Supplementary Material for: Sex differences in the neural song circuit and its relationship to song acoustic complexity in house wrens (Troglodytes aedon)

The song circuit in passerine birds is an outstanding model system for understanding the relationship between brain morphology and behavior, in part due to varying degrees of sex differences in structure and function across species. House wrens (Troglodytes aedon) offer a unique opportunity to advance our understanding of this relationship. Intermediate sex differences in song rate and complexity exist in this species compared to other passerines, and, among individual females, song complexity varies dramatically. Acoustic complexity in wild house wrens was quantified using a new machine learning approach. Volume, cell number, cell density, and neuron soma size were then measured for three song circuit regions, Area X, HVC (used as a proper name), and the robust nucleus of the arcopallium (RA), and one control region, the nucleus rotundus (Rt). For each song control area, males had a larger volume with more cells, larger somas, and lower cell density. Male songs had greater acoustic complexity than female songs, but these distributions overlapped. In females, increased acoustic complexity was correlated with larger volumes of and more cells in Area X and RA as well as larger soma size in RA. In males, song complexity was unrelated to morphology, although our methods may underestimate male song complexity. This is the first study to identify song control regions in house wrens and one of few examining individual variation in both sexes. Parallels between morphology and the striking variability in female song in this species provide a new model for understanding relationships between neural structure and function.