<p>Table S1. List of markers located on zebra finch Taeniopygia guttata chromosome 4a (Tgu4a), analyzed in the present study. Position on Tgu4a (P); gene ID (gTgu), locus tag (tTgu) and gene description (Description Tgu) in the zebra finch genome (taeGut3.2.4 at Ensembl); gene ID (tGga) and locus tag (tGga) in the chicken Gallus gallus genome (WASHUC2.1 at Ensembl). P gTgu tTgu Description Tgu gGga tGga 0.9 ENSTGUG00000002281 SYTL4 Synaptotagmin-like protein 4 ENSGALG00000006786 SYTL4 1.6 ENSTGUG00000002329 DIAPH2 Protein diaphanous homolog 2 ENSGALG00000006839 Q9DEH3_CHICK 2.7 ENSTGUG00000002396 PCDH11X Protocadherin-11 X-linked Precursor ENSGALG00000006851 Q9YGQ6_CHICK 4.0 ENSTGUG00000002423 DACH2 Dachshund homolog 2 ENSGALG00000006886 Q9PTH2_CHICK 4.4 ENSTGUG00000002466 ZNF711 Zinc finger protein 711 ENSGALG00000006929 O42424_CHICK 4.7 ENSTGUG00000002510 RPS6KA6 Ribosomal protein S6 kinase alpha-6 ENSGALG00000007097 RPS6KA6 5.3 ENSTGUG00000002582 ITM2A Integral membrane protein 2A (E25 protein) ENSGALG00000004107 Q5F3H7_CHICK 5.5 ENSTGUG00000002603 TAF9B Transcription initiation factor TFIID subunit 9B ENSGALG00000004187 TAF9 5.6 ENSTGUG00000002666 P2RY4 P2Y purinoceptor 4 ENSGALG00000004346 P2RY4 6.4 ENSTGUG00000002760 AR Androgen receptor ENSGALG00000004596 Q2ACE0_CHICK 6.8 ENSTGUG00000002846 ZC3H12B Zinc finger CCCH domain-containing protein 12B ENSGALG00000004713 Q5ZIQ0_CHICK 7.0 ENSTGUG00000002901 DDX26B Protein DDX26B ENSGALG00000006157 DDX26B 7.0 ENSTGUG00000002865 SLC9A6 Sodium/hydrogen exchanger 6 ENSGALG00000006180 SLC9A6 7.9 ENSTGUG00000003029 XP_002190721.1 Serine/threonine protein kinase MST4 ENSGALG00000006049 Q5ZMA7_CHICK 8.1 ENSTGUG00000003059 PLS3 Plastin-3 (T-plastin) ENSGALG00000005930 Q5ZI39_CHICK 8.4 ENSTGUG00000003141 WDR44 WD repeat-containing protein 44 (Rabphilin-11) ENSGALG00000005997 Q5ZLT9_CHICK 9.2 ENSTGUG00000003292 LONRF3 LON peptidase N-terminal domain and RING finger protein 3 ENSGALG00000008860 LONRF3</p><p>9.4 ENSTGUG00000003329 CXorf56 UPF0428 protein CXorf56 ENSGALG00000008652 CXorf56 9.5 ENSTGUG00000003338 LAMP2 Lysosome-associated membrane glycoprotein 2 Precursor ENSGALG00000008559 CUL4B 10.2 ENSTGUG00000003682 THOC2 THO complex subunit 2 ENSGALG00000008507 THOC2 11.6 ENSTGUG00000003899 SMARCA1 Probable global transcription activator SNF2L1 (EC3.6.1) ENSGALG00000008431 SMARCA1 (Nucleosome remodeling factor subunit