Supplementary Information for

New organelles illuminate the origins of Trichomonas hydrogenosomes and Giardia mitosomes

Leger, Kolisko, Kamikawa, et al.

Supplementary Table 1. Glycine cleavage system protein distribution in metabolically reduced MROs.

Supplementary Figure 1. PhyloBayes phylogeny of eukaryotes based on 159 proteins.

Supplementary Figure 2. RAxML phylogeny of eukaryotes based on 159 proteins.

Supplementary Figure 3. IQ-TREE phylogeny of eukaryotes based on 159 proteins.

Supplementary Figure 4. Single-gene phylogeny of Glycine cleavage system P protein P1 domain.

Supplementary Figure 5. Single-gene phylogeny of Glycine cleavage system P protein P2 domain.

Supplementary Figure 6. Single-gene phylogeny of Glycine cleavage system T protein.

Supplementary Figure 7. Single-gene phylogeny of Glycine cleavage system H protein.

Supplementary Figure 8. Single-gene phylogeny of GCS L protein.

Supplementary Figure 9. Single-gene phylogeny of HydE.

Supplementary Figure 10. Single-gene phylogeny of HydF.

Supplementary Figure 11. Single-gene phylogeny of HydG.

Supplementary Figure 12. Single-gene phylogeny of [FeFe]-hydrogenase, HydA.

Supplementary Figure 13. Single-gene phylogeny of SCS alpha subunit. Supplementary Figure 14. Single-gene phylogeny of SCS beta subunit.

Supplementary Figure 15. Single-gene phylogeny of ASCT 1B proteins.

Supplementary Figure 16. Single-gene phylogeny of ASCT 1C proteins.

Supplementary Figure 17. Single-gene phylogeny of ACS1.

Supplementary Figure 18. Single-gene phylogeny of ACS2.

Supplementary Figure 19. Single-gene phylogeny of eukaryotic Cardiolipin synthase CLS_cap.

Supplementary Figure 20. Single-gene phylogeny of aminoadipate-semialdehyde dehydrogenase.

Supplementary Table 1 - Glycine cleavage system protein distribution in metabolically reduced MROs

GCS 1 2 Survey Life styles Organisms MROs H T P L Discoba Stygiella incarcerata H Transcriptome Free-living + + + + Sawyeria marylandensis H Transcriptome Free-living + + + +

Fungi Encephalitozoon cuniculi M Genome Parasitic - - - - Piromyces sp. H Transcriptome Commensal - - - -

Pygsuia biforma H Transcriptome Free-living + + + +

Amoebozoa Entamoeba histolytica M Genome Parasitic - - - - Mastigamoeba balamuthi H Transcriptome Free-living + + + +

Rhizaria Mikrocytos mackini M Transcriptome Parasitic - - - -

Alveolata Cryptosporidium parvum M Genome Parasitic - - - -

Stramenopiles Cantina marsupialis H Transcriptome Free-living + + + +

1 Organisms with MROs lacking genomes, with highly reduced metabolic functions 2 hydrogenosome (H), and mitosome (M)

This table is based on Stairs et al. (2015) with additional, recently published data from Leger et al. (2016) and Noguchi et al. (2015) Trimastix marina Paratrimastix pyriformis Monocercomonoides sp. Excavata Tritrichomonas foetus Trichomonas vaginalis Pentatrichomonas hominis Carpediemonas membranifera Ergobibamus cyprinoides Aduncisulcus paluster Chilomastix cuspidata Chilomastix caulleri Kipferlia bialata Dysnectes brevis Giardia intestinalis 0.94 Trepomonas sp. Spironucleus vortens Spironucleus salmonicida Spironucleus barkhanus Malawimonas jakobiformis Malawimonas californiana Collodictyon triciliatum Acanthamoeba castellanii Mastigamoeba balamuthi Physarum polycephalum Amorphea Dictyostelium discoideum Thecamonas trahens Nuclearia simplex Spizellomyces punctatus Blastocladiella emersonii Rhizopus oryzae Ustiago maydis Neurospora crassa Sphaeroforma arctica Capsaspora owczarzaki Salpingoeca rosetta Monosiga brevicolis Homo sapiens 0.81 Drosophila melanogaster Trichoplax adhaerens Nematostella vectensis 0.54 Amphmedon queenlandica Stygiella incarcerata Andalucia godoyi Jakoba bahamensis Seculamonas ecuadorensis Excavata Jakoba libera Reclinomonas americana Histiona aroides Tsukubamonas globosa Naegleria gruberi Stachyamoeba lipophora Sawyeria marylandensis Peranema trichophorum Euglena gracilis Diplonema papillatum Trypanosoma brucei Phytomonas serpens Leishmania major Glaucocystis nostochinearum Telonema subtilis Cyanidioschyzon merolae Porphyridium cruentum Diaphoretickes Calliarthron tuberculosum Gracilaria changii 0.63 Chondrus crispus Guillardia theta Pavlova lutheri Prymnesium parvum Isochrysis galbana Emiliania huxleyi Oryza sativa Arabidopsis thaliana Micromonas sp. 0.62 Polytomella parva Chlamydomonas reinhardtii 0.62 Reticulomyxa filosa Cercomonas longicauda Bigelowiella natans Blastocystis hominis Phytophthora ramorum Thalassiosira pseudonana Phaeodactylum tricornatum Oxytricha trifallax Tetrahymena thermophila Paramecium tetraurelia Perkinsus marinus Oxyrrhis marina Karenia brevis Alexandrium tamarense Cryptosporidium parvum Toxoplasma gondii Sarcocystis neurona Theileria annulata Plasmodium falciparum

0.4

Supplementary figure 1

PhyloBayes phylogeny of eukaryotes based on 159 proteins. The analysis was performed on 94 taxa and 39,089 sites, under the CAT-GTR model incorporating among-site rate variation approximated by a discrete gamma distribution with four categories (CAT-GTR + Γ model). Bayesian posterior probabilities (BPPs) are shown on nodes when BPPs are smaller than 1.0. Andalucia godoyi 99 Stygiella incarcerata Jakoba bahamensis Seculamonas ecuadorensis Jakoba libera 96 Histiona aroides 83 Reclinomonas americana Tsukubamonas globosa Naegleria gruberi 84 Stachyamoeba lipophora 98 Sawyeria marylandensis Peranema trichophorum 98 Euglena gracilis Diplonema papillatum Trypanosoma brucei Leishmania major Phytomonas serpens Trimastix marina Monocercomonoides sp. Paratrimastix pyriformis Excavata Tritrichomonas foetus Trichomonas vaginalis Pentatrichomonas hominis Carpediemonas membranifera Ergobibamus cyprinoides Aduncisulcus paluster 88 Chilomastix caulleri Chilomastix cuspidata Kipferlia bialata Dysnectes brevis Giardia intestinalis 98 Spironucleus vortens Trepomonas sp. Spironucleus barkhanus Spironucleus salmonicida Malawimonas jakobiformis 87 Malawimonas californiana Collodictyon triciliatum Acanthamoeba castellanii 98 98 Mastigamoeba balamuthi Physarum polycephalum Amorphea Dictyostelium discoideum Thecamonas trahens 87 Nuclearia simplex 84 Blastocladiella emersonii Spizellomyces punctatus Rhizopus oryzae Neurospora crassa Ustiago maydis Sphaeroforma arctica Capsaspora owczarzaki Monosiga brevicolis Salpingoeca rosetta Amphmedon queenlandica 47 Nematostella vectensis Trichoplax adhaerens 41 Homo sapiens Drosophila melanogaster Toxoplasma gondii Sarcocystis neurona Plasmodium falciparum Theileria annulata Diaphoretickes Cryptosporidium parvum Perkinsus marinus Oxyrrhis marina Karenia brevis 98 Alexandrium tamarense Oxytricha trifallax 94 Paramecium tetraurelia Tetrahymena thermophila Blastocystis hominis Phytophthora ramorum Phaeodactylum tricornatum Thalassiosira pseudonana 49 Reticulomyxa filosa Bigelowiella natans Cercomonas longicauda Oryza sativa Arabidopsis thaliana 73 Micromonas sp. 51 Chlamydomonas reinhardtii 33 Polytomella parva Glaucocystis nostochinearum Guillardia theta Pavlova lutheri 53 Prymnesium parvum Emiliania huxleyi 84 Isochrysis galbana Telonema subtilis 78 Chondrus crispus Gracilaria changii 36 Calliarthron tuberculosum Porphyridium cruentum Cyanidioschyzon merolae

