Extended Data Fig. 1: Context-dependence of song sequencing in Drosophila melanogaster males (supplement to Fig. 1).

a, Male behaviors during courtship (modified from⁶⁵, including aspects from^13,66), with those focused in the present study highlighted in the grey box. b, Cumulative fraction of simple pulse (red), simple sine (blue), or complex (purple) bouts over time in recording for n = 20 wild-type male-female pairs (biological replicates). c, Distribution of pulse train duration, sine train duration, and the number of pulse-sine alternations in male song of n = 20 wild-type male-female pairs (biological replicates). d Chamber for behavioral experiments. Male courtship song was recorded using 16 microphones (green) tiling the chamber floor. Female (magenta) and male (blue) fly pose and tracks were estimated using SLEAP¹⁷. e Cumulative fraction of far (brown) and near (yellow) bouts over time in recording for n = 20 wild-type male-female pairs (biological replicates). f, Population-averaged probability to sing simple pulse, simple sine, or complex bouts relative to male forward velocity (mFV). Color code as in (a). g, Distribution of mFV near (yellow) and far (brown) from the female. h, The majority (91%) of final bouts (the last song bout prior to copulation) occur within 0.6 seconds preceding copulation. i, The majority (59%) of bouts immediately preceding copulation are complex (n = 23 wild-type pairs with copulation within a 20 minute recording). Song bouts are aligned to bout end. Time-resolved probability of pulse (red) and sine song (blue) (shown below song traces) rises prior to copulation. Black curve at the bottom shows the fraction of males that sing both pulse and sine song in the time prior to copulation. 80% of males sang both song modes within the final 1.5 seconds of song before copulation, suggesting complex bouts facilitate mating. b,e, mean ± mean absolute deviation from the mean. b,c,e,f,g, n = 20 recordings of male-female pairs (biological replicates).

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