Supplementary 1 (A) Forward and Reverse Primers Used for Amplification of CYP2C45 Segments

Supplementary 1 – (A) Forward and reverse primers used for amplification of CYP2C45 segments from barn owls (Tyto alba). Also included are expected amplicon sizes and PCR annealing temperatures. (B) Diagram of the barn owl (Tyto alba) CYP2C45 gene. The blue blocks indicate exon regions and their corresponding sizes; orange blocks indicate the introns; green and yellow arrowheads are the forward and reverse primers, respectively, used to amplify the six segments. Drawing is not to scale of actual sequence sizes. The remaining exons (8 and 9) and introns were not sequenced.

(A)

Segment / Primer sequences (5' - 3') / Amplicon sizes (bp) / Annealing temperatures (°C)
A / F: CTCCTGGTTTGCATTGCTTGCCT / 633 / 55
R: TGTCCTCTGGCAGCAAACTC
B / F: GGTGAAAGAAGCCTTGGTCGATC / 635 / 53.5
R: CAATGCTCCTCTTCCCCATCCCAAA
C / F: GCATTATTTTCAGCAACAACGAGGG / 517 / 55
R: TGTTGTTCATGTTGTTCATCAG
D / F: GACTATAAAGACAAGAAGTTCC / 664 / 60
R: GGAAGCAGTCAATGAAATCCTG
E / F: CAGGATTTCATTGACTGCTTCC / 659 / 61.5
R: GTGCTTGTTGTCTCCGTTCCAGC
F / F: GCTGGAACGGAGACAACAAGCAC / 589 / 61.5
R: GTGCTTGTTGTCTCCGTTCCAGC

(B)

Supplementary 2 – Summary of AICC statistics for models examining the relationship between various predictors and (a) total SGAR residue concentration, (b) presence/absence of any SGAR, (c) presence/absence of bromadiolone, and (d) presence/absence of toxicosis symptoms.

Model / AICC / Δ AICC / df / AICc weight
(a) Total SGAR residue Concentration
demographic + time.period / -78.2 / 0 / 5 / 0.86
demographic*time.period / -74.6 / 3.7 / 7 / 0.14
(b) Presence/absence of any SGAR
demographic + time.period / 154.7 / 0 / 5 / 0.82
demographic*time.period / 157.7 / 3 / 7 / 0.18
(c) Presence/absence of bromadiolone
demographic + time.period / 153.6 / 0 / 5 / 0.85
demographic*time.period / 157.1 / 3.5 / 7 / 0.15
(d) Presence/absence of toxicosis symptoms
[bromadiolone] + [brodifacoum]*[difethialone]*demographic + time.period / 42.5 / 0.0 / 15.0 / 0.9945
[bromadiolone]*[difethialone]*time.period + [brodifacoum] + demographic / 55.5 / 13.0 / 12.0 / 0.0015
[bromadiolone]*time.period + [brodifacoum] + [difethialone] + demographic / 56.7 / 14.1 / 12.0 / <0.001
[bromadiolone] + [brodifacoum]*[difethialone]*time.period + demographic / 57 / 14.4 / 9.0 / <0.001
[bromadiolone] + [brodifacoum] + [difethialone]*time.period + demographic / 57.5 / 15.0 / 9.0 / <0.001
[bromadiolone]*[difethialone] + [brodifacoum] + demographic + time.period / 58.5 / 16.0 / 9.0 / <0.001
[bromadiolone] + [brodifacoum] + [difethialone]*demographic + time.period / 58.6 / 16.1 / 10.0 / <0.001
[bromadiolone] + [brodifacoum]*demographic + [difethialone] + time.period / 59.1 / 16.6 / 10.0 / <0.001
[bromadiolone] + [brodifacoum] + [difethialone] + demographic + time.period / 59.4 / 16.8 / 9.0 / <0.001
[bromadiolone]*[brodifacoum]*demographic + [difethialone] + time.period / 59.4 / 16.9 / 8.0 / <0.001
[bromadiolone] + [brodifacoum]*time.period + [difethialone] + demographic / 60 / 17.5 / 12.0 / <0.001
[bromadiolone]*demographic*time.period + [brodifacoum] + [difethialone] / 61.2 / 18.6 / 9.0 / <0.001
[bromadiolone]*[brodifacoum] + [difethialone] + demographic + time.period / 61.6 / 19.0 / 9.0 / <0.001
[bromadiolone]*[brodifacoum]*time.period + [difethialone] + demographic / 61.7 / 19.1 / 10.0 / <0.001
[bromadiolone]*demographic + [brodifacoum] + [difethialone] + time.period / 62 / 19.5 / 15.0 / <0.001
[bromadiolone] + [brodifacoum]*[difethialone] + time.period + demographic / 62.5 / 20.0 / 14.0 / <0.001
[bromadiolone] + [brodifacoum] + [difethialone] + demographic*time.period / 62.8 / 20.2 / 10.0 / <0.001
[bromadiolone]*[brodifacoum]*[difethialone] + demographic + time.period / 64.9 / 22.4 / 12.0 / <0.001
[bromadiolone]*[difethialone]*demographic + [brodifacoum] + time.period / 65.9 / 23.4 / 15 / <0.001
[bromadiolone] + [brodifacoum] + [difethialone]*demographic*time.period / 66.4 / 23.9 / 40 / <0.001
[bromadiolone]*[brodifacoum]*[difethialone]*time.period*demographic / 67 / 24.4 / 15 / <0.001

Supplementary 3 – SNPs identified in the non-coding regions (introns) of the CYP2C45 gene and their associated nucleotide positions (starting from the start codon of exon 1). Highlighted in red are individuals categorized as “low AR tolerance”, and blue are “high AR tolerance”.

Nucleotide position
459 / 510 / 1023 / 1875 / 1913 / 1979 / 2662 / 3188 / 3223
L12-558 / R / Y / R / G / C / Y / K / Y / G
L12-559 / R / Y / G / G / C / T / K / Y / R
L12-709 / R / T / G / G / C / T / K / C / G
L12-736 / A / C / A / G / C / T / G / T / G
L12-753 / G / T / G / G / C / T / T / C / R
L12-727 / G / T / G / R / A / T / K / C / G
L12-737 / A / Y / R / G / C / T / G / T / G
L12-738 / R / T / G / G / C / T / K / C / R
L12-555 / R / Y / G / G / C / T / K / Y / R
L12-732 / G / T / G / G / A / T / K / C / G
L12-733 / R / C / A / G / C / T / G / Y / G
L12-752 / G / T / G / R / C / Y / T / C / G
L12-949 / R / Y / R / R / C / T / K / Y / G