PT - JOURNAL ARTICLE AU - David L. Stern AU - Jan Clemens AU - Philip Coen AU - Adam J. Calhoun AU - John B. Hogenesch AU - Ben Arthur AU - Mala Murthy TI - Reported <em>Drosophila</em> courtship song rhythms remain data analysis artifacts AID - 10.1101/140483 DP - 2017 Jan 01 TA - bioRxiv PG - 140483 4099 - http://biorxiv.org/content/early/2017/05/20/140483.short 4100 - http://biorxiv.org/content/early/2017/05/20/140483.full AB - From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we performed automated segmentation of songs, but failed to detect the proposed periodicity (1, 2). Kyriacou et al. (3) report that we failed to detect song rhythms because i) our flies did not sing enough and ii) our segmenter did not identify song pulses accurately. They 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, as well as the automatically segmented data, provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have re-examined our methods and analysis and find that our methods did not prevent detection of putative song periodicity. We therefore conclude that previous positive reports of song rhythms most likely resulted from inappropriate statistical analyses.Significance statement Previous studies have reported that male vinegar flies sing courtship songs with a periodic rhythm of approximately 60 seconds. Several years ago, we showed that we could not replicate this observation. Recently, the original authors have claimed that we failed to find rhythms because 1) our flies did not sing enough and 2) our software for detecting song was flawed. They claimed that they could detect rhythms in song annotated by hand. We report here that we cannot replicate their observation of rhythms in the hand-annotated data and that our original methods were not biased against detecting rhythms. We conclude that the original findings likely resulted from errors in the statistical analysis of songs.