Figure 2. Song Pulses Can Be Separated into P_fast and P_slow across Strains and Species, and Each Corresponds to a Distinct Wing Pose.

(A) Two-dimensional tSNE of all pulses detected by the automated segmenter reveals a clear separation of the detected pulses into P_slow (red) and P_fast (orange). Shown are 71,029 pulses from 47 males of the D. melanogaster strain NM91. Black waveforms correspond to the average waveform of all pulses classified as P_slow (top) or P_fast (bottom), respectively.

(B) Average waveforms (top) and spectra (bottom) of P_fast and P_slow for all eight D. melanogaster strains investigated (see STAR Methods for strain names).

(C) The P_fast versus P_slow distinction is evident upon inspection of individual pulses (top). Shown are 32 randomly selected, normalized pulses from NM91 males. Both pulse types are also separable using a linear dimensionality reduction technique–PCA (bottom). Plotted is the distribution of projection values of all 71,029 NM91 pulses onto the first principal component, which captures 55% of the variance. Pulses (top) and projection values (bottom) are colored by the pulse type they were classified as (see legend).

(D) Pulses driven by thermogenetic activation of P1 neurons [16] in either solitary males (purple), males courting a female (green), or males tethered and walking on a ball (pink). Flies on the ball experienced open-loop visual stimulation matching the natural statistics of female motion on the male retina during courtship [11]. In all three conditions, both pulse types occur, though with varying probability (left). Pulse shapes for P_slow (top right) and P_fast (bottom right) are nearly indistinguishable for all three conditions. Number of flies (pulses) is 10 (107,710) for solitary males, 11 (138,311) for males with female, and 34 (176,526) for tethered males.

(E) Pose of flies during the production of sine, P_slow, and P_fast. Shown is the average, mean subtracted frame at the time of the peak amplitude of sine and pulses, respectively (100 Hz frame rate; 25 px/mm). Frames were oriented such that the male faces rightward and flipped such that maximally extended wing is up. Both sine (left) and P_slow (middle) are produced with a strongly extended wing and P_fast (right) is produced with the wing held closer to the body. See legend for color map.

(F) Polar histograms of the angle of the maximally (top) and minimally (bottom) extended wing during sine (blue), P_slow (red), and P_fast (orange) production. Distribution of the maximal wing angles for sine and P_slow are similar (50°–70°). The distribution of P_fast angles has a main mode at 5°–30° and a smaller second mode overlapping with the angles observed for sine and P_slow. Minimal wing angles (bottom) overlap for all three song modes. Data in (E) and (F) are as follows: 99,181 ms sine, 56,386 P_slow pulses, and 47,241 P_fast pulses from 42 individuals of the D. melanogaster strain NM91.

(G) Phylogenetic tree for the four melanogaster species used (reproduced from [18]). The species that produce P_fast are highlighted in bold font.

(H) Average pulse waveforms for all strains analyzed, colored by species (see legend). Each strain of each species produces a slower symmetrical (P_slow, top) pulse type, and all species but D. sechellia produce a faster asymmetrical pulse (P_fast, bottom) type.

(I and J) Pulse frequency (I) and symmetry (J) for the pulse waveforms in (H) by pulse type. D. simulans and D. mauritiana pulses are faster than those of D. melanogaster. D. sechellia produces slower P_slow pulses than D. melanogaster and no P_fast. For N flies and pulses, see Figure S2A.

See also Figures S2 and S3.