0.2 Supplementary figure 2

RAxML phylogeny of eukaryotes based on 159 proteins. The analysis was performed on 94 taxa and 39,089 sites, under the LG model incorporating empirical amino acid frequencies and among-site rate variation approximated by a discrete gamma distribution with four categories (LG + Γ + F model). ML bootstrap values are shown on nodes when ML bootstrap values are smaller than 100. Andalucia godoyi Stygiella incarcerata Jakoba bahamensis Seculamonas ecuadorensis Jakoba libera 99 Histiona aroides 97 Reclinomonas americana Tsukubamonas globosa Naegleria gruberi Sawyeria marylandensis 99 Stachyamoeba lipophora Peranema trichophorum 72 Euglena gracilis Diplonema papillatum Leishmania major Phytomonas serpens Trypanosoma brucei Trimastix marina Paratrimastix pyriformis Monocercomonoides sp. Excavata Tritrichomonas foetus Trichomonas vaginalis Pentatrichomonas hominis Carpediemonas membranifera Ergobibamus cyprinoides Aduncisulcus paluster 99 Chilomastix cuspidata Chilomastix caulleri Kipferlia bialata Dysnectes brevis Giardia intestinalis Spironucleus barkhanus Spironucleus salmonicida Trepomonas sp. Spironucleus vortens Malawimonas jakobiformis 94 Malawimonas californiana Collodictyon triciliatum Nuclearia simplex Blastocladiella emersonii Spizellomyces punctatus Amorphea Neurospora crassa Ustiago maydis Rhizopus oryzae 99 Monosiga brevicolis Salpingoeca rosetta 87 Trichoplax adhaerens 71 Nematostella vectensis Drosophila melanogaster Homo sapiens Amphmedon queenlandica Capsaspora owczarzaki 99 Sphaeroforma arctica Thecamonas trahens Acanthamoeba castellanii Physarum polycephalum Dictyostelium discoideum Mastigamoeba balamuthi Toxoplasma gondii Sarcocystis neurona Plasmodium falciparum Theileria annulata Diaphoretickes Cryptosporidium parvum 99 Oxyrrhis marina Alexandrium tamarense Karenia brevis Perkinsus marinus Tetrahymena thermophila 96 Paramecium tetraurelia Oxytricha trifallax Thalassiosira pseudonana Phaeodactylum tricornatum Phytophthora ramorum Blastocystis hominis Bigelowiella natans Cercomonas longicauda Reticulomyxa filosa Arabidopsis thaliana Oryza sativa Chlamydomonas reinhardtii Polytomella parva 87 51 Micromonas sp. Prymnesium parvum 34 Isochrysis galbana Emiliania huxleyi 51 Pavlova lutheri Guillardia theta Glaucocystis nostochinearum Telonema subtilis 99 Calliarthron tuberculosum 55 Gracilaria changii 96 Chondrus crispus Porphyridium cruentum Cyanidioschyzon merolae

0.3 Supplementary figure 3

IQ-TREE phylogeny of eukaryotes based on 159 proteins. The analysis was performed on 94 taxa and 39,089 sites, under the C60 model incorporating empirical amino acid frequencies and among-site rate variation approximated by a discrete gamma distribution with four categories (C60 + Γ + F model). ML bootstrap values are shown on nodes when ML bootstrap values are smaller than 100. 326510657_Eukaryota_Viridiplantae_Streptophyta_Hordeum_vulgare_subsp__vulgare 193709318_Eukaryota_Metazoa_Arthropoda_Acyrthosiphon_pisum 41054671_Eukaryota_Metazoa_Chordata_Danio_rerio 432872891_Eukaryota_Metazoa_Chordata_Oryzias_latipes 658866763_Eukaryota_Metazoa_Chordata_Poecilia_reticulata 665799472_Eukaryota_Metazoa_Arthropoda_Microplitis_demolitor 195449363_Eukaryota_Metazoa_Arthropoda_Drosophila_willistoni 194902396_Eukaryota_Metazoa_Arthropoda_Drosophila_erecta 195499748_Eukaryota_Metazoa_Arthropoda_Drosophila_yakuba 678313140_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 403376783_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 145535149_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 145511750_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 551644570_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 528245459_Eukaryota_Kinetoplastida_Trypanosomatidae_Angomonas_deanei 154339095_Eukaryota_Kinetoplastida_Trypanosomatidae_Leishmania_braziliensis_MHOM/BR/75/M2904 342181850_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_congolense_IL3000 340054536_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_vivax_Y486 340054537_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_vivax_Y486 449475247_Eukaryota_Viridiplantae_Streptophyta_Cucumis_sativus

GCS P protein

470467303_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 448100465_Eukaryota_Fungi_Dikarya_Millerozyma_farinosa_CBS_7064 646299148_Eukaryota_Fungi_Dikarya_Botryobasidium_botryosum_FD-172_SS1 353239171_Eukaryota_Fungi_Dikarya_Piriformospora_indica_DSM_11827 330798849_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 470252808_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 281212642_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 544216783_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 545700668_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 546316948_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 546301853_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 3334198_Eukaryota_Viridiplantae_Streptophyta_Flaveria_anomala 1346117_Eukaryota_Viridiplantae_Streptophyta_Flaveria_pringlei 1346116_Eukaryota_Viridiplantae_Streptophyta_Flaveria_pringlei 569394604_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 294947352_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 300122016_Eukaryota_Blastocystis_Blastocystis 118375474_Eukaryota_Intramacronucleata_Oligohymenophorea_Tetrahymena_thermophila 290991213_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 298708947_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 219124701_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055/1 397566636_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_oceanica 223998052_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 574121161_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_astaci 673024746_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 669141080_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 641536477_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 325185825_Eukaryota_Albuginales_Albuginaceae_Albugo_laibachii_Nc14 635366108_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 568045997_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica 675198241_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_INRA-310 566020280_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P1569

100

gnl|Paratrimastix_pyriformis|879995 49 gnl|Trimastix_PCT|1244727 gnl|Chilomastix_cuspidata|395324 gnl|Carpediemonas_membranifera|2966 gnl|Kipferlia_bialata|580943 gnl|Aduncisulcus_paluster|88314 75 gnl|Ergobibamus_cyprinoides|535560 gnl|Dysnectes_brevis|920294

GCS P1

0.4

Supplementary figure 4

Single-gene phylogeny of Glycine cleavage system P protein P1 domain (294 taxa and 266 sites). Mitochondrial P protein sequences containing both P1 and P2 domains are highlighted in red. GCS P protein sequences of Metamonada containing only the P1 domain are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonads are recovered as a monophyletic group distantly related to mitochondrial P protein sequences. The GCS P1 tree showing detailed taxa and ML bootstrap support is also attached as part of Supplementary Data 2. 470467303_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 290991213_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 569394605_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa

449475247_Eukaryota_Viridiplantae_Streptophyta_Cucumis_sativus

678313140_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 403376783_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 118375474_Eukaryota_Intramacronucleata_Oligohymenophorea_Tetrahymena_thermophila 145511750_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 145535149_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 340054537_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_vivax_Y486 342181850_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_congolense_IL3000 154339095_Eukaryota_Kinetoplastida_Trypanosomatidae_Leishmania_braziliensis_MHOM/BR/75/M2904 528245459_Eukaryota_Kinetoplastida_Trypanosomatidae_Angomonas_deanei 294947352_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 300122016_Eukaryota_Blastocystis_Blastocystis 298708947_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 219124701_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055/1 397566636_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_oceanica 223998052_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 669141080_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 GCS P protein 641536477_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 673024746_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 574121161_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_astaci 635366108_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 325185825_Eukaryota_Albuginales_Albuginaceae_Albugo_laibachii_Nc14 566020280_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P1569 675198241_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_INRA-310 568045997_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica 330798849_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 470252808_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 281212642_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500

3334198_Eukaryota_Viridiplantae_Streptophyta_Flaveria_anomala 1346117_Eukaryota_Viridiplantae_Streptophyta_Flaveria_pringlei 1346116_Eukaryota_Viridiplantae_Streptophyta_Flaveria_pringlei 545700668_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 546316948_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 32394466_Eukaryota_Florideophyceae_Ceramiales_Griffithsia_japonica 544216783_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 569394604_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 551644570_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 448100465_Eukaryota_Fungi_Dikarya_Millerozyma_farinosa_CBS_7064 353239171_Eukaryota_Fungi_Dikarya_Piriformospora_indica_DSM_11827 646299148_Eukaryota_Fungi_Dikarya_Botryobasidium_botryosum_FD-172_SS1 41054671_Eukaryota_Metazoa_Chordata_Danio_rerio 658866763_Eukaryota_Metazoa_Chordata_Poecilia_reticulata 432872891_Eukaryota_Metazoa_Chordata_Oryzias_latipes 195449363_Eukaryota_Metazoa_Arthropoda_Drosophila_willistoni 194902396_Eukaryota_Metazoa_Arthropoda_Drosophila_erecta 195499748_Eukaryota_Metazoa_Arthropoda_Drosophila_yakuba 665799472_Eukaryota_Metazoa_Arthropoda_Microplitis_demolitor 326510657_Eukaryota_Viridiplantae_Streptophyta_Hordeum_vulgare_subsp__vulgare 193709318_Eukaryota_Metazoa_Arthropoda_Acyrthosiphon_pisum

gnl|Paratrimastix_pyriformis|873654 gnl|Trimastix_PCT|1242417 46 gnl|Chilomastix_cuspidata|394845 gnl|Kipferlia_bialata|584777 89 gnl|Dysnectes_brevis|920197 gnl|Ergobibamus_cyprinoides|545724 gnl|Carpediemonas_membranifera|2539 gnl|Aduncisulcus_paluster|83196 57 gnl|Ergobibamus_cyprinoides|545725

