Appendix S1 Additional information on genotyping performed in this study. We screened birds
at two introns on the Z chromosome, one in the CHD1Z gene (Fridolfsson & Ellegren 1999), the
other in the ADAMT56 gene (Backström et al. 2006). The CHD1Z gene is located between
nucleotide numbers 24736734 and 24736133 in the zebra finch genome; ADAMT56 is located
between 50815125 and 50815666 (genome.ucsc.edu, assembly July 2008).
We amplified fragments from ten males, five from each taxon sampled in allopatry
(coastal: Vancouver; inland: Wisconsin and Illinois; all caught later than 2007) using the primers
2550F (5’-GTTACTGATTCGTCTACGAGA-3’) and 2718R (5’ATTGAAATGATCCAGTGCTTG-3’) for CHD1Z and ADAMT56F (5’GGAGAGAATGGATTTCTGCC-3’) and ADAMT56R (5’-TGATTCCAGTCTAGGAAACG3’) for ADAMT56. PCR reactions included 1x PCR buffer (Invitrogen), 1.5 mM MgCl2
(Invitrogen), 0.2 mM dNTP mix (New England Biolabs), 0.5 μM forward and reverse primer,
0.04 units/μl Taq DNA polymerase (New England Biolabs), and 0.025 ng/μl genomic template
DNA, in a total volume of 10μl. The thermal cycling profile was 3 minutes at 94ºC followed by
35 cycles of 30 seconds at 94ºC, 30 seconds at 58ºC, and 45 seconds at 72ºC, ending with 5
minutes at 72ºC. Amplified fragments were sequenced bidirectionally by Genome Quebec
Innovation Center, using an Applied Biosystem's 3730xl DNA Analyzer.
A 680 bp region of the CHD1Z gene was amplified. There was an A/G polymorphism
220 bp from the 5’ end of the fragment. All of the coastal thrushes were homozygous for A in
this position; all of the inland thrushes were homozygous for G. The sequence of coastal thrushes
contains a cut site for the restriction enzyme XabI while the sequence of inland thrushes does
not. A 620bp region of the ADAMT56 gene was amplified. There were two T/C polymorphisms
200 and 480 bp from 5’ end of the fragment. All of the inland thrushes were homozygous for T
in both positions; all of the coastal thrushes were homozygous for C in both. The sequence of
inland thrushes contains a cut site for the restriction enzyme PsiI at 480 bp while the sequence of
coastal thrushes does not.
Using the same PCR reactions described above, we amplified the introns and digested 2μl
of the PCR product with 2 units of the restriction enzyme (XabI and PsiI) in its appropriate
buffer (New England Biolabs) and in a total volume of 6μl. Products were digested for 2 hours at
37ºC, and digested DNA was visualized by electrophoresis on 2% agarose gel stained with
SYBRSafe (Invitrogen). Digestion of CHD1Z cuts the coastal PCR product in two fragments
(220 and 480 bp); the inland PCR product remains intact. An individual carrying two copies of
the coastal sequence shows two bands; an individual carrying two copies of the inland sequence
shows one band; heterozygous individuals produce both cut and intact PCR products generating
3 bands. Digestion of ADAMT56 cuts the inland PCR product into two fragments (130 and 480
bp); the coastal PCR product remains intact. An individual carrying two copies of the inland
sequence shows two bands, an individual carrying two copies of the coastal sequence shows one
band, heterozygous individuals produce both cut and intact PCR products generating 3 bands.
Only males were used in this analysis. Original sequences were obtained from blood samples of
birds captured in Vancouver during the breeding season (allopatric coastal site) and tissue
samples from the Field Museum of Chicago. These tissue samples were collected in May (one in
autumn) in Wisconsin and Illinois (allopatric inland sites). We confirmed our PCR-RFLP
analysis by assaying birds at allopatric sites, including the Sunshine Coast and Vancouver
(allopatric coastal sites, n = 19 for CHD1Z and 17 for ADAMT56) and William’s Lake,
Kamloops and Kelowna (allopatric inland sites, n = 36 for CHD1Z and 27 for ADAMT56). Each
of these birds was homozygous for the corresponding allele.
1.
Backström, N., Qvarnström, A., Gustafsson, L. & Ellegren, H. (2006). Levels of linkage
disequilibrium in a wild bird population. Biol. Lett., 2, 435–438.
2.
Fridolfsson, A.K. & Ellegren, H. (1999). A simple and universal method for molecular sexing of
non-ratite birds. J.Avian Biol., 116–121.