X times the playback had a duration of 20 minutes in total. This design resembled the organization of natural song in this species. We presented each of the playbacks twice: once to a Flut/Let bird and once to a control bird, thereby alternating thepresentation order between the two groups. These pairs of groups were tested in close temporal proximity in order to rule out e.g. seasonal or male status effects. In addition, by this paired design we were able to control for effects of different source bird song. The simulated territorial intrusion (STI) experiments were performed by placing a stuffed decoy (male in full adult plumage protected by an inconspicuous cage, three different decoys used) into the center of the respective territories. A remote-controlled loudspeaker (Foxpro Scorpion, digital game caller, FOXPRO Inc. Lewistown, USA) was put underneath the decoy to play back the territorial song of a potential rival at a sound pressure level of 65 dB SPL at 1 m (as measured with a CEL 573.B1 Sound Level Analyzer). We only started an experiment when a male wasTestosterone Affects Song ModulationTable 2. Linear mixed model results for the effects of context and Flut/Let treatment on song output and structure in fall.elementtreatmentcontextinteractionCohens d [95 CI] placebo Flut/Let 2.4 [0.8, 3.9] 0.1 [21.0, 1.2] 21.3 [21.3, 1.0] 0.05 [21.1, 1.2] 20.7 [21.9, 0.5] 0.6 [20.6, 1.7] 22.6 [24.1, 20.9] 22.3 [23.7, 20.7] 2.1 [0.5, 3.6] 0.9 [20.3, 2.1]song rateF1,10 = 3.0 p = 0.F2,18 = 43.1 p,0.0001 F2,18 = 2.1 p = 0.1 F2,18 = 3.2 p = 0.06 F2,18 = 0.7 p = 0.5 F2,18 = 3.4 p = 0.06 F2,18 = 0.1 p = 0.9 F2,14 = 11.9 p,0.001 F2,16 = 26.1 p,0.0001 F2,16 = 2.0 p = 0.2 F2,16 = 3.9 p = 0.04 F2,14 = 3.8 p = 0.2.3 [0.8, 3.8] 0.8 [20.4, 2.9] 0.3 [20.8, 1.5] 0.7 [20.5, 1.8] 20.1 [21.2, 1.0] 20.2 [21.3, 1.0] 21.6 [23.0, 20.1] 24.2 [26.4, 21.9] 0.6 [20.6, 1.8] 0.7 [20.6, 1.9] combined: 1.0 [0.1,0.9]song durationF1,10 = 0.02 p = 0.duration AF1,10 = 4.3 p = 0.duration BF1,10 = 0.01 p = 0.duration CF1,10 = 2.2 p = 0.duration pause A-BF1,10 = 0.4 p = 0.no. of elements in AF1,10 = 2.4 p = 0.no. of elements in CF1,10 = 0.2 p = 0.freq bandwidth AF1,10 = 0.2 p = 0.max St well-characterized heme importer and exporter respectively. As shown in Figure frequency AF1,10 = 0.08 p = 0.freq bandwidth BF1,10 = 0.7 p = 0.F2,13 = 3.2 p = 0.08 F2,13 = 5.0 p = 0.02 F2,16 = 3.6 p = 0.05 F2,16 = 2.2 p = 0.21.6 [23.0, 20.2] 21.9 [23.3, 20.4] 1.0 [20.3, 2.3] 0.8 [20.5, 2.9]20.6 [21.8, 0.6] 20.8 [22.0, 0.5] 0.5 [20.7, 1.6] 0.3 [20.9, 1.4]max frequency BF1,10 = 1.5 p = 0.freq bandwidth CF1,10 = 0.03 p = 0.max frequency CF1,10 = 0.1 p = 0.Context is a within-subjects factor with three levels: before STI (spontaneously produced songs), during STI (playback and decoy present) and after STI (directly after Title Loaded From File removal of playback and decoy). Treatment is a between-subjects factor with two levels: placebo-implanted versus blocker-implanted males. Significant results are highlighted in bold. doi:10.1371/journal.pone.0052009.tsinging. The song was recorded 10 min prior to the start of the STI, during the 20 min STI and 10 min after the STI with a Sennheiser directional microphone (ME66/K6) connected to a Marantz solid state recorder PMD 660 (sampling frequency: 44.1 kHz; resolution: 16 bit).Data AnalysisThe song was analyzed using Avisoft-SASLab Pro software, version 4.51. Recordings were visualized in spectrograms (settings: sample rate 22,050 Hz, FFT = 256 points, Hamming-window, overlap: 50 ). We determined the number of