Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics Kristína Záhonová1,2, Sebastian Cristian Treitli1, Tien Le1, Ingrid Škodová-Sveráková2,3, Pavla Hanousková4, Ivan Čepička4, Jan Tachezy1, Vladimír Hampl1 1 Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic 2 Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice (Budweis), Czech Republic 3 Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia 4 Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic Corresponding authors: [email protected] (KZ); [email protected] (VH) S1 – S6 FIGURES Ceratiomyxa fruticulosa EF513175 58 Clastostelium recurvatum FJ766474 0.1 substitution/site 98 Protosporangium articulatum FJ792705 Multicilia marina AY268037 Dictyamoeba vorax KP864096 79 Ceratiomyxella tahitiensis FJ544419 Phalansterium solitarium AF280078 Angulamoeba microcystivorans KP864083 Heliamoeba mirabilis KP864095 Darbyshirella terrestris KP864087 80 Ischnamoeba montana KP864092 O Flamella beringiania KP219432 U 97 Telaepolella sp. KP864099 T Schizoplasmodiopsis amoeboidea EF513179 G R Planoprotostelium aurantium EU004604 100 O Protostelium mycophaga EU004603 U Polysphondylium pallidum AY040338 P 100 Dyctiostelium polycephalum AY040334 100 Dictyostelium discoideum AM168071 Cavostelium apophysatum EF513172 Soliformovum irregulare EF513181 Arcyria denudata AY643825 100 Trichia persimilis AY643826 100 Echinostelium arboreum AY842030 100 Stemonitis flavogenita AF239229 100 Protophysarum phloiogenum AY230189 98 Didymium nigripes AF239230 Rhizomastix libera JX157645 Rhizomastix elongata KP343633 91 Rhizomastixidae 100 Rhizomastix vacuolata KP343626 100 64 Rhizomastix bicoronata KP343636 Entamoeba polecki AF149913 Entamoeba coli AF149915 100 Entamoeba gingivalis D28490 78 Entamoeba equi DQ286371 52 93 Entamoeba bovis FN666248 Entamoebidae 96 Entamoeba histolytica X65163 77 Entamoeba hartmanni FR686379 94 91 Entamoeba invadens AF149905 100 Entamoeba ranarum AF149908 Mastigella eilhardi KJ879584 88 Mastigella erinacea KJ879586 Pelomyxa schiedti SKADARSKE 88 Pelomyxidae 100 Pelomyxa belevskii JX157662 99 Pelomyxa stagnalis JX157660 68 Pelomyxa palustris JX157661 Mastigamoeba errans JX157637 100 Mastigamoeba sp. AM114798 69 Mastigamoeba sp. JX157664 99 Mastigamoeba balamuthi L23799 71 95 Mastigamoeba punctachora JX157653 Mastigamoeba lenta JX157656 Mastigamoeba simplex AF421218 Mastigamoebidae 95 100 Mastigamoeba guttula JX157649 82 Mastigamoeba abducta JX157636 53 Endolimax piscium JX101953 56 Endolimax nana AF149916 100 Iodamoeba sp. JN635746 Archamoebae 96 Iodamoeba sp. JN635740 S1 Fig. Phylogenetic analysis of amoebozoan 18S rDNA. The Maximum Likelihood tree places Pelo- myxa schiedti in monophyletic Pelomyxidae group inside monophyletic Archamoebae. Standard bootstrap support values are shown when 50%. BUSCO Assessment Results Complete (C) and single-copy (S) Complete (C) and duplicated (D) Fragmented (F) Missing (M) P. schiedti transcriptome C: 83.2% [S: 47.2%, D: 36.0%], F: 2.0%, M: 14.8%, n: 303 P. schiedti predicted proteins C: 81.9% [S: 73.3%, D: 8.6%], F: 3.6%, M: 14.5%, n: 303 M. balamuthi predicted proteins C: 