97

GCS P2

1.0 Supplementary figure 5

Single-gene phylogeny of Glycine cleavage system P protein P2 domain (288 taxa and 291 sites). Mitochondrial P protein sequences containing both P1 and P2 domains are highlighted in red. GCS P protein sequences of Metamonada containing only the P2 domain are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences are recovered as a monophyletic group distantly related to mitochondrial P protein sequences. The GCS P2 tree showing detailed taxa and ML bootstrap support is also attached as part of Supplementary Data 2. GCS T protein

gnl|Kipferlia_bialata|590055 290991875_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 403367101_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 678346061_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 71409827_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_cruzi_strain_CL_Brener 557861002_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_cruzi_Dm28c 407837782_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_cruzi 32 281210648_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 330845252_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 66801565_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_discoideum_AX4 471226067_Eukaryota_Intramacronucleata_Oligohymenophorea_Ichthyophthirius_multifiliis 586731638_Eukaryota_Intramacronucleata_Oligohymenophorea_Tetrahymena_thermophila_SB210 118378042_Eukaryota_Intramacronucleata_Oligohymenophorea_Tetrahymena_thermophila 300124013_Eukaryota_Blastocystis_Blastocystis 300121816_Eukaryota_Blastocystis_Blastocystis 298712644_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 38481924_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_weissflogii 224006530_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 294896047_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294955718_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 673014032_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 669140642_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 641531749_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 695417962_Eukaryota_Peronosporales_Phytophthora_Phytophthora_sojae 635362869_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 675213457_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_INRA-310 567970118_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica 221057452_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_knowlesi_strain_H 470510820_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff gnl|Aduncisulcus_paluster|92012 41 gnl|Carpediemonas_membranifera|8642 gnl|Trimastix_PCT|1243061 gnl|Paratrimastix_pyriformis|880253 gnl|Ergobibamus_cyprinoides|534428 gnl|Chilomastix_cuspidata|394703 71 gnl|Dysnectes_brevis|926082 gnl|Kipferlia_bialata|583546 551649802_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 551544804_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 544210982_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 546316787_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 545701658_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 545700496_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 569413294_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 674912111_Eukaryota_Viridiplantae_Streptophyta_Brassica_napus 593781857_Eukaryota_Viridiplantae_Streptophyta_Phaseolus_vulgaris: 470114201_Eukaryota_Viridiplantae_Streptophyta_Fragaria_vesca_subsp__vesca 470363828_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864 586486358_Eukaryota_Metazoa_Chordata_Chrysochloris_asiatica 655834496_Eukaryota_Metazoa_Chordata_Oryctolagus_cuniculus 297671332_Eukaryota_Metazoa_Chordata_Pongo_abelii 646293744_Eukaryota_Fungi_Dikarya_Botryobasidium_botryosum_FD-172_SS1 443920474_Eukaryota_Fungi_Dikarya_Rhizoctonia_solani_AG-1_IA 471906132_Eukaryota_Fungi_Dikarya_Rhizoctonia_solani_AG-1_IB 511000252_Eukaryota_Fungi_Mucoromycotina_Mucor_circinelloides_f__circinelloides_1006PhL 671692085_Eukaryota_Fungi_Mucoromycotina_Absidia_idahoensis_var__thermophila 661175787_Eukaryota_Fungi_Mucoromycotina_Lichtheimia_corymbifera_JMRC_FSU_9682

0.4 Supplementary figure 6

Single-gene phylogeny of Glycine cleavage system T protein (288 taxa and 190 sites). Mitochondrial T protein sequences are highlighted in red. Metamonad sequences are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences show phylogenetic affinity to mitochondrial sequences. The T protein tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. GCS H protein

330804371_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 0 470261759_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 66806643_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_discoideum_AX4 281211111_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 8

300175727_Eukaryota_Blastocystis_Blastocystis_hominis_Blastocystis_hominis gnl_Ergobibamus_cyprinoides_557844_unnamed_protein_product gnl_Carpediemonas_membranifera_18094_unnamed_protein_product gnl_Carpediemonas_membranifera_1116_unnamed_protein_product 0 gnl_Carpediemonas_membranifera_22777_unnamed_protein_product gnl_Kipfelria_bialata_603136_unnamed_protein_product 0 gnl_Kipfelria_bialata_589992_unnamed_protein_product 20 gnl_Kipfelria_bialata_592784_unnamed_protein_product gnl_Kipfelria_bialata_577229_unnamed_protein_product gnl_Dysnectes_brevis_921267_unnamed_protein_product 0 gnl_Chilomastix_cuspidata_394760_unnamed_protein_product 0 gnl_Aduncisulcus_paluster_85079_unnamed_protein_product 298712643_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 641541279_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 669138736_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 673027680_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 635370810_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 325179991_Eukaryota_Albuginales_Albuginaceae_Albugo_laibachii_Nc14 397620001_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_oceanica 224003395_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 219111939_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055_1 553183964_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana_CCMP526 290985880_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG_M 551617016_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 672825193_Eukaryota_Fungi_Mortierellomycotina_Mortierella_verticillata_NRRL_6337 552918230_Eukaryota_Fungi_Glomeromycota_Rhizophagus_irregularis_DAOM_181602 213408016_Eukaryota_Fungi_Dikarya_Schizosaccharomyces_japonicus_yFS275 528319252_Eukaryota_Fungi_Dikarya_Schizosaccharomyces_cryophilus_OY26 19112961_Eukaryota_Fungi_Dikarya_Schizosaccharomyces_pombe_972h_ 313212247_Eukaryota_Metazoa_Chordata_Oikopleura_dioica 684159748_Eukaryota_Fungi_Dikarya_Exophiala_dermatitidis_NIH_UT8656 628274066_Eukaryota_Fungi_Dikarya_Capronia_coronata_CBS_617_96 549050119_Eukaryota_Fungi_Dikarya_Pyronema_omphalodes_CBS_100304 326473650_Eukaryota_Fungi_Dikarya_Trichophyton_tonsurans_CBS_112818 242806739_Eukaryota_Fungi_Dikarya_Talaromyces_stipitatus_ATCC_10500 627802785_Eukaryota_Fungi_Dikarya_Baudoinia_compniacensis_UAMH_10762 671683938_Eukaryota_Fungi_Mucoromycotina_Absidia_idahoensis_var__thermophila 511010218_Eukaryota_Fungi_Mucoromycotina_Mucor_circinelloides_f__circinelloides_1006PhL 384500799_Eukaryota_Fungi_Mucoromycotina_Rhizopus_delemar_RA_99_880 575473726_Eukaryota_Fungi_Chytridiomycota_Batrachochytrium_dendrobatidis_JAM81 331215111_Eukaryota_Fungi_Dikarya_Puccinia_graminis_f__sp__tritici_CRL_75_36_700_3 636603501_Eukaryota_Fungi_Dikarya_Trametes_versicolor_FP_101664_SS1 540386660_Eukaryota_Fungi_Dikarya_Cryptococcus_neoformans_var__grubii_H99 58262332_Eukaryota_Fungi_Dikarya_Cryptococcus_neoformans_var__neoformans_JEC21 597858119_Eukaryota_Metazoa_Nematoda_Ancylostoma_ceylanicum 560140126_Eukaryota_Metazoa_Nematoda_Haemonchus_contortus 118348286_Eukaryota_Intramacronucleata_Oligohymenophorea_Tetrahymena_thermophila 678311558_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 403333119_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 261331496_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_brucei_gambiense_DAL972 594146702_Eukaryota_Kinetoplastida_Trypanosomatidae_Phytomonas_sp__isolate_Hart1 528244034_Eukaryota_Kinetoplastida_Trypanosomatidae_Strigomonas_culicis 514692046_Eukaryota_Choanoflagellida_Salpingoecidae_Salpingoeca_rosetta 589947462_Eukaryota_Metazoa_Chordata_Peromyscus_maniculatus_bairdii 507623067_Eukaryota_Metazoa_Chordata_Octodon_degus 319997258_Eukaryota_Dinophyceae_Gymnodiniales_Karlodinium_veneficum 294875663_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 524898272_Eukaryota_Metazoa_Mollusca_Aplysia_californica 65 gnl_Trimastix_PCT_1241548_unnamed_protein_product gnl_Paratrimastix_pyriformis_888371_unnamed_protein_product 449672450_Eukaryota_Metazoa_Cnidaria_Hydra_vulgaris

390335598_Eukaryota_Metazoa_Echinodermata_Strongylocentrotus_purpuratus 552819238_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 545709259_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 544210717_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 551650956_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 340374383_Eukaryota_Metazoa_Porifera_Amphimedon_queenslandica 470525390_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 168056222_Eukaryota_Viridiplantae_Streptophyta_Physcomitrella_patens 565493248_Eukaryota_Viridiplantae_Streptophyta_Capsella_rubella 297823331_Eukaryota_Viridiplantae_Streptophyta_Arabidopsis_lyrata_subsp__lyrata 674949363_Eukaryota_Viridiplantae_Streptophyta_Brassica_napus

123469487_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl_Trichomonas_vaginalis_861861_unnamed_protein_product 123388076_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 9 gnl_Trichomonas_vaginalis_847475_unnamed_protein_product

0.3 Supplementary figure 7

Single-gene phylogeny of Glycine cleavage system H protein (342 taxa and 102 sites). Mitochondrial H protein sequences are highlighted in red. Metamonad sequences are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences other than T. vaginalis and S. salmonicida show phylogenetic affinity to mitochondrial sequences. The H protein tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 82593766_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_yoelii_yoelii_17XNL 577151510_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_petteri GCS L protein 669195506_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_vinckei

290995925_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG_M 209876490_Eukaryota_Apicomplexa_Coccidia_Cryptosporidium_muris_RN66 551652903_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 569383692_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 569383691_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 569405978_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa

300121010_Eukaryota_Blastocystis_Blastocystis

gnl_Pentatrichomonas_hominis_648526 gnl_Pentatrichomonas_hominis_633489 528214552_Eukaryota_Kinetoplastida_Trypanosomatidae_Strigomonas_culicis

