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 BAB12024.1 Mastigamoeba balamuthi PFO1 AIW52600.1 100 Pelomyxa schiedti Pelo9624 100 Mastigamoeba balamuthi PNO AGA37390.1 100 Pelomyxa schiedti Pelo2126 100 Entamoeba invadens XP_004259380.1 91 Entamoeba invadens XP_004183052.1 100 Entamoeba dispar XP_001733938.1 76 100 Entamoeba nuttalli XP_008855741.1 99 CAA90575.1 pyruvate oxidoreductase Entamoeba histolytica 100 Entamoeba histolytica XP_657019.2 Lingula anatina XP_013418497.1 100 Capitella teleta ELT95712.1 Blastocystis hominis XP_012893603.1 100 Blastocystis sp. ATCC 50177-Nand II OAO15556.1 98 Blastocystis sp. subtype 4 XP_014524937.1 Blastocystis sp. subtype 4 XP_014528599.1 100 100 Blastocystis sp. ATCC 50177-Nand II OAO13241.1 100 Blastocystis hominis XP_012896511.1 100 Blastocystis hominis XP_012895001.1 100 Blastocystis sp. subtype 4 XP_014527203.1 Blastocystis sp. ATCC 50177-Nand II ACD10931.1 100 100 OAO15020.1 pyruvate Blastocystis sp. ATCC 50177-Nand II 100 Blastocystis hominis XP_012897901.1 100 Blastocystis hominis XP_012893814.1 Guillardia theta XP_005835303.1 100 Symbiodinium microadriaticum OLQ00553.1 100 Symbiodinium microadriaticum OLP84795.1 100 Symbiodinium microadriaticum OLQ10457.1 100 Symbiodinium microadriaticum OLP94941.1 Thalassiosira pseudonana XP_002286672.1 100 Fistulifera solaris GAX22782.1 100 Fistulifera solaris GAX19812.1 Blastocystis sp. ATCC 50177-Nand II OAO12670.1 100 Blastocystis hominis XP_012899163.1 77 100 Blastocystis hominis XP_012894411.1 Blastocystis hominis XP_012894564.1 Piromyces finnis ORX45931.1 100 Piromyces sp. E2 OUM67866.1 97 Anaeromyces robustus ORX69317.1 S4 Fig. Phylogenetic analysis of PFO enzymes. The Maximum Gonium pectorale KXZ51159.1 Likelihood phylogenetic tree identified a PFO version putatively 93 100 Gonium pectorale KXZ50713.1 100 Chlamydomonas reinhardtii XP_001701208.1 100 Volvox carteri f. nagariensis XP_002949465.1 operating in Pelomyxa schiedti MRO. Hydrogenosomal PFO 100 100 Volvox carteri f. nagariensis XP_002947533.1nagariensis Scenedesmus sp. NREL 46B-D3 KAF6254486.1 copies of Mastigamoeba balamuthi are marked with stars. 100 Monoraphidium neglectum XP_013901127.1 Number in parenthesis shows number of species in the collapsed 99 Raphidocelis subcapitata GBF87641.1 100 Chlorella variabilis XP_005847443.1 clade. Ultrafast bootstrap support values are shown when 75%. 100 Chlorella sorokiniana PRW44392.1 99 Micractinium conductrix PSC69871.1 A

NifU_N FdhD

B 1 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 546 Identity

Thermotoga maritima WP_010865327 NifU_N

Mastigamoeba balamuthi cytosolic NifU_N Fer2_BFD NifU AGA37363 Mastigamoeba balamuthi hydrogenosomal NifU_N Fer2_BFD NifU AGA37365 Pelomyxa schiedti Fer2_BFD Pelo14273 NifU_N NifU

Pelomyxa schiedti Pelo19958 NifU_N Fer2_BFD NifU

Pelomyxa schiedti Pelo10620 NifU_N FdhD

120 140 160 180 200 220 Identity Thermotoga maritima WP_010865327 NifU_N

Mastigamoeba balamuthi cytosolic NifU_N AGA37363 Mastigamoeba balamuthi hydrogenosomal NifU_N AGA37365 Pelomyxa schiedti Pelo14273 NifU_N

