Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

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

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Institute of Evolutionary Biology Faculty of Biology Biological and Chemical Research Centre University of Warsaw Warsaw Poland

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

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Leander, B. S., Lax, G., Karnkowska, A. & Simpson, A. G. B. Euglenida in Handbook of the Protists (Slamovits, C. H., Archibald, J. M., Simpson, A. G. B.) (Springer International Publishing, 2017).

Lax, G. et al. Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs. Mol. Phylogenet. Evol.159 (2021). PubMed

Lax G, Lee WJ, Eglit Y, Simpson A. Ploeotids represent much of the phylogenetic diversity of euglenids. Protist. 2019;170:233–257. doi: 10.1016/j.protis.2019.03.001. PubMed DOI

Gibbs SP. The comparative ultrastructure of the algal chloroplast. Ann. N. Y. Acad. Sci. 1970;175:454–473. doi: 10.1111/j.1749-6632.1970.tb45167.x. DOI

Lefort-Tran M, Pouphile M, Freyssinet G, Pineau B. Signification structurale et fonctionnelle des enveloppes chloroplastiques d’Euglena: Etude immunocytologique et en cryofracture. J. Ultrastruct. Res. 1980;73:44–63. doi: 10.1016/0022-5320(80)90115-X. PubMed DOI

Turmel M, et al. 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

Leander BS. Did trypanosomatid parasites have photosynthetic ancestors? Trends Microbiol. 2004;12:251–258. doi: 10.1016/j.tim.2004.04.001. PubMed DOI

Hannaert V, et al. Plant-like traits associated with metabolism of Trypanosoma parasites. Proc. Natl. Acad. Sci. USA. 2003;100:1067–1071. doi: 10.1073/pnas.0335769100. PubMed DOI PMC

Marin B. Origin and fate of chloroplasts in the euglenoida. Protist. 2004;155:13–14. doi: 10.1078/1434461000159. PubMed DOI

Busse I, Preisfeld A. Systematics of primary osmotrophic euglenids: a molecular approach to the phylogeny of Distigma and Astasia (Euglenozoa) Int. J. Syst. Evol. Microbiol. 2003;53:617–624. doi: 10.1099/ijs.0.02295-0. PubMed DOI

Gawryluk RMR, et al. Morphological identification and single-cell genomics of marine diplonemids. Curr. Biol. 2016;26:3053–3059. doi: 10.1016/j.cub.2016.09.013. PubMed DOI

Flegontova O, et al. Extreme diversity of diplonemid eukaryotes in the ocean. Curr. Biol. 2016;26:3060–3065. doi: 10.1016/j.cub.2016.09.031. PubMed DOI

Gibson, W. Kinetoplastea. (2016) in Handbook of the Protists (Slamovits, C. H., Archibald, J. M., Simpson, A. G. B.) (Springer International Publishing, 2017).

Aurrecoechea C, et al. EuPathDB: the eukaryotic pathogen genomics database resource. Nucleic Acids Res. 2017;45:581–591. doi: 10.1093/nar/gkw1105. PubMed DOI PMC

Hallick RB, et al. Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res. 1993;21:3537–3544. doi: 10.1093/nar/21.15.3537. PubMed DOI PMC

Dobáková E, Flegontov P, Skalický T, Lukeš J. Unexpectedly streamlined mitochondrial genome of the euglenozoan Euglena gracilis. Genome Biol. Evol. 2015;7:3358–3367. doi: 10.1093/gbe/evv229. PubMed DOI PMC

Ebenezer TETE, et al. Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol. 2019;17:1–23. doi: 10.1186/s12915-019-0626-8. PubMed DOI PMC

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

Kasiborski BA, Bennett MS, Linton EW. The chloroplast genome of Phacus orbicularis (Euglenophyceae): an initial datum point for the Phacaceae. J Phycol. 2016;52:404–411. doi: 10.1111/jpy.12403. PubMed DOI

Karnkowska A, Bennett MS, Triemer RE. Dynamic evolution of inverted repeats in Euglenophyta plastid genomes. Sci. Rep. 2018;8:1–10. doi: 10.1038/s41598-018-34457-w. PubMed DOI PMC

Novák Vanclová, A. M. G. et al. Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid. New Phytol.225, 1578–1592 (2020). PubMed

Hammond MJ, et al. A uniquely complex mitochondrial proteome from Euglena gracilis. Mol. Biol. Evol. 2020;37:2173–2191. doi: 10.1093/molbev/msaa061. PubMed DOI PMC

Kanehisa M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 2019;28:1947–1951. doi: 10.1002/pro.3715. PubMed DOI PMC