82.8% [S: 80.2%, D: 2.6%], F: 3.0%, M: 14.2%, n: 303 0 20 40 60 80 100 %BUSCOs S2 Fig. BUSCO analysis of the Pelomyxa schiedti transcriptome and predicted proteins. Completeness of P. schiedti datasets were assessed using the odb9_eukaryota dataset and compared with completeness of predicted proteins from Mastigamoeba balamuthi. Aquifex aeolicus AAC07071.1 0.5 substitution/site Leptospira interrogans AAC35416.1 prokaryotes Borreliella burgdorferi AAC66887.1 1 substitution/site Thermoplasma acidophilum AAC41460.1 Halobacterium salinarum AAC41461.1 100 Mycobacterium avium paratuberculosis CAA42063.1 Francisella tularensis AAA69561.1 100 Escherichia coli AAC73125.1 100 75 Haemophilus influenzae AAC22889.1 96 Neorickettsia sennetsu AAC27487.1 97 Rickettsia prowazekii CAA14651.1 99 Rhodopseudomonas sp. No.7 BAA19796.1 Alphaproteobacteria 100 Agrobacterium fabrum CAA60592.1 100 Sinorhizobium meliloti AAA64925.1 Arabidopsis thaliana AAO00750.1 100 Solanum tuberosum Q08276.1 96 87 mitochondrial Plasmodium falciparum BAB17688.1 100 Cryptosporidium parvum AAP59793.1 98 Eimeria tenella CAA87086.1 Entamoeba histolytica AAG16651.1 96 79 Encephalitozoon cuniculi CAA10035.1 100 Antonospora locustae AAC47660.1 Saccharomyces cerevisiae AAA63792.1 100 88 Schizosaccharomyces pombe AAA35314.1 97 Caenorhabditis elegans NP_504291.1 100 Drosophila melanogaster AAA28628.1 97 Homo sapiens AAA67526.1 Trichomonas vaginalis AAB09772.1 Giardia intestinalis BAB84357.1 Leishmania major P12076.3 100 Trypanosoma cruzi AAA30215.1 91 Pelomyxa schiedti Pelo10550 100 Mastigamoeba balamuthi m51a1_g1166+comp4411_c0_seq1_1604_224 Giardia lamblia XP_001709475.1 Pelomyxa schiedti Pelo19959 75 Plasmodium falciparum XP_001352050.1 ER Zea mays AAC49900.1 100 Drosophila melanogaster AAA28626.1 Pelomyxa schiedti Pelo11577 75 100 Pelomyxa schiedti Pelo9677 100 Mastigamoeba balamuthi m51a1_g7032 100 Mastigamoeba balamuthi m51a1_g5004 Mastigamoeba balamuthi m51a1_g11528 Trypanosoma brucei brucei AAC37174.1 Mastigamoeba balamuthi m51a1_g14298 99 Mastigamoeba balamuthi m51a1_g7769 98 cytosolic 99 Mastigamoeba balamuthi m51a1_g7770 Pelomyxa schiedti Pelo13846 82 95 Pelomyxa schiedti Pelo9585 97 Pelomyxa schiedti Pelo16822 85 Pelomyxa schiedti Pelo177 Pisum sativum CAA67867.1 83 Pelomyxa schiedti Pelo14211 Pelomyxa schiedti Pelo5973 99 98 Pelomyxa schiedti Pelo16289 100 Mastigamoeba balamuthi m51a1_g8470 100 Pelomyxa schiedti Pelo16012 100 Mastigamoeba balamuthi m51a1_g4637 100 Pelomyxa schiedti Pelo5717 100 Pelomyxa schiedti Pelo16407 100 Mastigamoeba balamuthi m51a1_g4194 98 Mastigamoeba balamuthi m51a1_g12510 Giardia lamblia EAA38588.1 S3 Fig. Phylogenetic Trichomonas vaginalis AAB52423.1 analysis of HSP70 Leishmania major P14834.1 proteins. The Maximum 100 Trypanosoma cruzi AAA30205.1 Likelihood tree documents Babesia bovis AAF14194.1 that one of the Pelomyxa 100 Theileria annulata A44985 schiedti HSP70 sequence 100 Cryptosporidium parvum AAC25925.1 98 is related to mitochondrial Plasmodium falciparum NP_704366.1 orthologues from other Blastocladiella emersonii AAA65099.1 Saccharomyces cerevisiae AAC37398.1 eukaryotes. Ultrafast boot- 98 100 Schizosaccharomyces pombe BAA25322.1 81 strap support values are Caenorhabditis elegans AAA28078.1 shown when 75%. 