Pelomyxa schiedti Pelo19958 NifU_N

Pelomyxa schiedti Legend Pelo10620 NifU_N PLP (pyridoxal-5’-phosphate) ring phosphate-binding residue active site

C 1 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 746 Identity Thermotoga maritima WP_004082208 Aminotran_5

Mastigamoeba balamuthi cytosolic Aminotran_5 AGA37364 Mastigamoeba balamuthi hydrogenosomal Aminotran_5 AGA37366 Pelomyxa schiedti Pelo6206 Aminotran_5

Pelomyxa schiedti Pelo13211 Aminotran_5

Pelomyxa schiedti Pelo3572 Aminotran_5

Pelomyxa schiedti Pelo594 Aminotran_5

Pelomyxa schiedti Pelo19960 Aminotran_5

Pelomyxa schiedti Pelo19961 Aminotran_5

Pelomyxa schiedti Pelo14142 Aminotran_5 RHODANESE_3

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 Identity Thermotoga maritima WP_004082208 Aminotran_5

Mastigamoeba balamuthi cytosolic Aminotran_5 AGA37364 Mastigamoeba balamuthi hydrogenosomal Aminotran_5 AGA37366 Pelomyxa schiedti Pelo6206 Aminotran_5

Pelomyxa schiedti Pelo13211 Aminotran_5

Pelomyxa schiedti Pelo3572 Aminotran_5

Pelomyxa schiedti Pelo594 Aminotran_5

Pelomyxa schiedti Aminotran_5 Pelo19960

Pelomyxa schiedti Aminotran_5 Pelo19961

Pelomyxa schiedti Pelo14142 Aminotran_5

S5 Fig. Sequences of Pelomyxa schiedti components of NIF system. (A) The diagram depicts P. schiedti protein Pelo10620 composed of a NifU N-terminal domain fused to a FdhD (formate dehydrogenase accessory sulfurtransferase) C-terminal domain as determined by InterProScan. (B-C) Sequence alignment of NifU (B) and NifS (C) proteins from P. schiedti and Mastigamoeba balamuthi in comparison with bacterial homologues from Thermotoga maritima. The amino acid residues necessary for the func- tion of NifU and NifS are labeled according to the legend. Arabidopsis thaliana Q8LG88.2 99 Cavenderia fasciculata XP_004358737.1 A 100 Oryza sativa A2Z2I9 B Acytostelium subglobosum XP_012759079.1 Mus musculus Q9JHI4.2 1 substitution/site 1 substitution/site 98 100 Tieghemostelium lacteum KYQ91532.1 100 Rattus rattus Q07782.1 Dictyostelium purpureum XP_003287896.1 97 Anopheles gambiae XP_308709.4 90 96 95 Caenorhabditis elegans P32739.3 Polysphondylium violaceum KAF2072773.1 95 Caenorhabditis elegans Q93655.3 Acanthamoeba castellanii XP_004347766.1 Trichomonas vaginalis XP_001301538.1 96 Acytostelium subglobosum XP_012751245.1 Trypanosoma brucei Q383G2 100 Cavenderia fasciculata XP_004360107.1 100 Leishmania major Q4Q7W9 87 Pelomyxa schiedti Pelo12297 100 Trypanosoma cruzi Q4DX24 98 Penicillium rubens CAP93562.1 Mastigamoeba balamuthi m51a1_g9104 Yarrowia lipolytica XP_504618.1 98 Entamoeba invadens XP_004258805.1 100 PAPS Eremothecium gossypii NP_986175.2 100 Entamoeba dispar XP_001735481.1 transporter 99 100 Candida glabrata XP_ 446074.1 Entamoeba histolytica XP_649800.1 100 Kluyveromyces lactis Q6CL30 94 94 100 Saccharomyces cerevisiae EDV12253.1 100 Entamoeba nuttalli XP_008855545.1 Chloroflexus aurantiacus WP_012258584.1 Juglans regia XP_018815559.1 99 Geobacter sulfurreducens WP_010941508.1 100 Trebouxia sp. A1-2 KAA6428683.1 Deinococcus geothermalis WP_011530521.1 Chlamydomonas