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

Milanowski R, Karnkowska A, Ishikawa T, Zakrys B. Distribution of conventional and nonconventional introns in tubulin (α and β) genes of euglenids. Mol. Biol. Evol. 2014;31:584–593. doi: 10.1093/molbev/mst227. PubMed DOI PMC

Harada R, et al. Inventory and evolution of mitochondrion-localized Family A DNA Polymerases in Euglenozoa. Pathogens. 2020;9:257. doi: 10.3390/pathogens9040257. PubMed DOI PMC

Záhonová K, et al. Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses. Sci. Rep. 2018;8:1–15. PubMed PMC

Garin S, Levi O, Cohen B, Golani-Armon A, Arava YS. Localization and RAN binding of mitochondrial aminoacyl tRAN synthetases. Genes (Basel). 2020;11:1–20. doi: 10.3390/genes11101185. PubMed DOI PMC

Tanaka Y, et al. Glucan synthase-like 2 is indispensable for paramylon synthesis in Euglena gracilis. FEBS Lett. 2017;591:1360–1370. doi: 10.1002/1873-3468.12659. PubMed DOI

Morales J, et al. Novel mitochondrial Complex II isolated from Trypanosoma cruzi is composed of 12 peptides including a heterodimeric. J. Biol. Chem. 2009;284:7255–7263. doi: 10.1074/jbc.M806623200. PubMed DOI PMC

Gawryluk, R. & Gray, M. W. A split and rearranged nuclear gene encoding the iron-sulfur subunit of mitochondrial succinate dehydrogenase in Euglenozoa. 7, 1–7 (2009). PubMed PMC

Kalscheuer R, Steinbüchel A. A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 2003;278:8075–8082. doi: 10.1074/jbc.M210533200. PubMed DOI

Tomiyama T, et al. Wax ester synthase/diacylglycerol acyltransferase isoenzymes play a pivotal role in wax ester biosynthesis in euglena gracilis. Sci. Rep. 2017;7:1–13. doi: 10.1038/s41598-017-14077-6. PubMed DOI PMC

Nakazawa, M. et al. Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis. Comp. Biochem. Physiol. - B Biochem. Mol. Biol.141, 445–452 (2005). PubMed

Gawryluk RMRR, Chisholm KA, Pinto DM, Gray MW. Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants. J. Proteomics. 2014;109:400–416. doi: 10.1016/j.jprot.2014.07.005. PubMed DOI

Pennisi R, et al. Molecular evolution of alternative oxidase proteins: a phylogenetic and structure modeling approach. J. Mol. Evol. 2016;82:207–218. doi: 10.1007/s00239-016-9738-8. PubMed DOI

Shigeoka S, Onishi T, Nakano Y, Kitaoka S. Requirement for vitamin B1 for growth of Euglena gracilis. J. Gen. Microbiol. 1987;133:25–30.

Shigeoka S, et al. Occurrence and subcellular distribution of thiamine pyrophosphokinase isozyme in Euglena gracilis. Agric. Biol. Chem. 1987;51:2811–2813.

Russell AG, Watanabe YI, 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

Yoshida Y, et al. De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics. 2016;17:1–10. doi: 10.1186/s12864-015-2294-6. PubMed DOI PMC

Breckenridge DG, Watanabe Y, Greenwood SJ, Gray MW, Schnare MN. U1 small nuclear RNA and spliceosomal introns in Euglena gracilis. Proc. Natl. Acad. Sci. USA. 1999;96:852–856. doi: 10.1073/pnas.96.3.852. PubMed DOI PMC

Ebel C, Frantz C, Paulus F, Imbault P. Trans-splicing and cis-splicing in the colourless euglenoid, Entosiphon sulcatum. Curr. Genet. 1999;35:542–550. doi: 10.1007/s002940050451. PubMed DOI

Canaday J, Tessier LH, Imbault P, Paulus F. Analysis of Euglena gracilis alpha-, beta- and gamma-tubulin genes: introns and pre-mRNA maturation. Mol. Genet. Genomics. 2001;265:153–160. doi: 10.1007/s004380000403. PubMed DOI

Vesteg M, et al. A possible role for short introns in the acquisition of stroma-targeting peptides in the flagellate Euglena gracilis. DNA Res. 2010;17:223–231. doi: 10.1093/dnares/dsq015. PubMed DOI PMC

Milanowski R, Gumińska N, Karnkowska A, Ishikawa T, Zakryś B. Intermediate introns in nuclear genes of euglenids—are they a distinct type? BMC Evol. Biol. 2016;16:49. doi: 10.1186/s12862-016-0620-5. PubMed DOI PMC

