Echolocating animals use echolocation to navigate and to forage. This special sensory system is essential for their survival and reproduction. Although many behavioral and physiological studies exist on animal echolocation, little is known about the underlying molecular mechanisms. This study employs theoretical and analytical evolutionary methods and combines functional experiments to explore the molecular mechanisms of echolocation in bats. Hearing genes are paramount because echolocating animals must emit the high-frequency sounds to echolocate. Their sensitivity for high-frequency sounds is much higher than that of non-echolocating animals. First, evolutionary analyses and functional experiments determine that the hearing gene prestin is involved in vertebrate high-frequency hearing. Motor proteins encoded by prestin form the molecular basis of cochlear amplification in mammals, which is responsible for the highest hearing sensitivity and frequency selectivity. The ability to detect high-frequency sounds varies enormously among different species. From fishes to tetrapods, and from lower mammals to higher mammals, the detection of high-frequency sounds improves. Based on this evolutionary phenomenon, we hypothesized that prestin might be under positive selection for the detection of sound signals to adapt their surroundings. To test this hypothesis, we compare and analyze the orthologs of prestin from different groups of vertebrates. Signals of positive selection occur in the most recent common ancestor (MRCA) of tetrapods, mammals, therians, and placentals, respectively. Functional experiments fail to refute this hypothesis and reveal the functional divergence of voltage-dependent nonlinear capacitance of prestin. Second, we identify the first echolocating gene—prestin. We obtain coding sequences of prestin from the dolphin and bats to conduct the evolutionary analyses. Phylogenetic reconstructions based on the amino acid sequences of prestin cluster dolphins and echolocating bats. However, when we use synonymous sites to re-construct the phylogenetic tree, the topology is consistent with that of species phylogeny, which suggests that the functional amino acids and not the evolutionary history of prestin cause these two types of echolocating mammals to cluster together. Evolutionary analyses further show that prestin experienced parallel evolution in dolphin and echolocating bats and we identify 11 parallel-evolved amino acids. We verify functional convergence of prestin between the dolphin and echolocating bats, and by using experiments determine that parallel sites drive functional convergence. Finally, using methods of comparative genomics, we find the hearing gene KCNQ4, a gene that co-expresses with prestin in the outer hair cells of mammalian cochlea, also experienced parallel evolution in echolocating bats. Thus, it may be the second gene related to bat echolocation. The evolutionary trajectories of the parallel sites suggest independent gains of higher frequency hearing in echolocating bats. The identification of KCNQ4 further deepens our understanding of the molecular mechanisms of bat echolocation. In summary, we explore the molecular mechanisms of echolocation in bats by applying theories and methods of evolutionary and experimental biology to provide new insight for investigating the relationships between genotypes and complex phenotypes.
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