Euglenophytes are a familiar algal group with green alga-derived secondary plastids, but the knowledge of euglenophyte plastid function and evolution is still highly incomplete. With this in mind we sequenced and analysed the transcriptome of the non-photosynthetic species Euglena longa. The transcriptomic data confirmed the absence of genes for the photosynthetic machinery, but provided candidate plastid-localised proteins bearing N-terminal bipartite topogenic signals (BTSs) of the characteristic euglenophyte type. Further comparative analyses including transcriptome assemblies available for photosynthetic euglenophytes enabled us to unveil salient aspects of the basic euglenophyte plastid infrastructure, such as plastidial targeting of several proteins as C-terminal translational fusions with other BTS-bearing proteins or replacement of the conventional eubacteria-derived plastidial ribosomal protein L24 by homologs of archaeo-eukaryotic origin. Strikingly, no homologs of any key component of the TOC/TIC system and the plastid division apparatus are discernible in euglenophytes, and the machinery for intraplastidial protein targeting has been simplified by the loss of the cpSRP/cpFtsY system and the SEC2 translocon. Lastly, euglenophytes proved to encode a plastid-targeted homolog of the termination factor Rho horizontally acquired from a Lambdaproteobacteria-related donor. Our study thus further documents a substantial remodelling of the euglenophyte plastid compared to its green algal progenitor.

Department of Biology and Ecology Faculty of Natural Sciences Matej Bel University 974 01 Banská Bystrica Slovakia

Department of Biology Faculty of Natural Sciences University of ss Cyril and Methodius in Trnava 917 01 Trnava Slovakia

Department of Genetics Faculty of Natural Sciences Comenius University 842 15 Bratislava Slovakia

Department of Parasitology Faculty of Science Charles University BIOCEV Prague Czech Republic

Institute of Parasitology Biology Centre CAS 370 05 České Budějovice Czech Republic

Life Science Research Centre Department of Biology and Ecology and Institute of Environmental Technologies Faculty of Science University of Ostrava 701 00 Ostrava Czech Republic

University of South Bohemia Faculty of Science 370 05 České Budějovice Czech Republic

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Leander, B. S., Lax, G., Karnkowska, A. & Simpson, A. G. B. Euglenida in Handbook of the Protists (eds John M. Archibald et al.), 1–42 (Springer International Publishing, 2017).

Campbell DA, Thomas S, Sturm NR. Transcription in kinetoplastid protozoa: why be normal? Microbes Infect. 2003;5:1231–1240. doi: 10.1016/j.micinf.2003.09.005. PubMed DOI

Clayton CE. Gene expression in kinetoplastids. Curr Opin Microbiol. 2016;32:46–51. doi: 10.1016/j.mib.2016.04.018. PubMed DOI

Ebenezer, T. E. et al. Unlocking the biological potential of Euglena gracilis: evolution, cell biology and significance to parasitism. bioRxiv, 10.1101/228015, (2017).

Hoffmeister M, et al. Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions. J Biol Chem. 2004;279:22422–22429. doi: 10.1074/jbc.M400913200. PubMed DOI

Frantz C, Ebel C, Paulus F, Imbault P. Characterization of trans-splicing in Euglenoids. Curr Genet. 2000;37:349–355. doi: 10.1007/s002940000116. PubMed DOI

Liang XH, Haritan A, Uliel S, Michaeli S. Trans and cis splicing in trypanosomatids: mechanism, factors, and regulation. Eukaryot Cell. 2003;2:830–840. doi: 10.1128/EC.2.5.830-840.2003. PubMed DOI PMC

Ebenezer TE, Carrington M, Lebert M, Kelly S, Field MC. Euglena gracilis genome and transcriptome: Organelles, nuclear genome assembly strategies and initial features. Adv Exp Med Biol. 2017;979:125–140. doi: 10.1007/978-3-319-54910-1_7. PubMed DOI

O’Neill EC, et al. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry. Mol Biosyst. 2015;11:2808–2820. doi: 10.1039/C5MB00319A. PubMed DOI

Yoshida Y, et al. De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics. 2016;17:182. doi: 10.1186/s12864-016-2540-6. PubMed DOI PMC

Keeling PJ, et al. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 2014;12:e1001889. doi: 10.1371/journal.pbio.1001889. PubMed DOI PMC

Jackson C, Knoll AH, Chan CX, Verbruggen H. Plastid phylogenomics with broad taxon sampling further elucidates the distinct evolutionary origins and timing of secondary green plastids. Sci Rep. 2018;8:1523. doi: 10.1038/s41598-017-18805-w. PubMed DOI PMC