99 154420811_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3

gnl_Trichomonas_vaginalis_855420

gnl_Pentatrichomonas_hominis_641513 145550163_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4_2 145543566_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4_2 340059892_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_vivax_Y486 123975551_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 401427397_Eukaryota_Kinetoplastida_Trypanosomatidae_Leishmania_mexicana_MHOM_GT_2001_U1103 452769520_Eukaryota_Kinetoplastida_Trypanosomatidae_Leptomonas_pyrrhocoris gnl_Trichomonas_vaginalis_871638 544214030_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 546308624_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 545710970_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 471230218_Eukaryota_Intramacronucleata_Oligohymenophorea_Ichthyophthirius_multifiliis 471236496_Eukaryota_Intramacronucleata_Oligohymenophorea_Ichthyophthirius_multifiliis 678330436_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 403346640_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax gnl_Aduncisulcus_paluster_210933 403372042_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 678337190_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 676387517_Eukaryota_Pelagophyceae_Aureococcus_Aureococcus_anophagefferens 556759874_Eukaryota_Metazoa_Chordata_Pantholops_hodgsonii 299469809_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 585112196_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 219116322_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055_1 397576396_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_oceanica 528249914_Eukaryota_Kinetoplastida_Trypanosomatidae_Strigomonas_culicis 241645399_Eukaryota_Metazoa_Arthropoda_Ixodes_scapularis 224013650_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 gnl_Dysnectes_brevis_957947 566021407_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P1569 570981898_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P10297 449474614_Eukaryota_Viridiplantae_Streptophyta_Cucumis_sativus 570325023_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P1976 498977504_Eukaryota_Metazoa_Arthropoda_Ceratitis_capitata 551605552_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 641530302_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 669150834_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 325188453_Eukaryota_Albuginales_Albuginaceae_Albugo_laibachii_Nc14 635362235_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 557139426_Eukaryota_Apicomplexa_Coccidia_Eimeria_tenella 608650849_Eukaryota_Apicomplexa_Gregarinasina_Gregarina_niphandrodes 656187255_Eukaryota_Apicomplexa_Aconoidasida_Babesia_bigemina 672258934_Eukaryota_Apicomplexa_Coccidia_Toxoplasma_gondii_p89 557737356_Eukaryota_Apicomplexa_Coccidia_Toxoplasma_gondii_VEG 384495270_Eukaryota_Fungi_Mucoromycotina_Rhizopus_delemar_RA_99_880 575476766_Eukaryota_Fungi_Chytridiomycota_Batrachochytrium_dendrobatidis_JAM81 gnl_Dysnectes_brevis_955406 672818725_Eukaryota_Fungi_Mortierellomycotina_Mortierella_verticillata_NRRL_6337 552909835_Eukaryota_Fungi_Glomeromycota_Rhizophagus_irregularis_DAOM_181602 595486653_Eukaryota_Fungi_Glomeromycota_Rhizophagus_irregularis_DAOM_197198w 630208956_Eukaryota_Fungi_Dikarya_Moniliophthora_roreri_MCA_2997 597938090_Eukaryota_Fungi_Dikarya_Serpula_lacrymans_var__lacrymans_S7_9 630359871_Eukaryota_Fungi_Dikarya_Gloeophyllum_trabeum_ATCC_11539 465795370_Eukaryota_Fungi_Dikarya_Malassezia_sympodialis_ATCC_42132 589279240_Eukaryota_Fungi_Dikarya_Tremella_mesenterica_DSM_1558 576990320_Eukaryota_Fungi_Dikarya_Rhizoctonia_solani_AG_3_Rhs1AP 557996453_Eukaryota_Fungi_Dikarya_Pseudozyma_brasiliensis_GHG001 19114408_Eukaryota_Fungi_Dikarya_Schizosaccharomyces_pombe_972h_ 213407356_Eukaryota_Fungi_Dikarya_Schizosaccharomyces_japonicus_yFS275 401625928_Eukaryota_Fungi_Dikarya_Saccharomyces_arboricola_H_6 259146172_Eukaryota_Fungi_Dikarya_Saccharomyces_cerevisiae_EC1118 gnl_Dysnectes_brevis_955485 448104498_Eukaryota_Fungi_Dikarya_Millerozyma_farinosa_CBS_7064 326530778_Eukaryota_Viridiplantae_Streptophyta_Hordeum_vulgare_subsp__vulgare 615438098_Eukaryota_Fungi_Dikarya_Colletotrichum_fioriniae_PJ7 636592069_Eukaryota_Fungi_Dikarya_Setosphaeria_turcica_Et28A 477587302_Eukaryota_Fungi_Dikarya_Bipolaris_maydis_ATCC_48331 Stygiella_incarcerata_____ANM86801_1 Mastigamoeba_balamuthi_____AJE29367_1 47600753_Eukaryota_Euglenida_Euglenales_Euglena_gracilis Mastigamoeba_balamuthi_____AJE29366_1 gnl_Paratrimastix_pyriformis_883331 470514645_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 470266597_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 470267275_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 66802500_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_discoideum_AX4 330795096_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 145352044_Eukaryota_Viridiplantae_Chlorophyta_Ostreococcus_lucimarinus_CCE9901 196017865_Eukaryota_Metazoa_Placozoa_Trichoplax_adhaerens 303285081_Eukaryota_Viridiplantae_Chlorophyta_Micromonas_pusilla_CCMP1545 556745728_Eukaryota_Metazoa_Chordata_Pantholops_hodgsonii 569377009_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 556748518_Eukaryota_Metazoa_Chordata_Pantholops_hodgsonii 255085931_Eukaryota_Viridiplantae_Chlorophyta_Micromonas_sp__RCC299 552813474_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 633909477_Eukaryota_Viridiplantae_Chlorophyta_Helicosporidium_sp__ATCC_50920 556742469_Eukaryota_Metazoa_Chordata_Pantholops_hodgsonii 356565179_Eukaryota_Viridiplantae_Streptophyta_Glycine_max 475592887_Eukaryota_Viridiplantae_Streptophyta_Aegilops_tauschii 326517553_Eukaryota_Viridiplantae_Streptophyta_Hordeum_vulgare_subsp__vulgare 545357906_Eukaryota_Viridiplantae_Chlorophyta_Coccomyxa_subellipsoidea_C_169 675353328_Eukaryota_Viridiplantae_Chlorophyta_Auxenochlorella_protothecoides 302846791_Eukaryota_Viridiplantae_Chlorophyta_Volvox_carteri_f__nagariensis 159474092_Eukaryota_Viridiplantae_Chlorophyta_Chlamydomonas_reinhardtii 694540145_Eukaryota_Fonticula_Fonticula

514483999_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864 470293148_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864 308467098_Eukaryota_Metazoa_Nematoda_Caenorhabditis_remanei 560138635_Eukaryota_Metazoa_Nematoda_Haemonchus_contortus 560134328_Eukaryota_Metazoa_Nematoda_Haemonchus_contortus 308803422_Eukaryota_Viridiplantae_Chlorophyta_Ostreococcus_tauri 156408155_Eukaryota_Metazoa_Cnidaria_Nematostella_vectensis 552823530_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 307190023_Eukaryota_Metazoa_Arthropoda_Camponotus_floridanus 391325117_Eukaryota_Metazoa_Arthropoda_Metaseiulus_occidentalis 302850331_Eukaryota_Viridiplantae_Chlorophyta_Volvox_carteri_f__nagariensis 340368218_Eukaryota_Metazoa_Porifera_Amphimedon_queenslandica 544216024_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 37362210_Eukaryota_Metazoa_Chordata_Danio_rerio 551569810_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 41393167_Eukaryota_Metazoa_Chordata_Danio_rerio 115752588_Eukaryota_Metazoa_Echinodermata_Strongylocentrotus_purpuratus 551643784_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 167536777_Eukaryota_Choanoflagellida_Codonosigidae_Monosiga_brevicollis_MX1 545701519_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 514686110_Eukaryota_Choanoflagellida_Salpingoecidae_Salpingoeca_rosetta 546314787_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus

0 gnl_Paratrimastix_pyriformis_877475 194476572_Eukaryota_Euglyphida_Paulinellidae_Paulinella_chromatophora gnl_Trimastix_marina_1250070 gnl_Dysnectes_brevis_933805 1 156778113_Eukaryota_Heterolobosea_Psalteriomonadidae_Sawyeria_marylandensis gnl_Carpediemonas_membranifera_7776 gnl_Kipferlia_bialata_588892 gnl_Aduncisulcus_paluster_103285 gnl_Chilomastix_cuspidata_402937 gnl_Ergobibamus_cyprinoides_538656 gnl_Ergobibamus_cyprinoides_556441 528223876_Eukaryota_Kinetoplastida_Trypanosomatidae_Strigomonas_culicis gnl_Ergobibamus_cyprinoides_537343 gnl_Ergobibamus_cyprinoides_538658 528270210_Eukaryota_Kinetoplastida_Trypanosomatidae_Angomonas_deanei gnl_Ergobibamus_cyprinoides_556442 528276597_Eukaryota_Kinetoplastida_Trypanosomatidae_Angomonas_deanei gnl_Chilomastix_cuspidata_458051

551673789_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 551630928_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712

294929694_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294951339_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983