eustigma GAX77752.1 100 Deinococcus radiodurans WP_010888050.1 100 Volvox carteri f. nagariensis XP_002957829.1 Desulfovibrio alaskensis WP_011366777.1 100 100 100 Desulfopila aestuarii WP_073611492.1 96 Tetrabaena socialis PNH11818.1 Prochlorococcus marinus WP_012006895.1 96 Chlamydomonas reinhardtii XP_001693350.1 100 Microcystis aeruginosa NCR65667.1 Planoprotostelium fungivorum PRP81223.1 100 Acidobacteria bacterium MBA3633332.1 95 Phytophthora sojae XP_009527785.1 Balneola sp. MAO64682.1 97 Phytophthora fragariae KAE8897698.1 100 Psychroflexus halocasei WP_093241220.1 100 100 Psychroflexus sediminis WP_093365502.1 100 Phytophthora rubi KAE8972961.1 Aliifodinibius halophilus WP_165266886.1 100 Achlya hypogyna OQR87465.1 86 Actinobacteria bacterium HAM21572.1 99 Aphanomyces euteiches KAF0738165.1 92 100 Isoptericola jiangsuensis WP_170023580.1 99 Aphanomyces astaci XP_009843699.1 100 76 Serinicoccus sediminis WP_130011465.1 100 Rhodothermus profundi WP_072714492.1 Aphanomyces invadans XP_008866918.1 100 Rhodothermus marinus WP_161541558.1 Calocera viscosa KZO93361.1 Cyanobacteria bacterium QS_7_48_42 PSP02956.1 Pneumocystis jirovecii XP_018230116.1 100 97 91 100 Cyanobacteria bacterium QS_8_64_29 PSP16165.1 Lachancea dasiensis SCU98192.1 Bacteroidetes bacterium NBB84892.1 Coleophoma cylindrospora RDW57992.1 Rhodothermaceae bacterium NNF58461.1 99 90 100 Coleophoma crateriformis RDW91165.1 95 Rhodothermaceae bacterium ISCAR-4553 WP_166976191.1 96 Rhodothermales bacterium NND72020.1 100 Arthrobotrys oligospora XP_011125417.1 Planctomycetes bacterium RLT19116.1 100 Dactylellina haptotyla EPS37814.1 100 Planctomycetes bacterium RLT10804.1 100 99 Arthrobotrys entomopaga KAF3903703.1 Oceanicoccus sagamiensis WP_085756993.1 Jimgerdemannia flammicorona RUP45728.1 100 Spongiibacteraceae bacterium MBN48368.1 Mucor circinelloides EPB87258.1 Nitrosococcus oceani WP_002810411.1 95 100 95 Pleurocapsa sp. PCC_7319 WP_019507594.1 Mucor lusitanicus KAF1800512.1 Rhodopirellula sp. NQV27617.1 Lobosporangium transversale XP_021878398.1 100 Moraxellaceae bacterium PCI43004.1 96 Rhizophagus clarus GBB97305.1 Chromohalobacter canadensis WP_097022528.1 97 Gigaspora margarita KAF0474119.1 97 Gammaproteobacteria bacterium LSUCC0112 WP_134839788.1 100 95 100Gigaspora rosea RIB20826.1 100 Marinimicrobium WP_024462082.1 95 91 100 endosymbiont of Bathymodiolus puteoserpentis WP_174483485.1 Smittium mucronatum OLY79715.1 100 100 Glutamicibacter soli WP_161449450.1 Smittium culicis OMJ17861.1 Glutamicibacter WP_138178270.1 98 Piptocephalis cylindrospora RKP11551.1 100 Paeniglutamicibacter gangotriensis WP_007270563.1 99 Thamnocephalis sphaerospora RKP06229.1 96 Glutamicibacter sp. HCM95353.1 90 100 100 Glutamicibacter arilaitensis WP_013348749.1 Syncephalis pseudoplumigaleata RKP22513.1 100 Gammaproteobacteria bacterium PIQ42578.1 Mastigamoeba balamuthi m51a1_g7059 Thiothrix nivea WP_002710359.1 Porphyridium purpureum KAA8494193.1 100 100 Thiofilum flexile WP_020559741.1 Planoprotostelium fungivorum PRP82230.1 Pseudomonas mendocina WP_012018336.1 