DNA was amplified from single extractions to minimize variation in DNA quality and concentration. Negative controls were run to prevent scoring artifact bands.
Amplified fragment length polymorphism analyses Amplified fragment length polymorphism analyses were carried out according to Vos et al. (1995) with some modifications. In short, 0.5 µg genomic DNA was incubated for 1 hour at 37 ºC with 5 units Eco RI and 10 units Mse I in 20 µL reaction buffer REact3 (Invitrogen, Life Technologies) and 50 ng/µL BSA. Thereafter, a cocktail was added to this mixture containing 2.5 pmol Eco RI adapter (Vos et al. 1995), 25 pmol Mse I adapter (Vos et al. 1995), 0.5 µL 10 x T4 DNA ligation buffer (Promega), 0.5 unit T4 DNAligase (Promega), and dH2O to a total volume of 5 µL. Incubation was continued for 3 hours at 37 ºC. The reaction mixture was diluted tenfold in dH2O.
Preamplification PCR was performed with Eco RI primer ET (5-GACTGCGTACCAATTCT-3) and Mse I primer MC (5-GATGAGTCCTGAGTAAC-3), using the following temperature profile: 94 ºC for 2 min, followed by 20 cycles of 94 ºC for 30 sec, 56 ºC for 30 sec and 72 ºC for 1 min and a final elongation at 72 ºC for 10 min. The preamplification PCR product was diluted tenfold and stored at -20 ºC. In the second selective amplification, the PCR included 94 ºC for 2 min, a 12 cycle touchdown procedure of 94 ºC for 30 sec, 60 ºC - 0.7 ºC/cycle for 30 sec and 72 ºC for 1 min followed by 23 cycles of 94 ºC for 30 sec, 56 ºC for 30 sec, 72 ºC for 1 min, and elongation at 72 ºC for 10 min. Primers with three selective nucleotides were used in four combinations: ETAG*/MCGA, ETAG*/MCTG, ETCT */MCGA and ETCT */MCTG. The Eco RI primers (E) were labeled with FAM at the 5-end. The PCR products were run on 5% polyacrylamide gels for 4 hours on an ABI 377 automated DNA sequencer (Applied Biosystems) using filter set A. All samples were run with an internal lane standard (2500 Rox). Amplified fragment length polymorphism data were analyzed using GeneScan 3.1.2 and Genotyper 2.5. The choice of specific markers (bands) was based on their utility for species and hybrid identification. Initially, ten individuals of each species were screened for suitable markers. Markers with the most pronounced differences in frequency between the two species were selected and scored for all individuals. DNA from toe pads of museum specimens showed clear patterns of degraded DNA (few and short bands only) and were excluded from further AFLP analyses. Amplified fragment length polymorphisms were scored for presence or absence of the selected bands. Results of all bands were also pooled into a principle coordinate analysis (PCO) using MultiVariate Statistical Package 3.0 (1998, Kovach Computing Services, Anglesey, Wales; <http://www.kovcomp.co.uk/mvsp/index.html>). Thus, genetic variability of sampled individuals was summarized into a few major components (PCO1, PCO2, etc). The Doh assignment test (Paetkau et al. 1995, 1997) was used for species assignments based on presence/absence of bands. Further, assignment scores were used to indicate the likelihood that an individual was assigned to a certain species (according to Doh assignment calculator). Differences in likelihood scores between Spotted Owls and Barred Owls reflected how much more likely it was that an individual was assigned to Barred Owl than to Spotted Owl, or vice versa.