Supporting Information

Sebé-Pedrós et al. 10.1073/pnas.1002257107

Daphnia pulex 5 Integrin α Daphnia pulex 2 -/ - Capitella sp. 2 Capitella sp. 1 Daphnia pulex 1 Lottia gigantea 2 Amphimedon queenslandica 1 Geodia cydonium Drosophila melanogaster PS5 Drosophila melanogaster scab Drosophila melanogaster PS4 Podocoryne carnea Acropora millepora Homo sapiens 9 Homo sapiens 4 Daphnia pulex 4 Nematostella vectensis Homo sapiens 10 Homo sapiens 11 Homo sapiens 1 Homo sapiens 2 Amastigomonas sp. Homo sapiens 5 Homo sapiens 8 30/ 0.99 Homo sapiens IIb Homo sapiens V Lottia gigantea 1 Amphimedon queenslandica 4 Amphimedon queenslandica 2 Amphimedon queenslandica 3 Amphimedon queenslandica 5 Drosophila melanogaster inflated Drosophila melanogaster mew Daphnia pulex 3 Homo sapiens 6 Homo sapiens 7 Capsaspora owczarzaki 3 Capsaspora owczarzaki 4 Capsaspora owczarzaki 2 Capsaspora owczarzaki 1 Kordia algicida Monosiga brevicollis XP_001749484 Chloroherpeton thalassium Flavobacteriales bacterium Gloeobacter violaceus 88/ 1.00 Nostoc punctiforme Stigmatella aurantiaca Other FG-GAP containing proteins Homo sapiens GPI-PL Nematostella vectensis GPI-PL Capitella sp. GPI-PL Proterospongia sp. GPI-PL

Capsaspora owczarzaki GPI-PL GPI-PLs

Fig. S1. Maximum likelihood tree of the integrin α homolog and other proteins containing FG-GAP repeats. Alignment has been done using the only common region between all these proteins, which are three consecutive FG-GAP repeats. The taxon sampling includes all of the integrin α homologs here described and a wide representation of metazoan homologs. The putative integrin α from Monosiga brevicollis and some FG-GAP repeat-containing bacteria proteins obtained when blasting the M. brevicollis sequence have also been included, together with several glycosylphosphatidylinositol phospholipases. The tree is rooted using the midpoint-rooted tree option. Statistical support was obtained by RAxML with 1,000-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probabilities. BV values are <50% for most branches. Both values are only shown for the some external key branches. The general topology is the same for Bayesian and maximum likelihood analyses.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 1of8 Deuterostomia Homo sapiens 6 Gallus gallus 6 Xenopus tropicalis 6 Homo sapiens 3 Gallus gallus 3 Xenopus tropicalis 3 Homo sapiens 5 Gallus gallus 5 Xenopus tropicalis 5 Homo sapiens 8 Gallus gallus 8 Xenopus tropicalis 8 Homo sapiens 2 Gallus gallus 2 Xenopus tropicalis 2 Homo sapiens 7 Xenopus tropicalis 7 Homo sapiens 1 Gallus gallus 1 Xenopus tropicalis 1 Anopheles gambiae Protostomia Daphnia pulex 2 Daphnia pulex 1 Lottia gigantea 3 Capitella sp 2 Capitella sp 1 Lottia gigantea 2 Biomphalaria glabrata Lottia gigantea 1 Strongylocentrotus purpuratus C Deuterostomia Strongylocentrotus purpuratus G Strongylocentrotus purpuratus L-like Strongylocentrotus purpuratus L Acropora millepora 2 Cnidaria Nematostella vectensis 1 Nematostella vectensis 2 Podocoryne carnea Trichoplax adhaerens 2 Placozoa Trichoplax adhaerens 1 Acropora millepora 1 Cnidaria Nematostella vectensis 3 Nematostella vectensis 4 Nematostella vectensis 5 Amphimedon queenslandica 1 Porifera Ophlitaspongia tenuis Suberites domuncula Geodia cydonium Amphimedon queenslandica 2 Amphimedon queenslandica 4 Amphimedon queenslandica 3 Amphimedon queenslandica 5 Amphimedon queenslandica 6 Amphimedon queenslandica 7 3 95.5/1.00 Capsaspora owczarzaki Filasterea 63.5/ - Capsaspora owczarzaki 2 59.8/0.90 Capsaspora owczarzaki 1 Capsaspora owczarzaki 4 84.2/1.00 84.4/1.00 Amastigomonas sp. Apusozoa Trichodesmium erythraeum Cyanobacteria

Fig. S2. Maximum likelihood tree of the integrin β protein. The tree is rooted using the midpoint-rooted tree option. Statistical support was obtained by RAxML with 1,000-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probabilities (BPP). Both values are shown on key branches. A black dot indicates BV >90% and BPP >0.95.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 2of8 Amastigomonas EG-ADC--TSYTSCSACISSAD--QCGWCFTPGS------AGCQAASTS---TCAAPDFLNPMSEVQSQSTTS S------VMTPTDV Homo --MNLQPIFWIGLISSVC C------Cow_Ib1 TS-DPC--SAFTICEDCINNAALTMCGWCGDHGQ------CQSAAGT----CS--NWQSP------ASRVLGTSGSTP------EVSPTTT Cow_Ib2 -----C--TDILTCNECITGDSIYGCSWCVADGT------CRSPSDNALL-CPNPAGVLNPTSNIR-STTNVANADGVV------EIRPTTA Amastigomonas ------XEATRTTHRRHV------Cow_Ib3 VDPNTC--AALTDCKSCVNNLNIAGCGWCAQDGT------CRSAQG----DCPAASYHNPS------STYTAAPISGTP------EIYPAAA -MAAKRVLALLALALLLCL------Ophlitaspongia (sponge) LG-RLC--NAQVSCGDCIFSSP--NCVWCADQNY------NGTRCYAIGDPDFENLCSGMEQNPMGEI---TDTVDPPLQDLT------QVSPSRV Cow_Ib1 Capitella (annelid) ------QRTCGSCVSAGP--LCGWCQQEGF---DATNHERCDTTDNLALHGCDEHYMVFPEHNITYDSELKPRNAEGG Q------EAIQLSPQKV Cow_Ib2 ----MKLFAALLLLLALAS------Homo (vertebrate) TDENRCLKANAKSCGECIQAGP--NCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDIHQIQPQQL 123 4 567 Cow_Ib3 MRCWTALLAAVALMLALSASSAD AAVAPPTTICNAVDEYGGGMTLTCPTGQTISAIVFASYGLPQPSPFTVGTTPCSSMAYDPCNSRSSRSVVESFCLGQ- Integrin beta domain ------MLLLVSCISSVE------Cow_Ib4 Trichodesmium ------KVFLRLNDPLSFVVTVTPG P-IPLDLVLVEDLSGSMSDDVNTLRALAR D-MVTRVRTEI------QADTNFGIAGFIDKPAEPIAWNR D--- NTE Amastigomonas 89 C Cow_Ib1 NVFLRAGQPLLVTVSVTPQPCKPVDLYLLMDFSGSMDDDLTKVRSLAQ P-LATKVTQLCTDTSNPTCSNTNCARLGFGSFLEKPAYPMGRWTTSGWIKDS Cow_Ib2 VVELRKGVPQTVSIVVTIPQRKEVEFFYLFDLSGSMGDDLRNVKNLGN N-LRDKMQSLCRGSSSIS---SDCHYWRLGSHVDRPNGPFGGSGDYEFR IEG Cow_Ib3 NVTLRYNIPVVVPITVTVSTSKPLDLFILLDVSATMADDLATLKSFTQTAFSNSINK NCNGGTAPPSG-SECAYVRFGTFVDKPVVPLGGPTDWNFKIAS Ophlitaspongia (sponge) SINVRPGQPQTFNLNVRPARNFPIDLYLLMDLSYSMRDDLDNLKQLGA D-LAASIVGL------STNFRIGFGSFVEKVVAPFTTLDVRFQQ NPC Capitella (annelid) SIKLRPNDAFKFSVNFRQAENYPVDLYYVMDLSNSMSDDKSKLAELGD L-LSEEMGKI------TKNFHLGFGSFVDKETMPYISTVPTKLVS PC ------VFAQTD--E-NRCLK------ANAKSCGECIQAGP--NCGWCTNSTFLQEGMPTSARCDDLEALKKKGC P-PDDIE Homo (vertebrate) VLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGT D-LMNEMRRI------TSDFRIGFGSFVEKTVMPYISTTPAKLR NPC Homo 8 Amastigomonas ------GGGGTG--EGADC------TSYTSCSACISSAD--QCGWC------FTPGSAGCQAASTST---CA-APDFL

