The current functional model of the anuran larynx includes four pairs of laryngeal muscles. Their contractions do not account, however, for the behavioral control of call complexity observed in male túngara frogs (Engystomops pustulosus), which optionally add a secondary note with distinct harmonic structure to their advertisement call. Examination of the túngara frog's laryngeal morphology through dissection, microtomography, and resin histology has revealed that the m. dilatator laryngis is divided into two separate bundles (superficial and deep). The superficial bundle closely matches the typical description of the m. dilatator laryngis and is well positioned to open the glottis. The deep bundle is exclusively innervated by the short laryngeal nerve and has an attachment to the fibrous mass, an internal laryngeal structure necessary for complex call production. This attachment indicates a separate role for the deep bundle in controlling the complexity of the call. Based on physical separation, exclusive attachments, distinct fiber orientation, exclusive innervation, and potential action, this study recognizes the deep bundle of the m. dilatator laryngis as a separate muscle. It also revalidates the name m. arylabialis which had been previously used to describe it. The split of the m. dilatator laryngis into two muscles results in a laryngeal innervation pattern that closely matches that of mammals. This study identified a novel laryngeal muscle in túngara frogs, a potential mechanism for the control of call complexity, and revealed new evidence of homologies between the laryngeal structures of amphibians and mammals.

Male túngara frogs can add a distinctive note ("chuck”) to their mating call. Production of the chuck involves vibrating a pair of laryngeal fibrous masses that is attached to the vocal cords. The muscular control of this mechanism remains unknown. Recent studies revealed a split in the laryngeal dilator muscle, which unveiled the deep dilator as a novel laryngeal muscle with unique attachments, innervation, and (likely) function. The deep dilator may position the fibrous masses for chuck production. The goals of this study were 1) to confirm the innervation of the novel muscle through electrophysiology; and 2) to determine the action of each laryngeal muscle (including the deep dilator), in isolation and in combination with one another, to elucidate the control of laryngeal function. I stimulated 32 combinations of the five laryngeal muscles electrically with 3-5 repetitions. Using suction glass electrodes, I stimulated the branches of the laryngeal nerves in excised larynges maintained in saline solution and filmed the resulting movements to measure their displacement due to stimulation. The results showed that the novel muscle is exclusively innervated by the short laryngeal nerve, a condition equivalent to that of the mammalian posterior cricoarytenoid muscle, responsible for opening the vocal cords. Also, contraction of the deep dilator muscle is required and sufficient to produce lateral displacement of the fibrous masses and, therefore, to create a chuck. This identifies the deep dilator as a key element in the evolution of call complexity in túngara frogs. Clarifying the mechanism that controls the addition of chucks to the túngara frog call is an important step in understanding the evolution of signal complexity in animal communication systems. The recognition of the mechanism may allow comparative studies to be made that can reveal why complex calling evolved in the túngara frog lineage while not in others.