Turmel M, Gagnon MC, O’Kelly CJ, Otis C, Lemieux C. The chloroplast genomes of the green algae Pyramimonas, Monomastix, and Pycnococcus shed new light on the evolutionary history of prasinophytes and the origin of the secondary chloroplasts of euglenids. Mol Biol Evol. 2009;26:631–648. doi: 10.1093/molbev/msn285. PubMed DOI

Vanclová, A. M. G., Hadariová, L., Hrdá, Š. & Hampl, V. Chapter Nine - Secondary Plastids of Euglenophytes in Advances in Botanical Research Vol. 84 (ed. Yoshihisa Hirakawa), 321–358 (Academic Press, 2017).

Durnford, D. G. & Schwartzbach, S. D. Protein targeting to the plastid of Euglena in Euglena: Biochemistry, cell and molecular biology Vol. 979 (eds Steven D. Schwartzbach & Shigeru Shigeoka), 183–205 (Springer International Publishing, 2017). PubMed

Durnford DG, Gray MW. Analysis of Euglena gracilis plastid-targeted proteins reveals different classes of transit sequences. Eukaryot Cell. 2006;5:2079–2091. doi: 10.1128/EC.00222-06. PubMed DOI PMC

Kořený L, Oborník M. Sequence evidence for the presence of two tetrapyrrole pathways in Euglena gracilis. Genome Biol Evol. 2011;3:359–364. doi: 10.1093/gbe/evr029. PubMed DOI PMC

Lakey B, Triemer R. The tetrapyrrole synthesis pathway as a model of horizontal gene transfer in euglenoids. J Phycol. 2017;53:198–217. doi: 10.1111/jpy.12491. PubMed DOI

Markunas CM, Triemer RE. Evolutionary history of the enzymes involved in the Calvin-Benson cycle in euglenids. J Eukaryot Microbiol. 2016;63:326–339. doi: 10.1111/jeu.12282. PubMed DOI

Marin B, Palm A, Klingberg M, Melkonian M. Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure. Protist. 2003;154:99–145. doi: 10.1078/143446103764928521. PubMed DOI

Gockel G, Hachtel W. Complete gene map of the plastid genome of the nonphotosynthetic euglenoid flagellate Astasia longa. Protist. 2000;151:347–351. doi: 10.1078/S1434-4610(04)70033-4. PubMed DOI

Hadariová L, Vesteg M, Birčák E, Schwartzbach SD, Krajčovič J. An intact plastid genome is essential for the survival of colorless Euglena longa but not Euglena gracilis. Curr Genet. 2017;63:331–341. doi: 10.1007/s00294-016-0641-z. PubMed DOI

Záhonová K, Füssy Z, Oborník M, Eliáš M, Yurchenko V. RuBisCO in non-photosynthetic alga Euglena longa: divergent features, transcriptomic analysis and regulation of complex formation. PLoS ONE. 2016;11:e0158790. doi: 10.1371/journal.pone.0158790. PubMed DOI PMC

Webster DA, Hackett DP, Park RB. The respiratory chain of colorless algae: III. Electron microscopy. J Ultrastruct Res. 1967;21:514–523. doi: 10.1016/S0022-5320(67)80154-0. PubMed DOI

Nudelman MA, Rossi MS, Conforti V, Triemer RE. Phylogeny of Euglenophyceae based on small subunit rDNA sequences: Taxonomic implications. J Phycol. 2003;39:226–235. doi: 10.1046/j.1529-8817.2003.02075.x. DOI

Záhonová K, et al. Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase. Sci Rep. 2018;8:5239. doi: 10.1038/s41598-018-23575-0. PubMed DOI PMC

Russell AG, Watanabe Y, Charette JM, Gray MW. Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya. Nucleic Acids Res. 2005;33:2781–2791. doi: 10.1093/nar/gki574. PubMed DOI PMC

Nagai M, Yoneda Y. Small GTPase Ran and Ran-binding proteins. Biomol Concepts. 2012;3:307–318. doi: 10.1515/bmc-2011-0068. PubMed DOI

Záhonová K, et al. A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis. FEBS Lett. 2014;588:783–788. doi: 10.1016/j.febslet.2014.01.034. PubMed DOI

Maier UG, et al. Massively convergent evolution for ribosomal protein gene content in plastid and mitochondrial genomes. Genome Biol Evol. 2013;5:2318–2329. doi: 10.1093/gbe/evt181. PubMed DOI PMC