songs by visual inspection and selected songs of su.X times the playback had a duration of 20 minutes in total. This design resembled the organization of natural song in this species. We presented each of the playbacks twice: once to a Flut/Let bird and once to a control bird, thereby alternating thepresentation order between the two groups. These pairs of groups were tested in close temporal proximity in order to rule out e.g. seasonal or male status effects. In addition, by this paired design we were able to control for effects of different source bird song. The simulated territorial intrusion (STI) experiments were performed by placing a stuffed decoy (male in full adult plumage protected by an inconspicuous cage, three different decoys used) into the center of the respective territories. A remote-controlled loudspeaker (Foxpro Scorpion, digital game caller, FOXPRO Inc. Lewistown, USA) was put underneath the decoy to play back the territorial song of a potential rival at a sound pressure level of 65 dB SPL at 1 m (as measured with a CEL 573.B1 Sound Level Analyzer). We only started an experiment when a male wasTestosterone Affects Song ModulationTable 2. Linear mixed model results for the effects of context and Flut/Let treatment on song output and structure in fall.elementtreatmentcontextinteractionCohens d [95 CI] placebo Flut/Let 2.4 [0.8, 3.9] 0.1 [21.0, 1.2] 21.3 [21.3, 1.0] 0.05 [21.1, 1.2] 20.7 [21.9, 0.5] 0.6 [20.6, 1.7] 22.6 [24.1, 20.9] 22.3 [23.7, 20.7] 2.1 [0.5, 3.6] 0.9 [20.3, 2.1]song rateF1,10 = 3.0 p = 0.F2,18 = 43.1 p,0.0001 F2,18 = 2.1 p = 0.1 F2,18 = 3.2 p = 0.06 F2,18 = 0.7 p = 0.5 F2,18 = 3.4 p = 0.06 F2,18 = 0.1 p = 0.9 F2,14 = 11.9 p,0.001 F2,16 = 26.1 p,0.0001 F2,16 = 2.0 p = 0.2 F2,16 = 3.9 p = 0.04 F2,14 = 3.8 p = 0.2.3 [0.8, 3.8] 0.8 [20.4, 2.9] 0.3 [20.8, 1.5] 0.7 [20.5, 1.8] 20.1 [21.2, 1.0] 20.2 [21.3, 1.0] 21.6 [23.0, 20.1] 24.2 [26.4, 21.9] 0.6 [20.6, 1.8] 0.7 [20.6, 1.9] combined: 1.0 [0.1,0.9]song durationF1,10 = 0.02 p = 0.duration AF1,10 = 4.3 p = 0.duration BF1,10 = 0.01 p = 0.duration CF1,10 = 2.2 p = 0.duration pause A-BF1,10 = 0.4 p = 0.no. of elements in AF1,10 = 2.4 p = 0.no. of elements in CF1,10 = 0.2 p = 0.freq bandwidth AF1,10 = 0.2 p = 0.max frequency AF1,10 = 0.08 p = 0.freq bandwidth BF1,10 = 0.7 p = 0.F2,13 = 3.2 p = 0.08 F2,13 = 5.0 p = 0.02 F2,16 = 3.6 p = 0.05 F2,16 = 2.2 p = 0.21.6 [23.0, 20.2] 21.9 [23.3, 20.4] 1.0 [20.3, 2.3] 0.8 [20.5, 2.9]20.6 [21.8, 0.6] 20.8 [22.0, 0.5] 0.5 [20.7, 1.6] 0.3 [20.9, 1.4]max frequency BF1,10 = 1.5 p = 0.freq bandwidth CF1,10 = 0.03 p = 0.max frequency CF1,10 = 0.1 p = 0.Context is a within-subjects factor with three levels: before STI (spontaneously produced songs), during STI (playback and decoy present) and after STI (directly after removal of playback and decoy). Treatment is a between-subjects factor with two levels: placebo-implanted versus blocker-implanted males. Significant results are highlighted in bold. doi:10.1371/journal.pone.0052009.tsinging. The song was recorded 10 min prior to the start of the STI, during the 20 min STI and 10 min after the STI with a Sennheiser directional microphone (ME66/K6) connected to a Marantz solid state recorder PMD 660 (sampling frequency: 44.1 kHz; resolution: 16 bit).Data AnalysisThe song was analyzed using Avisoft-SASLab Pro software, version 4.51. Recordings were visualized in spectrograms (settings: sample rate 22,050 Hz, FFT = 256 points, Hamming-window, overlap: 50 ). We determined the number of songs by visual inspection and selected songs of su.