0.6 0.6

Supplementary figure 8

Single-gene phylogeny of GCS L protein (1031 taxa and 166 sites). Mitochondrial L protein sequences are highlighted in red. Metamonad sequences are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences, including trichomonad hydrogenosomal L proteins, are recovered as a monophyletic group, but do not show particular phylogenetic affinity to mitochondrial L protein sequences. The L protein tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 290976728_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 76 470487651_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 551580404_Eukaryota_Mastigamoebidae_Mastigamoeba_Mastigamoeba_balamuthi 302834102_Eukaryota_Viridiplantae_Chlorophyta_Volvox_carteri_f__nagariensis 585105754_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 585105753_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 577705671_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 HydE 59 577705673_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 gnl|Carpediemonas_membranifera|1486 gnl|Aduncisulcus_paluster|80434 gnl|Dysnectes_brevis|920273 gnl|Trepomonas_sp|1154325 99 540206600_Eukaryota_Hexamitidae_Hexamitinae_Trepomonas_sp__PC1 410719250_Eukaryota_Hexamitidae_Hexamitinae_Spironucleus_salmonicida gnl|Spironucleus_salmonicida|1114861 gnl|Spironucleus_vortens|701237 gnl|Spironucleus_vortens|701246 gnl|Ergobibamus_cyprinoides|557952 gnl|Ergobibamus_cyprinoides|536811 gnl|Paratrimastix_pyriformis|883763 gnl|Paratrimastix_pyriformis|880989 gnl|Trimastix_PCT|1265358 123495487_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|840793 gnl|Trichomonas_vaginalis|850854 100 123974893_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 123478185_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|822262

Supplementary figure 9 0.4

Single-gene phylogeny of HydE (139 taxa and 210 sites). Metamonada sequences are highlighted in light blue. HydE sequences of other eukaryotes are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences are recovered as a monophyletic group, and the metamonad clade shows phylogenetic affinity to eukaryotic sequences. The HydE tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. HydF

553187047_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana_CCMP526 gnl|Spironucleus_vortens|739569 98 gnl|Spironucleus_salmonicida|1115951 410719252_Eukaryota_Hexamitidae_Hexamitinae_Spironucleus_salmonicida gnl|Trepomonas_sp|1143734 540206621_Eukaryota_Hexamitidae_Hexamitinae_Trepomonas_sp__PC1 gnl|Trichomonas_vaginalis|854277 123436442_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Aduncisulcus_paluster|80611 gnl|Dysnectes_brevis|921301 585105758_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 553193001_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana_CCMP526 552838007_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 577705673_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 302834102_Eukaryota_Viridiplantae_Chlorophyta_Volvox_carteri_f__nagariensis 159466536_Eukaryota_Viridiplantae_Chlorophyta_Chlamydomonas_reinhardtii 577705673_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 552838007_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis gnl|Trimastix_PCT|1270860 gnl|Carpediemonas_membranifera|6790 gnl|Ergobibamus_cyprinoides|534889 gnl|Ergobibamus_cyprinoides|569893 gnl|Dysnectes_brevis|999466 gnl|Kipferlia_bialata|597863

0.4

Supplementary figure 10

Single-gene phylogeny of HydF (163 taxa and 252 sites). Metamonada sequences are highlighted in light blue. HydF sequences of other eukaryotes are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences show phylogenetic affinity to mitochondrial sequences. The HydF tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data S2. HydG gnl|Aduncisulcus_paluster|79075 gnl|Ergobibamus_cyprinoides|535333 gnl|Kipferlia_bialata|581085 41 gnl|Carpediemonas_membranifera|3475 gnl|Dysnectes_brevis|922991 gnl|Spironucleus_vortens|810841 gnl|Spironucleus_vortens|733324 gnl|Spironucleus_vortens|704075 63 gnl|Spironucleus_salmonicida|1116467 85 410719254_Eukaryota_Hexamitidae_Hexamitinae_Spironucleus_salmonicida 540206630_Eukaryota_Hexamitidae_Hexamitinae_Trepomonas_sp__PC1 gnl|Trepomonas_sp|1132885 gnl|Trepomonas_sp|1132877 gnl|Pentatrichomonas_hominis|636989 gnl|Pentatrichomonas_hominis|634563 79 123438557_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|846576 123448856_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|865552 gnl|Paratrimastix_pyriformis|880279 gnl|Trimastix_PCT|1247514 156778119_Eukaryota_Heterolobosea_Psalteriomonadidae_Sawyeria_marylandensis gnl|Kipferlia_bialata|581087 290976824_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 553187049_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana_CCMP526 585105755_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 470487725_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 577705675_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 159466244_Eukaryota_Viridiplantae_Chlorophyta_Chlamydomonas_reinhardtii 302833844_Eukaryota_Viridiplantae_Chlorophyta_Volvox_carteri_f__nagariensis

0.5

Supplementary figure 11

Single-gene phylogeny of HydG (202 taxa and 365 sites). Metamonada sequences are highlighted in light blue. Sequences of other eukaryotes are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences show phylogenetic affinity to other eukaryotic homologues. The HydG tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 83

gnl_Carpediemonas_membranifera_11231 430133105_Eukaryota_Mastigamoebidae_Mastigamoeba_Mastigamoeba_balamuthi 56 Periplasmic-type

471205129_Eukaryota_Entamoeba_Entamoeba 167392731_Eukaryota_Entamoeba_Entamoeba 67474180_Eukaryota_Entamoeba_Entamoeba gnl_Trimastix_marina_1248458 gnl_Paratrimastix_pyriformis_879145 86 gnl_Paratrimastix_pyriformis_880819 HydA

164414630_Eukaryota_Intramacronucleata_Litostomatea_Epidinium_ecaudatum

gnl_Spironucleus_salmonicida_1115505 410719268_Eukaryota_Hexamitidae_Hexamitinae_Spironucleus_salmonicida gnl_Trichomonas_vaginalis_820616 123485532_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 22 123494938_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 91 gnl_Trichomonas_vaginalis_871190 gnl_Trichomonas_vaginalis_855522 123455670_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 164414618_Eukaryota_Intramacronucleata_Litostomatea_ gnl_Trichomonas_vaginalis_835956 Diploplastron_affine

gnl_Dysnectes_brevis_989883

164414624_Eukaryota_Intramacronucleata_Litostomatea_Entodinium_caudatum

224013016_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 gnl_Chilomastix_cuspidata_456244 0 gnl_Dysnectes_brevis_949907 gnl_Ergobibamus_cyprinoides_538894 JZ548321_JZ548856_JZ549056_Breviata_anathema@JZ554214 gnl_Chilomastix_caulleryi_484249 JZ549642_FM205809_JZ550715_JZ552821_JZ548490_JZ547978_JZ554109_JZ553038_Breviata_anathema@JZ549953 19572308_Eukaryota_Heterolobosea_Psalteriomonadidae_Psalteriomonas_lanterna 156778101_Eukaryota_Heterolobosea_Psalteriomonadidae_Sawyeria_marylandensis Pygsuia_biforma@Hyd1 Stygiella_incarcerata_Eukaryota@Hyd1 Stygiella_incarcerata_Eukaryota@Hyd4 gnl_Trichomonas_vaginalis_833606 gnl_Tritrichomonas_foetus_905533 563403977_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_gallinae gnl_Trichomonas_vaginalis_838444 341872749_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_gallinae 158258984_Eukaryota_Trichonymphida_Teranymphidae_Pseudotrichonympha_grassii 158258986_Eukaryota_Spirotrichonymphida_Holomastigotoididae_Holomastigotoides_mirabile gnl_Tritrichomonas_foetus_913433 206604104_Eukaryota_Tritrichomonadida_Dientamoebidae_Histomonas_meleagridis gnl_Trichomonas_vaginalis_816421 164414634_Eukaryota_Intramacronucleata_Litostomatea_Ophryoscolex_caudatus 164414628_Eukaryota_Intramacronucleata_Litostomatea_Epidinium_ecaudatum gnl_Trichomonas_vaginalis_850536 1345094_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis gnl_Trichomonas_vaginalis_828867 gnl_Chilomastix_cuspidata_448656 51947501_Eukaryota_Intramacronucleata_Armophorea_Nyctotherus_ovalis gnl_Trimastix_marina_1252981 gnl_Chilomastix_caulleryi_484248 Pygsuia_biforma@HydCysJ3_1 gnl_Trichomonas_vaginalis_833237 gnl_Tritrichomonas_foetus_915216 158258982_Eukaryota_Trichonymphida_Teranymphidae_Pseudotrichonympha_grassii gnl_Trichomonas_vaginalis_831106 gnl_Trichomonas_vaginalis_827377 gnl_Trichomonas_vaginalis_853283 430133032_Eukaryota_Mastigamoebidae_Mastigamoeba_Mastigamoeba_balamuthi Pygsuia_biforma@HydCysJ3_2 Pygsuia_biforma@HydCysJ2 430133140_Eukaryota_Mastigamoebidae_Mastigamoeba_Mastigamoeba_balamuthi Pygsuia_biforma@Hyd4 Stygiella_incarcerata_Eukaryota@Hyd2 300123855_Eukaryota_Blastocystis_Blastocystis 187438956_Eukaryota_Blastocystis_Blastocystis Stygiella_incarcerata_Eukaryota@Hyd3 gnl_Trimastix_marina_1243673 gnl_Dysnectes_brevis_927957 gnl_Chilomastix_cuspidata_393158 gnl_Dysnectes_brevis_935442 gnl_Aduncisulcus_paluster_82107 gnl_Carpediemonas_membranifera_14921 gnl_Carpediemonas_membranifera_3225 514694628_Eukaryota_Choanoflagellida_Salpingoecidae_Salpingoeca_rosetta 552827006_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 552833909_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis 553187038_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana_CCMP526 577705669_Eukaryota_Viridiplantae_Chlorophyta_Tetraselmis_sp__GSL018 12581498_Eukaryota_Viridiplantae_Chlorophyta_Acutodesmus_obliquus Chlamydomonas_moewusii@AAT90438 290983098_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG_M 387233129_Eukaryota_Fungi_Neocallimastigomycota_Neocallimastix_frontalis 19548180_Eukaryota_Fungi_Neocallimastigomycota_Piromyces_sp__E2 23664246_Eukaryota_Fungi_Neocallimastigomycota_Neocallimastix_frontalis gnl_Carpediemonas_membranifera_13838 gnl_Dysnectes_brevis_923282 gnl_Carpediemonas_membranifera_7443 gnl_Kipferlia_bialata_579976 gnl_Dysnectes_brevis_932251 gnl_Chilomastix_cuspidata_394010 gnl_Dysnectes_brevis_933522 gnl_Aduncisulcus_paluster_88564 gnl_Ergobibamus_cyprinoides_534878 gnl_Ergobibamus_cyprinoides_535256 gnl_Spironucleus_barkhanus_660137 gnl_Spironucleus_barkhanus_664408 gnl_Spironucleus_salmonicida_1114456 gnl_Spironucleus_vortens_801752 gnl_Dysnectes_brevis_932052 253741867_Eukaryota_Hexamitidae_Giardiinae_Giardia_intestinalis_ATCC_50581 gnl_Giardia_intestinalis_573293 gnl_Spironucleus_vortens_801742 gnl_Trepomonas_sp_1169372 gnl_Trepomonas_sp_1127458 gnl_Trepomonas_sp_1127492 67482769_Eukaryota_Entamoeba_Entamoeba 158634508_Eukaryota_Retortamonadidae_Retortamonas_Retortamonas_sp__Vale gnl_Spironucleus_salmonicida_1112516 gnl_Spironucleus_salmonicida_1116124 gnl_Spironucleus_barkhanus_660622 gnl_Spironucleus_barkhanus_665279 gnl_Spironucleus_salmonicida_1112082 gnl_Spironucleus_salmonicida_1115363 gnl_Spironucleus_barkhanus_661083 gnl_Spironucleus_barkhanus_659602 gnl_Trepomonas_sp_1177443 gnl_Trepomonas_sp_1142682 gnl_Trepomonas_sp_1142670 gnl_Trepomonas_sp_1143796 gnl_Spironucleus_vortens_749706 2.0 gnl_Trepomonas_sp_1146908 gnl_Trepomonas_sp_1146896 gnl_Trepomonas_sp_1146902 gnl_Trepomonas_sp_1118950 gnl_Spironucleus_salmonicida_1113476 gnl_Spironucleus_vortens_810057 gnl_Spironucleus_vortens_810068 gnl_Spironucleus_vortens_707075 gnl_Spironucleus_vortens_718917 gnl_Spironucleus_vortens_796310 gnl_Spironucleus_vortens_753540 gnl_Spironucleus_vortens_791241 gnl_Spironucleus_vortens_796299 gnl_Spironucleus_vortens_806397 gnl_Spironucleus_vortens_773806 gnl_Spironucleus_vortens_798418 gnl_Spironucleus_vortens_808773 gnl_Spironucleus_vortens_767979 gnl_Spironucleus_vortens_735449 gnl_Spironucleus_vortens_728027 gnl_Spironucleus_vortens_758885 gnl_Spironucleus_vortens_795239