Figueroa-Martinez F, Nedelcu AM, Smith DR, Reyes-Prieto A. The plastid genome of Polytoma uvella is the largest known among colorless algae and plants and reflects contrasting evolutionary paths to nonphotosynthetic lifestyles. Plant Physiol. 2017;173:932–943. doi: 10.1104/pp.16.01628. PubMed DOI PMC

Kamikawa R, et al. Proposal of a twin arginine translocator system-mediated constraint against loss of ATP synthase genes from nonphotosynthetic plastid genomes. Mol Biol Evol. 2015;32:2598–2604. doi: 10.1093/molbev/msv134. PubMed DOI

Suzuki S, Endoh R, Manabe RI, Ohkuma M, Hirakawa Y. Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca. Sci Rep. 2018;8:940. doi: 10.1038/s41598-017-18378-8. PubMed DOI PMC

Habib S, Vaishya S, Gupta K. Translation in organelles of apicomplexan parasites. Trends Parasitol. 2016;32:939–952. doi: 10.1016/j.pt.2016.07.005. PubMed DOI

Mailu BM, et al. Plasmodium apicoplast Gln-tRNAGln biosynthesis utilizes a unique GatAB amidotransferase essential for erythrocytic stage parasites. J Biol Chem. 2015;290:29629–29641. doi: 10.1074/jbc.M115.655100. PubMed DOI PMC

Sheppard K, et al. From one amino acid to another: tRNA-dependent amino acid biosynthesis. Nucleic Acids Res. 2008;36:1813–1825. doi: 10.1093/nar/gkn015. PubMed DOI PMC

Gile GH, Moog D, Slamovits CH, Maier UG, Archibald JM. Dual organellar targeting of aminoacyl-tRNA synthetases in diatoms and cryptophytes. Genome Biol Evol. 2015;7:1728–1742. doi: 10.1093/gbe/evv095. PubMed DOI PMC

Ban N, et al. A new system for naming ribosomal proteins. Curr Opin Struct Biol. 2014;24:165–169. doi: 10.1016/j.sbi.2014.01.002. PubMed DOI PMC

Adams KL, Daley DO, Whelan J, Palmer JD. Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts. Plant Cell. 2002;14:931–943. doi: 10.1105/tpc.010483. PubMed DOI PMC

Bieri P, Leibundgut M, Saurer M, Boehringer D, Ban N. The complete structure of the chloroplast 70S ribosome in complex with translation factor pY. EMBO J. 2017;36:475–486. doi: 10.15252/embj.201695959. PubMed DOI PMC

Bubunenko MG, Schmidt J, Subramanian AR. Protein substitution in chloroplast ribosome evolution. A eukaryotic cytosolic protein has replaced its organelle homologue (L23) in spinach. J Mol Biol. 1994;240:28–41. doi: 10.1006/jmbi.1994.1415. PubMed DOI

Takagi M, Absalon MJ, McLure KG, Kastan MB. Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell. 2005;123:49–63. doi: 10.1016/j.cell.2005.07.034. PubMed DOI

Zhang M, Zhang J, Yan W, Chen X. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability. Oncotarget. 2016;7:78255–78268. PubMed PMC

Chan RL, Keller M, Canaday J, Weil JH, Imbault P. Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein. EMBO J. 1990;9:333–338. doi: 10.1002/j.1460-2075.1990.tb08115.x. PubMed DOI PMC

Enomoto T, Sulli C, Schwartzbach SD. A soluble chloroplast protease processes the Euglena polyprotein precursor to the light harvesting chlorophyll a/b binding protein of photosystem II. Plant Cell Physiol. 1997;38:743–746. doi: 10.1093/oxfordjournals.pcp.a029229. DOI

Koziol AG, Durnford DG. Euglena light-harvesting complexes are encoded by multifarious polyprotein mRNAs that evolve in concert. Mol Biol Evol. 2008;25:92–100. doi: 10.1093/molbev/msm232. PubMed DOI

Nowitzki U, Gelius-Dietrich G, Schwieger M, Henze K, Martin W. Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide. Eur J Biochem. 2004;271:4123–4131. doi: 10.1111/j.1432-1033.2004.04350.x. PubMed DOI

Zhang H, Lin S. Complex gene structure of the the form II RuBisCO in the dinoflagellate Prorocentrum minimum (Dinophyceae) J Phycol. 2003;39:1160–1171. doi: 10.1111/j.0022-3646.2003.03-055.x. DOI

Benz JP, et al. Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated into the chloroplast redox poise. Plant Cell. 2009;21:3965–3983. doi: 10.1105/tpc.109.069815. PubMed DOI PMC