100 Drosophila melanogaster AAA28625.1 100 Mus musculus AAA37864.1 prokaryotes (55) 1 substitution/site Streblomastix strix KAA6397532.1 100 Paratrimastix pyriformis ABW76110.1 Beauveria bassiana KGQ06091.1 100 Trichuris trichiura CDW60499.1 100 Ephemera danica KAF4533642.1 99 Drosophila eugracilis XP_017085656.1 100 Drosophila elegans XP_017118400.1 100 Drosophila kikkawai XP_017018290.1 99 Stygiella incarcerata ANM86733.1 100 Stygiella incarcerata ANM86735.1 89 99 Stygiella incarcerata ANM86732.1 Streblomastix strix KAA6395081.1 90 Spironucleus salmonicida AFV80067.1 94 Spironucleus salmonicida AFV80064.1 Giardia lamblia XP_001708704.1 98 100 100 Spironucleus salmonicida AFV80063.1 Mastigamoeba balamuthi PFO6 AGA37396.1 100 Mastigamoeba balamuthi PFO2 AAM53401.1 97 Giardia lamblia XP_001708948.1 100 Retortamonas sp. Vale ABW76111.1 99 Spironucleus salmonicida AFV80066.1 100 Spironucleus salmonicida AFV80065.1 Stygiella incarcerata ANM86734.1 Sawyeria marylandensis ABU95423.1 97 Acanthamoeba castellanii XP_004336994.1 99 100 Acanthamoeba castellanii XP_004339046.1 99 Acanthamoeba castellanii XP_004339045.1 100 Tritrichomonas foetus OHT14860.1 Tritrichomonas foetus OHT08363.1 100 100 Tritrichomonas foetus ARM19877.1 100 Tritrichomonas foetus OHS98215.1 100 Trichomonas vaginalis XP_001314811.1 100 Trichomonas vaginalis XP_001582360.1 100 Trichomonas vaginalis XP_001321321.1 100 Trichomonas vaginalis XP_001311861.1 Mastigamoeba balamuthi PFO3 AGA37395.1 100 Pelomyxa schiedti Pelo3613 92 Mastigamoeba balamuthi PFO5 AIW52602.1 100 Pelomyxa schiedti Pelo847 100 Mastigamoeba balamuthi PFO4 AIW52601.1 100 Pelomyxa schiedti Pelo7196 100 Pelomyxa schiedti Pelo10870 100 Pelomyxa schiedti Pelo129 Stygiella incarcerata ANM86867.1 98 Vitrella brassicaformis CEM14662.1 100 Cryptosporidium muris XP_002140958.1 100 Cryptosporidium andersoni OII76317.1 100 Cryptosporidium felis KAF7456279.1 100 Cryptosporidium ubiquitum XP_028873320.1 100 Cryptosporidium parvum Q968X7.1 99 Cryptosporidium meleagridis POM83206.1 79 Cryptosporidium parvum XP_625673.1 Cryptosporidium tyzzeri TRY52171.1 97 Cryptosporidium hominis PPS93056.1 100 Cryptosporidium hominis OLQ17293.1 Perkinsus marinus XP_002781654.1 100 Perkinsus marinus XP_002771617.1 90 91 Symbiodinium microadriaticum OLQ01481.1 Perkinsus olseni KAF4667627.1 100 100 Perkinsus marinus XP_002774160.1 100 Perkinsus marinus XP_002772046.1 100 Perkinsus marinus XP_002764792.1 100 Perkinsus olseni KAF4696005.1 99 100 Perkinsus olseni KAF4688717.1 100 Perkinsus olseni KAF4696006.1 Perkinsus chesapeaki KAF4666657.1 100 Perkinsus marinus XP_002765111.1 99 Perkinsus olseni KAF4689951.1 100 Perkinsus olseni KAF4673500.1 100 Perkinsus olseni KAF4673501.1 100 Perkinsus marinus XP_002772896.1 Perkinsus chesapeaki KAF4666655.1 100 100 Perkinsus olseni KAF4689950.1 100 Perkinsus marinus XP_002765113.1 100 Perkinsus marinus XP_002772895.1 99 Perkinsus marinus XP_002765114.1 93 Peranema trichophorum ABL11191.1 100 Euglena gracilis