Acytostelium subglobosum XP_012755775.1 100 Gynuella sunshinyii WP_044619115.1 100 Halomonas utahensis WP_077529361.1 100 Heterostelium album XP_020436201.1 100 Marinobacter bohaiensis WP_111495795.1 97 Dictyostelium discoideum XP_642050.1 96 Polycyclovorans algicola WP_029890580.1 100 Tieghemostelium lacteum KYR03171.1 Sandaracinaceae bacterium RZO50136.1 Pelomyxa schiedti Pelo15406 Chlamydomonas reinhardtii XP_001690491.1 100 Pelomyxa schiedti Pelo10791 MCF transporter 100 Thalassiosira pseudonana XP_002295214.1 100 100 Phaeodactylum tricornutum XP_002182977.1 Anneissia japonica XP_033104648.1 99 Thalassiosira pseudonana XP_002291049.1 87 Pogona vitticeps XP_020659802.1 Gracilariopsis chorda PXF40826.1 100 100 Lacerta agilis XP_033029497.1 100 97 Chondrus crispus XP_005715956.1 97 Zootoca vivipara XP_034996844.1 100 Cyanidioschyzon merolae XP_005537069.1 98 100 Esox lucius XP_010881619.1 99 Porphyridium purpureum KAA8494383.1 100 Porphyridium purpureum KAA8494544.1 Salmo salar XP_014018743.1 100 Chlamydomonas reinhardtii XP_001697494.1 Anguilla anguilla XP_035242959.1 Pelomyxa schiedti Pelo14136 100 Arvicanthis niloticus XP_034359984.1 90 100 98 Pelomyxa schiedti Pelo6108 Lontra canadensis XP_032719224.1 Entamoeba histolytica 2 XP_654527.1 100 100 100 100 Entamoeba invadens 2 EIN_236900 Odobenus rosmarus divergens XP_004397623.1 Entamoeba histolytica 5 XP_657578.1 Gonapodya prolifera KXS11659.1 100 100 Entamoeba histolytica 4 XP_655928.2 Acanthamoeba castellanii XP_004367594.1 100 Entamoeba invadens 5 EIN_034050 Rhizoclosmatium globosum ORY29923.1 97 Entamoeba histolytica 1 XP_649603.1 Synchytrium microbalum XP_031025039.1 100 Entamoeba invadens 1 EIN_111870 99 99 Entamoeba histolytica 3 XP_654503.1 99 Powellomyces hirtus TPX58605.1 100 Entamoeba invadens 3 EIN_092780 100 Spizellomyces palustris TPX69239.1 100 100 Spirochaetaceae bacterium TVR66671.1 Spizellomyces punctatus XP_016613084.1 Alkalitalea saponilacus WP_079558865.1 Allomyces macrogynus KNE57442.1 100 Mariniphaga sp. NQU87806.1 Basidiobolus meristosporus ORX96882.1 Candidatus Marinimicrobia bacterium RKY48783.1 100 99 100 candidate division KSB1 bacterium NQT27563.1 Basidiobolus meristosporus ORX78565.1 100 Gemmatimonadetes bacterium RMH74284.1 Mortierella verticillata KFH67669.1 98 Calditrichaeota bacterium NOY59347.1 94 Coemansia reversa PIA17100.1 98 Bacteroidetes bacterium HDL19101.1 Absidia glauca SAM04867.1 100 candidate division KSB1 bacterium NQT26441.1 100 Rhizophagus irregularis XP_025175553.1 100 candidate division KSB1 bacterium RQW01313.1 100 100 Calditrichaeota bacterium RPI03489.1 Glomus cerebriforme RIA90225.1 100 candidate division KSB1 bacterium RQW05736.1 99 Gigaspora rosea RIB16551.1 99 Proteobacteria bacterium HFH49989.1 100 Diversispora epigaea RHZ71065.1 Calditrichaeota bacterium RQV94033.1 Conidiobolus coronatus KXN69980.1 100 Calditrichaeota bacterium NOX90291.1 Serendipita vermifera PVG04728.1 100 100 Caldithrix abyssi WP_006930600.1 99 Calditrichaeota bacterium RMF56933.1 100 Panaeolus cyanescens PPR06755.1 99 90 100 Candidatus