Supplementary figure 12

Single-gene phylogeny of [FeFe]-hydrogenase, HydA (493 taxa and 231 sites). Metamonada sequences are highlighted in light blue. Eukaryotic homologues are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. The HydA tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 551669814_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 260831053_Eukaryota_Metazoa_Chordata_Branchiostoma_floridae 225713784_Eukaryota_Metazoa_Arthropoda_Lepeophtheirus_salmonis 514694910_Eukaryota_Choanoflagellida_Salpingoecidae_Salpingoeca_rosetta 646299511_Eukaryota_Fungi_Dikarya_Botryobasidium_botryosum_FD-172_SS1 405121827_Eukaryota_Fungi_Dikarya_Cryptococcus_neoformans_var__grubii_H99 SCS alpha 58269516_Eukaryota_Fungi_Dikarya_Cryptococcus_neoformans_var__neoformans_JEC21 156778115_Eukaryota_Heterolobosea_Psalteriomonadidae_Sawyeria_marylandensis 291001767_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 391348139_Eukaryota_Metazoa_Arthropoda_Metaseiulus_occidentalis 325303392_Eukaryota_Metazoa_Arthropoda_Amblyomma_variegatum 571521756_Eukaryota_Metazoa_Arthropoda_Apis_mellifera 40 328775991_Eukaryota_Metazoa_Arthropoda_Apis_mellifera 17550194_Eukaryota_Metazoa_Nematoda_Caenorhabditis_elegans 525588068_Eukaryota_Fungi_Dikarya_Penicillium_oxalicum_114-2 549048692_Eukaryota_Fungi_Dikarya_Pyronema_omphalodes_CBS_100304 315048319_Eukaryota_Fungi_Dikarya_Microsporum_gypseum_CBS_118893 470512228_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 569441288_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 330844712_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 780694_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_discoideum 66805061_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_discoideum_AX4 298705781_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 223999547_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 695406447_Eukaryota_Peronosporales_Phytophthora_Phytophthora_sojae 675193089_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_INRA-310 570985810_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P10297 635364518_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 635364517_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 669146736_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 574113872_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_astaci 673034508_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 593704347_Eukaryota_Viridiplantae_Streptophyta_Phaseolus_vulgaris 15237260_Eukaryota_Viridiplantae_Streptophyta_Arabidopsis_thaliana 21593483_Eukaryota_Viridiplantae_Streptophyta_Arabidopsis_thaliana 403351684_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 678345260_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 471222061_Eukaryota_Intramacronucleata_Oligohymenophorea_Ichthyophthirius_multifiliis 145533787_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 145479775_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 551535487_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 551618882_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 401423573_Eukaryota_Kinetoplastida_Trypanosomatidae_Leishmania_mexicana_MHOM/GT/2001/U1103 557860395_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_cruzi_Dm28c 407846446_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_cruzi 300176826_Eukaryota_Blastocystis_Blastocystis 300120129_Eukaryota_Blastocystis_Blastocystis 300176246_Eukaryota_Blastocystis_Blastocystis 294887515_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294935591_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294931297_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 557176499_Eukaryota_Apicomplexa_Coccidia_Eimeria_mitis 357017637_Eukaryota_Apicomplexa_Coccidia_Eimeria_tenella 557233042_Eukaryota_Apicomplexa_Coccidia_Eimeria_necatrix 545711901_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 544211383_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 546320835_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus gnl|Carpediemonas_membranifera_454|3974_0 83 gnl|Carpediemonas_membranifera|4367 gnl|Ergobibamus_cyprinoides|556226 gnl|Aduncisulcus_paluster|81534 gnl|Kipferlia_bialata|579998 gnl|Kipferlia_bialata|603948 gnl|Kipferlia_bialata|579161 29 gnl|Kipferlia_bialata|604007 506968203_Eukaryota_Metazoa_Arthropoda_Coptotermes_formosanus 206604100_Eukaryota_Tritrichomonadida_Dientamoebidae_Histomonas_meleagridis gnl|Pentatrichomonas_hominis|644092 84 gnl|Trichomonas_vaginalis|869690 gnl|Tritrichomonas_foetus|891258 1710865_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis 123393950_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|871101 gnl|Trichomonas_vaginalis|835638 123501690_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Tritrichomonas_foetus|910801 gnl|Tritrichomonas_foetus|894319 gnl|Tritrichomonas_foetus|904065 gnl|Tritrichomonas_foetus|917489 gnl|Tritrichomonas_foetus|899981 gnl|Tritrichomonas_foetus|917023 gnl|Tritrichomonas_foetus|904055 gnl|Tritrichomonas_foetus|917101 gnl|Tritrichomonas_foetus|917021 gnl|Tritrichomonas_foetus|910800 gnl|Tritrichomonas_foetus|917100