Gumińska, N., Płecha, M., Zakryś, B. & Milanowski, R. Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis. PLoS Genet.14, (2018). PubMed PMC

Žihala, D., Salamonová, J. & Eliáš, M. Evolution of the genetic code in the mitochondria of Labyrinthulea (Stramenopiles). Mol. Phylogenet. Evol.152, 106908 (2020). PubMed

Lavrov DV, et al. Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code. Mol. Biol. Evol. 2013;30:865–880. doi: 10.1093/molbev/mss274. PubMed DOI

Van Hellemond JJ, Bakker BM, Tielens AGM. Energy metabolism and its compartmentation in Trypanosoma brucei. Adv. Microb. Physiol. 2005;50:199–226. doi: 10.1016/S0065-2911(05)50005-5. PubMed DOI

Tucci S, Vacula R, Krajcovic J, Proksch P, Martin W. Variability of wax ester fermentation in natural and bleached euglena gracilis strains in response to oxygen and the elongase inhibitor flufenacet. J. Eukaryot. Microbiol. 2010;57:63–69. doi: 10.1111/j.1550-7408.2009.00452.x. PubMed DOI

Müller M, et al. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol. Mol. Biol. Rev. 2012;76:444–495. doi: 10.1128/MMBR.05024-11. PubMed DOI PMC

Saidha T, Stern AI, Lee DH, Schiff JA. Localization of a sulphate-activating system within Euglena mitochondria. Biochem. J. 1985;232:357–365. doi: 10.1042/bj2320357. PubMed DOI PMC

Hamza I, Dailey HA. One ring to rule them all: Trafficking of heme and heme synthesis intermediates in the metazoans. Biochim et Biophys Acta - Mol Cell Res. 2012;1823:1617–1632. doi: 10.1016/j.bbamcr.2012.04.009. 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

Cenci U, et al. Heme pathway evolution in kinetoplastid protists. BMC Evol. Biol. 2016;16:109. doi: 10.1186/s12862-016-0664-6. PubMed DOI PMC

Woo YH, et al. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. Elife. 2015;4:1–41. doi: 10.7554/eLife.06974. PubMed DOI PMC

Cihláø, J., Füssy, Z., Horák, A. & Oborník, M. Evolution of the tetrapyrrole biosynthetic pathway in secondary algae: Conservation, redundancy and replacement. PLoS One11, (2016). PubMed 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

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Brown, C. T. et al. The khmer software package: enabling efficient nucleotide sequence analysis. F1000Research4, (2015). PubMed PMC

Li W, Godzik A. Cd-hit: A fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006;22:1658–1659. doi: 10.1093/bioinformatics/btl158. PubMed DOI

Sharp PM, Li WH. The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987;15:1281–1295. doi: 10.1093/nar/15.3.1281. PubMed DOI PMC

Crooks, G. E. et al. VARNA: Interactive drawing and editing of the RNA secondary structure. 25, 1974–1975 (2009). PubMed PMC

Crooks GE, Hon G, Chandonia J, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–1190. doi: 10.1101/gr.849004. 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-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinforma. Appl. NOTE. 2009;25:1972–197310. doi: 10.1093/bioinformatics/btp348. PubMed DOI PMC

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. doi: 10.1093/bioinformatics/btu033. PubMed DOI PMC

Emanuelsson O, Nielsen H, Brunak S, Von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 2000;300:1005–1016. doi: 10.1006/jmbi.2000.3903. PubMed DOI

Fukasawa Y, et al. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol. Cell. Proteomics. 2015;14:1113–1126. doi: 10.1074/mcp.M114.043083. PubMed DOI PMC

Li L, Stoeckert CJ, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003;13:2178–2189. doi: 10.1101/gr.1224503. PubMed DOI PMC

Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A. C. & Kanehisa, M. KAAS: An automatic genome annotation and pathway reconstruction server. Nucleic Acids Res.35, (2007). PubMed PMC

Müllner AN, Angeler DG, Samuel R, Linton EW, Triemer RE. Phylogenetic analysis of phagotrophic, phototrophic and osmotrophic euglenoids by using the nuclear 18S rDNA sequence. Int. J. Syst. Evol. Microbiol. 2001;51:783–791. doi: 10.1099/00207713-51-3-783. PubMed DOI

Criscuolo A, Gribaldo S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 2010;10:210. doi: 10.1186/1471-2148-10-210. PubMed DOI PMC

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

Euglenozoan kleptoplasty illuminates the early evolution of photoendosymbiosis