Protist, Vol. 170, 125682, November 2019 http://www.elsevier.de/protis Published online date 5 September 2019 ORIGINAL PAPER Taxon-rich Multigene Phylogenetic Analyses Resolve the Phylogenetic Relationship Among Deep-branching Stramenopiles a,b c,d c,1 Rabindra Thakur , Takashi Shiratori , and Ken-ichiro Ishida a Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan b Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA 01003, USA c Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan d Marine Biodiversity and Environmental Assessment Research Center (BioEnv), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan Submitted August 20, 2018; Accepted August 28, 2019 Monitoring Editor: Hervé Philippe Stramenopiles are one of the major eukaryotic assemblages. This group comprises a wide range of species including photosynthetic unicellular and multicellular algae, fungus-like osmotrophic organ- isms and many free-living phagotrophic flagellates. However, the phylogeny of the Stramenopiles, especially relationships among deep-branching heterotrophs, has not yet been resolved because of a lack of adequate transcriptomic data for representative lineages. In this study, we performed multigene phylogenetic analyses of deep-branching Stramenopiles with improved taxon sampling. We sequenced transcriptomes of three deep-branching Stramenopiles: Incisomonas marina, Pseudophyl- lomitus vesiculosus and Platysulcus tardus. Phylogenetic analyses using 120 protein-coding genes and 56 taxa indicated that Pl. tardus is sister to all other Stramenopiles while Ps. vesiculosus is sister to MAST-4 and form a robust clade with the Labyrinthulea. The resolved phylogenetic relationships of deep-branching Stramenopiles provide insights into the ancestral traits of the Stramenopiles. © 2019 Elsevier GmbH. All rights reserved. Key words: Stramenopiles; evolution; phylogeny; transcriptome data; MAST. Introduction prised of unicellular algae and seaweeds, as well as many heterotrophic lineages, including fungus- Stramenopiles are among the largest eukaryotic like osmotrophic organisms, intestinal parasites clades, comprising a phototrophic lineage com- and diverse free-living phagotrophic flagellates (Cavalier-Smith and Chao 2006; Cavalier-Smith 1 and Scoble 2013; Moriya et al. 2000; Riisberg Corresponding author; fax +81 298 53 4533 e-mail [email protected] (K.-i. Ishida). et al. 2009; Shiratori et al. 2015). Stramenopiles are https://doi.org/10.1016/j.protis.2019.125682 1434-4610/© 2019 Elsevier GmbH. All rights reserved. 2 R. Thakur et al. currently separated into three phyla: The Ochro- Moreover, except for MAST-3 and 4, most of the phyta, Pseudofungi and Bigyra (Cavalier-Smith deep-branching MAST lineages lack data for multi- and Chao 2006; Cavalier-Smith and Scoble 2013; gene phylogenetic analyses. Leonard et al. 2018; Ruggiero et al. 2015). The To better understand the phylogeny and char- Ochrophyta include all phototrophic clades (e.g., acter evolution of Stramenopiles, we perform Eustigmatophyceae, Phaeophyceae, and Raphi- taxon-rich multigene phylogenetic analyses, includ- dophyceae) and the Pseudofungi include two ing MASTs and species of uncertain phylogenetic fungus-like taxa (Oomycetes, Hyphochytrea) plus position. We sequenced transcriptomes of a a clade of free-living flagellates (Bigyromonadea). recently described deep-branching stramenopile, Monophyly of the Ochrophyta and Pseudofungi is Platysulcus tardus, and two MASTs, Incisomonas robustly supported by morphological and ultrastruc- marina (MAST-3) and Pseudophyllomitus vesicu- tural similarities, as well as molecular phylogenetic losus (MAST-6) and extended the multigene analyses using small subunit ribosomal RNA (SSU phylogenetic analyses of the Stramenopiles with rRNA) gene (Cavalier-Smith and Chao 2006; Leipe the improved taxon sampling. Our new phylogeny et al. 1996). The non-monophyletic Bigyra include provides additional information to understand the the rest of the deep-branching heterotrophic Stra- character evolution of Stramenopiles. menopiles (e.g., the Bikosea, Labyrinthulea, and Opalinea); however, the monophyly and the rela- tionships among internal branches are unclear in Results the SSU rRNA gene phylogenies (Cavalier-Smith and Chao 2006; Cavalier-Smith and Scoble 2013; Dataset Construction Ruggiero et al. 2015). To resolve the phylogeny of deep-branching Stra- Stramenopiles also include approximately 18 menopiles, we sequenced transcriptomes of three deep-branching environmental lineages termed deep-branching lineages: Incisomonas marina MAST (MArine STramenopile), that are related (i.e. MAST-3), Pseudophyllomitus vesiculosus (i.e. to bigyran lineages (Massana et al. 2004, MAST-6), and Platysulcus tardus (Platysulcidae). 