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

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

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

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).

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

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

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.

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)

SYTL4 0.9 HQ415821 HQ415822 HQ415823 HQ415824 HQ415825

DIAPH2 1.6 HQ415826 HQ415827 HQ415828 HQ415829 HQ415830

PCDH11X 2.7 HQ415831 HQ415832 HQ415833 HQ415834 HQ415835

DACH2 4 HQ415836 HQ415837 HQ415838 HQ415839

ZNF711 4.4 HQ415840 HQ415841 HQ415842 HQ415843 HQ415844

RPS6KA6 4.7 HQ415845 HQ415846 HQ415847 HQ415848 HQ415849

ITM2A 5.3 HQ415850 HQ415851 HQ415852 HQ415853

TAF9B 5.5 HQ415854 HQ415855 HQ415856 HQ415857

P2RY4 5.6 HQ415858 HQ415859 HQ415860 HQ415861 HQ415862

AR 6.4 HQ415863 HQ415864 HQ415865 HQ415866 HQ415867

ZC3H12B 6.8 HQ415868 HQ415869 HQ415870 HQ415871 HQ415872

DDX26B 7 HQ415873 HQ415874 HQ415875 HQ415876 HQ415877

SLC9A6 7 HQ415878 HQ415879 HQ415880 HQ415881 HQ415882

XP_ 7.9 HQ415883 HQ415884 HQ415885 HQ415886 HQ415887 002190721.1 PLS3 8.1 HQ415888 HQ415889 HQ415890 HQ415891 HQ415892

WDR44 8.4 HQ415893 HQ415894 HQ415895 HQ415896 HQ415897

LONRF3 9.2 HQ415898 HQ415899 HQ415900 HQ415901 HQ415902

CXorf56 9.4 HQ415903 HQ415904 HQ415905 HQ415906 HQ415907

LAMP2 9.5 HQ415908 HQ415909 HQ415910 HQ415911 HQ415912

THOC2 10.2 HQ415913 HQ415914 HQ415915 HQ415916 HQ415917

SMARCA1 11.6 HQ415918 HQ415919 HQ415920 HQ415921 HQ415922

RAB39B 11.8 HQ415923 HQ415924 HQ415925 HQ415926 HQ415927

IRS4 12.1 HQ415928 HQ415929 HQ415930 HQ415931

HMGB3 13 HQ415932 HQ415933 HQ415934 HQ415935 HQ415936

MAMLD1 13.2 HQ415937 HQ415938 HQ415939 HQ415940 HQ415941

XP_ 16.5 HQ415942 HQ415943 HQ415944 HQ415945 HQ415946 002195404.1 MTMR8 16.8 HQ415947 HQ415948 HQ415949 HQ415950

SOX3 18.8 HQ415951 HQ415952 HQ415953 HQ415954 HQ415955

GABRE 19.2 HQ415956 HQ415957 HQ415958 HQ415959

TEX11 20.1 HQ415960 HQ415961 HQ415962 HQ415963

HTR2C 20.6 HQ415964 HQ415965 HQ415966 HQ415967 HQ415968