Phylogenomics of non-model ciliates based on transcriptomic analyses

Abstract

Ciliates are one of the oldest living eukaryotic unicellular organisms, widely distributed in the waters around the world. As a typical marine oligotrich ciliate, Strombidium sulcatum plays an important role in marine food webs and energy flow. Here we report the first deep sequencing and analyses of RNA-Seq data from Strombidium sulcatum. We generated 42,640 unigenes with an N50 of 1,451 bp after de novo assembly and removing rRNA, mitochondrial and bacteria contaminants. We employed SPOCS to detect orthologs from S. sulcatum and 17 other ciliates, and then carried out the phylogenomic reconstruction using 127 single copy orthologs. In phylogenomic analyses, concatenated trees have similar topological structures with concordance tree on the class level. Together with phylogenetic networks analysis, it aroused more doubts about the placement of Protocruzia, Mesodinium and Myrionecta. While epiplasmic proteins are known to be related to morphological characteristics, we found the potential relationship between gene expression of epiplasmic proteins and morphological characteristics. This work supports the use of high throughput approaches for phylogenomic analysis as well as correlation analysis between expression level of target genes and morphological characteristics.

Keywords

Strombidium / protozoa / transcriptome / non-model ciliate / phylogenomic analysis

References

[1] Adl SM, Simpson AG, Lane CE (2012) The revised classification of eukaryotes. J Eukaryot Microbiol59: 429−493

[2] Agatha S (2004) A cladistic approach for the classification of oligotrichid ciliates (Ciliophora: Spirotricha). Acta Protozool43: 201

[3] Allali K, Dolan J, Rassoulzadegan F (1994) Culture characteristics and orthophosphate excretion of a marine oligotrich ciliate, Strombidium sulcatum, fed heat-killed bacteria. Mar Ecol Prog Ser105: 159

[4] Ane C, Larget B, Baum DA, Smith SD, Rokas A (2007) Bayesian estimationof concordanceamong genetrees. Mol Biol Evol24: 1575

[5] Aubusson-Fleury A, Bricheux G, Damaj R (2013) Epiplasmins and epiplasm in Paramecium: the building of a submembraneous cytoskeleton. Protist164: 451−469

[6] Aury J-M, Jaillon O, Duret L (2006) Global trends of wholegenome duplications revealed by the ciliate Paramecium tetraurelia. Nature444: 171−178

[7] Baum DA (2007) Concordance trees, concordance factors, and the exploration of reticulate genealogy. Taxon56: 417−426

[8] Bernard C, Rassoulzadegan F (1990) Bacteria or microflagellates as a major food source for marine ciliates: possible implications for the microzooplankton. Mar Ecol Prog Ser64: 147−155

[9] Bryant D, Moulton V (2004) Neighbor-net: an agglomerative method for the construction of phylogenetic networks. Mol Biol Evol21: 255−265

[10] Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol17: 540−552

[11] Christaki U, Dolan JR, Pelegri S, Rassoulzadegan F (1998) Consumption of picoplankton-size particles by marine ciliates: effects of physiological state of the ciliate and particle quality. Limnol Oceanogr43: 458−464

[12] Corliss JO (1979) The ciliated protozoa. Characterization, classification and guide to the literature. Pergamon, Oxford

[13] Coyne RS, Hannick L, Shanmugam D (2011) Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control. Genome Biol12: R100

[14] Curtis DS, Phillips AR, Callister SJ, Conlan S, McCue LA (2013) SPOCS: software for predicting and visualizing orthology/paralogy relationships among genomes. Bioinformatics29: 2641−2642

[15] Dale T, Lynn DH (1998) Stomatogenesis of the ciliate genus Strombidinopsis with an improved description of S. spiniferum and S. acuminatum. J Eukaryot Microbiol45: 210−217

[16] Damaj R, Pomel S, Bricheux G, Coffe G, Viguès B, Ravet V, Bouchard P (2009) Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia. BMC Evol Biol9: 125

[17] Darriba D, Taboada GL, Doallo R, Posada D (2011) ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics27: 1164−1165

[18] Dolan JR, Sall N, Metcalfe A, Gasser B (2003) Effects of turbulence on the feeding and growth of a marine oligotrich ciliate. Aquat Microbial Ecol31: 183−192

[19] Dragesco J, Dragesco-Kernéis A, Fryd-Versavel G (1986) Ciliés libres de l’Afrique intertropicale: introduction à la connaissance et à l’étude des Ciliés. IRD Editions

[20] Eddy SR (2009) A new generation of homology search tools based on probabilistic inference. Genome Inform23: 205−211

[21] Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res32: 1792−1797

[22] Eisen JA, Coyne RS, Wu M (2006) Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol4: e286

[23] Fauré-Fremiet E (1962) Le genre Paranassula Kahl (Ciliata, Cyrtophorina). Cah Biol Mar3: 61−77

