TABLE S1. Data characterized for this study. rDNA 3 - - Culture 3 - etK sp70cyt rc5 f1a f2 ps22a ps23a Lineage Taxon accession # Lab sec61 SSU 14 40S Actin Atub Btub E E G H Hsp90 M R R T SUM Cercomonadida Heteromita globosa 50780 Katz 1 1 Cercomonadida Bodomorpha minima 50339 Katz 1 1 Euglyphida Capsellina sp. 50039 Katz 1 1 1 1 4 Gymnophrea Gymnophrys sp. 50923 Katz 1 1 2 Cercomonadida Massisteria marina 50266 Katz 1 1 1 1 4 Foraminifera Ammonia sp. T7 Katz 1 1 2 Foraminifera Ovammina opaca Katz 1 1 1 1 4 Gromia Gromia sp. Antarctica Katz 1 1 Proleptomonas Proleptomonas faecicola 50735 Katz 1 1 1 1 4 Theratromyxa Theratromyxa weberi 50200 Katz 1 1 Ministeria Ministeria vibrans 50519 Katz 1 1 Fornicata Trepomonas agilis 50286 Katz 1 1 Soginia “Soginia anisocystis” 50646 Katz 1 1 1 1 1 5 Stephanopogon Stephanopogon apogon 50096 Katz 1 1 Carolina Tubulinea Arcella hemisphaerica 13-1310 Katz 1 1 2 Cercomonadida Heteromita sp. PRA-74 MBL 1 1 1 1 1 1 1 7 Rhizaria Corallomyxa tenera 50975 MBL 1 1 1 3 Euglenozoa Diplonema papillatum 50162 MBL 1 1 1 1 1 1 1 1 8 Euglenozoa Bodo saltans CCAP1907 MBL 1 1 1 1 1 5 Alveolates Chilodonella uncinata 50194 MBL 1 1 1 1 4 Amoebozoa Arachnula sp. 50593 MBL 1 1 2 Katz lab work based on genomic PCRs and MBL (Marine Biological Laboratory) data are all from EST analyses. Culture accession number is ATTC unless noted. GenBank accession numbers for new sequences (including paralogs) are GQ377645-GQ377715 and HM244866-HM244878. TABLE S2. Lineages based on Patterson 1999 with updates from Adl et al., 2005. Lineages Clade Genes Lineages Clade Genes Acanthamoebidae Amoebozoa 15 Nucleariidae Opisthokonta 5 Acantharea Rhizaria 1 Opisthokont Opisthokonta 16 Allantion Rhizaria 1 Oxymonadida Excavata 5 Allas Rhizaria 1 Parabasalids Excavata 16 Alveolates Alveolates 16 Paradinium Rhizaria 1 Ancyromonas* - 5 Paramonas Stramenopiles 1 Apusomonads - 6 Paramyxea - 1 Breviata - 12 Pelomyxa Amoebozoa 2 Centroheliozoa - 7 Phaeodarea Rhizaria 1 Cercomonadida Rhizaria 10 Phalansterium Amoebozoa 2 Chlamydomyxa Stramenopiles 1 Plamsmodiophorida Rhizaria 4 Chlorarachniophytes Rhizaria 14 Pleurostomum Excavata 1 Collodictyonidae - 1 Polycystinea Rhizaria 2 Cryothecomonas Rhizaria 1 Proleptomonas Rhizaria 4 Cryptomonads Cryptomonads 10 Red algae Red algae 14 Desmothoracids Rhizaria 2 Soginia Excavata 5 Dimorphid Rhizaria 4 Stephanopogon Excavata 5 Ebriid Rhizaria 1 Sticholonche Rhizaria 1 Ellobiopsids Alveolates 1 Stramenopiles Stramenopiles 16 Entamoebidae Amoebozoa 16 Telonema - 4 Euglenozoa Excavata 16 Thaumatomonads Rhizaria 4 Euglyphida Rhizaria 4 Theratromyxa Rhizaria 1 Eumycetozoa Amoebozoa 16 Trichosphaerium Amoebozoa 4 Flabellinea Amoebozoa 2 Trimastix Excavata 16 Foraminifera Rhizaria 10 Tubulinea Amoebozoa 14 Diplomonadida Excavata 16 Vampryellidae Rhizaria 1 Carpediemonas Excavata 5 Viridaeplantae Viridaeplantae 16 Glaucophytes Glaucophytes 16 Xenophyophores Rhizaria 1 Gromia Rhizaria 3 Actinophryids Stramenopiles Gymnophrea Rhizaria 5 Biomyxa - Haplosporidia Rhizaria 2 Caecitellus Stramenopiles Haptophytes Haptophytes 15 Coelosporidium - Heterolobosea Excavata 16 Copromyxids - Hyperamoeba Amoebozoa 4 Discocelis Stramenopiles Jakobids Excavata 14 Fonticula - Kathablepharids Cryptomonads 7 Gymnosphaerida - Leukarachnion Stramenopiles 4 Komokiacea - Malawimonas Excavata 15 Luffisphaera - Mastigamoebidae Amoebozoa 16 Nephridiophagids Opisthokonta Mesomycetozoa Opisthokonta 15 Phagodinium - Metopion Rhizaria 1 Pseudodendromonads Stramenopiles Metromonas Rhizaria 1 Pseudospora - Ministeria Opisthokonta 8 Retortamonads Excavata Multicilia Amoebozoa 1 Spironemidae - These eukaryotic lineages, defined by ultrastructural identities (Patterson, 1999), have proven robust in analyses with multiple datatypes (Parfrey et al. 2006; Simpson and Patterson 2006; Yoon et al. 2008). The lone exception is ‘ramicristates’, which is now divided between Amoebozoa and Rhizaria. Thus, lineages within Amoebozoa and Euglyphida are based on the classification of Adl et al. 2005. Clade = Higher-level grouping into which lineage has been subsummed (e.g. (Pawlowski and Burki 2009; Simpson and Patterson 2006)). Genes = number of genes in this study. *The identity of this taxon was recently clarified and Planomonas is a junior synonym (Heiss et al. 2010). See Table S4 for more detail on genes included in analysis. TABLE S3. Protein-coding genes used in this study Homolo Gene name Alternate gene names Gene IDs Taxa Grf4 (General regulatory factor4) tyrosine 3-monooxygenase/tryptophan 14-3-3 5-monooxygenase activation protein 100743 87 40S 40S ribosomal protein S4 90857 82 Actin actin, beta 110648 197 Atub tubulin, alpha 4a 68496 158 Btub tubulin, beta 4 55952 161 eukaryotic translation elongation factor 1 Ef1α alpha 1 105313 98 EF2 eukaryotic translation elongation factor 2 100816 86 Enolase enolase 1 68183 84 Grc5 60S ribosomal protein L10 68830 87 Hsp70cyt heat shock 70kDa protein 8 68524 94 Hsp90 heat shock protein 90kDa alpha (cytosolic) 74306 130 methionine