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Background Species-specific advertisement calls are the main mechanism of transmitting information between individuals in anuran amphibians and are therefore indispensable for anuran survival and reproduction. Survey methods that monitor these calls can be used for rapid species recognition, behavioral experiments, and conservation monitoring. In this study, we described in detail 10 call parameters from three sympatric species in the genus Microhyla and analyzed the differences in call parameter among these species to provide a basis for systematic monitoring, acoustic analysis and taxonomic study of this genus. Methods The quantitative analyses of temporal and spectral call parameters were used in our study for the advertisement calls of three sympatric Microhyla species (M. beilunensis, M. fissipes and M. heymonsi) in Zhejiang Province, East China. Results Our results showed the following: (1) Significant differences existed among the three sympatric Microhyla species in call duration (CD), call interval (CI), number of pulses (NP), pulse rate, call intensity (CIT), dominant frequency (DF) and frequency of the first to fourth formants (F1, F2, F3 and F4). (2) Some spectral parameters (DF, F1 and F3) were negatively correlated with the body size of the vocalizing individuals in each species. (3) The coefficients of variation within individuals (CVw) for CIT, DF and F1–F4 were smaller than 5%, whereas the CVW for CI was larger than 10% in each species. (4) The principal component analysis and discriminant function analysis showed that call parameters could distinguish the three Microhyla species. (5) The phylogenetic generalized least squares analysis showed that phylogenetic relationships affected CD and NP against snout-vent length (SVL), DF and NP against CD, and NP against DF, but not of DF against SVL; based on the phylogenetic analysis, CD and NP were not related to SVL, but DF was negatively related to SVL.

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