2.0

Supplementary figure 13

Single-gene phylogeny of SCS alpha subunit (367 taxa and 224 sites). Mitochondrial SCS alpha subunit sequences are highlighted in red. Metamonada sequences are highlightedin light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences show phylogenetic affinity to mitochondrial sequences. The SCS alpha subunit protein tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 164608346_Eukaryota_Blastocystis_Blastocystis 300176556_Eukaryota_Blastocystis_Blastocystis 569378677_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 290982641_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 528273850_Eukaryota_Kinetoplastida_Trypanosomatidae_Angomonas_deanei 554930268_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_rangeli_SC58 554942829_Eukaryota_Kinetoplastida_Trypanosomatidae_Trypanosoma_rangeli_SC58 676390591_Eukaryota_Pelagophyceae_Aureococcus_Aureococcus_anophagefferens 551564241_Eukaryota_Isochrysidales_Noelaerhabdaceae_Emiliania_huxleyi_CCMP1516 397620382_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_oceanica 661892614_Eukaryota_Viridiplantae_Streptophyta_Coffea_canephora 565372297_Eukaryota_Viridiplantae_Streptophyta_Solanum_tuberosum 83284007_Eukaryota_Viridiplantae_Streptophyta_Solanum_tuberosum 551644323_Eukaryota_Cryptophyta_Pyrenomonadales_Guillardia_theta_CCMP2712 546324370_Eukaryota_Florideophyceae_Gigartinales_Chondrus_crispus 22 544218181_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 545709500_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 470530757_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff 330843612_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 470267193_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum SCS beta 281208774_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 403340375_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 678320671_Eukaryota_Intramacronucleata_Spirotrichea_Stylonychia_lemnae 156778117_Eukaryota_Heterolobosea_Psalteriomonadidae_Sawyeria_marylandensis 290993522_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 290971634_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 70952059_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_chabaudi_chabaudi 669197687_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_vinckei 577150834_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_petteri 294871826_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294930372_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 294881825_Eukaryota_Perkinsida_Perkinsidae_Perkinsus_marinus_ATCC_50983 691794855_Eukaryota_Fungi_Dikarya_Trametes_cinnabarina 667524471_Eukaryota_Fungi_Dikarya_Stachybotrys_chartarum_IBT_40293 666403884_Eukaryota_Fungi_Dikarya_Stachybotrys_chartarum_IBT_7711 505785967_Eukaryota_Metazoa_Chordata_Sorex_araneus 466032630_Eukaryota_Metazoa_Chordata_Orcinus_orca 507939656_Eukaryota_Metazoa_Chordata_Condylura_cristata 298712066_Eukaryota_Phaeophyceae_Ectocarpales_Ectocarpus_siliculosus 585105693_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 585105692_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 219117017_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055/1 223993443_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 635366075_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 325186785_Eukaryota_Albuginales_Albuginaceae_Albugo_laibachii_Nc14 695442616_Eukaryota_Peronosporales_Phytophthora_Phytophthora_sojae 301107704_Eukaryota_Peronosporales_Phytophthora_Phytophthora_infestans_T30-4 566008584_Eukaryota_Peronosporales_Phytophthora_Phytophthora_parasitica_P1569 673053173_Eukaryota_Saprolegniales_Saprolegniaceae_Aphanomyces_invadans 669165626_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_diclina_VS20 641530836_Eukaryota_Saprolegniales_Saprolegniaceae_Saprolegnia_parasitica_CBS_223_65 608667481_Eukaryota_Apicomplexa_Gregarinasina_Gregarina_niphandrodes 403352555_Eukaryota_Intramacronucleata_Spirotrichea_Oxytricha_trifallax 145485378_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 145521150_Eukaryota_Intramacronucleata_Oligohymenophorea_Paramecium_tetraurelia_strain_d4-2 330831784_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 481023481_Eukaryota_Dictyosteliida_Acytostelium_Acytostelium_subglobosum 281209693_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 514698388_Eukaryota_Choanoflagellida_Salpingoecidae_Salpingoeca_rosetta 167520005_Eukaryota_Choanoflagellida_Codonosigidae_Monosiga_brevicollis_MX1 672824882_Eukaryota_Fungi_Mortierellomycotina_Mortierella_verticillata_NRRL_6337 470305762_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864 gnl|Kipferlia_bialata|578047 91095067_Eukaryota_Metazoa_Arthropoda_Tribolium_castaneum 307183642_Eukaryota_Metazoa_Arthropoda_Camponotus_floridanus 307210402_Eukaryota_Metazoa_Arthropoda_Harpegnathos_saltator gnl|Ergobibamus_cyprinoides|550514 gnl|Ergobibamus_cyprinoides|538836 gnl|Ergobibamus_cyprinoides|550188 gnl|Ergobibamus_cyprinoides|547439 gnl|Ergobibamus_cyprinoides|548840 gnl|Aduncisulcus_paluster|80427 gnl|Carpediemonas_membranifera_454|2866_3 gnl|Carpediemonas_membranifera_454|2185_5 gnl|Carpediemonas_membranifera|2957 gnl|Carpediemonas_membranifera_454|9954_5 gnl|Carpediemonas_membranifera_454|8706_2 gnl|Carpediemonas_membranifera_454|2558_2 gnl|Carpediemonas_membranifera_454|4342_1 22 gnl|Carpediemonas_membranifera_454|7616_1 gnl|Carpediemonas_membranifera_454|8853_1 506968737_Eukaryota_Metazoa_Arthropoda_Coptotermes_formosanus 506967965_Eukaryota_Metazoa_Arthropoda_Coptotermes_formosanus gnl|Trichomonas_vaginalis|854010 2351685_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis 2351687_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis gnl|Trichomonas_vaginalis|834197 gnl|Trichomonas_vaginalis|828865 2351689_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis gnl|Tritrichomonas_foetus|904011 gnl|Tritrichomonas_foetus|911708 gnl|Pentatrichomonas_hominis|641109 gnl|Tritrichomonas_foetus|903082 gnl|Tritrichomonas_foetus|900770 gnl|Tritrichomonas_foetus|899973 gnl|Tritrichomonas_foetus|892754 gnl|Tritrichomonas_foetus|904012 gnl|Tritrichomonas_foetus|911709 gnl|Ergobibamus_cyprinoides|550189 gnl|Ergobibamus_cyprinoides|547437 gnl|Tritrichomonas_foetus|901987 gnl|Tritrichomonas_foetus|918037 gnl|Tritrichomonas_foetus|914269 gnl|Tritrichomonas_foetus|892999 gnl|Tritrichomonas_foetus|903108 gnl|Tritrichomonas_foetus|892091 gnl|Tritrichomonas_foetus|906307 gnl|Tritrichomonas_foetus|906834 gnl|Tritrichomonas_foetus|917783 gnl|Tritrichomonas_foetus|918228 gnl|Tritrichomonas_foetus|912749 gnl|Tritrichomonas_foetus|918179 gnl|Tritrichomonas_foetus|906305 gnl|Tritrichomonas_foetus|917784 gnl|Tritrichomonas_foetus|906835 gnl|Tritrichomonas_foetus|904067

0.7

Supplementary figure 14

Single-gene phylogeny of SCS beta subunit (429 taxa and 258 sites). Mitochondrial SCS beta subunit sequences are highlighted in red. Metamonada sequences are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences show phylogenetic affinity to mitochondrial sequences. The SCS beta subunit tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. 552834509_Eukaryota_Viridiplantae_Chlorophyta_Chlorella_variabilis ASCT 1B

95 gnl|Kipferlia_bialata|593231 gnl|Aduncisulcus_paluster|80906 gnl|Carpediemonas_membranifera|13334

528894078_Eukaryota_Fungi_Cryptomycota_Rozella_allomycis_CSF55

300122151_Eukaryota_Blastocystis_Blastocystis 300176285_Eukaryota_Blastocystis_Blastocystis 545705899_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 545702780_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria 585107800_Eukaryota_Eustigmatophyceae_Eustigmatales_Nannochloropsis_gaditana 321455201_Eukaryota_Metazoa_Arthropoda_Daphnia_pulex 668456612_Eukaryota_Metazoa_Arthropoda_Anopheles_sinensis 391333054_Eukaryota_Metazoa_Arthropoda_Metaseiulus_occidentalis

569397009_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 569378660_Eukaryota_Foraminifera_Reticulomyxidae_Reticulomyxa_filosa 290990913_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 290983648_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 470362640_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864

470421821_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str__Neff

156348345_Eukaryota_Metazoa_Cnidaria_Nematostella_vectensis

0.3

Supplementary figure 15

Single-gene phylogeny of ASCT 1B proteins (188 taxa and 279 sites). Mitochondrial ASCT 1B sequences are highlighted in red. Metamonada sequences are highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. Metamonad sequences are recovered as a monophyletic group. The ASCT 1B protein tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. ASCT 1C

gnl|Kipferlia_bialata|610481 gnl|Kipferlia_bialata|585226 gnl|Kipferlia_bialata|610480 97 300121644_Eukaryota_Blastocystis_Blastocystis 300120579_Eukaryota_Blastocystis_Blastocystis

290994174_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 584129190_Eukaryota_Fungi_Dikarya_Fusarium_verticillioides_7600 517319609_Eukaryota_Fungi_Dikarya_Fusarium_fujikuroi_IMI_58289 587693926_Eukaryota_Fungi_Dikarya_Fusarium_oxysporum_Fo47 281200883_Eukaryota_Dictyosteliida_Polysphondylium_Polysphondylium_pallidum_PN500 470244882_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_fasciculatum 330794075_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 123498752_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 90 gnl|Trichomonas_vaginalis|847719 gnl|Tritrichomonas_foetus|915181 gnl|Pentatrichomonas_hominis|631437 gnl|Pentatrichomonas_hominis|653206 gnl|Trichomonas_vaginalis|826316 123417742_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|867927 gnl|Tritrichomonas_foetus|898679 123975034_Eukaryota_Trichomonadida_Trichomonadidae_Trichomonas_vaginalis_G3 gnl|Trichomonas_vaginalis|814639

0.4

Supplementary figure 16

Single-gene phylogeny of ASCT 1C proteins (143 taxa and 408 sites). Metamonada sequences are highlighted in light blue. Mitochondrial ASCT 1C protein sequences are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. The ASCT 1C protein tree, showing detailed taxa and ML bootstrap support, is also attached as partof Supplementary Data 2. ACS1

Kipfelria_bialata@601207 Kipfelria_bialata@579953 Dysnectes_brevis@929219 97 Dysnectes_brevis@948404 Chilomastix_caulleri@481506 Chilomastix_cuspidata@393293 Chilomastix_caulleri@521862 Chilomastix_caulleri@486133 Chilomastix_caulleri@481507 Giardia_intestinalis_ATCC_50581@253745065_Eukaryota_Hexamitidae_Giardiinae Giardia_lamblia_ATCC_50803@159111022_Eukaryota_Hexamitidae_Giardiinae Spironucleus_barkhanus@27983596_Eukaryota_Hexamitidae_Hexamitinae Spironucleus_salmonicida@410719318_Eukaryota_Hexamitidae_Hexamitinae Trepomonas_sp_PC1@AGV05441_Eukaryota_Hexamitidae_Hexamitinae Spironucleus_vortens@730912 Spironucleus_vortens@783979 Spironucleus_vortens@813133 Spironucleus_vortens@813203 Spironucleus_vortens@789855 Spironucleus_vortens@787767 Spironucleus_vortens@744616 Spironucleus_vortens@761843 Spironucleus_vortens@751053 Spironucleus_vortens@761188 Spironucleus_vortens@813297 Spironucleus_vortens@809075 Chilomastix_cuspidata@436406