adenosyltransferase II alpha, MetK S-adenosylmethionine synthetase 38112 77 Rps22a Rps15a (ribosomal protein S15A) 111120 77 ribosomal protein S23 Rps23a 40S ribosomal protein S23 799 86 Tsec61 Sec61 alpha 1 subunit 55537 77 Gene: The protein-coding genes included in this study, with the abbreviation corresponding to the data matrix. Alternate gene names are taken from HomoloGene. HomoloGene IDs are from www.ncbi.nlm.nih.gov/HomoloGene/. Taxa: number of taxa with protein-coding gene sequences in our analysis. TABLE S4. Details on individual genes by taxa Lineage based on Patterson 1999, Supergroup Taxon rDNA 3 - updated with Adl et. - 3 al., 2005 - SSU 14 40S Actin Atub Btub Ef1a Ef2 Enolase Grc5 Hsp70cyt Hsp90 MetK Rps22a Rps23a Tsec61 Sum Amoebozoa Entamoebidae Entamoeba histolytica 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Amoebozoa Eumycetozoa Dictyostelium discoideum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Phreatamoeba balamuthi Amoebozoa Mastigamoebidae (Mastigamoeba) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Archaeplastida’ Glaucophytes Cyanophora paradoxa 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Archaeplastida’ Viridaeplantae Arabidopsis thaliana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Archaeplastida’ Viridaeplantae Ginkgo biloba 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Archaeplastida’ Viridaeplantae Oryza sativa 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Archaeplastida’ Viridaeplantae Physcomitrella patens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Cryptosporidium parvum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Paramecium tetraurelia 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Plasmodium berghei 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Tetrahymena thermophila 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Toxoplasma gondii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Alveolates Theileria parva 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Stramenopiles Phaeodactylum tricornutum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 ‘Chromalveolata’ Stramenopiles Thalassiosira pseudonana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Euglenozoa Leishmania major 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Euglenozoa Trypanosoma brucei 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Diplomonadida Giardia lamblia ATCC 50803 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Heterolobosea Naegleria gruberi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Jakobids Seculamonas ecuadoriensis' 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Excavata Parabasalids Trichomonas vaginalis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Trimastix pyriformis ATCC Excavata Trimastix 50562 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Caenorhabditis elegans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Candida albicans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Ciona intestinalis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Drosophila melanogaster 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Gallus gallus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Homo sapiens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Saccharomyces cerevisiae 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Schizosaccharomyces pombe 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Opisthokonta Opisthokonta Nematostella vectensis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 Amoebozoa Acanthamoebidae Acanthamoeba castellanii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Amoebozoa Eumycetozoa Physarum polycephalum 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Archaeplastida’ Viridaeplantae Chlamydomonas reinhardtii 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Archaeplastida’ Viridaeplantae Micromonas pusilla 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Archaeplastida’ Viridaeplantae Volvox carteri 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Chromalveolata’ Alveolates Perkinsus marinus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Chromalveolata’ Haptophytes Emiliania huxleyi 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Chromalveolata’ Haptophytes Isochrysis galbana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Chromalveolata’ Stramenopiles Aureococcus anophagefferens 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 ‘Chromalveolata’ Stramenopiles Phytophthora infestans 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Excavata Jakobids Reclinomonas americana 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Excavata Malawimonas Malawimonas jakobiformis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Opisthokonta Opisthokonta Branchiostoma floridae 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Opisthokonta Opisthokonta Monosiga brevicollis 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 Lineage based on Patterson 1999, rDNA 3 Supergroup Taxon - - updated with Adl et.