[24] Foissner W (1993) Colpodea (Ciliophora). In: Balogh scientific books. Gustav Fischer Verlag, Stuttgart, p 798

[25] Foissner W, Müller H, Agatha S (2007) A comparative fine structural and phylogenetic analysis of resting cysts in oligotrich and hypotrich Spirotrichea (Ciliophora). Eur J Protistol43: 295−314

[26] Fu L, Niu B, Zhu Z, Wu S, Li W (2012) CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics28: 3150−3152

[27] Gentekaki E, Kolisko M, Boscaro V (2014) Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages. Mol Phylogenet Evol78: 36−42

[28] Gordon A, Hannon G (2010) Fastx-toolkit. FASTQ/A short-reads preprocessing tools (unpublished).

[29] Gould SB, Tham W-H, Cowman AF, McFadden GI, Waller RF (2008) Alveolins, a new family of cortical proteins that define the protist infrakingdom Alveolata. Mol Biol Evol25: 1219−1230

[30] Grabherr MG, Haas BJ, Yassour M (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol29: 644−652

[31] Grant JR, Lahr DJ, Rey FE (2012) Gene discovery from a pilot study of the transcriptomes from three diverse microbial eukaryotes: Corallomyxa tenera, Chilodonella uncinata, and Subulatomonas tetraspora. Protist Genomics1: 3−18

[32] Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximumlikelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol59: 307−321

[33] Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser41: 95−98

[34] Hall BG (2011) Phylogenetic trees made easy. A how-to manual. Sinauer, Sunderland

[35] Honts JE, Williams NE (2003) Novel cytoskeletal proteins in the cortex of Tetrahymena. J Eukaryot Microbiol50: 9−14

[36] Huson DH (1998) SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics14: 68−73

[37] Huson DH, Bryant D (2006) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol23: 254−267

[38] Huson DH, Rupp R, Scornavacca C (2010) Phylogenetic networks. Concepts, algorithms and applications. Cambridge University Press, Cambridge

[39] Huttenlauch I, Stick R (2003) Occurrence of articulins and epiplasmins in protists. J Eukaryot Microbiol50: 15−18

[40] Huttenlauch I, Geisler N, Plessmann U, Peck RK, Weber K, Stick R (1995) Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization. J Cell Biol130: 1401−1412

[41] Huttenlauch I, Peck RK, Stick R (1998) Articulins and epiplasmins: two distinct classes of cytoskeletal proteins of the membrane skeleton in protists. J Cell Sci111: 3367−3378

[42] Inc S (2007) SPSS 16.0 for Windows. SPSS Inc, Chicago

[43] Iseli C, Jongeneel CV, Bucher P (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc Int Conf Intell Syst Mol Biol99: 138−148

[44] Kim S-J, Min G-S (2009) Taxonomic study of poorly-known marine pleurostomatid ciliates of Litonotus paracygnus and L. pictus (Ciliophora: Pleurostomatida) from Korea. Anim Syst Evol Divers25: 167−178

[45] Kim SY, Yang EJ, Gong J, Choi JK (2010a) Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with phylogenetic analyses based on small subunit rRNA gene sequences. J Eukaryot Microbiol57: 460−467

[46] Kim YO, Kim SY, Lee WJ, Choi JK (2010b) New Observations on the Choreotrich Ciliate Strombidinopsis acuminata Fauré-Fremiet 1924, and Comparison with Strombidinopsis jeokjo Jeong et al., 2004. J Eukaryot Microbiol57: 48−55

[47] Kloetzel JA, Baroin-Tourancheau A, Miceli C, Barcetta S, Farmar J, Banerjee D, Fleury-aubusson A (2003a) Plateins: a novel family of signal peptide-containing articulins in euplotid ciliates. J Eukaryot Microbiol50: 19−33

[48] Kloetzel JA, Baroin-Tourancheau A, Miceli C, Barchetta S, Farmar J, Banerjee D, Fleury-Aubusson A (2003b) Cytoskeletal proteins with N-terminal signal peptides: plateins in the ciliate Euplotes define a new family of articulins. J Cell Sc116: 1291−1303

[49] Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol10: R25

[50] Larget BR, Kotha SK, Dewey CN, Ane C (2010) BUCKy: gene tree/ species tree reconciliation with Bayesian concordance analysis. Bioinformatics26: 2910−2911

[51] Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform12: 323

[52] Li L, Thorsten S, Kyoon SM, Al-Rasheid Khaled AS, Al-Khedhairy Bdulaziz A, Song W (2010) Protocruzia, a highly ambiguous ciliate (Protozoa; Ciliophora): very likely an ancestral form for Heterotrichea, Colpodea or Spirotrichea? With reevaluation of its evolutionary position based on multigene analyses. Sci China Life Sci53: 131−138