Cow_Ib1 ------GQARAQ--TSDPC------SAFTICEDCINNAALTMCGW C------GDHGQCQSAAGT------CS---NWQ Cow_Ib2 ------SA------YGQAC------TDILTCNECITGDSIYGCSW C------VADGTCRSPSDNALL---CPNPAGVL Amastigomonas SPLANQDCW-NYEYMGWLAAASDENLFFDALENSLENHG N----KDWPESMSEGAFHAAVCTARSGWRS-----NSRHIMLISTDADFHIAGDSELIGVY Cow_Ib1 TYTGGTSLPTNHAFRARLPLSYNLGQFVTLVQGISGIQG N----IDTPENMIDGMLQAILCQKLIDWPSFNTTYQPRRLMLMLTDDTTHFRKE GYMAGIV Cow_Ib3 QTCTITNTNNAAQGGGGFNSYFNDPCQNVVKRLYVSALCVDPNTCAALTDCKSCVNNLNIAGCGW C------AQDGTCRSAQGD------CP-AASYH Cow_Ib2 IASGDDRSFGSF------GAFSTALGNAD-TEWG----NDFPESQYSSMLQSLLCVN---WNP-----ARRHILLLATDATGHMEFDSN RLSSA ------VSALTVEDCTALELQSCNACLSK T--SSCGWCKST------GACMLNTDGTAQ---CPQ-ASWI Cow_Ib3 ------QYAASSAFSRDFSIARSAVQEP T-ASTNGYSVNDRPNSFLDAAVQAA LCSN---WDP-----THRHMLLVVTDAPFHFAGD AEALSSA Cow_Ib4 Ophlitaspongia (sponge) LNRNDIVCEPTYSYRHIISLTDDADEFNDLVQEQ M-ISGN----QDLPEGGFDGFLQSLLCTNLIGWRD-----VSRKLLLYITDAGFHFAGDGKLGGLI Trichodesmium ------Capitella (annelid) -----NGCAAPYAFRNQLPLTDDTSKFKTKVERT Q-ISGN----LDGPEGGFDAIMQAVSCNNEIGWRE-----TARKLLIYSSDAPYHFAGD GKLGGIV Homo (vertebrate) --TSEQNCTTPFSYKNVLSLTNKGEVFNELVGKQ R-ISGN----LDSPEGGFDAIMQVAVCGSLIGWRN-----VTR-LLVFSTDAGFHFAGDGKLGGIV 9 10

NPRGSKDIKKNKNVTNRSKGTAEKLKPEDIHQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYL M L Y MK LENVKSLGTD-LMNEMRRITSD- Amastigomonas NPHSRLCEDAVGGDGYGPGMA------EDY--PSASLIRDALLRQNVVPIFAVTAGDGGNSRFDNIQLYQDIVNEWGF G-FVYTLESDSSN Homo D S S DD Cow_Ib1 EPFPYKC--YVPDSAFTLPSTNTNAVNRFIDDASTKYD Y--PSFSQLKNALIENNIVPIFGSTA T------GINQRTFQDLVTALGFG-QSGTLATNSDN 11 Amastigomonas NP------MSEVQSQSTTSSV------MTPTDVKVFLRLNDPLSFVVTVTPGP I-PLDLVLVEDLSGSMSDDVNTLRALARD-MVTRVRTEIQAD Cow_Ib2 TAFPQRRALQKCHVTPG---TASSSSNFANTINAATLDHEIPSWPQIKAAFLDKNVVPILAITA Y------SSNNDHYDSFINALGFG-GRAGLSGDSSN Cow_Ib3 PHAVYKYAQQRCFTGTDAVWASTE------YEY--PTLGLLSAALLSEFVVPAFIVT N------AASNNIYTSLVNQLGFGRADLSLASDSNN Cow_Ib1 SP------ASRVLGTSGSTPE------VSPTTTNVFLRAGQPLLVTVSVTPQPCKPVDLYLL MDFSGSMDDDLTKVRSLAQP-LATKVTQLCTDT Ophlitaspongia (sponge) LPHQSIC--RLPNSGPHTGSVPVEYMD------AELFDY--PSVGQIAQALREQDIIPIFAA Q------RDAREFYDVLAAEIGEGASTGTLASDSSN NPTSN---IRSTTNVANADGVVE------IRPTTAVVELRKGVPQTVSIVVTIPQRKEVEFFYL F L G MG LRNVKNLGNN-LRDKMQSLCRGS Capitella (annelid) TPNDGHC--HLDDDGLYSASLS------QDY--PSIGQLSKIISDKKVNVIFAV T------KSRVPIYRLLGDFIEGS-TVGELANDSSN Cow_Ib2 D S S DD Homo (vertebrate) LPNDGQC--HLENNMYTMSHY------YDY--PSIAHLVQKLSENNIQTIFAV T------EEFQPVYKELKNLIPKS-AVGTLSANSSN Cow_Ib3 NPS------STYTAAPISGTPE------IYPAAANVTLRYNIPVVVPITVTVSTSKPLDLFIL LDVSATMADDLATLKSFTQTAFSNSINKNCNGG 11 Cow_Ib4 NPTGQ---LLPNNVPGASAGAFQPSPSAS V--FSPANGTAQLRMGREFTFDITVTPVQYPPVDFYLL LDASSGMFSDIHTLQQLRTNITEWL A------MAKKTKSSITNIVDRKGGLNPDVVDVTLVPGDNVTFDITAKVTKKSSTKLPLDLVFL S L G YG LPVLQDLVPK-LVSSVRDI---- Trichodesmium D S S DD Amastigomonas IIDAIVTAYTVTAETVT L-IEAADTRDWV--KSITPTG--G------YQNALLGVAYPLTVNMLASH G---DENVVEQILLTSFGFGSVNINATTVY DC 1 1 1 22 Cow_Ib1 LVQLLSDAYNSIASTIVAAVQSNGNEGF I--SAISPAAAVG------YTNVVVGTTYKFNFTLLYDGT R--ARTDGNTIIVTFVGFSAVSIVVNLVNDC 2 Cow_Ib2 VLSVIESVYNQIVGTIKPQLFDNGMDKF V--RSLTPAA--G------YTGLSRGDSRTFTLVLEDDELSIGYNIAAADVRVVFLGLGESRITLV P-STC Cow_Ib3 FLALFDAAYAGVANNVLARVGDTDAARF V--SQISPST------ATTSPATFSITLLATANTQSITSATSTLEVVFP GVGSAFLSIKL-ADC Ophlitaspongia (sponge) VVELVRQQYNTISQRVIFDQEPVPGVSVVINPT SCPGGVIEDGI----CTGLQIERVAEFDVTVSITECTPELLFQEISIPLRVVGFG E-LEIVLNALCR Capitella (annelid) VVALVRDNYNKISSTVEL-KADGNEDVFVSFRAKCGEN--EDFTESAKCDKLRIGQNVTFEVSLKVTSCPEDPAQRRKSFNIYPVGLTEKLTVEVDLICE Homo (vertebrate) VIQLIIDAYNSLSSEVILENGKLSEGVTISYKS YCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKC---PKKDSDSFKIRPLGFTEEVEVILQY ICE ------FRIGFGSFV KTVMPYISTTPAKLRNPCTSE Q--NCTTPFSYKNVL---SLTNKGEVFNELVGKQRIS G L S GGFDAIMQVAVCGSL 12 13 14 15 Homo E N D PE Amastigomonas TN------FGIAGFIDKPAEPIA------WNRDCNTESPLANQDCWNYEYMGWLAAASDENLFFDALENSLENH GNKDWPESMSEGAFHAAVCTAR SNPTCSNTNCARLGFGSFL KPAYPMGRWTTSGWIKDSTYT G--GTSLPTNHAFRARLPLSYNLGQFVTLVQGIS GIQG I T NMIDGMLQAILCQKL 1 Cow_Ib1 E N D PE Amastigomonas DCSAALC-LETGGQQCN-GQG------TCECGVCTCASTNFTGPGCEC---D-----VNAPCD----PP-CVN---GECVCGQCVC------SDG-- Cow_Ib2 SS--IS-SDCHYWRLGSHVDRPNGPFG--GSGDYEFRIE-----GIASGDDRSF------G-SFGAFSTALGNADTEW GN-DFPESQYSSMLQSLLC--- Cow_Ib1 NCA-GLC17----PVNKCN-GHG------TCLCGRCTCDD-GWTGETCTC--NS-----DPMACPSYNGAV-CNGILRGTCQCGTCVC------NEG-- Cow_Ib2 NCPVGTC----P-GACS-SPG------TASCECGRCNCAP-GWSGPTCSC--NN-----PAGTCP----NA-CSG--RGTCVCGACQC------NAG-- Cow_Ib3 TAPPSG-SECAYVRFGTFVDKPVVPLG--GPTDWNFKIASQY--AASSAFSRDF------SIARSAVQEPTASTNGYS VN-DRPNSFLDAAVQAALC--- Cow_Ib3 KVPL--C----D-DACA-ALDCNVNGTTAVNCDCGKCVCLP-EWTGATCSCLRAN-----NNLPCPVVNGAD-CSG--RGSCLCGKCEC------DIG------SQSPSVRFGLGVFV KPVQPF------TDPDVAWPPANLPTQNVQHLLNFTSDPLEFHAA L-QAVQVSS L A SVLDALSQIALCPSI Ophlitaspongia (sponge) CECEDTESP--NDPACS-GSG------TLSCGQCVCNP-DNFGEFCQCDATN---PNAQLDCPAGDSNIQCTG--RGTCLCGICDC------DPG-- Cow_Ib4 E D D PN Capitella (annelid) CDCESPELEQRNSDRCN-GTG------TYECGACTCAE-GAYGKKCECSASNLESDDYEASCKRTNTSEVCEG--RGQCLCGRCECYPITPGDPSRK Trichodesmium ------QPNSQFGLASYIDKPKDPFG--GPKDFVYRMESAITKSRT D------FQKAMDDLKIGNGN-DGPEAQLEALMQLALREKE Homo (vertebrate) CECQSEGIPE--SPKCHEGNG------TFECGACRCNE-GRVGRHCECSTDEVNSEDMDAYCRKENSSEICSN--NGECVCGQCVCRKRDNTNEI-- 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3 3 3 31 Integrin beta cysteine stalk 3