SNF2L)(ATP- dependent helicase SMARCA1)(SWI/SNF-related matrix- associated actin-dependent regulator of chromatin subfamily A member 1) 11.8 ENSTGUG00000004029 RAB39B Ras-related protein Rab-39B ENSGALG00000008384 RAB39B 12.1 ENSTGUG00000004277 IRS4 Insulin receptor substrate 4 ENSGALG00000008107 IRS4 13.0 ENSTGUG00000004568 HMGB3 High mobility group protein B3 ENSGALG00000009071 HMGB3 13.2 ENSTGUG00000004663 MAMLD1 Mastermind-like domain-containing protein 1 ENSGALG00000009098 CXorf6 16.5 ENSTGUG00000005499 XP_002195404.1 Hypothetical protein LOC100232337 ENSGALG00000007530 LOC428714</p><p>16.8 ENSTGUG00000005560 MTMR8 Myotubularin-related protein 8 ENSGALG00000007590 MTMR8_CHICK 18.8 ENSTGUG00000006268 SOX3 Transcription factor SOX-3 ENSGALG00000007243 CH03_CHICK 19.2 ENSTGUG00000006374 GABRE Gamma-aminobutyric acid receptor subunit epsilon precursor ENSGALG00000020292 GBRG4_CHICK (GABA(A) receptor subunit epsilon 20.1 ENSTGUG00000007329 TEX11 Testis expressed 11 ENSGALG00000005775 TEX11</p><p>Table S2. Primer sequences (Seq 5’-3’) and respective annealing temperatures (Ta) for each of the selected loci located at different positions on Tgu4a (P). The type of marker (t) is indicated: intron (int) or coding sequence only (cds). </p><p>Primer P (Mb) t Seq 5’-3’ Ta SYTL4 F 0.9 int GGTGGAGATCCAYCGGCAGCGG 63-58 [57] SYTL4 R ATGCTGACCATGCTCCCCAGY DIAPH2 F 1.6 int GTGARAARAAAGCAGCAGAATTTTCCA 58-54 [53] DIAPH2 R TCTTTGATTTTCTCCTCTCGTTTCTGM PCDH11X F 2.7 cds TGGCCAGAACATTGGGGATGGTG 60-55 [53] PCDH11X R GGGGCCTGGGGTCTTGAAGC DACH2 F 4.0 int GGCTGGACAGGCCTTGCCTC 60-55 [53] DACH2 R TCCACAGCCACTTTCAGCAGGC ZNF711 F 4.4 cds GGGGAAAAGCCATACCAGTGTCAGCAC 61-57 [56] ZNF711 R GGCCTCTYTGTGGTGCCTCATTATR RPS6KA6 F 4.7 int GAAGGCCTATTCTTTCTGTGGT 56-52 [50] RPS6KA6 R TCGATCTTTACCTTGAAATGGY ITM2A F 5.3 int CACAAGGTGTACCGTGGKGA 58-54 [53] ITM2A R AAGAGATCCATCAGATTYCTTGG TAF9B F 5.5 int GCCGCGCGTCATCAACCAGA 61-57 [56] TAF9B R GTGCGGCACTGGATCGCCA P2RY4 F 5.6 cds GGKCTGCCCCTCAACTCCTGGGC 61-57 [56] P2RY4 R GCCKCTTGGCCATCAGGCAGTAGCAC AR F 6.4 int GGCCTGAAGAACCAGAAGCTCTTTGA 58-54 [53] AR R TCTTGGGAACCTGCACAGAGATGAT ZC3H12B F 6.8 cds AGCCCGSAAGTCCGAGGCCA 61-57 [56] ZC3H12B R CTCCTGGACAACTGGACCGTGCT DDX26B F 7.0 int TGAAGCCACAGGCATTCAGRAA 58-54 [53] DDX26B R GCAACAGGAACACTRTGAAGGC SLC9A6 F 7.0 int GCCTTTGCAGTYACCTGCTATGGGATA 61-57 [56] SLC9A6 R TCAGCGGCGCAGAGGTTCAAG XP002190721.1F 7.9 int