[53] Li J, Liu W, Gao S, Warren A, Song W(2013) Multigene-based analyses of the phylogenetic evolution of oligotrich ciliates, with consideration of the internal transcribed spacer 2 secondary structure of three systematically ambiguous genera. Eukaryot Cell12: 430−437

[54] Lynn DH (2008) Ciliated protozoa: characterization, classification, and guide to the literature. Springer, Dordrecht

[55] Lynn DH, Montagnes DJS, Dale T, Gilron GL, Strom SL (1991) A reassessment of the genus Strombidinopsis (Ciliophora, Choreotrichida) with descriptions of four new planktonic species and remarks in its taxonomy and phylogeny. J Mar Biol Assoc UK71: 597−612

[56] Marrs JA, Bouck GB (1992) The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins. J Cell Biol118: 1465−1475

[57] McManus GB, Xu D, Costas BA, Katz LA (2010) Genetic identities of cryptic species in the Strombidium stylifer/apolatum/oculatum cluster, including a description of Strombidium rassoulzadegani n. sp. J Eukaryot Microbiol57: 369−378

[58] Modeo L, Petroni G, Rosati G, Montagnes DJ (2003) A multidisciplinary approach to describe protists: redescriptions of Novistrombidium testaceum Anigstein 1914 and Strombidium inclinatum Montagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia). J Eukaryot Microbiol50: 175−189

[59] Montagnes DJ, Taylor F, Lynn D (1990) Strombidium inclinatum n. sp. and a reassessment of Strombidium sulcatum Claparède and Lachmann (Ciliophora). J Protozool37: 318−323

[60] Morrison DA (2011) An Introduction to phylogenetic networks. RJR Productions, Uppsala

[61] Nahon P, Coffe G, Guyader H, Darmanaden-Delorme J, Jeanmaire-Wolf R, Clerot J-C, Adoutte A (1993) Identification of the epiplasmins, a new set of cortical proteins of the membrane cytoskeleton in Paramecium. J Cell Sci104: 975−990

[62] Philippe H, Snell EA, Bapteste E, Lopez P, Holland PW, Casane D (2004) Phylogenomics of eukaryotes: impact of missing data on large alignments. Mol Biol Evol21: 1740−1752

[63] Pomel S, Diogon M, Bouchard P, Pradel L, Ravet V, Coffe G, Viguès B (2006) The membrane skeleton in Paramecium: molecular characterization of a novel epiplasmin family and preliminary GFP expression results. Protist157: 61−75

[64] Qi J, Zhao F (2011) inGAP-sv: A novel scheme to identify and visualize structural variation from paired end mapping data. Nucleic Acids Res39: W567−W575

[65] Qi J, Zhao F, Buboltz A, Schuster SC (2010) inGAP: An integrated nextgeneration genome analysis pipeline. Bioinformatics26: 127−129

[66] Resources NCfG (2014) Marine Microbial Eukaryote Transcriptome Sequencing Project

[67] Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics19: 1572−1574

[68] Song W, Wilbert N (1997) Morphological investigations on some free living ciliates (Protozoa, Ciliophora) from China Sea with description of a new hypotrichous genus Hemigastrostyla nov. gen. Archiv für Protistenkunde148: 413−444

[69] Song W, Wang M, Warren A (2000) Redescriptions of three marine ciliates, Strombidium elegans Florentin, 1901, Strombidium sulcatum Claparède & Lachmann, 1859 and Heterostrombidium paracalkinsi Lei, Xu & Song, 1999 (Ciliophora, Oligotrichida). Eur J Protistol36: 327−342

[70] Swart EC, Nowacki M, Shum J (2012) The Oxytricha trifallax mitochondrial genome. Genome Biol Evol4: 136−154

[71] Swart EC, Bracht JR, Magrini V (2013) The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol11: e1001473

[72] Tamar H (1992) Four marine species of Mesodinium (Ciliophora: Mesodiniidae) II. Mesodinium pulex Clap. & Lachm., 1858. Archiv für Protistenkunde141: 284−303

[73] Wiadnyana NN, Rassoulzadegan F (1989) Selective feeding of Acartia clausi and Centropages typicus on microzooplankton. Mar Ecol Prog Ser53: 37−45

[74] Williams NE (2004) The epiplasm gene EPC1 influences cell shape and cortical pattern in Tetrahymena thermophila. J Eukaryot Microbiol51: 201−206

[75] Xiong J, Lu X, Zhou Z (2012) Transcriptome analysis of the model protozoan, Tetrahymena thermophila, using deep RNA sequencing. PLoS One7: e30630

[76] Yang Y, Smith SA (2013) Optimizing de novo assembly of short-read RNA-seq data for phylogenomics. BMC Genomics14: 328

RIGHTS & PERMISSIONS

2015 Higher Education Press and Springer-Verlag Berlin Heidelberg