Brocadia sp. WS118 TVM03170.1 82 Postia placenta XP_024341308.1 100 Planctomycetes bacterium RBG_16_41_13 OHB70029.1 82 Piloderma croceum KIM90161.1 candidate division Zixibacteria bacterium TFH62206.1 100 Dendrothele bispora THV04676.1 100 Geobacter sp. M18 WP_015719308.1 Calderihabitans maritimus WP_088555116.1 Yarrowia lipolytica XP_505035.1 Tortispora caseinolytica ODV89226.1 Campylobacter jejuni AAX33832.1 100 Aquificae bacterium NPA52247.1 Pichia kudriavzevii XP_029321648.1 100 Persephonella marina WP_012676880.1 Metschnikowia bicuspidata XP_018715041.1 100 98 100 Aquificae bacterium NPA16811.1 Meyerozyma sp. JA9 RLV87792.1 98 Balnearium lithotrophicum WP_142935281.1 99 100 100 Meyerozyma guilliermondii XP_001485805.1 100 Persephonella sp. IF05-L8 WP_029520541.1 Bacteroidetes bacterium PCH92130.1 98 Candida albicans KHC53548.1 98 93 Prosthecochloris aestuarii WP_012506299.1 99 Debaryomyces fabryi XP_015468287.1 100 Chlorobi bacterium NOY50625.1 100 Debaryomyces hansenii XP_002770777.1 Bacteroidetes bacterium RLD83627.1 92 Bacteroidetes bacterium 4572_128 OQX96976.1 100 Bacteroidetes bacterium GWF2_38_335 OFY36551.1 86 Bacteroidetes bacterium NQT76752.1 98 Bacteroidetes bacterium GWA2_30_7 OFX44550.1 78 Bacteroidetes bacterium GWF2_33_38 OFY21265.1 99 95 Ancylomarina subtilis WP_130306493.1 Marinilabilia salmonicolor WP_106153004.1 100 Carboxylicivirga sp. M1479 WP_143303520.1 98 Marinilabiliaceae bacterium JC017 WP_130642794.1 S6 Fig. Phylogenetic analysis of transporters involved in the 100 Chlorobi bacterium NPA36999.1 Lactobacillus sp. CBA3606 WP_105448221.1 sulfate activation pathway. (A) The phylogenetic analysis did not 100 Lactobacillus garii WP_125072278.1 100 Lactobacillus paraplantarum WP_131509675.1 resolve which one of the sodium/sulfate symporters of Pelomyxa 78 100 Lactobacillus plantarum WP_076661448.1 Anaerobacillus alkalidiazotrophicus WP_084788055.1 schiedti is related to the Entamoeba histolytica mitosomal trans- 100 Alkalihalobacillus halodurans WP_010899525.1 100 Bacillus sp. FJAT-45385 WP_088103472.1 100 Metabacillus litoralis WP_066335803.1 porter. (B) The Maximum Likelihood phylogenetic tree confirms 100 Cytobacillus firmus WP_159346166.1 98 100 Bacillus sp. ZZV12-4809 WP_159866473.1 one P. schiedti transporter as PAPS (3’-phosphoadenosine 5’-phos- 100 Cytobacillus firmus WP_082139047.1 100 Alkalihalobacillus bogoriensis WP 026672233.1 phosulfate) transporter, while two others belong to a broader mito- Bacillus sp. FJAT-45385 WP_088102112.1 100 Sinobaca qinghaiensis WP_120192872.1 chondrial carrier family of transporters. Experimentally proven 100 Alkalihalobacillus akibai WP_035667754.1 93 Desulfuribacillus alkaliarsenatis WP_069643402.1 mitosomal transporters of E. histolytica are marked with stars. 90 Bacillus thuringiensis WP_097912362.1 Bacillus salarius WP_125553532.1 97 Bacillus vietnamensis WP_148954132.1 Ultrafast bootstrap support values are shown when 75%. 97 Salicibibacter halophilus WP_142090428.1 98 Bacillus taeanensis WP_113806732.1 100 Bacillus oryziterrae WP_017756531.1