23 Amastigomonas WKGDTCECDSKPC-QG--GGNCNG--HGVCEC-GVCNCTTG------WTQSD---CSC------TLSPCPGT----PACSG--NGLC------TPC- Homo IGWR----- NVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSH Y------YDY- PSIAHLVQKLSENNIQTIFAVTEEF Cow_Ib1 FAGPACECPTTGC-PTSTGDVCSG--HGTCNC-GVCTCDAA------WNSTSAIDCSC----PTVAPGCMKPSGSGVDCEN--HGSC------KCN SGWRS---- NSRHIMLIST ADFHIAGDSELIGVYNPHSRL C-- EDAVGGDGYGPGMAE------DY- PSASLIRDALLRQNVVPIFAVTAGD Cow_Ib2 FTGASCEC-IDSC-PTFNGVRCNG--QGTCVC-GQCQCNAG------YNGTA---CEC---AASATVTCASL----NSCSG--HGVCTEA---STSGSCI Amastigomonas D Cow_Ib3 YTGDACNCPVAAC-QD----NCNG--NGQCIC-GNCVCNDG------YFGPT---CNCFAGLDSSSGSCPVGSNSLE-CSGASHGSCDTSIINPQNNVCS Cow_Ib1 IDWPSFNTTYQPRRLMLMLTDDTTHFRKEGYMAGIVEPFPYKCYVPDSAFTLPSTNTNAVNRFIDDASTKYD Y--PSFSQLKNALIENNIVPIFGSTATG Ophlitaspongia (sponge) FFGQACECDERDCINQDSGLLCSG--RGSCGCDGRCTCNVEPVSQLPYNGDLNV-CEC------TPNTQNCRDPTNRTSICN------Capitella (annelid) YDGSFCECNNYAC-DFSGGELCGGPSRGICKC-RECQCLPG------FSGAA---CDC------PTSQETCRAKESGL-MCN------Cow_Ib2 VNWNP---- ARRHILLLATDATGHMEFDSNRLSSATAFPQRRA L-QKCHVTPGTASSSSNFANTINAATLDHEIPSWPQIKAAFLDKNVVPILAITAYS Homo (vertebrate) YSGKFCECDNFNC-DRSNGLICGG--NGVCKC-RVCECNPN------YTGSA---CDC------SLDTSTCEA-SNGQ-ICN------SNWDP---- THRHMLLVVT APFHFAGDAEALSSA-PHAVYKYAQQRCFTGTDAVWAST E------YEY- PTLGLLSAALLSEFVVPAFIVTNAA 30 31 32 33 34 35 36 37 38 39 40 41 Cow_Ib3 D Cow_Ib4 INWRS----DARQIVFVLTNDGYHLAMDGLRARIETPFVPTCQLVQ Q------PSGAFVAPMTTHDY--PSVAEFVRAFSPSGVVPIFGVPASR IGFRK-----KSRRVVVLST ANYHKAGDGKKAGIKTPNNGDTVLDGK P-AGTGE------DY--PSIDQVRDALQEAGIVPIFAVT G-- 45 67 8 9 10 11 12 Trichodesmium D Amastigomonas GTCECQAGWTHPPPPAPQDCSCSTIPC--FCS------GHGNCEC-GSCVCE-ANWI-GSDCSCNN-VSCPIDPVTGTECGGGQ------ECVCG- 1 Cow_Ib1 -QCTCRPGYTGT------YCDTPVLPCPNSCS------GHGNCTA-GSCVCE-PGWT-GSSCSCST--VCP------DNCSGHG------TCQCG- 2 Cow_Ib2 ATCRCNIGWSGP------KCDCSSQCANTDCNP------PRGQCVC-GQCQCA-TGWDPATNCSCST-ASCPRDQ-NNVECGGIGTDPLSHASACTCDK Cow_Ib3 GRCNCQSGWSGP------TCECSTNICDGGCNELCSELPGGCGTCNC-GACQCA-LGYDPATNCKCLLGATCPVDE-NGDLCGGAG-----QSTGCTCG- Ophlitaspongia (sponge) ------GRGACGCDGSCECE-DPYF-GQFC------ELCSGSE------ICFDT- Capitella (annelid) ------GKGRCRC-GECICDKDDYYTGRTC------EDCPV------CP-G- Homo (vertebrate) ------GRGICEC-GVCKCT-DPKFQGQTC------EMCQT------CL-G- 42 43 44 45 46 47 48 Homo QPVYKELKNLIPK SAVGTLSA------NSSNVIQLIIDAYNSLSSE V-- ILENG----- KLSEGVTI--SYKSYC------KNGV GGNSRFDNIQLYQDIVNEWGF G-FVYTLESDSSNIIDAIVTAYTVTAET V-TLIEAADTRDWVKSIT PTG--- GYQNALLGVAYPLTVNML A-- SHGD Amastigomonas 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Cow_Ib1 INQ------RTFQDLVTALGFG-QSGTLATNSDNLVQLLSDAYNSIASTIVAAVQSNGNEGFI SAISPAAVGYTNVVVGTTYKFNFTLLY DGTRA- Amastigomonas ECVCAEGFTGPFCNCSTNLCPTTGNGACN---GHGSC-EC-GECTCESGFGGI-ACQCDASLPCPGSPQ---CSGHGVC--DRTDQCG-CVCDNGWTTAP SNN------DHYDSFIN LGFG-GRAGLSGDSSNVLSVIES VYNQIVGTIKPQLFDNGMDKFVRSLTPA A-GYTGLSRGDSRTFTLVLEDDELSIGY Cow_Ib1 VCRCQAGYSGLNCACN-NNCPSRGGLTCS---GHGQCNNCDGTCTCDVGYQSKPDCSCS-DAPCPNG-----CSGHGTC------TCGVCTCSGLYT--- Cow_Ib2 A Cow_Ib2 TCVCKGGWTGPACNCS-TRCPG----NCN---GHGTC-NC-GVCQCDSGWSGV-DCKCS-SNTCPVGTNGLECSGFGTC------ICGQCVCDALHA--- Cow_Ib3 SNN----IYTSLVNQLGFGRADLSLASDSNNFLALFDAAYAGVANNVLARVGDTDAARFVSQISPS T--- ATTS------PATFSITLLATANTQSI Cow_Ib3 VCQCTSAWTGPACNCSATPCND----NCNVNQGGGQC-VC-GQCVCNAGWTGE-TCGCPVGTTCPTDSNGVTCGGQGVCQTDSAATCGKCKCNPGYT--- Ophlitaspongia (sponge) ------NCD---SNRDCANCALDIIVQMVETTSVMEFFANAESNPNLP EGSMVSFDSENNAMQVVLPQ------Cow_Ib4 PHLS-WAYGRP-NGLVEQLGHG-AVVQLDERATNLMLRLQQGYRAVANS V- HIYDPSGNAIVRRVQPSSASVLDVTGAQVATSVTVSVTLFTPTRLTAP Capitella (annelid) ------KCD---ENKACVQC------Trichodesmium ----- NQVRNYKKLVDKLGF G-TVERLSRDSSNLVKVVTEGLEEVFSD L-TIVPQSDEFGYIKSIKPT T---- YENVRPGQSRTFEVK L------Homo (vertebrate) ------VCA---EHKECVQC------49 50 51 2