ATGCATCCRATGAGAGTTCTGTT 56-52 [50] XP002190721.1R TGTGCTTCAGRAGTTCTTTTGC PLS3 F 8.1 int YGAGGACCTGGGTGATGGTCA 58-54 [53] PLS3 R ACTGCCAAGCTTGTGCTGATAGT WDR44 F 8.4 int AGCAGATCTTCTTGATCTTTCCTGGT 58-54 [53] WDR44 R AGCAGTGACAAAGTCTATATGCTGGA LONRF3 F 9.2 int TGAAGAGGAAATAGCAGAGCTTTCCA 58-54 [53] LONRF3 R GGACATGGGACTGTAGGATAAGCC CXorf56 F 9.4 int TTCATTGTYGATGGTGCTGTSGTCAA 60-55 [53] CXorf56 R AACTTGCCCATGTCTTTGGT LAMP2 F 9.5 int TGTGGAGCTGCTTTCACMAGCAAACT 60-55 [53] LAMP2 R AGGCACATAGGTTGGCCARTAACCCA THOC2 F 10.2 int TGGAAGCCATGACTGGTGGAGA 60-55 [53] THOC2 R AGCAGGCASAGTGGAAGGGCAA SMARCA1 F 11.6 int TCAGAGACGCTGCAATACTCTCAT 56-52 [50] SMARCA1 R GCATCTTTYTTTCCAGAGTTYTCAGCA RAB39B F 11.8 cds GGTCCATCACCAGAGCCTACTACAGGA 61-57 [56] RAB39B R GGGACAAACCCGCTCTTTACCCCT IRS4 F 12.1 cds RCCTCCCTGAGGCCTGGCAC 61-57 [56] IRS4 R GCCGGCCCAAATTCATCCTTGCAGT HMGB3 F 13.0 int YCCCAATGCCCCAAARCGACC 60-55 [53] HMGB3 R TCACCATCACTGAGGTTGTTCCACA MAMLD1 F 13.2 cds AGTCTGTGGACCAGGAGCTGCAAG 61-57 [56] MAMLD1 R GGGGTGCTGCTGGAAGTCATGC XP002195404.1F 16.5 cds MGTGCCCTGGCTGTGGGC 61-57 [56] XP002195404.1R AGCTTMAGGATCCCACCAGCTACCAC MTMR8 F 16.8 int AGGAGTGGATTGCAATGGGCCAYAA 61-57 [56] MTMR8 R GGAACTGCTGCATSAGCTGCCA SOX3 F 18.8 cds AAATACCGGCCCCGGAGGAA 61-57 [56] SOX3 R GTTTATATGTGAGTTAGTGGTACGGTR GABRE F 19.2 int AYATGGTGAGCCGCATCTGG 58-54 [53] GABRE R GCAGCTGGAGCAGACATTCAG TEX11 F 20.1 int CAAACTGCTGCTGGAGCATGG 61-57 [56] TEX11 R TTTGCTTAGCRAGCAGCTCCCTCTT HTR2C F 20.6 cds GCCCAAGCAGAGGAGGAGCAGC 61-57 [56] HTR2C F TCCCTGGTTGCATYTCAAGGCCCTC Table S3. List of samples (in alphabetical order) used in the phylogenetic analysis (Phy) and dating of the neo-sex chromosome origin (D). GenBank accession numbers of sequences of cytochrome b and myoglobin genes are indicated for each species. Scientific name cyt b Phy D myo Phyl D Accession no Accession no</p><p>Abroscopus superciliaris DQ008515.1 X X DQ008567.1 X X Acanthisitta chloris AY325307.1 X EU739911.1 X Acrocephalus arundinaceus AJ004252.1 X X DQ008530.1 X X Acrocephalus dumetorum AJ004773.1 X X AY887682.1 X X Aegithalos caudatus AY228044.1 X X AY228281.1 X X Aegithalos concinnus DQ008519.1 X X DQ008570.1 X X Alauda arvensis AY228047.1 X X AY228284.1 X X Alcippe morrisonia DQ008483.1 X X DQ008535.1 X X Andropadus latirostris DQ008508.1 X X DQ008560.1 X X Apalis flavida DQ008496.1 X X DQ008547.1 X X Apalis thoracica DQ008497.1 X X DQ008548.1 X X