Figure 1. Drosophila melanogaster Courtship Song Comprises Three, Not Two, Modes.

(A) During courtship, the male chases the female and produces a courtship song through unilateral wing vibrations. Shown are male (gray) and female (magenta) traces during courtship. Song recording (right) with background noise (gray) and signals (black) from a wild-type D. melanogaster male is shown.

(B) Non-overlapping signal chunks (25 ms duration) were extracted (top) and then normalized (middle) to scale all signals to the same peak amplitude and to flip the sign of signal waveforms such that the largest peak prior to the pulse center is always positive. Variation in signal amplitude or sign could be due to the male’s position relative to the microphone. Here, we show a random subset of 86 out of all 21,104 chunks. After clustering, signals produced by males during courtship fall into four classes: non-song noises (gray), sine (blue), P_slow (red), and P_fast (orange).

(C) We reduced the dimensionality of 21,104 waveforms from 47 individual males of the D. melanogaster strain NM91 using t-distributed stochastic neighbor embedding (tSNE), and the resulting two-dimensional distribution (density color coded; see color bar) of signals was partitioned using the watershed algorithm. This yielded seven clusters, which upon inspection of the waveforms, correspond to noise, sine song (three clusters), and two distinct pulse song modes—P_slow (two clusters) and P_fast (see text labels). Thick white lines mark the main mode boundaries after cluster consolidation (see D), and thin white lines mark the submodes within each main mode.

(D) Hierarchical clustering of the average waveform for each watershed cluster (centroids, thick black lines) supports the grouping of clusters into 4 main modes. Horizontal distance in the tree (left) corresponds to the dissimilarity between cluster centroids. Thin colored lines (right) show individual waveforms for each cluster. The waveforms corresponding to non-song noises (gray) are highly heterogeneous and have in common only the peak in energy to which they were aligned. Three sine song clusters (blue) mainly differ in frequency and constitute a continuum of waveforms. Note the second sine cluster is heterogeneous. Two P_slow clusters (red) differ only in their asymmetry, with more weight on either the negative lobe leading (bottom) or lagging (top) relative to the main positive peak. P_fast (orange) joins all fast and biphasic pulses.

(E) Similarity between all pulse waveforms (left) or all sine waveforms (right) obtained from the watershed clustering (Figure 1C). Similarity was computed by correlating all pulse or sine waveforms with the centroids for one of the clusters (the waveform used for correlation is thicker in the inset). Distributions do not strongly depend on which of the clusters was chosen as a template for correlation. The distribution for the pulse clusters is bimodal: the two P_slow clusters strongly overlap and have high correlation values with the centroid, indicating that they belong to a single song mode. The P_fast pulses exhibit projection values onto the P_slow centroid symmetrically distributed around 0, indicating very low similarity with the P_slow pulses and supporting its classification into a distinct pulse song mode. By contrast, the distribution for all three sine song clusters (right) is skewed toward high values, and all three distributions overlap, indicating that sine song constitutes a single song mode.

See also Figure S1.