28 29 30 31 32 33 34 35 36 37 38 39 40 41 Amastigomonas DGTEDCSCSTTPCATS----CQP--PFGNCE--CNKCTCNTTYIDSLPCTVD------C-GAGECVKVVDKDCGMTMQCSCPNDPANP Cow_Ib1 --GADCSCWNVGCNSASN--CNSALGHGSCN--CSQCVCTGNYTPETECLCDRTEH-CAGWDGTSE----CSGHGTCV-SDATQCH---QCKCDADA-QG Cow_Ib2 --GPACGCVKGVCPSVGGVRCN---G-GDCDPICGICTCPPGKT-GPACDCDTVAHPCPTGNSTSGVVLPCSGQGTCLQSSATQCG---ICLCNRDPLTG NGTGENGRKC--- SNISIGDEVQFEISIT Cow_Ib3 --GENCTCQNRPCPFTSNGLCN---GHGSCQ--CGVCVCDEGFV-GSKCDCNAGSKPCPA--SSDGVA--CSGNGVCLHVDSSTCG---VCQCDRDPVRN Homo Ophlitaspongia (sponge) ------GQCPL------CDA------Amastigomonas ENVVEQILLTSFGFGSVNINATTVYDCDCS Capitella (annelid) ------TAHKTGDLTEDYCK------ANCTHVDVVPYV------RTDGNTIIVTFVGFSAVSIVVNLVNDC NC- Homo (vertebrate) ------RAFNKGE-KKDTCT------QECSYFNITKVE------Cow_Ib1 52 53 Cow_Ib2 NIAAADVRVVFLGLGESRITLVPS T-CNCP Cow_Ib3 TSATSTLEVVFPGVGSAFLSIKLA D-CKVP 42 43 44 45 46 47 48 49 50 51 52 53 5455Cow_Ib4 VTLGLRASGQPASFPI I-VSDGALVACTSG Amastigomonas VPVTPG-ECPCMQDAEGKDLCGAANCFEANSVNCTARCGVCECKPGVIGDS--CLCDPGTEAPAC-DSPCLNGGSCVPSAGKRDPVTGALIAGCVW SCQ Cow_Ib1 SPLYSGASCNCSLVECGGETINGFKCNQAAGNGEC-ICGTCHCKNNFTGPS--CECG-- PC---- SHDCGEHGTCV-C-G------TCV Trichodesmium GITDLDASQKDRLSLEVLGYGETKVNVTPI Cow_Ib2 TPLYTGSDCSCPTSGC- IKVGGQQCNYP- NGECDGCK-CKCKPGYGTPETGCSCKIGVTCPVNLQNQTCSNHGTCDTCNG------VCI Cow_Ib3 LPLWNGLNCNCSTVGC- PISGGIPCG-R- HGSCGACGVCTCDEGYTGAD- CSCKVE-ACPI-INGRECNGHGTCG-CFG------QCV Ophlitaspongia (sponge) ------GAVIINGTERADYQIDGEMAV RCEEV------Capitella (annelid) ------EDLPDRRLCKF------Homo (vertebrate) ------SRDKLPQPVQPDPVSHCKE------54

56 57 58 59 60 61 62 63 64 65 Amastigomonas CVPPFSGPS-CADCDVTNLDCPLACRAQTCGDCFLDSGSTDPRLQECGWCVSDSGNSSC-- IKRAECSASNGVVVD-ACPVA- PTPAPTLLENKSVFY Cow_Ib1 CAPFWGPPGQCTWCDVTAVNNT- CPHDSCTNYTSCGSCTDHNDRGCMWCEDLGKCYNNTNGNVKNVCTGTVIRSNG-DCPF-- LGDLPV---- GTTI Cow_Ib2 CEAGYTGR--- LCNRP-- DP- DP-- CGAATNCDACSRRSNPGCVWCDDNNLCMAKSAAIAE- CYAKYA- NG-TCPTSGLSDEAKA---- GIAG Cow_Ib3 CEAGWYDQG-CIFCNATILGDS- CPGAICSNQTSCSNCTRL- PECAWCSAGGFCTYNESAIAG- CSGPIL- HGQACSVD- SETVDV---- GVVT Ophlitaspongia (sponge) ------RDGCVYRYFVGIAATTDNFTAVHVEYARSCPDQQGGTAPWI------Capitella (annelid) ------RDEDDCDFYFTYVY-- MSNQVQAQRTKECPKP- VNVLAI------Homo (vertebrate) ------KDVDDCWFYFTYSVNGNNEVMVHVVENP ECPTG- PDIIPI------55 56