Bathmocercus cerviniventris DQ008494.1 X X DQ008545.1 X X Batis mixta DQ011862.1 X X DQ011860.1 X Bradypterus baboecala DQ008473.1 X X DQ008525.1 X X Bradypterus tacsanowskius DQ008474.2 X X Calcarius lapponicus AY228050.1 X X AY228287.1 X X Camaroptera brachyura DQ008498.1 X X DQ008549.1 X X Campylorhynchus fasciatus AY228051.1 X X AY228288.1 X X Certhia familiaris DQ008524.1 X X DQ011861.1 X X Cettia cetti DQ008509.1 X X DQ008561.1 X X Cettia diphone DQ008510.1 X X DQ008562.1 X X Chaetops frenatus AY228052.1 X X AY228289.1 X X Chlorocichla flaviventris AY228053.1 X X AY228290.1 X X Chloropeta natalensis DQ008523.1 X X Chrysomus cyanopus AY228046.1 X X AY228283.1 X X Cisticola juncidis DQ008493.1 X X DQ008544.1 X X Coccothraustes coccothraustes AY228055.1 X X AY228292.1 X X Coracina lineata FJ821115.1 X EU273398.1 X Corvus corone AY228087.1 X X AY228327.1 X X Criniger pallidus DQ008507.1 X X DQ008559.1 X X Cyanistes caeruleus DQ474042.1 X X Delichon urbica DQ008517.1 X X DQ008568.1 X Delichon urbica urbica DQ008568.1 X Donacobius atricapilla DQ008481.1 X X DQ008533.1 X X Eopsaltria australis AY064273.1 X X AY064732.1 X X Erithacus rubecula AY228058.1 X X AY228296.1 X X Ficedula hypoleuca AY228062.1 X X AY228300.1 X X Fringilla montifringilla AY495390.1 X EU739966.1 X Garrulax chinensis DQ008484.1 X X DQ008536.1 X X Graminicola bengalensis DQ008480.1 X X DQ008532.1 X X Heliolais erythropterus DQ008499.1 X X DQ008550.1 X X Hippolais icterina DQ008479.1 X X DQ008531.1 X X Hirundo rustica DQ008516.1 X X AY064258.1 X X Hypsipetes mcclellandii DQ008506.1 X X DQ008558.1 X X Leptopoecile sophiae DQ008518.1 X X DQ008569.1 X X Locustella certhiola DQ008476.1 X X DQ008528.1 X X Locustella fluviatilis DQ008475.1 X X DQ008527.1 X X Macrosphenus kretschmeri DQ008574.1 X X * Taeniopygia guttata sequence retrieved from genome build</p><p>Table S4. Primer sequences (Seq 5’-3’) for specific indel areas on selected loci in the great reed warbler. Position (P) on zebra finch chromosome 4a is indicated for each locus.</p><p>Primer P (Mb) Seq 5’-3’ DIAPH2_indel F 1.6 CTGCTGGTCTGGTCTAAGTTGGGA DIAPH2_indel R GTTCTTCCCTCCAAGAGAACACTGC RPS6KA6_indel F 4.7 AGGCACCTGAAAAATCCTGTGGTGT RPS6KA6_indel R CCCAGCAAGTGCAGAAGTGGCA THOC2_indel F 10.2 CCTGCTCATCAGTAAAACCCTCTGG THOC2_indel R GTGCATCACAGCACCTCATGTTCAG SMARCA1_indel F 11.6 AGTCACAGCAGTATGTTTAGCAGCAC SMARCA1_indel R CCTCGTGGTTTCAGTGCCTACAATTA