Online Resource 9. Figure S8: Amino acid alignments.

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1 Online Resource 9 This file contains amino acid alignments of the Long Oskar and Lasp-binding domains from 18 Drosophilids including the D. virilis allele from Webster et al. (1994) using the PRANK MSA, and the results of multiple PAML analyses of these alignments. The following elements comprise this Additional File: Figure S8: Amino acid alignments. Table S14. Log likelihood values and parameter estimates under branch site models of Oskar domains. Ahuja & Extavour Online Resource 9, Page 1 of 5

2 Figure S6. Amino acid alignment of Long Osk and Lasp binding region using the D. virilis allele from Webster et al (1994) and the PRANK MSA. Positively selected sites with BEB probability = 0.95 are indicated in red, BEB = 0.98 in blue. Long Osk domain MATYRSAHSEPYTSKSNS------N----SNSSSSSRLTALRKRLTTCLQQWCQHL---- MATYRSAHSEPYTSKSNS------N----SNSSSSSRLTALRKRLTTCLQQWCQHL---- MATFRSE----FNSVPNT------H----PD-----RLTAFRKKLTTCFHQWRQKL---- MATFRSE----FNSVPNT------Y----NQ-----RLTALKKKLTTCFQQWRQQLLKLH MAAVTSE----FPSKPISYTST--NTSAKTY-----YLKSVKKRVTTCFQQLRDKL---- MATVTSE----FPSKPISYTST--NTSAKTY-----YLKSVKKRLTTCFQQLREKL---- MATVTSE----FPSKPISYTST--NTSAKTY-----YLKSVKKRLTTCFQQLREKL---- MAAVTSE----FPSKPISYTST--NTSAKTY-----YLKSVKKRVTTCFQQLREKL---- MAAVTSE----FPSKPISCTST--NTSAKSN-----YLKSVKKRVTTCFQQLREKL---- MAAVRSE----FPSEPISYTNT--NTSNKSN-----CLRSVKKRVTTCLQQLREKL---- MAAVGSE----FPSEPISCRNT--NTPAKPD-----RLRSVKKRVTTCLQQLREKL---- MATVTSE----FPSKPISYTNTNTNTSTKTY-----CLRSVKRRVTTCLQQLREKL---- MAAVRSE----FPSEPYSNTNN--NTSTKTD-----CLRSVKKRVTTCLKQLCEKL---- MAAVRSE----FPSEPNSNTNT--NTSTKTD-----RLRSVKNRVTTCLKQLCEKL---- MAAVRSE----FPSKPNSNTYT--NS--KFD-----DLRSVKKRVTTCLKKLCEKL---- MAAVRSE----FPSESNS------NPSTKAD-----SLRSLKKHVATCWHNLCKKL---- MAAVTSE----LPREPTT------TTNTKSD-----RLRAVKKRVTTCLQQWCQKL---- MAAVTSE----LPREPTT------TTNTKSD-----RLRAVKKRVTTCLQQWCQKL QKPASFQKCKSG-PNHQFYKIFEEVFDSK QKPASFQKCKSG-PNHQFYKIFEEVFDSK Q HF-----KH-DYS QQQQQPKQQQQQQQHQSQHQHQQQKQKQRGSE HFYNIFVRA-DCS QSSGSFRKSSSS----CLNQIFVRS-DFS QPFGSFRKSSSN----RLYKIFVKS-DFS QPFGSFRKSSSSS---RLYKIFVKS-DFS QASGSFRKSSTS----RLYKIFVKA-DFA QASGSFRKSSPS----RLYKIFVKA-DFA PASGSFRKSSSS----RFYKIFVKA-DFS PAAGSFRKSSTG----RFYQVFVRA-DFS PATGSFRKSSTS----RFHRFFIKS-DFS QAPGSFRKSSSS----RFYKIFVKA-DYS QATGSFRKSSSS----RFYKIFVKA-DYS QAPGSFRKSSSS----RFHKIFVRA-DYS QQPASFRKSSHS----RFYKIFLKA-DCS QQPASFRKSSSS----RFYRLFVKA-DYT QQPASFRKSSSS----RFYRIFVKA-DYT Ahuja & Extavour Online Resource 9, Page 2 of 5