2009, 2014). Recent phylogenomic analyses using The filtered and assembled sequences yielded genomic and transcriptomic data have shed light 44,382; 116,491; and 31,779 transcript contigs on the ambiguous phylogeny of the Bigyra and from I. marina, Ps. vesiculosus, and Pl. tar- indicated that these lineages separate into two sub- dus, respectively. We used the transcriptomic and clades, the Opalozoa and the Sagenista (Burki genomic data of 47 Stramenopiles and 9 out- et al. 2016; Derelle et al. 2016; Noguchi et al. groups (Alveolates and Rhizaria) from the Marine 2016). The Opalozoa includes various free-living Microbial Eukaryotes Transcriptome Sequencing phagotrophic flagellates (Bikosea, Placididea, and Project (MMETSP) and GenBank. This dataset Nanomonadea) and intestinal parasites (Opali- includes representatives from nine classes of nata and Blastocystis). The Sagenista include Ochrophytes (Bacillariophyceae, Bolidophyceae, the Labyrinthulea, a clade of phagotrophic and Chrysophyceae, Dictyochophyceae, Eustigmato- osmotrophic protists with extracellular filaments, phyceae, Pelagophyceae, Phaeophyceae, Raphi- and an uncultured environmental lineage MAST-4, dophyceae, and Xanthophyceae) and six classes of whose cellular structure remains unknown. Despite heterotrophic Stramenopiles (Bikosea, Placididea, the power of large-scale phylogenomic analyses, Blastocystea, Labyrinthulea, Bigyromonadea, and the positions of the Opalozoa and Sagenista are Oomycetes). We generated a final dataset of 56 not consistent among published analyses, and the species in 120 gene alignments, containing 34,706 phylogenetic validity of the Bigyra remains uncer- amino acid positions, with missing characters and tain (Burki et al. 2016; Derelle et al. 2016; Noguchi genes of 16.6% and 12.07%, respectively (Sup- et al. 2016). plementary Material Tables S3–S5, Fig. S1). The Another remaining problem for the phylogeny of average missing characters and genes of three the Bigyra is that available genomic and transcrip- newly sequenced taxa were 13.22% and 7.2%, tomic data do not represent all the major lineages. respectively. Although novel deep-branching Stramenopiles that are likely closely related to, or included within, Phylogenetic Results of Stramenopiles the Bigyra have been described, some of them lack data for large-scale phylogenetic analysis to We performed Maximum-Likelihood (ML) analy- resolve their position (Cavalier-Smith and Scoble sis under the best-fitting model (LG + C60+F + G) 2013; Gómez et al. 2011; Shiratori et al. 2017). and Bayesian inference (BI) under the site- Taxon-rich Multigene Phylogenetic Analyses Resolve the Phylogenetic Relationship Among Deep-branching Stramenopiles 3 Aureococcus anophagefferens Pelagomonas subviridis Pelagomonas calceolata Pelagophyceae Aureoumbra lagunens Chrysocystis fragilis Rhizochromulina marina Diatomi Pseudopedinella elastica Dictyophyceae Florenciella sp. Dictyocha speculum sta Bolidomonas pacifica Bolidomonas sp. Bolidophyceae Och Pseudonitzschia sp. rophyta Phaeodactylum tricornutum Bacillariophyceae Thalassiosira oceanica Thalassiosira pseudonana Gyrista Mallomonas sp. Synurophyceae Dinobryon sp. Spumella elongata Chrysophyceae Chrysista Paraphysomonas bandaiensis Paraphysomonas vestita 71 Chattonella subsalsa Heterosigma akashiwo Raphidophycea Vauchaeria litorea Xanthophyceae Ectocarpus siliculosus 89/1.00 Phaeophyceae Nannochloropsis gaditana Eustigmatophyceae Aphanomyces invadans Saprolegnia parasitica Pseu Albugo sp. Oomycetes d Phytophthora sp. ofun 90/0.92 Phytophthora infestans 87/1.00 g Phytophthora parasitica i Developayella elegans Bigyromonadea Aplanochytrium sp. Aplanochytrium stocchinoi Labyrinthulea Aurantiochytrium limacinum Sagenista Schizochytrium aggregatum MAST-4 89/0.98 Eogyrea Pseudophyllomitus vesiculosus (MAST-6) 96/1.00 Bicosoicida sp. Bicoecida Cafeteria roenbergensis Anoecida Bikosia Opalozoa Cantina marsupialis 'Cafeteria' (Placidida) Placididea 96/1.00 Wobblina lunata Incisomonas marina (MAST-3) Nanomonadea Placidozoa 99/1.00 Blastocystis hominis Opalinata Blastocystis sp. Platysulcus tardus Platysulcea Cryptosporidium parvum Plasmodium falciparum Toxoplasma gondii ALVEOLATA Perkinsus marinus Paramecium tetraurelia Tetrahymena thermophila Bigelowiella natans Plasmodiophora brassicae RHIZARIA Reticulomyxa filosa Figure 1. The phylogenetic tree of Stramenopiles inferred from the dataset (56 taxa/120 genes). The topology is Maximum-likelihood (ML) tree under the best-fitting model (LG + C60+F + G) with both ML bootstrap percentage (BP) values and Bayesian posterior probabilities (PP). BP < 70% and PP < 0.92 are