Experimental and statistical reevaluation provides no evidence for courtship song rhythms.

From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we previously performed automated segmentation of songs but failed to detect the proposed rhythm [Arthur BJ, et al. (2013) 11:11; Stern DL (2014) 12:38]. Kyriacou et al. [Kyriacou CP, et al. (2017) 114:1970-1975] report that we failed to detect song rhythms because () our flies did not sing enough and () our segmenter did not identify many of the song pulses. Kyriacou et al. manually annotated a subset of our recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with their original reports. We cannot replicate this finding and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have reexamined our methods and analysis and find that our automated segmentation method was not biased to prevent detection of putative song periodicity. We conclude that there is no evidence for the existence of courtship song rhythms.