Chigri F, et al. Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32. Proc Natl Acad Sci USA. 2006;103:16051–16056. doi: 10.1073/pnas.0607150103. PubMed DOI PMC

Kikuchi S, et al. A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane. Plant Cell. 2009;21:1781–1797. doi: 10.1105/tpc.108.063552. PubMed DOI PMC

Hauenstein M, Christ B, Das A, Aubry S, Hortensteiner S. A role for TIC55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during plant senescence. Plant Cell. 2016;28:2510–2527. doi: 10.1105/tpc.16.00630. PubMed DOI PMC

Maier UG, Zauner S, Hempel F. Protein import into complex plastids: Cellular organization of higher complexity. Eur J Cell Biol. 2015;94:340–348. doi: 10.1016/j.ejcb.2015.05.008. PubMed DOI

Lee DW, Lee J, Hwang I. Sorting of nuclear-encoded chloroplast membrane proteins. Curr Opin Plant Biol. 2017;40:1–7. doi: 10.1016/j.pbi.2017.06.011. PubMed DOI

Braun NA, Davis AW, Theg SM. The chloroplast Tat pathway utilizes the transmembrane electric potential as an energy source. Biophys J. 2007;93:1993–1998. doi: 10.1529/biophysj.106.098731. PubMed DOI PMC

Träger C, et al. Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms. Plant Cell. 2012;24:4819–4836. doi: 10.1105/tpc.112.102996. PubMed DOI PMC

Ziehe D, Dünschede B, Schünemann D. From bacteria to chloroplasts: evolution of the chloroplast SRP system. Biol Chem. 2017;398:653–661. doi: 10.1515/hsz-2016-0292. PubMed DOI

Skalitzky CA, et al. Plastids contain a second sec translocase system with essential functions. Plant Physiol. 2011;155:354–369. doi: 10.1104/pp.110.166546. PubMed DOI PMC

Li Y, Martin JR, Aldama GA, Fernandez DE, Cline K. Identification of putative substrates of SEC. 2, a chloroplast inner envelope translocase. Plant Physiol. 2017;173:2121–2137. doi: 10.1104/pp.17.00012. PubMed DOI PMC

Nishimura K, Kato Y, Sakamoto W. Chloroplast proteases: Updates on proteolysis within and across suborganellar compartments. Plant Physiol. 2016;171:2280–2293. PubMed PMC

Nakai M, Sugita D, Omata T, Endo T. Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942. Biochem Biophys Res Commun. 1993;193:228–234. doi: 10.1006/bbrc.1993.1613. PubMed DOI

Yusa F, Steiner JM, Löffelhardt W. Evolutionary conservation of dual Sec translocases in the cyanelles of Cyanophora paradoxa. BMC Evol Biol. 2008;8:304. doi: 10.1186/1471-2148-8-304. PubMed DOI PMC

Chen C, MacCready JS, Ducat DC, Osteryoung KW. The molecular machinery of chloroplast division. Plant Physiol. 2018;176:138–151. doi: 10.1104/pp.17.01272. PubMed DOI PMC

Miyagishima SY, Nakamura M, Uzuka A, Era A. FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division. Front Plant Sci. 2014;5:459. doi: 10.3389/fpls.2014.00459. PubMed DOI PMC

Chi W, He B, Mao J, Jiang J, Zhang L. Plastid sigma factors: Their individual functions and regulation in transcription. Biochim Biophys Acta. 2015;1847:770–778. doi: 10.1016/j.bbabio.2015.01.001. PubMed DOI

Kriner MA, Sevostyanova A, Groisman EA. Learning from the leaders: Gene regulation by the transcription termination factor Rho. Trends Biochem Sci. 2016;41:690–699. doi: 10.1016/j.tibs.2016.05.012. PubMed DOI PMC

Parks, D. H. et al. A proposal for a standardized bacterial taxonomy based on genome phylogeny. Nat Biotechnol., 10.1038/nbt.4229, (2018). PubMed

Anantharaman K, et al. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat Commun. 2016;7:13219. doi: 10.1038/ncomms13219. PubMed DOI PMC

Cao H, et al. Delta-proteobacterial SAR324 group in hydrothermal plumes on the South Mid-Atlantic Ridge. Sci Rep. 2016;6:22842. doi: 10.1038/srep22842. PubMed DOI PMC

Chitsaz H, et al. Efficient de novo assembly of single-cell bacterial genomes from short-read data sets. Nat Biotechnol. 2011;29:915–921. doi: 10.1038/nbt.1966. PubMed DOI PMC