Amastigomonas GGIAA- AAVLILAILAMIIYKIFIWKRDKQLWAKFNAQQ D-- WGLDANPLYKDAFDQHENPIYAENADVDDRGAAFF Cow_Ib1 GVFVGVFGGIIVAAIAALVALKLINGMRDRREWGKFEAEREKSRWKGDDNPLFKASTTEYSNPLYNDS GK*------Cow_Ib2 G- VG- GGLAAAGLLALLAYKLYGMLMDKREWQKFEQGRQASQWKSDNNPLFQSSVKETENPLYQGDRSH *------A. Whole Cow_Ib3 GAAVG- AALGLAFLIGLIALKIVHTIADRREWEKFQRDKESMRWKGDDNPLFKSSTKEFDNPLYNASHQQ *------B. Integrin β extracellular domain Ophlitaspongia (sponge) -IAISIIIPLIVLGLLLLLLLKGLLLLWDVVEVRKFEREIKNAKYTKNENPLYRSATKDYQNPLYG K------Capitella (annelid) - VLGVIAGIILVGLALLLIWKLFVTIHDRREFANFEKERQNARWEMAENPIYKQATSTFKNPTYANK G------Homo (vertebrate) - VAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKW DTGENPIYKSAVTTVVNPKYEG K------Integrin β Transmembrane Integrin beta cytoplasmatic domain

Fig. S3. (A) Illustrative alignment of the whole integrin β, showing the integrin β domain, integrin stalk, transmembrane region, and cytoplasmic tail. Integrin homologs of Capsaspora owczarzaki (except integrin β-4, which is too derived), Amastigomonas sp., Homo sapiens, Capitella sp., and Ophlitaspongia tenuis are shown. The 56 conserved cysteines in metazoans are highlighted in green, whereas the 65 extra cysteines speciﬁc to protistan integrins are highlighted in red (see main text for more details). (B) Integrin β domain alignment for H. sapiens, Amastigomonas sp., C. owczarzaki, and Trichodesmium erythraeum to show in more detail the speciﬁc cation-binding motifs of Fig. 1 in main text, that is MIDAS (yellow, 1), ADMIDAS (blue, 2), and LIMB (red, 3). Orange and green means an amino acid shared by two motifs (indicated by the numbers below).

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 3of8 Homo sapiens 4 Deuterostomia Gallus gallus 4 Xenopus tropicalis 4 A. α-Actinin B. c-Src Deuterostomia Homo sapiens Homo sapiens 1 Gallus gallus 1 Xenopus tropicalis Xenopus tropicalis 1 Protostomia Drosophila melanogaster Homo sapiens 3 Lottia gigantea 1 Xenopus tropicalis 3 Xenopus tropicalis 2 Lottia gigantea 2 Cnidaria Gallus gallus 2 Hydra magnipapillata Homo sapiens 2 Porifera Amphimedon queenslandica 1 Biomphalaria glabrata Protostomia

Lottia gigantea 68/1.00 Amphimedon queenslandica 2

Capitella sp. Amphimedon queenslandica 3 Daphnia pulex Amphimedon queenslandica 4 Drosophila melanogaster Filasterea Cnidaria Capsaspora owczarzaki 2 89/1.00 Nematostella vectensis Placozoa Trichoplax adhaerens Capsaspora owczarzaki 1 95/1.00 Porifera Amphimedon queenslandica Choanoflagellata Proterospongia sp. Monosiga brevicollis Choanoflagellata 100/1.00 100/1.00 Proterospongia sp. Monosiga brevicollis 2 87/1.00 Filasterea Capsaspora owczarzaki Monosiga brevicollis 1 Dictyostelium discoideum Amoebozoa Monosiga brevicollis 3 Dictyostelium purpureum 75/0.98 Entamoeba hystolitica Homo sapiens Abl

Acanthamoeba castellanii Gallus gallus Abl Allomyces macrogynus Fungi 100/1.00 Xenopus laevis Abl Spizellomyces punctatus Drosophila melanogaster Abl 100/1.00 100/1.00 Batrachochytrium dendrobatidis Cryptococcus neoformans Strongylocentrotus purpuratus Abl Schizosaccharomyces pombe Abl kinases Nematostella vectensis Abl Yarrowia lipolytica Aspergillus fumigatus Amphimedon queenslandica Abl Apusozoa Amastigomonas sp. Capsaspora owczarzaki Abl1 Other eukaryotes Histomonas meleagridis 2 Monosiga brevicollis Abl1 Histomonas meleagridis 1 Monosiga brevicollis Abl2 Trichomonas vaginalis Histomonas meleagridis 3

Fig. S4. Maximum likelihood tree of (A) α-actinin and (B) c-Src. The α-actinin tree (A) is rooted using bikont eukaryotes as an outgroup. The c-Src tree (B)is rooted using the closely related Abl kinase family as an outgroup. Statistical support was obtained by RAxML with 100-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probabilities (BPP). Both values are shown in key branches. A black dot indicates BV >90% and BPP >0.95.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 4of8 A. vinculin Homo sapiens Deuterostomia B. talin Deuterostomia Homo sapiens 1 Gallus gallus Gallus gallus 1 Xenopus tropicalis Homo sapiens 2 Gallus gallus 2 Branchiostoma floridae Xenopus tropicalis 1 Lottia gigantea Protostomia Xenopus tropicalis 2 Capitella sp. Branchiostoma floridae Drosophila melanogaster Strongylocentrotus purpuratus Protostomia Daphnia pulex Capitella sp. 73/1.00 Lottia gigantea Nematostella vectensis Cnidaria Daphnia pulex Trichoplax adhaerens Placozoa D. melanogaster Amphimedon queenslandica Porifera Trichoplax adhaerens Placozoa Cnidaria Monosiga brevicollis Choanoflagellata 72/1.00 Nematostella vectensis 1 Nematostella vectensis 2 Proterospongia sp. Porifera Filasterea 52/0.67 Amphimedon queenslandica Capsaspora owczarzaki Monosiga brevicollis 2 Fungi 100/1.00 Spizellomyces punctatus 1 100/1.00 Monosiga brevicollis 1 Spizellomyces punctatus 2 Proterospongia sp. 96/1.00 Capsaspora owczarzaki B. dendrobatidis 100/1.00 Allomyces macrogynus 71/1.00 Allomyces macrogynus 2 Amastigomonas sp. Apusozoa Amoebozoa Allomyces macrogynus 1 Dictyostelium purpureum 1 Apusozoa Amastigomonas sp. Dictyostelium discoideum 1

Dictyostelium discoideum Amoebozoa D. purpureum 2 D. discoideum 2 Dictyostelium purpureum Acanthamoeba castellanii 1 Acanthamoeba castellanii Acanthamoeba castellanii 2