GABRE_indel F 19.2 TGTTGTCTTGGTCACTCCAGTTGTTT GABRE_indel R AGTTTCACCTTCAAAAAGCCCTTCTGT Table S5. GenBank accession numbers of representative sequences including informative sites for each locus in the great reed warbler (GRW); the common whitethroat (CW); the skylark (SL); the goldcrest (GC) and the blue tit (BT). In the cases in which ambiguities could not be resolved by direct sequencing, a partial sequence was submitted. Locus tag (tTgu4a) and position of the zebra finch orthologues are indicated. tTgu4a P GRW CW SL GC BT (Mb)</p><p>SYTL4 0.9 HQ415821 HQ415822 HQ415823 HQ415824 HQ415825</p><p>DIAPH2 1.6 HQ415826 HQ415827 HQ415828 HQ415829 HQ415830</p><p>PCDH11X 2.7 HQ415831 HQ415832 HQ415833 HQ415834 HQ415835</p><p>DACH2 4 HQ415836 HQ415837 HQ415838 HQ415839</p><p>ZNF711 4.4 HQ415840 HQ415841 HQ415842 HQ415843 HQ415844</p><p>RPS6KA6 4.7 HQ415845 HQ415846 HQ415847 HQ415848 HQ415849</p><p>ITM2A 5.3 HQ415850 HQ415851 HQ415852 HQ415853</p><p>TAF9B 5.5 HQ415854 HQ415855 HQ415856 HQ415857</p><p>P2RY4 5.6 HQ415858 HQ415859 HQ415860 HQ415861 HQ415862</p><p>AR 6.4 HQ415863 HQ415864 HQ415865 HQ415866 HQ415867</p><p>ZC3H12B 6.8 HQ415868 HQ415869 HQ415870 HQ415871 HQ415872</p><p>DDX26B 7 HQ415873 HQ415874 HQ415875 HQ415876 HQ415877</p><p>SLC9A6 7 HQ415878 HQ415879 HQ415880 HQ415881 HQ415882</p><p>XP_ 7.9 HQ415883 HQ415884 HQ415885 HQ415886 HQ415887 002190721.1 PLS3 8.1 HQ415888 HQ415889 HQ415890 HQ415891 HQ415892</p><p>WDR44 8.4 HQ415893 HQ415894 HQ415895 HQ415896 HQ415897</p><p>LONRF3 9.2 HQ415898 HQ415899 HQ415900 HQ415901 HQ415902</p><p>CXorf56 9.4 HQ415903 HQ415904 HQ415905 HQ415906 HQ415907</p><p>LAMP2 9.5 HQ415908 HQ415909 HQ415910 HQ415911 HQ415912</p><p>THOC2 10.2 HQ415913 HQ415914 HQ415915 HQ415916 HQ415917</p><p>SMARCA1 11.6 HQ415918 HQ415919 HQ415920 HQ415921 HQ415922</p><p>RAB39B 11.8 HQ415923 HQ415924 HQ415925 HQ415926 HQ415927</p><p>IRS4 12.1 HQ415928 HQ415929 HQ415930 HQ415931</p><p>HMGB3 13 HQ415932 HQ415933 HQ415934 HQ415935 HQ415936</p><p>MAMLD1 13.2 HQ415937 HQ415938 HQ415939 HQ415940 HQ415941</p><p>XP_ 16.5 HQ415942 HQ415943 HQ415944 HQ415945 HQ415946 002195404.1 MTMR8 16.8 HQ415947 HQ415948 HQ415949 HQ415950</p><p>SOX3 18.8 HQ415951 HQ415952 HQ415953 HQ415954 HQ415955</p><p>GABRE 19.2 HQ415956 HQ415957 HQ415958 HQ415959</p><p>TEX11 20.1 HQ415960 HQ415961 HQ415962 HQ415963</p><p>HTR2C 20.6 HQ415964 HQ415965 HQ415966 HQ415967 HQ415968</p>