DOI: 10.5061/dryad.gf1vhhn0b
title: Glissandra oviformis n. sp.: A novel predatory flagellate illuminates the character evolution within the eukaryotic clade CRuMs

description: Culturing protists offers a powerful approach to exploring eukaryotic
 diversity, especially for deep-branching lineages. In this study, we
 cultured and described a novel protist species, named Glissandra oviformis
 n sp., within the poorly studied and unclassified genus Glissandra. While
 an SSU rDNA gene phylogeny failed to resolve its phylogenetic placement in
 the eukaryotic tree, a phylogenomic analysis of 340 proteins indicated G.
 oviformis as a deep-branching member of the CRuMs clade. Prior to this
 study, this clade consisted of diverse heterotrophic amoeba and
 flagellates and lacks clear synapomorphies. Ultrastructural observations
 revealed that G. oviformis shares the characteristics with some CRuMs
 members, including the pellicle underlying the plasma membrane and an
 internal sleeve surrounding the central pair of the axoneme at the
 flagellar transitional region. Our findings suggest potential shared
 characteristics and synapomorphies for CRuMs and contribute to a deeper
 understanding of the character evolution within CRuMs.
description: In this study, we cultured and described a novel protist species,
 <em>Glissandra oviformis</em> n. sp., within the poorly
 studied and unclassified genus <em>Glissandra</em>. To
 determine the phylogenetic position of <em>G. oviformis</em>,
 we conducted phylogenetic analyses using an SSU rDNA dataset and a
 340-protein dataset. Furthermore, for the results based on the 340-protein
 dataset, we assessed the phylogenetic position of <em>G.
 oviformis</em> using fast-site removal analysis.
description: # *Glissandra oviformis* n. sp.: A novel predatory flagellate illuminates
 the character evolution within the eukaryotic clade CRuMs
 [https://doi.org/10.5061/dryad.gf1vhhn0b](https://doi.org/10.5061/dryad.gf1vhhn0b) ## Description of the data and file structure ### Files and variables **File: 340protein-dataset.zip**\ **Description:** Contains raw data of protein sequences used in the 340-protein dataset. **File: SRT312_SSU_phylogeny.phy**\ **Description:** Dataset for SSU rDNA phylogenetic analysis. **File: 132Taxa340Genes88397AAposi.fst**\ **Description:** Dataset for 340-protein phylogenetic analysis. **File: FastSiteRemoval.zip**\ **Description:** Contains datasets for fast-site removal analysis. #### SSU rDNA phylogenetic dataset The SSU rDNA sequence of *G. oviformis* was aligned with those of 190 phylogenetically diverse eukaryotes using MAFFT v7.520 (Katoh and Standley 2013). After removing ambiguously aligned positions using BMGE v1.12 (Criscuolo and Gribaldo 2010) with default settings, 1,137 nucleotide positions remained.  #### 340-protein dataset To elucidate the phylogenetic position of *G. oviformis*, we prepared a phylogenomic alignment by updating an existing one comprising 351 proteins (Harada et al. 2024). For each protein, we added homologous sequences retrieved by BLASTp (with an E-value cut-off of 10⁻30) from the newly generated *G. oviformis* protein sequences (see above), as well as sequences from the provorans, anaeroamoebids, and other organisms (a total of 50 species, including *G. oviformis* added, described in Table S1). Individual single-protein alignments were generated using MAFFT v7.520 with the L-INS-i algorithm, followed by manual correction and automated exclusion of ambiguously aligned positions using BMGE v1.12. Each single-protein alignment was subjected to a preliminary phylogenetic analysis using FASTTREE v2.1.11 (Price et al. 2010) under the LG + Γ model. The resulting approximately maximum-likelihood trees with SH-like local supports were examined to identify alignments with aberrant phylogenetic signals that strongly disagreed with well-established monophyletic groups in the eToL, including Opisthokonta, Amoebozoa, Alveolata, Stramenopiles, Rhizaria, Rhodophyta, Chloroplastida, Glaucophyta, Haptophyta, Cryptista, Jakobida, Euglenozoa, Heterolobosea, Diplomonadida, Parabasalia, and Malawimonadida. Eleven out of the 351 single-protein alignments were found to violate these criteria and were excluded from further phylogenomic analyses. The remaining 340 single-protein alignments were concatenated into a single phylogenomic alignment comprising 132 taxa with 88,397 unambiguously aligned amino acid positions.  #### Fast site removal We evaluated the impact of fast-evolving positions within the 340-protein alignment. Substitution rates for each position were calculated on the ML tree by IQ-TREE v2.2.2.6, and four sets of sub-alignments were created from the original alignment by sequentially removing the top 20%, 40%, 60%, and 80% fastest-evolving positions. The original alignment and four sub-alignments were individually subjected to the ultra-fast bootstrapping approximation under the LG + Γ + F + C20 model using IQ-TREE v 2.2.2.6.