C. ILK-ankyrin repeats D. ILK-kinase domain Homo sapiens Deuterostomia Deuterostomia Homo sapiens Xenopus laevis Gallus gallus Gallus gallus Branchiostoma ﬂoridae Capitella sp. Protostomia Xenopus laevis Lottia gigantea Drosophila melanogaster Branchiostoma ﬂoridae Daphnia pulex Nematostella vectensis Cnidaria Protostomia 85/1.00 Daphnia pulex Trichoplax adhaerens Placozoa 97/0.99 Amphimedon queenslandica Porifera Drosophila melanogaster 78/0.99 Capsaspora owczarzaki Filasterea B. dendrobatidis Fungi Capitella sp. Spizellomyces punctatus D. discoideum TKL Lottia gigantea TKL group kinases D. purpureum TKL Placozoa Oryza sativa MAP3K Trichoplax adhaerens MAP3K kinases Vitis vinifera MAP3K Filasterea Arabidopsis thaliana MAP3K 100/1.00 C. owczarzaki 70/1.00 Ricinus communis MAP3K Apusozoa Amastigomonas sp. Oryza sativa EDR1 EDR1 kinases Hordeum vulgare EDR1 Cnidaria Nematostella vectensis Triticum aestivum EDR1 Danio rerio TNNI3K TNNI3 interacting kinases Amphimedon queenslandica Porifera Homo sapiens TNNI3K Mus musculus TNNI3K B. dendrobatidis Fungi Gallus gallus TNNI3K Strongylocentrotus purpuratus TNNI3K Spizellomyces punctatus Brugia malayi TNNI3K Aligned area Aligned area

Ankyrin repeats Kinase domain Ankyrin repeats Kinase domain

Fig. S5. Maximum likelihood tree of (A) vinculin protein, (B) talin protein, (C) integrin-linked kinase (ILK) protein based on the ankyrin repeats, and (D) ILK protein using the kinase domain. For each tree, statistical support was obtained by RAxML with 100-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probability (BPP). Both values are shown in key branches. A black dot indicates BV >90% and BPP >0.95. Trees in A and B are rooted using the Amoebozoa as the outgroup. Tree in C is rooted using the midpoint-rooted tree option. Tree in D is rooted using several closely related kinase families as an outgroup.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 5of8 A. Parvin Deuterostomia B. PINCH Homo sapiens γ Homo sapiens 1 Deuterostomia

Xenopus tropicalis 1 Gallus gallus γ

Xenopus tropicalis 2 Xenopus tropicalis γ

Gallus gallus 1 Homo sapiens β Homo sapiens 2 Gallus gallus β Gallus gallus 2

Xenopus tropicalis β Branchiostoma ﬂoridae Protostomia Homo sapiens α Daphnia pulex

Gallus gallus α Drosophila melanogaster

Capitella sp. Branchiostoma ﬂoridae Protostomia Lottia gigantea Capitella sp. Cnidaria Nematostella vectensis 100/1.00 Lottia gigantea Placozoa Trichoplax adhaerens 79/1.00 Porifera Drosophila melanogaster Amphimedon queenslandica

Filasterea Daphnia pulex 82/1.00 Capsaspora owczarzaki Fungi 73/1.00 Cnidaria Batrachochytrium dendrobatidis Nematostella vectensis 89/1.00 Placozoa Allomyces macrogynus Trichoplax adhaerens Amastigomonas sp. Apusozoa Porifera Amphimedon queenslandica Amoebozoa Acanthamoeba castellanii 1 Filasterea Capsaspora owczarzaki Acanthamoeba castellanii 2 91/0.92 Fungi Batrachochytrium dendrobatidis Dictyostelium discoideum

Apusozoa Dictyostelium purpureum Amastigomonas sp.

Entamoeba hystolitica

Fig. S6. Maximum likelihood tree of (A) the parvin protein and (B) the PINCH (particularly interesting Cys-His-rich) protein. The parvin tree is rooted using the midpoint-rooted tree option, whereas the PINCH tree is rooted using Amoebozoa as an outgroup. For each tree, statistical support was obtained by RAxML with 100-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probability (BPP). Both values are shown in key branches. A black dot indicates BV >90% and BPP >0.95.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 6of8 A Homo (vertebrate) MSGVSEPLSRVKLGTLRRPEGPAEPMVVVPVDVEKEDVRILKVCFYSNSFNPGKNFKLVKCTVQTEIREIITSILLSGRIGPNIRLA--ECYGLRLKHMK Drosophila (insect) MNTAGATSQPPPTKNEI------NSEEYLIHVHM------PNKSFKAVRFNVKETVFHVIRRTV--EDLGTDGRTPSIQRYACRMLNMI Capitella (annelid) M------DKAILKVHL------PNGGFNVVKCGDATDIKDIVQLVV--GRLAAGQRNYK-ASYALRLTHTV Nematostella (cnidarian) ------LKVYL------SNGDSRSVKCGEATDIKGIIHLVI--GSLGADPILIG-DYYGIMLEHVN Amphimedon (sponge) MAVSTDRVA------DLHVIRVLL------VNGDSRSVRLDENTDVADIVYYIL--SRLRANLEVAP-HLFSILLEHTV Capsaspora M------EGANLRVHL------ETGDVKAVKYAANTTVQDIINIMG--LKIGLQSV----AHFGLYLEHSD

Homo (vertebrate) SDEIHWLHPQMTVGEVQDKY------EC---LHVEAE------WRYDLQIRYLPEDFMESLKEDRTTLLYFYQQLRNDYMQRYASKV Drosophila (insect) TKEVIWLARSTSMQKVLSHILTPGCSNVDC---PNNQSELDEVLLEHGRRITDNRVWRVELRVRYVPNNIQELFEEDKATCFYYFNQVKEDFIQANVTAI Capitella (annelid) STESYWLHSDLTMYQVRQKY------ES---LHPADE------WRYELRVRYLPKSFHDLLAKDRVTFYYFYDQVWNDYMKHIAEKT Nematostella (cnidarian) TEEAFWLSCKSTVSEVRSKH------EA---LYPADQ------WKYLLRVRYLPLDYRDLYQKDKVTFFYLYDQVKNDYLQHKSEDV Amphimedon (sponge) SGEYHWLSSGYSVLDLITSH------CG---SRPLHE------WR------Capsaspora AAQSQWLSPLRPVATVEAFY------QSKTQIFVTSS------WKYTFRVRFLPKNAVNLYSKDKTVFSYLHQQLCRFLKQGKFDDI FERM domai Homo (vertebrate) SEGMALQLGCLELRRFFKDMPHNALDKKSNFELLEKEVGLDLFFPKQMQENL--KPKQFRKMIQQTFQQYASLREEECVMKFFNTLAPFANIDQETY-RC Drosophila (insect) DTEVAVQLCCLGIRHYFKNITVKAPDKKQHIDYIEKEIGFKSFLPQSVIATS--KPKNLKKLIQVGYKKVYNYNDIEYLTRFFDLLKNIYLTNFEQF-SV Capitella (annelid) EQDVAIRLGCIEIKRIFKHMQQNALEKKSNMEFLEKEIGLKRFLPKKILDSV--KVKQLRKLVQHTFKQYATLSEEECVFKFFETLAEVWRFDQENF-KC Nematostella (cnidarian) EEEKSFRLGALEMRRYFKDMPQIALDKKSNFEYIEKEVGLTKFIPKTILENI--KSKVLRKTIHGYFRQYSTLTEEECCCKFFELLSTVYRYDLETF-KC Amphimedon (sponge) ------KEYGFEKFFKKSFLQSARSKKKNLSKMIQSTFQQYESLTSDGCIFQFFAILGRFNPLDIDKFHNC Capsaspora AEQSLVQLAGAEIVRRCLGMASH----KVNLDYFEKEIGFSEILSEGILHRY--KVKELKKLLSPHCKEYEKLSEDECKLVFFKTLQEHSELGLTTF-KV