Tully BJ, Graham ED, Heidelberg JF. The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans. Sci Data. 2018;5:170203. doi: 10.1038/sdata.2017.203. PubMed DOI PMC

Hadariová L, Vesteg M, Hampl V, Krajčovič J. Reductive evolution of chloroplasts in non-photosynthetic plants, algae and protists. Curr Genet. 2018;64:365–387. doi: 10.1007/s00294-017-0761-0. PubMed DOI

Huang J, Yue J. Horizontal gene transfer in the evolution of photosynthetic eukaryotes. J Syst Evol. 2013;51:13–29. doi: 10.1111/j.1759-6831.2012.00237.x. DOI

Mackiewicz P, Bodyl A, Moszczynski K. The case of horizontal gene transfer from bacteria to the peculiar dinoflagellate plastid genome. Mob Genet Elements. 2013;3:e25845. doi: 10.4161/mge.25845. PubMed DOI PMC

Maruyama S, Suzaki T, Weber AP, Archibald JM, Nozaki H. Eukaryote-to-eukaryote gene transfer gives rise to genome mosaicism in euglenids. BMC Evol Biol. 2011;11:105. doi: 10.1186/1471-2148-11-105. PubMed DOI PMC

Cramer M, Myers J. Growth and photosynthetic characteristics of Euglena gracilis. Archiv Mikrobiol. 1952;17:384–402. doi: 10.1007/BF00410835. DOI

Mateášiková-Kováčová B, et al. Nucleus-encoded mRNAs for chloroplast proteins GapA, PetA, and PsbO are trans-spliced in the flagellate Euglena gracilis irrespective of light and plastid function. J Eukaryot Microbiol. 2012;59:651–653. doi: 10.1111/j.1550-7408.2012.00634.x. PubMed DOI

Schmieder R, Edwards R. Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PLOS ONE. 2011;6:e17288. doi: 10.1371/journal.pone.0017288. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Simpson JT, et al. ABySS: a parallel assembler for short read sequence data. Genome Res. 2009;19:1117–1123. doi: 10.1101/gr.089532.108. PubMed DOI PMC

Robertson G, et al. De novo assembly and analysis of RNA-seq data. Nat Methods. 2010;7:909–912. doi: 10.1038/nmeth.1517. PubMed DOI

Grabherr MG, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–652. doi: 10.1038/nbt.1883. PubMed DOI PMC

Xie Y, et al. SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads. Bioinformatics. 2014;30:1660–1666. doi: 10.1093/bioinformatics/btu077. PubMed DOI

Huang X, Madan A. CAP3: A DNA sequence assembly program. Genome Res. 1999;9:868–877. doi: 10.1101/gr.9.9.868. PubMed DOI PMC

Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015;31:3210–3212. doi: 10.1093/bioinformatics/btv351. PubMed DOI

Altschul SF, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Eddy SR. A new generation of homology search tools based on probabilistic inference. Genome Inform. 2009;23:205–211. PubMed

Marchler-Bauer A, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45:D200–D203. doi: 10.1093/nar/gkw1129. PubMed DOI PMC

Larkin MA, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. doi: 10.1093/bioinformatics/btm404. PubMed DOI

Hiller K, Grote A, Scheer M, Munch R, Jahn D. PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res. 2004;32:W375–379. doi: 10.1093/nar/gkh378. PubMed DOI PMC

Petsalaki EI, Bagos PG, Litou ZI, Hamodrakas SJ. PredSL: a tool for the N-terminal sequence-based prediction of protein subcellular localization. Genomics Proteomics Bioinformatics. 2006;4:48–55. doi: 10.1016/S1672-0229(06)60016-8. PubMed DOI PMC

Krogh A, Larsson B, von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305:567–580. doi: 10.1006/jmbi.2000.4315. PubMed DOI

Kearse M, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–1649. doi: 10.1093/bioinformatics/bts199. PubMed DOI PMC

Blum T, Briesemeister S, Kohlbacher O. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction. BMC Bioinformatics. 2009;10:274. doi: 10.1186/1471-2105-10-274. PubMed DOI PMC

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–1973. doi: 10.1093/bioinformatics/btp348. PubMed DOI PMC

Larsson A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. doi: 10.1093/bioinformatics/btu531. PubMed DOI PMC

Goodstadt L, Ponting CP. CHROMA: consensus-based colouring of multiple alignments for publication. Bioinformatics. 2001;17:845–846. doi: 10.1093/bioinformatics/17.9.845. PubMed DOI

Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268–274. doi: 10.1093/molbev/msu300. PubMed DOI PMC

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