3 Ahuja & Extavour Online Resource 9, Page 3 of 5 V-FGDKTKYIRQFKKIFKG----N KD L----- V-FGDKTKYIRQFKKIFKG----N KD L----- T-LRAKSICWQKFKKIYTA----T KL I----- I-LSEKVTYLRKFKRIFTA----RQNLASARTAATAASAKQPVLGRC I----- A-CGE------RFRKIFKSARK-T EL PELWK- A-CGE------RFQKIFKSARK-T ES PELWK- A-CGE------RFQKIFKSARK-T ES PELWK- A-CGE------RFQRIFKSARK-T ES PELWK- A-CGE------RFQRIFKSARK-T QS PELWK- A-FGE------RFQKIFKSARK-T ES PELWK- A-FGE------RFRKIFKSARK-T ES PELWK- D-FSE------KLQKIFKSARK-S ES PELRR- G-FREKSKFVRQFQKIFKSARK-P ES PELWK- G-LREKSKFLRQLQKIYKSTRK-T ES PELWK- ----DCSKFVRRFQKIFKSARK-T ES PESWK- ASFPEKSKFLRQFHRIFKAGRK-S EGGGEPDPEPELWKE A-FGDKFKSVRQFHRIFKAGRK-G KE P----- A-IGDKFKSVRQFHKIFKAGRKKS KE P A-KKSEPGRVVLVLVPEKPVDDTEE-----SQK-KQLQLVARLFSSTLISAKEE A-KKSEPGRVVLVLVPEKPVDDTEE-----SQK-KQLQLVARLFSSTLISAKEE S-Q EE-----VEE-QQLQIVATLFSSTLIS-KYT G-Q EE QQLQIVASLFSSTLIS-KHS--- VPLVAH ELT--SRQSS-QQLQVVARLFSSTQIS-TKE--- VPLVAH ELT--SRQSS-QQLQIVARLFSSTQIS-TKE--- VPLVAH ELT--SRQSS-QQLHIVARLFSSTQIS-TKE--- VPLA-H DLTTTSRQSS-QQLRIVARLFSSTQIS-TRE--- VPLAAH DLTN-GRQTS-QQLRIVARLFSSTQIS-TKE--- VPLA-H D--V-TGQSG-QQLQIVARLFSSTLIS-TKE--- VPLA-H D--V-TGPSS-QHLQIVARLFSSTLIS-TKE--- VPLSTH DQQT-SGQSS-QQLQIVARLFSSTLIS-TKE--- VPLA-H D--V-TGQNS-QQLQIVARLFSSTLVS-TEE--- VPLA-H D--V-TGQSS-QQLQIVTRLFSSTLIS-TEE--- VPLA-H D--V-TRQSSIHQLQIVARLFSSTQIS-TEE--- VPLA-D EQ-----RSQ-EQLQIVARLFSSTLIS-TKEEEI ---A-E SV-----DER-QQLQFVARLFSSTLIS-TEE A-E SV-----DER-QQLQFVARLFSSTLIS-TEE--- I SC-SY--NSS I SC-SY--NSS S SK-NS--SSC S SA-SI--TSS ITY-NSNS---NTSE NN ITY-SSNN---NTSE TN ITY-SSNS---NTSE SN ITYTNSNN-----SSYSFRRRKSE-SH--RSN ITYSTSNN---NTSSYSFRSRKSE-SC--RSN IHC-N-NN------SYSYKSRKSE-SY--SSN ISY-S-NH------RYSCRSGKSE-SY--SSN ITF-N-NH---TKNSFSYKCRKSE-GY--SSN ITY-HTNI---N----SYKSRKSQ-SY--DCD ITY-NTNNKNNN----SCKSRKSHKSY--KSS IIY---NN---I----SFNSRKSQ-SH--SSN ITT-SRSS---NCNT-SYTRRKSQ-SYCSTST I-----YG---N------SSRKSQ-SF--NSN I-----YG---NGKT-SYSSRKSQ-SY--NSN