Homo (vertebrate) (...)---PDETLR--RPGGPQYGIAREDVVLNRILGEGFFGEVYEGVYT------NHKGEKINVAVKTCKKDCTLDNKEKF Drosophila (insect) (...)-GLLEGEGDS-TPTVRNYELDRSLITPSAKIGVGQFGDVYVGTYTLPKLGKGKNLAGNGKNSNSDQRNADSRPDVIQVAIKTCKANDDPEKTENF Capitella (annelid) (...)--VEEEGDY--STPGIDYEIERSSVDLDEILGEGQFGDVHRGSYS------DADGNKIAVAIKTCKVDCEDSRADSF Nematostella (cnidarian) (...)---VDDGDYAEAMAAKDYEIPRNKINLGPIIGQGQFGDVHKGTFK------SLDNPNMPVAIKTCK---NPDTREKF Amphimedon (sponge) (...)----KDPDR--LMKSLGRPLSPTDIMLADRLGEGQFGDVHKGILY------PDTTEEVAVAVKTCKPDSAPEERVKF Capsaspora (...)----DDKLDCDT------IFRDQLTIARVIGEGQFGSVNEGVWT------RPDNTNVPVAIKTCKNNVSREVQREF Tyrosine kinase domain Homo (vertebrate) MSEAVIMKNLDHPHIVKLIGIIEEE-PTWIIMELYPYGELGHYLERNKNSLKVLTLVLYSLQICKAMAYLESINCVHRDIAVRNILVASPECVKLGDFGL Drosophila (insect) LAEAYIMQKFDHPHIIRLIGICSVM-PIWIVMELAKLGELRAYLKTNSERLSHGTLLKYCYQLSTALSYLESKKFVHRDIAARNVLVSSPTCVKLADFGL Capitella (annelid) LEEARIMQQFEHPHIIKLLGLCLDS-PIWIIMELAQLGEMRAFLQSNKHRLRLDMLIMYCYQLSTALSYLESKNFVHRDIAARNVLVSSEDCVKLADFGL Nematostella (cnidarian) LEEAYIMKQFDHPHIIKLIGVCMED-NFFIVMELAAFGEMRTYLQKHRGLINHEMLLDYIFQLSTAMSYLESKNFVHRDIAARNVLVCSHKCVKLADFGL Amphimedon (sponge) LQEAAIMKQFNHPHIVKLFGVVTQGLRTYIVMELAPLGQLRQYLLLNGESISQDILLNYIKQLCSAMVHLESKNYVHRDIAARNILLLSPDKIKLSDFGL Capsaspora LAEATLMSKLNHPHIVRILGVSLSS-PILIVCELVPLGSLRNYLLDNKPSLNLKMLLGYNWQIVSAMSYLEHVKVIHRDLATRNILVATRQVVKLTDFGL

Homo (vertebrate) SRYIEDEDYYKASVT-RLPIKWMSPESINFRRFTTASDVWMFAVCMWEILSFGKQPFFWLENKDVIGVLEKGDRLPKPDLCPPVLYTLMTRCWDYDPSDR Drosophila (insect) SRWVSDQSYYHSTPTVALPIKWMSPESINFRRFTTASDVWMFGVCIWEILMLGVKPFQGVKNSDVILKLENGERLPLPPNCPPRLYSLMSQCWAYEPLKR Capitella (annelid) SRGVNEQSYYKATKG-KLPIKWMAPESINFRRFTTASDVWMFGVCMWEILMYGVKPFQGVKNNDVIGKIEAGERLAFPPNCPASLYNLMNLCWSYEPSRR Nematostella (cnidarian) SRWVEEQAYYKASKG-KLPIKWMAPESINFRRFTSASDAWMFGVCIWEILMYGIKPFQGVKNNDVIGKIEQGERLALPPNCPPALYHLMTECWSYEPSKR Amphimedon (sponge) SRWLEEADFYVASRG-KLPIKWMAPESINFRRFTGSSDVWMFGVCCWEILMRGVKPFMSIKNDEVIGKLERGERLPLPPDCPPSLFNIMNHCWQYEPEER Capsaspora SRVLTEDDIYTATGG-KMPVKWMAPESINYRTFTTATDVWSFGVCMWEIMSYGEKPYPQLQNSDVIDHLESGARLQCPDDCPDSLYRVMHDCWAYKPEDR

Homo (vertebrate) PRFTELVCSLSDVYQMEKD------IAMEQERNARYRTP------KILEPTAFQEP- Drosophila (insect) PNFKRIKETLHEILIEDSI------NSSETLKREQRKVA------SMSWIGSD-DID Capitella (annelid) KSFGDVKSYLREIFSEERQ------IQEEQARMDGRRVQ------SWGSCGSDEEAP Nematostella (cnidarian) PSFQYIKTRLSVIVQEERF------QSEERLRRESRRIN------SMSVDLARME-- Amphimedon (sponge) PSFTDIEQQLCVILEQEKH------FNALAVSGRIRGYEVP------DRPHVPIGRVPSGGAHRPPH Capsaspora PSFRELYDRMMQVIAEERIEIQGVTTSHLRKSTSEAEMRAQTKRIVESVRGSQSSIAMSSLTAPASDSGRSKSPRASRLTHQQSLPASAFASMGDEPHVL

Homo (vertebrate) (...) --NVMELVRAVLELKNELCQLPPEGYVVVVKNVGLTLRKLIGSVDDLLPSLP----SSSRTEIEGTQ----KLLNKDLAELINKMRLAQQNAVTS Drosophila (insect) (...) --ATTLVVKSIMALSQGVEKANTEGYLELVKNVGVKLRNLLTSVDKISIIFP----AQALKEVQMAH----QVLSKDMHELVSAMRLAQQYSDTT Capitella (annelid) (...) --STTNVVRAVMDMSKGVHQAKADQYVELVKNVGLQLKSLLASVDDQVIHLP----QSCHEEVEMAH----KVLSSDMAQLIEAMRLAQSYSTTL Nematostella (cnidarian) (...)--NTTSVVQTVIDLNTGLPFARPEDYPQFVKSIGLALRGLLSEVDGEIQDLP----QSSHKEIEMAH----KVLSADMAELINSMKLAQKYSTTT Amphimedon (sponge) (...) --CTTGVVQSIVELSTKLPAARPGDYVDLVKGVGVSLRQLLVKVDSSLDMIP----EHTHSKVKMAH----KVLSSDMTQLVKSMKELQENYQSF Capsaspora (...) QDKVVAVMKSVMDLKESVRLNETSAFVDMVRVIVMCAGEVMDMVDPVMACLADLNDSQVAPEVHIALADDVSLLKVDIEKLVE--RTGQATQFSP Focal_AT domain Homo (vertebrate) LSEECKRQMLTASHTLAVDAKNLLDAVDQAKVLANLAHPPAE------Drosophila (insect) LDCEYRKSMLSAAHVLAMDAKNLFDVVDSIRQRYQHLFP-PSATKETSCSSSFEST------SGSIVTEPVNDLGGYIKTSTSGDLLQNTGI Capitella (annelid) MDNEYRRGMLKAAHVLAMDSKNLLDSVDNARRLTESTSSEPLQHSRTNSSASVEGACKIDPALEALPQDFAASVTLDPSSSPSPEPDSNGTQNSREDT-- Nematostella (cnidarian) LDQEYRRGMLSAGHVLAVDAKHLFDVVDTAR------Amphimedon (sponge) LLDHYQKQMLQSANIIAVNSKHLLEAFSQARRSRRHKR------Capsaspora QAHLFQAEMLDAAFKLAVDAKKLCDRITKF------

B Deuterostomia Homo sapiens 2

Xenopus tropicalis 2

Homo sapiens 1

Xenopus tropicalis 1

Gallus gallus Protostomia Lottia gigantea

Capitella sp.

