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Appendix 1. Tissues sampling, extraction procedures, and polymerase chain reaction
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conditions used to amplify modern and historic marbled murrelet DNA at 16 microsatellite
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loci.
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Tissue sampling at museums was conducted as cleanly as possible by bleaching surfaces
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and flaming sampling equipment. To screen for contamination at the sampling stage, we
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collected one “sampling” negative control for every two specimens by opening empty sample
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tubes and exposing them to the museum environment for approximately the same time it took to
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sample a specimen.
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DNA was extracted from modern blood samples using Qiagen’s DNAeasy extraction kit
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according to protocols recommended by the manufacturer. DNA extractions from museum
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tissue were conducted using a phenol-chloroform extraction procedure (Sambrook et al. 1989) in
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a “clean” laboratory in which no modern DNA or PCR products were allowed. The clean lab
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was physically isolated from laboratories containing PCR product and personnel showered and
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changed into clean clothes immediately prior to entering the lab. Surfaces were UV-sterilized
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and bleached and all pipettes and plastic-ware were UV-sterilized prior to laboratory work.
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PCR reactions for both historic and modern samples were carried out with fluorescently
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labeled primers. PCR reactions for modern samples were carried in a volume of 10.0 μl with the
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following reagents: 1 μl of DNA template, 0.5 μM forward primer, 0.5 μM reverse primer, 0.24
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mM each dNTP, 1.5 mM MgCl2, 0.25 U Taq DNA polymerase (Qiagen), and 1.0 μl 10x reaction
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buffer (Qiagen). The number of cycles varied among loci (see Table S1), each step containing a
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30 sec denaturing step at 94°C, a 30 sec annealing step at temperatures listed in Table S1, and a
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30 sec extension step at 72°C. All reactions consisted of a 3 min initial denaturing step at 94°C
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and a 10 min final extension step at 72 °C. PCR reactions for historic samples were carried out
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in a volume of 12.5μl with the following reagents: approximately 1.5-5 μl of DNA template, 0.5
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μM forward primer, 0.5 μM reverse primer, 0.24 mM each dNTP, 0.25 μl 100x BSA, 2.0-3.0
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mM MgCl2, and 0.625 U Taq DNA polymerase (Roche), 1.25 μl 10x reaction buffer (Roche).
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The number of cycles varied among loci (see Table S1), each step containing a 30-45 sec
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denaturing step at 94°C, a 30-45 sec annealing step at temperatures listed in Appendix S1, and a
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30-45 sec extension step at 72°C. All reactions consisted of a 5 min initial denaturing step at
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94°C and a 10 min final extension step at 72 °C. Eleven negative controls were included per 96-
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well PCR plate including negative controls from extractions described above. Amplification
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products from both modern and historic sources were sized by capillary electrophoresis on an
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ABI 3730 sequencer (Applied Biosystems, Inc.), and alleles were scored using program
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GENEMAPPER Ver. 4.0 software (Applied Biosystems, Inc.).
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For historic samples, primers for three loci were modified from Rew et al. (2006) to
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reduce the size of amplicons because we had limited success amplifying DNA fragments greater
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than 200 base pairs (see Table S1). Each allele detected in modern samples was amplified at
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least once with the both the original and modified primers in a subsample of the modern samples
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to cross-reference allele scores between time periods.
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Historic samples were amplified at least twice at each locus to assess the repeatability of
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allele calling and estimate the rate of allelic dropout (Taberlet et al. 1996; Wandeler et al. 2007).
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Single-locus genotypes that did not agree between amplifications were amplified again until
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allele scoring appeared unambiguous. Ninety-four percent of historic genotypes agreed between
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the first and second amplifications, and ambiguous genotypes were generally resolvable after a
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third amplification. Ninety-seven percent of single-locus genotypes derived from 24 randomly
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selected and re-extracted historic samples were the same as derived from the original
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amplifications. The 3% of genotypes that did not agree appeared to be the result of allelic
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dropout in the positive controls, and not due to genotyping errors in the original dataset.
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REFERENCES
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Rew, M. B., Peery, M. Z., Beissinger, S. R., Berube, M., Lozier, J. D., Rubidge, E. M. &
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Palsboll, P. J. 2006 Cloning and characterization of 29 tetranucleotide and two
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dinucleotide polymorphic microsatellite loci from the endangered marbled murrelet
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(Brachyramphus marmoratus). Molecular Ecology Notes 6, 241-244.
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56
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Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989 Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press.
Taberlet, P., Griffin, S., Goossens, B., Questiau, S., Manceau, V., Escaravage, N., Waits, L. P. &
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Bouvet, J. 1996 Reliable genotyping of samples with very low DNA quantities using
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PCR. Nucleic Acids Research 24, 3189-3194.
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Wandeler, P., Hoeck, P. E. A. & Keller, L. F. 2007 Back to the future: museum specimens in
population genetics. Trends in Ecology & Evolution 22, 634-642.
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