4 Lasp-binding domain IE--DQ-AGPLQQLARAAAESNWCYQDNWNHLKNCYQ R------ANIF----A IE--DQ-AGPLQQLARAAAESNWCYQDNWNHLKNCYQ R------ANIF----A -----K-ITPVQHLATAAMAPFGVYQDNWKHLNNQYQLSQLATQN------SNQFSCIRS -----K-ISKLQPLATAAAMSNDVYQDNWKHLNNQYQLPQLNAPK------NNIHSHIAS IA--TR-PVSLHQMANEAAESNWCYQDNWKHLNNFYQQASVNAPK--MPVPINIY----S IA--TR-PASLHQMANEAAESNWCYQDNWKHLNNFYQQASVNAPQ--MPVPINIY----S IP--TR-PASLHQMANEAAESNWCYQDNWKHLNNFYQQASVNAPQ--MPVPINIY----S SA--TRQPASLHQMANEAAESNWCYQDNWKHLNNFYQQSSVNAPK--MPVPVNIY----S TS--TR-PASLHQMANEAAESNWCYQDNWKHLNNFYQQASVNAPK--MPVPINIY----S IN--TK-AAPLQQLAKEAAESNWCYQDNWNHLNNFYQQSRVNEPQ--MPVPINIY----S VD--TR-AAPLQQLAKEAAESNWCYQDNWNHLNNIYQRSSVSEPQIQLPVPINIY----S MSMHTR-AAPLQQLAKVAAESNWCYQDNWKHLNNFYQQSSVNEAQ--RPVPINIY----T IT--AK-TTPLHQLAKEAAESNWCYQDNWKHLNNFYQQSSANEPQ--RPVPINIY----S AT--TK-AAPLQQLAKEAAESNWCYQDNWKHLNNFYQQSSANEPQ--MPVPINIY----S IA--TK-AAPLPQLAKEAAESNWCYQDNWKHLNNYYQQSSANEPQ--IPVPINIY----T IN--TR-SAALPHMAKAAAESNWCYQDNWNHLKNIYQQSSGNAPH--MHEPNQIY----T IN--AN-SATLQKLAKAAGESNWCYQDNWNHLNNFYQQSNVNGHQ--NQVPPNIY----V IK--TN-SASLQKLAKAAGESNWCYQDNWNHLNNFYQQNNVNGPQ--NPVPPNIY----I -DPPEK-P-PPLPA-QE-N-HL-RNSN S----QN -DPPEK-P-PPLPA-QE-N-HL-RNSN S----QN -DPVQL-QIAPHPP-EQ-Q-H QQ -NPAQL-Q-AALPA-E PDAPEE-P-INLAPPGH-QPSC-RTQS QKTE-PT -DAPEE-P-THLAAPGQ-QPSCCRTQS KKTE-PT -DAPEE-P-THLAAPGQ-QPSCCRTQS KKPE-PT -DAPEE-R-IHLDAPGH-QPSC-RTQN QKTQQTT -DAPEE-R-IQVDAPGH-QPSC-RTQN QKTQHPT -DATEE-P-IQLDAPEH-HPHC-QNHNENQNPNQN-QNTK-QN -DAPEE-P-NQMNVPQH-HPLC---HNRNQNPSQN-QQAK-QD -DALEDLP-IQLDNPEH-HPNC-KNNN QNIK-QN -DPPEE-R-TQFAVPEL-PPHC-RNPN QNTK-QN -DATEE-R-TQFAVPEP-RPCG-KNHN QNNK-PN -DATEE-P-NQFAVPEH-LPPC-KNHK QNTK-PN -DPPEL-S-NHLANPELIKPPC-KNHN S----PN -DTPDR-Q-TQVTDPE--Q-HC-QNPH PS----QN -DTPDQ-P-TQVTDPEL-L-HC-QNRN PS----QN Ahuja & Extavour Online Resource 9, Page 4 of 5

5 Table S13. Log likelihood and parameter estimates from branch site models for Oskar using PRANK MSA alignments of 18 Drosophilids including the D. virilis allele sequenced in Webster et al. (1994). Long Osk (PRANK) Lasp (PRANK) ω values under Model: MA ω values under Model: MA fix Site Class Proportion Background D. virilis l Proportion Background D. virilis l a b a b Ahuja & Extavour Online Resource 9, Page 5 of 5

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