<p> SUPPLEMENTARY MATERIAL</p><p>Adaptive evolution of gamete-recognition proteins in birds and mammals</p><p>Sofia Berlin, Qu Lujiang 1 and Hans Ellegren</p><p>Supplementary Table 1-3, Supplementary Figure 1 and 2 + some other information Supplementary Table 1. Avian Genbank accession numbers.</p><p>Gene Chicken Zebra finch Mallard Turkey Quail CD9 AB032767 EF191917 Acrosin XR_026893 ZP1 NM_204683 AB251639 AB253774 AB061520 ZP2 NM_00103909 ZP4 NM_204879 ZPAX NM_001045837</p><p>Trees that were used in the PAML4 analyses of the mammalian orthologues (topologies based on Murphy et al. (2001)): </p><p>CD9: ((((Homo_sapiens,Pan_troglodytes),Macaca_mulatta),Callithrix_jacchus),(Rattus_norvegicus,Mus_musculus),((Sus_scrofa,Bos_taurus),</p><p>(Felis_catus,Phoca_vitulina)))</p><p>ZP1: (((Homo_sapiens,Pan_troglodytes),Macaca_mulatta),Equus_caballus,(Mesocricetus_auratus,(Mus_musculus,Rattus_norvegicus)))</p><p>ZP2: (((Equus_caballus,(Felis_catus,(Canis_familiaris,Mustela_erminea))),(Bos_taurus,Sus_scrofa)),(Callithrix_jacchus,(Homo_sapiens,</p><p>Macaca_mulatta)),(Oryctolagus_cuniculus,(Mesocricetus_auratus,(Mus_musculus,Rattus_norvegicus))))</p><p>ZP4: ((Macaca_mulatta,(Homo_sapiens,Pan_troglodytes)),(Oryctolagus_cuniculus,(Mesocricetus_auratus,Rattus_norvegicus)),((Sus_scrofa,</p><p>Bos_taurus),(Mustela_erminea,Felis_catus)))</p><p>Supplementary Table 2. Primers combinations and tissue used for amplification of bird sequences. </p><p>CD9 Acrosin ZP1 ZP2 ZP4 ZPAX</p><p>(spleen) (testis) (ovary) (ovary) (ovary) (ovary) Mallard A/B C/D - C/D, K/L, M/N, C/D, E/F C/D</p><p>O/P Pigeon A/B - - A/B, C/D, E/F E/F, G/H Guineafowl A/B A/B C/D A/B, C/D, E/F, A/B, C/D A/B, C/D, E/F,</p><p>G/H, G/H, I/J Pheasant C/D C/D A/B, C/D, E/F C/D, I/J, K/L; A/B, C/D C/D, E/F, G/H, I/J</p><p>M/N Red grouse A/B - - - - - Turkey A/B - - A/B, C/D, E/F, A/B, C/D A/B, C/D, E/F,</p><p>G/H, G/H, I/J Quail A/B A/B - A/B, C/D, E/F, A/B, C/D A/B, E/F, G/H, I/J</p><p>G/H,</p><p>Primer sequences: C: CD9-cons-for: AAGTGCATCAARTACTTGCTC (22) CD9 D: CD9-cons-rev: CTTTCTGCGGATAGCACAG A: CD9-for: ATGCCTGTCAAAGGAGGCAC (1) B: CD9-rev: CAGCTCTTAGACCATYTCTC (662) Acrosin F: ZP2c-rev: GGGTCATTGCGGTTCAGGAG A: Acro-for: GATGGTGCTGCTGCTGCCC G: ZP2d-for: ATCCTTCTAAGCTACCCAG B: Acro-rev: GGGCTGCCATTGGGCATTA H: ZP2d-rev: ACCACATTTGCCATCAAGG C: AcroN-for: GGCACGGGRCACATSTGTGG I: ZP2Na-for: GACCTGCCTGCAGGACAGGC D: AcroN-rev: AYATGCAGGAGCTCCTGGAGC J: ZP2Na-rev: GCATTCTTGACTCCACGGTC K: ZP2Nb-for: GCCTTCACTGCCACTGGAG ZP1 L: ZP2Nb-rev: GGTCCCACAYCCRCTCAGTG A: ZP1Na-for: AGCYGCTCCCTGCTGCTGC M: ZP2Nc-for: TAGCTGCTGTATGCACCCAGG B: ZP1Na-rev: GACCACTAGCGTGGGTCATCC N: ZP2Nc-rev: GCAGTGGAAGTAGACTAGGC C: ZP1Nd-for: CCCAAGCAGGTCTTTTTCAC O: ZP2Nd-for: GGCTGGAAGTGAAGGCTTTTG D: ZP1Nd-rev: CACTGTGTGACGGGAAAGTG P: ZP2Nd-rev: TCTTCTCTTCAGGCATTTAAG E: ZP1Ne-for: CAGCCCTACAACCTGGACA F: ZP1Ne-rev: CTGGTAGTGGCTGGGGAAG ZP4 A: ZP4a-for: TGTGGCCAAGGGAGCTTGC ZP2 B: ZP4a-rev: AAGTGCTGTCCCTAGTGACAG A: ZP2a-for: CTGTTCTTGGCCCCTGGTG C: ZP4b-for: CAGGTGACTGGAGACCAGG B: ZP2a-rev: GGTAGGCAACAGTCATGTGTG D: ZP4b-rev: GGGCCTTTGCTGGAAACAC C: ZP2b-for: TCTACACTGCGGCACTGAAGC E: ZP4Na-for: GGCTGGGAAGGGAATGCTTCC D: ZP2b-rev: GCTAGCCTGAACTCACTGTC F: ZP4Na-rev: CCCTGGATGCCACCAACTC E: ZP2c-for: CTAGTCCTGGACACGCTCAG ZPAX F: ZPAXb-rev: GGAAGGCACTGTATTAGCTCTG A: ZPAXa-for: TAGGAATACTCATCTCTGC F: ZPAXc-for: TCTCAATTTCCTGCAGATTTCC B: ZPAXa-rev: ATCATCCACTGCTGTTTGTA G: ZPAXc-rev: GCAGTTCAACTTCAAAGTACA C: ZPAXstar-for: TATGTGAAACCAACTACATGG H: ZPAXd-for: GAATACATGTGGAACAAGTAG D: ZPAXstar-rev: TGTTCCAAGAACAGGTTGAT I: ZPAXd-rev: CACTGCAGTGCAAATATAACT E: ZPAXb-for: TAGGTGCAGAAGGTGGCTCTT </p><p>Murphy, W. J., E. Eizirik, S. J. O'Brien, O. Madsen, M. Scally, C. J. Douady, E. Teeling, O. A. Ryder, M. J. Stanhope, W. W. de Jong, and M. S. Springer. 2001. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294:2348-2351. Supplementary Table 3. Number of base pairs analysed for each species and gene.</p><p>Gene Chicken Zebra finch Mallard Pigeon Guinea fowl Pheasant Red grouse Turkey Quail CD9 672 672 641 637 641 576 640 640 641 Acrosin 1335 459 630 - 693 647 - - 894 ZP1 2802 915 2814 - 497 1167 - 2802 2802 ZP2 2061 1542 1350 - 1995 1667 - 1995 1998 ZP4 1629 1411 535 1280 1317 1285 - 1317 1320 ZPAX 2718 1926 614 1245 2468 1332 - 2468 1874 Supplementary Figure 1. Amino acid alignment of chicken and human Acrosin. Positively selected sites are indicated in bold.</p><p>Chicken ------Human ------VEMLPTAILLVLAVSVVAKDN </p><p>Chicken ------GTRVVGGTDAPQGAWPWIVSLQSTWYVGTG--H Human ATCDGPCGLRFRQNPQGGVRIVGGKAAQHGAWPWMVSLQIFT-YNSHRYH </p><p>Chicken ICGGSLITPQWVLTAAHCFDHATPDTPWHVVIGGHDLKR-----LGPEAV Human TCGGSLLNSRWVLTAAHCFVGKNNVHDWRLVFGAKEITYGNNKPVKAPLQ </p><p>Chicken VRNVIRIIPHEYYHRNNMANDIALLELDQPVQCSYYIQLACVPDASLRVS Human ERYVEKIIIHEKYNSATEGNDIALVEITPPISCGRFIGPGCLPHFKAGLP </p><p>Chicken ELTD-CYVSGWGHMGLRSLQEYVEPYRVLQEAKVQLIDLNICNSSNWYAG Human RGSQSCWVAGWGYIE----EKAPRPSSILMEARVDLIDLDLCNSTQWYNG </p><p>Chicken AVHIHNVCAGYPQGGIDTCQGDSGGPLMCKDKTADYFWLIGVTSWGKG?? Human RVQPTNVCAGYPVGKIDTCQGDSGGPLMCKDSKESAYVVVGITSWGVGCA </p><p>Chicken ???QPGVYASTQYFPLWILVQMGLLPAEAPTTTPYPVYISSSYQRPKPTY Human RAKRPGIYTATWPYLNWIASKIGSNALRMIQ------</p><p>Chicken SSPFRPCPFPRQKLLDFFNLLQELLQGLRGKKA Human ------</p><p>Supplementary Figure 2. Amino acid alignment of chicken and human ZP2. Positively selected sites are indicated in bold.</p><p>Chicken ------RGRLLLLLLFGFLLFLAPGASGEWDL Human ACRQRGGSWSPSGWFN--AGWSTYRSISLFFALVTSVNSIDVSQLVNPAF </p><p>Chicken SESMTCLQDRLELELPRELGNYTWHVRAVDVSGEEMMSCEHAVDYEKLLL Human PGTVTCDEREITVEFPSSPGTKKWHASVVDPLGLDMPNCTYILDPEKLTL </p><p>Chicken SALLVNCTSLEHGQYQLRLLLLLNGTAGEERNVTYSAHCSAAHGDEIIAP Human RATYDNCTRRVHGGHQMTIRVMNNSAALRHGAVMYQFFCPAMQVEE--TQ </p><p>Chicken LFVGETNCTKDSMAVTFP--GPSLSDEHLV---QVAVLTGTLTIDDGIKV Human GLSASTICQKDFMSFSLPRVFSGLADDSKG---TKVQMGWSIEVGDGARA </p><p>Chicken HQLSLGEAMQHGYSFLADG-HHLVFQAAFTATGVVSYKHNHKALYTAALK Human KTLTLPEAMKEGFSLLIDN-HRMTFHVPFNATGVTHYVQGNSHLYMVSLK </p><p>Chicken LMYGPPEHRLTVESRMLCVPGP-VFCNTTHMTVAIPAFPGTLMAVAVEDE Human LTFISPGQKVIFSSQAICAPDP-VTCNATHMTLTIPEFPGKLKSVSFENQ </p><p>Chicken TIPMDQLQDKGITLKTTVGVELHVSRRVLKSTLHGESCPRVQSYLSSLKL Human NIDVSQLHDNGIDLEATNGMKLHFSKTLLKTKL-SEKCLLHQFYLASLKL </p><p>Chicken TFHFHEETVAMVMHPQCPCDQLTPIA--AACTRDGYMDFEVLAGSTTPPL Human TFLLRPETVSMVIYPECLCESPVSIVTGELCTQDGFMDVEVYSYQTQPAL </p><p>Chicken VLDTLRLRDPTCKPASRSPLNDRAWFHVPLSGCGTRYWLEGEKIMYENEV Human DLGTLRVGNSSCQPVFEAQSQGLVRFHIPLNGCGTRYKFEDDKVVYENEI </p><p>Chicken RALRSDSVLHRISRDSEFRLAVLCSFSNGDASVSVRVDNPPPLAASTNQG Human HALWTDFPPSKISRDSEFRMTVKCSYSRNDMLLNINVESLTPPVASVKLG </p><p>Chicken PLSLILLSYPEDSYRQPYHDDQYPIVRYLQQPIFMEVQVLNRNDPNLYLQ Human PFTLILQSYPDNSYQQPYGENEYPLVRFLRQPIYMEVRVLNRDDPNIKLV </p><p>Chicken LDDCWATALEDPTSLPQWNIVVDGCEYEQDSYRTVFHPVGHGVSYPNYRQ Human LDDCWATSTMDPDSFPQWNVVVDGCAYDLDNYQTTFHPVGSSVTHPDHYQ </p><p>Chicken RLEVKAFAFVSGDKALPGLVYFHCSVLICSRFQLDSPLCTARCPRLPRRK Human RFDMKAFAFVSEAHVLSSLVYFHCSALICNRLSPDSPLCSVTCPVSSRHR </p><p>Chicken RGSGMLGA-SSVVSLQGPVLLVPHGWAAA------Human RATGATEAEKMTVSLPGPILLLSDDSSFRGVGSSD---LKASGSSGEKSR </p><p>Chicken ------RGGTLLSKVVWAA------VTATAVGVFSLTAIMLLFMDLL Human SETGEEVGSRGAMDTKGHKTAG-----DVGSKAVAAVAAFAGVVATLGFI </p><p>Chicken KCLKRR------Human YYLYEKRTVSNH-</p>