62/0.99 Drosophila melanogaster

Nematostella vectensis Cnidaria

Trichoplax adhaerens Placozoa 100/1.00 Amphimedon queenslandica Porifera

Monosiga brevicollis Choanoﬂagellata

Capsaspora owczarzaki Filasterea Homo sapiens EGFR EGF receptor kinases Xenopus laevis EGFR

Lottia gigantea EGFR

Drosophila melanogaster EGFR

Amphimedon queenslandica EGFR

Fig. S7. (A) An illustrative focal adhesion kinase (FAK) alignment showing the different functional domains. (B) Maximum likelihood tree of the FAK protein with EGFR kinase family, based on the kinase domain. Tree is rooted using the EGFR kinase family as an outgroup. Statistical support was obtained by RAxML with 100-bootstrap replicates (bootstrap value, BV) and Bayesian posterior probability (BPP). Both values are shown in key branches. A black dot indicates BV >90% and BPP >0.95.

Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 7of8 153 168 198 238 METAZOA Homo NKMDSTEPPYSQKRYE ... GDNMLEPSANMPWFKGWKVTR------KDGNASGTTLLEALDCILPP Drosophila NKMDSTEPPYSEARYE ... GDNMLEPSEKMPWFKGWSVER------KEGKAEGKCLIDALDAILPP Brugia NKMDSTEPAFSEARFN ... GDNMLEPSPNMPWFKGWNVER------KEGNASGKTLLEALDAVIPP Lottia NKMDSTEPPYSESRFD ... GDNMLEKSQKMPWWKQWKIEQKD-EKGNMQTVTGETLSDALDSIQPP Dugesia NKMDSTEPPFSEPRFD ... GDNMIDESSNMPWYKGWEITRKN-AKKEEIKTTGRTLLDALDSLEPP Trichoplax NKMDSTEPPYSEARYN ... GDNMIEESTNMKWFKGWSVER------KEGNASGKTLFEALDAILPP Nematostella NKMDSTEPPYSEARFK ... GDNMLEKSENMPWFKQWTIERVDPATKKEANASGVTLFEGLDSILPP Geodia NKMDSTEPPYSQARYD ... GDNMLEESPNMKWFKGWNVER------KEGNASGKTLFNPLDSILPP METAZOAN ALLIES Monosiga NKMDSTEPPYSESRFN ... GDNMIEASEKLPWYKGWEITR------KDGNAKGKTLLEALDAIIPP Corallochytrium NKMDSIK--YSKDRFD ... GDNMIEASTNMDWYKGWE------KDGSVGGKTLIEALDAVSPP Capsaspora NKMDSIK--FAEERYN ... GDNMLEASENMPWFKGWTIER------KEGNASGKTLIEALDAISPP Ministeria NKMDSIK--YDEARFT ... GDNMLDASTNMPWYKGWEVDRD-----KNGKASGKTLIDALDAVLPP Amoebidium NKMDSIK--FAQDRFN ... GDNMVEPTDNMPWYKGWEVER------KEGNATGKTLLEALDAILPP Ichthyophonus NKMDSVK--YSEDRFK ... GDNMVAPTENMPWYKGWTCER------KEGNTSGFTLLEALDNIQAP FUNGI Ustilago NKMDTTK--YSEDRFN ... GDNMIEPTKEMPWYKGWERET------KAGKVSGKTLLDAIDAIEPP Neurospora NKMDTTQ--WSQTRFE ... GDNMLEPSTNCPWYKGWEKET------KAGKATGKTLLEAIDAIEPP Mucor NKMDTTK--WSQDRYN ... GDNMLDESTNMPWFKGWNKET------KAGSKTGKTLLEAIDAIEPP Allomyces NKMDMVD--WSEARFK ... GDNLLTPSANMPWYQGWSRQSK-----DGTVKTGMTLIEAMDAVDPP OPISTHOKONTA Batrachochytrium NKMDTNK--WSEERFN ... GDNMLEPSANMPWFKGWTKET------KAGTSTGKTLLNAIDSIEAP Spizellomyces NKMDSDPAPYKKERYD ... GDNLLKKSEKMSWYQGQEVTAL-----SGKKVKVHTLLDALNDFEMP Glugea NKVDTIDEKNRISRFD ... GINIVEKGDKFEWFKGWKPVSG-----AG--DSIFTLEGALNSQIPP FUNGI ALLIES Fonticula NKMDSCQ--YSEARFT ... GDNMIEPTTNMSWWKGFEITR------GSAKLTGLTLLDALNHIEPP Nuclearia NKMDTCK--YSEERFN ... GDNMLEATPNMPWFKNWEIER------KSGKVTGKTLVDALDAIEPP APUSOZOA Amastigomonas NKMDADSVQFSQQRFE ... GDNMLEPSSNMSWWT------GPTLLEALDSIKAP Planomonas NKMDDKSVNYSKARFD ... GDNMTEPSANMPWYS------GPTLLGALDACEVP Apusomonas NKMDDKTVKYSKDRYE ... GDNMMEPSPQMGWWK------GGTLLEALDAITPP AMOEBOZOA Entamoeba NKMDAIQ--YKQERYE ... GDNMIEPSTNMPWYK------GPTLIGALDSVTPP Dictyostelium NKMDEKSTNYSQARYD ... GDNMLERSDKMEWYK------GPTLLEALDAIVEP Physarum NKMDEKSVNWSQARYD ... GDNMLEKSANLPWYK------GPTLLEALDQITEP Acanthamoeba NKMDNVN--WAENRYN ... GDNMVDRTDKMPWYK------GPTLLEALDDIKPP PLANTAE Arabidopsis NKMDATTPKYSKARYD ... GDNMIERSTNLDWYK------GPTLLEALDQINEP Porphyra NKMDDKNVNWSKERYE ... GDNMLEKSTNMPWYK------GPCLLEALDNCDPP HETEROKONTA Phytophthora NKMDDSSVMYGQARYE ... GDNMIDRSSNMPWYK------GPYLLEALDNLNAP ALVEOLATA Toxoplasma NKMDSCN--YSEDRFN ... GDNMVEKSTNMSWYK------GKTLVEALDTMEAP Paramecium NKMDEKTVNYAQGRYD ... GDNMLEKSANFGWYK------GPTLLEALDAVTPP EUGLENOZOA Euglena NFKDDKTVKYSQARYE ... GDNMIEASENMGWYK------GLSLIGALDNLEPP Leishmania NKMDDKTVTYAQSRYD ... GDNMIEKSDNMPWYK------GPTLLDALGMLEPP HETEROLOBOSEA Naegleria NKFDDTSVNYAEKRYD ... GDNMIEKSDKMGWYK------GPCLLDALDNLIEP Acrasis NKMDDKSVQYKEDRYK ... GDNMLEKSTNMPWYK------GPTLLEALDALEPP PARABASALIDEA Trichomonas NKMDDKTVNYNKARFD ... GDNMTEKSPNMPWYN------GPYLLEALDSLQPP DIPLOMONIDIDA Giardia NKMDDGQVKYSKERYD ... GDNIMEKSDKMPWYE------GPCLIDAIDGLKAP OXYMONADIDA Dinenympha NKMDDKSVNWAESRYN ... GDNMLDRSTNMPWYK------DPILFDALDLLEVP ARCHAEA Sulfolobus NKMDLTEPPYDEKRYK ... GDNITHRSENMKWYN------GPTLEEYLDQLELP Thermoplasma NKMDATEPPFSEKRFN ... GDNVTKPSPNMPWYK------GPSLLQALDAFKVP

Fig. S8. Schematical alignment, based on the one shown by Steenkamp et al. (1), of a portion of the EF-1α gene showing the synapomorphic indel of opisthokonts. Amastigomonas sp. is shown in bold.

1. Steenkamp ET, Wright J, Baldauf SL (2006) The protistan origins of animals and fungi. Mol Biol Evol 23:93–106.

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Sebé-Pedrós et al. www.pnas.org/cgi/content/short/1002257107 8of8