0.3

Dysnectes MGKLNYLLKPKTVAVIGASGNSKKVGYSVM Giardia MGKLSFLTNPASVAIIGASPNTGKVGNTVV

S. salmonicida MGKLAFFTNPTSIAVIGASSAAGKVGYTVV

Supplementary figure 17

Single-gene phylogeny of ACS1 (176 taxa and 601 sites). Metamonad sequences are highlighted in light blue, except for the cytosolic Giardia sequences, which are shown in green. Alphaproteobacterial sequences are highlighted in purple. N-terminal alignments of Giardia, Spironucleus, and Dysnectes are also shown. The N-terminal regions of the Dysnectes protein is almost identical in length and sequence to the cytosolic homologues of Spironucleus and Giardia, and lending support to a hypothesized cytosolic localization for this protein. The ACS1 tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. Some sequences listed in Supplementary Data 1 are not included in the phylogeny because they are identical to other sequences from the same organisms already present in the phylogeny. gnl_Trimastix_PCT_1241743_unnamed_protein_product ACS2 24 gnl_Trimastix_PCT_1243966_unnamed_protein_product gnl_Trimastix_PCT_1246148_unnamed_protein_product gnl_Trimastix_PCT_1264460_unnamed_protein_product gnl_Trimastix_PCT_1242464_unnamed_protein_product 300175837_Eukaryota_Blastocystis_Blastocystis_hominis_Blastocystis_hominis 300123828_Eukaryota_Blastocystis_Blastocystis_hominis_Blastocystis_hominis 50 gnl_Carpediemonas_membranifera_6359_unnamed_protein_product gnl_Ergobibamus_cyprinoides_537447_unnamed_protein_product gnl_Aduncisulcus_paluster_80898_unnamed_protein_product 544214583_Eukaryota_Bangiophyceae_Cyanidiales_Cyanidioschyzon_merolae_strain_10D 545704929_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria gnl_Ergobibamus_cyprinoides_547776_unnamed_protein_product

224012040_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335

290973168_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG_M

410719280_Eukaryota_Hexamitidae_Hexamitinae_Spironucleus_salmonicida gnl_Spironucleus_salmonicida_1112340_unnamed_protein_product hydrogenosomal ACS

608664235_Eukaryota_Apicomplexa_Gregarinasina_Gregarina_niphandrodes 209879069_Eukaryota_Apicomplexa_Coccidia_Cryptosporidium_muris_RN66 457875367_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_cynomolgi_strain_B 221059978_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_knowlesi_strain_H 156101812_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vivax_Sal_1 672196873_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_inui_San_Antonio_1 124809263_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_falciparum_3D7 641578789_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_reichenowi 574992906_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_falciparum_Palo_Alto_Uganda 68068033_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_berghei_ANKA 675225547_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_berghei_ANKA 564276911_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_yoelii_17X 675231223_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_yoelii 82594846_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_yoelii_yoelii_17XNL 669200957_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_vinckei 577147988_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_vinckei_petteri 675219664_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_chabaudi_chabaudi 70953624_Eukaryota_Apicomplexa_Aconoidasida_Plasmodium_chabaudi_chabaudi

471206427_Eukaryota_Entamoeba_Entamoeba_invadens_Entamoeba_invadens_IP1 9502268_Eukaryota_Entamoeba_Entamoeba_histolytica_Entamoeba_histolytica 167383747_Eukaryota_Entamoeba_Entamoeba_dispar_Entamoeba_dispar_SAW760 67481881_Eukaryota_Entamoeba_Entamoeba_histolytica_Entamoeba_histolytica_HM_1_IMSS

0.5

255597642_Eukaryota_Viridiplantae_Streptophyta_Ricinus_communis

219119927_Eukaryota_Bacillariophyta_Bacillariophyceae_Phaeodactylum_tricornutum_CCAP_1055_1 223996235_Eukaryota_Bacillariophyta_Coscinodiscophyceae_Thalassiosira_pseudonana_CCMP1335 498975309_Eukaryota_Metazoa_Arthropoda_Ceratitis_capitata

Supplementary figure 18

Single-gene phylogeny of ACS2 (449 taxa and 184 sites). Metamonada sequences are highlighted in light blue. ACS2 sequences of other eukaryotes are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. Hydrogenosomal ACS in Spironucleus salmonicida is distantly related to other metamonad homologues, and thus there is no phylogenetic evidence of MRO localization for other metamonad ACS2 homologues. The ACS2 tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2. Cardiolipin synthase with one CDP-alcohol phosphatidyltransferase domain

Aspergillus clavatus Blastocystis hominis Cryptococcus neoformans Acanthamoeba castellanii Drosophila melanogaster Homo sapiens Pan troglodytes Rattus norvegicus Cafeteria roenbergensis Wobblia lunata Cafeteria sp. Caron Lab Isolate Albugo laibachii Pythium aphanidermatum Pythium arrhenomanes Pythium irregulare Pythium ultimum Pythium ultimum var. sporangiiferum Pythium iwayamai Pythium vexans Phytophthora ramorum Phytophthora parasitica Phytophthora infestans Phytophthora capsici Phytophthora cinnamomi var. cinnamomi Hyaloperonospora parasitica Phytophthora sojae Achlya hypogyna Saprolegnia parasitica Saprolegnia declina Pseudopedinella elastica Dictyocha speculum Nannochloropsis gaditana Nannochloropsis gaditana Pygsuia biforma 90 Guillardia theta Galdieria sulphuraria Cyanidioschyzon merolae Aplanochytrium kerguelense Schizochytrium aggregatum Aurantiochytrium limacinum Thraustochytrium sp. LLF1b Mallomonas sp. CCMP3275 Spumella elongata Dinobryon sp. UTEXLB2267 Thalassiosira oceanica Thalassiosira pseudonana Phaeodactylum tricornutum Phaeodactylum tricornutum Pseudonitzschia multiseries Fragilariopsis cylindrus Fragilariopsis cylindrus Developayella elegans Ectocarpus siliculosus Aureococcus anophagefferens Phaeomonas parva Heterosigma akashiwo Chattonella subsalsa Ostreococcus tauri Aureoumbra lagunensis Citrus sinensis Arabidipsis thaliana Theobroma cacao Chlorella variabilis Carpediemonas membranifera

0.3

Supplementary figure 19

Single-gene phylogeny of eukaryotic Cardiolipin synthase CLS_cap (84 taxa and 143 sites). Carpediemonas membranifera is highlighted in light blue. Alphaproteobacterial sequences are highlighted in purple. The Cardiolipin synthase tree with ML bootstrap support is also attached as part of Supplementary Data 2. aminoadipic semialdehyde dehydrogenase

545703768_Eukaryota_Bangiophyceae_Cyanidiales_Galdieria_sulphuraria Trimastix_PCT_1241204 100 301114667_Eukaryota_Peronosporales_Phytophthora_Phytophthora_infestans_T30-4 635366347_Eukaryota_Albuginales_Albuginaceae_Albugo_candida 873219254_Vitrella brassicaformis CCMP3155 831773621_Acytostelium subglobosum LB1 330799253_Eukaryota_Dictyosteliida_Dictyostelium_Dictyostelium_purpureum 403364162_Oxytricha trifallax 678307619_Stylonychia lemnae 954194993_Pseudocohnilembus persalinus 118389426_Tetrahymena thermophila SB210 471225697_Ichthyophthirius multifiliis 470423162_Eukaryota_Longamoebia_Acanthamoeba_Acanthamoeba_castellanii_str._Neff 290996812_Eukaryota_Heterolobosea_Schizopyrenida_Naegleria_gruberi_strain_NEG-M 675368607_Eukaryota_Metazoa_Arthropoda_Stegodyphus_mimosarum 334325259_Eukaryota_Metazoa_Chordata_Monodelphis_domestica 395510582_Eukaryota_Metazoa_Chordata_Sarcophilus_harrisii 167526746_Eukaryota_Choanoflagellida_Codonosigidae_Monosiga_brevicollis_MX1 470306577_Eukaryota_Ichthyosporea_Capsaspora_Capsaspora_owczarzaki_ATCC_30864 909132165_Allomyces macrogynus ATCC 38327 1027006937_Fungi_Spizellomyces punctatus 575471682_Eukaryota_Fungi_Chytridiomycota_Batrachochytrium_dendrobatidis_JAM81 672822614_Eukaryota_Fungi_Mortierellomycotina_Mortierella_verticillata_NRRL_6337

0.2

Supplementary figure 20

Single-gene phylogeny of aminoadipate-semialdehyde dehydrogenase (59 taxa and 472 sites). The aminoadipate-semialdehyde dehydrogenase sequence of Trimastix is highlighted in light blue. Mitochondrial sequences are highlighted in red. Alphaproteobacterial sequences are highlighted in purple. The aminoadipate-semialdehyde dehydrogenase tree, showing detailed taxa and ML bootstrap support, is also attached as part of Supplementary Data 2.