Supplementary Material for: Pose analysis in free-swimming adult zebrafish, Danio rerio: “fishy” origins of movement design

Introduction
Movement requires maneuvers that generate thrust to either make turns or move the body forward in physical space. The computational space for perpetually controlling the relative position of every point on the body surface can be vast. We hypothesize the evolution of efficient design for movement that minimizes active (neural) control by leveraging the passive (reactive) forces between the body and the surrounding medium at play. To test our hypothesis, we investigate the presence of stereotypical postures during free-swimming in adult zebrafish, Danio rerio.
Methods
We perform markerless tracking using DeepLabCut, a deep learning pose estimation toolkit, to track geometric relationships between body parts. To identify putative clusters of postural configurations obtained from twelve freely behaving zebrafish, we use unsupervised multivariate time-series analysis (B-SOiD machine learning software).
Results
When applied to single individuals, this method reveals a best-fit for 36 to 50 clusters in contrast to 86 clusters for data pooled from all 12 animals. The centroids of each cluster obtained over 14,000 sequential frames recorded for a single fish represent an apriori classification into relatively stable “target body postures” and inter-pose “transitional postures” that lead towards and away from a target pose. We use multidimensional scaling of mean parameter values for each cluster to map cluster-centroids within two dimensions of postural space. From a post-priori visual analysis, we condense neighboring postural variants into 15 superclusters or core body configurations. We develop a nomenclature specifying the anteroposterior level/s (upper, mid and lower) and degree of bending.
Conclusion
Our results suggest that constraining bends to mainly three levels in adult zebrafish preempts the neck, fore- and hindlimb design for maneuverability in land vertebrates.