Selection on vocal output affects laryngeal morphology in rats

Although laryngeal morphology often reflects adaptations for vocalization, the structural consequences of selection for particular aspects of vocal behavior remain poorly understood. In this study, we investigated the effects of increased ultrasonic calling in pups on the adult larynx morphology in selectively bred rat lines. Laryngeal morphology was assessed using multiple techniques: mineralized cartilage volumes were compared in 3D-models derived from microCT scans, internal structure was compared using clearing and staining procedures combined with microscopy, cellular structure was compared using histology and microscopy, and element composition was assessed with scanning energy dispersive X-ray spectroscopy. Our results show that adult rats from lines bred to produce ultrasonic calls at higher rates as pups have shorter vocal folds and a more mineralized thyroid cartilage compared to rats bred to produce ultrasonic calls at lower rates. The change in vocal fold length appears to account for differences in low-frequency calls in these two rat lines. We suggest that the observed increases in mineralization of the thyroid cartilage in the high-ultrasound lineage provide increased reinforcement of the laryngeal structure during ultrasonic call production. Our findings therefore demonstrate an effect of selection for vocal behavior on laryngeal morphology, with acoustic consequences.