
 Maryland sequencing center or Genewiz (New Jersey, USA). Adapters and
 low-quality regions were removed using Trimmomatic. Trimmed reads were
 assembled using rnaSPAdes v3.13. In the case of O. luteus K-0444 and P.
 flagellatus RCC22, total RNA was extracted with TRI Reagent® (TR 118)
 (Molecular Research Center, Inc., Cincinnati, USA), following standard
 procedures. Sequencing libraries were prepared by Macrogen Inc. (Seoul,
 South Korea) using TruSeq Stranded mRNA LT Sample Prep Kit and
 transcriptome sequencing was performed with the Illumina NovaSeq 6000
 platform in pair-end mode (2×151bp and 2×101bp for O. luteus and P.
 flagellatus, respectively). De novo transcriptome assemblies for O. luteus
 and P. flagellatus were obtained using Trinity v2.1.1. RNA of C.
 australica EC13 was extracted with a Purelink Plant RNA kit
 (ThermoFisher). Sequencing libraries were prepared and sequenced on
 Illumina HiSeq 2500 (Novogene, Hong Kong) and assembled with rnaSPAdes
 v3.13 Trinity 2.4.0. Transcripts were translated to protein sequences
 using TransDecoder (https://github.com/TransDecoder/). WinstonCleaner
 (https://github.com/kolecko007/WinstonCleaner) was used to identify and
 remove lowly expressed transcripts and cross-contamination from the
 assembled transcriptomes. Completeness of the genome or all transcriptomes
 generated as part of this study were assessed using BUSCO v. 5.5.0 with
 the Stramenopile gene set (Table S1). 
description: # Multiple plastid losses within photosynthetic stramenopiles revealed by
 comprehensive phylogenomics
 [https://doi.org/10.5061/dryad.1g1jwsv5v](https://doi.org/10.5061/dryad.1g1jwsv5v) **Description of the data and file structure** Uploaded to this repository contains concatenated fasta or phylip files used to generate the phylogenies shown in the manuscript, genome scaffolds,  and all transcriptomic assemblies, ortholog selection, paralog selection, and alignments ### Files and variables #### File: Ochro\_pseduo\_all.fas **Description:** Fasta file of the Ochro dataset. This fasta file was used to construct Figure 1 #### File: Stram\_all.phy **Description:** Phylip file of the stram dataset. This phylip file was used to construct  Figure S2 #### File: Picophagus\_genome\_scaffolds.fasta **Description:** Fasta file of Picophagus genome scaffold contigs #### File: 18S\_aligned.fasta **Description:** 18S rRNA alignment of 259 ochrophyte sequences.  #### File: 18S\_aligned\_trimmed.fasta **Description:** 18S rRNA of 259 ochrophyte sequences that has been trimmed by eye to a final length of 1,820 positions. This was the alignment that was used to construct the ML tree shown in figure S1 #### File: Act.fas **Description:** fasta file from the Act dataset. This fasta file was used to construct Figure S4 #### File: Stram\_PF\_release.tar.gz **Description:** This compressed file contains all assemblies, ortholog selections, paralog selections, and alignments. #### File: glutamyl.dataset\_trimmed.aln **Description:** Aligned and trimmed fasta file of mitochondrion-targeted glutamyl-tRNA synthetase. This file was used to create glutamylRS.tree.coloured.pdf #### File: DPD\_focused.dataset\_untrimmed\_unaligned.fasta **Description:**  untrimmed, unaligned fasta file of diaminopimelate decarboxylase that includes ochrophyte homologs #### File: DPD\_global.linsi\_untrimmed.aln **Description:** Aligned, but untrimmed broad scale phylogenetic analysis of diaminopimelate decarboxylase (DAPDC). #### File: DPD\_global.dataset.linsi\_trimmed.aln **Description:** Aligned, and trimmed broad scale phylogenetic analysis of diaminopimelate decarboxylase (DAPDC). This file was used to create figure S8. #### File: DPD\_global.dataset\_untrimmed\_unaligned.fas **Description:** Unaligned, and untrimmed broad scale phylogenetic analysis of diaminopimelate decarboxylase (DAPDC). #### File: serylRS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of mitochondrion-targeted seryl-tRNA synthetase #### File: FAS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of a putative FAS I identified in several ochrophytes, including *Picophagus flagellatus* #### File: glycylRS.dataset\_untrimmed\_unaligned.fasta **Description:** untrimmed and unaligned fasta file of glycine-tRNA synthetase #### File: valylRS.dataset\_trimmed.aln **Description**: Trimmed and aligned fasta file of organellar and cytoplasmic ValRSs. This file was used to create Figure 5 #### File: glycylRS.dataset.trimmed.aln **Description:** trimmed and aligned fasta file of glycine-tRNA synthetase. This file was used to generate glycylRS.tree.coloured.pdf #### File: threonylRS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of mitochondrion-targeted threonyl-tRNA synthetase #### File: threonylRS\_dataset.trimmed.aln **Description:** Trimmed and aligned fasta file of mitochondrion-targeted threonyl-tRNA synthetase. This file was used to create  threonylRS.tree.coloured.pdf #### File: FAS.dataset\_trimmed.aln **Description:** Trimmed and aligned fasta file of a putative FAS I. This file was used to create Fig4_FASI-publication.pdf #### File: valylRS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of organellar and cytoplasmic ValRSs #### File: DPD\_focused.dataset.linsi\_untrimmed.aln **Description:** untrimmed, but aligned fasta file of diaminopimelate decarboxylase that includes ochrophyte homologs #### File: glutamylRS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of glutamyl-tRNA synthetase #### File: aspartylRS.dataset.trimmed.aln **Description:**  Trimmed and aligned fasta file of organellar and cytoplasmic aspartyl-tRNA synthetases. This file was used to create aspartylRS.tree.coloured.pdf #### File: serylRS\_dataset.trimmed.aln **Description:** Trimmed and aligned fasta file of mitochondrion-targeted seryl-tRNA synthetase. This file was used to create serylRS.tree.coloured.pdf.  #### File: threonylRS.tree.coloured.pdf **Description:** ML tree of mitochondrion-targeted threonyl-tRNA synthetase #### File: leucylRS.dataset\_untrimmed\_unaligned.fasta **Description:** Untrimmed and unaligned fasta file of mitochondrion-targeted leucyl-tRNA synthetase #### File: glycylRS.tree.coloured.pdf **Description:** ML tree of glycine-tRNA synthetase #### File: leucylRS.dataset\_trimmed.aln **Description:** Trimmed and aligned fasta file of mitochondrion-targeted leucyl-tRNA synthetase. This file was used to create leucylRS.tree.coloured.pdf #### File: leucylRS.tree.coloured.pdf **Description:** ML tree of mitochondrion-targeted leucyl-tRNA synthetase #### File: glutamylRS.tree.coloured.pdf **Description:** ML tree of mitochondrion-trageted glutamyl-tRNA synthetase #### File: serylRS.tree.coloured.pdf **Description:** ML tree of mitochondrion-targeted seryl-tRNA synthetase.   #### File: AA\_comp\_plots.zip **Description:** compressed folder containing individual amino acid composition box and whisker plots  #### File: aspartylRS.dataset.untrimmed.unaligned.fasta **Description:** Untrimmed and unaligned organellar and cytoplasmic aspartyl-tRNA synthetases #### File: DPD\_focused.dataset.linsi\_trimmed.aln **Description:** trimmed, and aligned fasta file of diaminopimelate decarboxylase that includes ochrophyte homologs. This file was used to create Fig S9 #### File: Ochrophyte\_testing.R  **Description:** R file that contains code to recreate the fast site removal, heterotacheous, random gene resampling, ANOVA, and amino acid composition plots #### File: aspartylRS.tree.coloured.pdf **Description:** ML tree of organellar and cytoplasmic aspartyl-tRNA synthetases ## Code/software R  studio (Version 2024.04.0+735) was used to write the code that is in Ochrophyte_testing.R The files included in StramPFrelease.tar.gz can be used in Phylofisher 1.2. The ortholog and paralog selections included can be used in working_dataset_constructor.py  ## Access information Other publicly accessible locations of the data: * All raw reads have been deposited on NCBI Sequence read archive (SRA) Data was derived from the following sources: * Data was derived from culture collection banks. Monocultures were grown according to culture bank recommendations. Cells were harvested once they reached mid-exponential phase and flash frozen in liquid nitrogen until RNA or DNA extraction. RNA or DNA was extracted following manufactures protocols and sent for library prep and sequencing.