Sexual and interspecific differences in biofluorescence of two hylid frog species from Mexico

Biofluorescence, the re-emission of absorbed light at longer wavelengths, has been increasingly reported in amphibians, yet methodological inconsistencies limit comparative analyses. We reviewed 20 studies documenting amphibian biofluorescence across 29 families and 237 species, revealing a predominance of ultraviolet and royal blue excitation sources and a general lack of standardization in data collection. In this study, we developed a cost-effective, image-based method for quantifying fluorescence using ImageJ, and applied it to two sympatric treefrog species from Mexico, Charadrahyla taeniopus and Rheohyla miotympanum . Individuals were photographed under royal blue light (440–460 nm) and analyzed for fluorescence intensity and spatial distribution across dorsal and ventral surfaces. Both species exhibited biofluorescence, with significant interspecific and sex-based differences. Charadrahyla taeniopus displayed higher dorsal and ventral brightness, and males showed greater ventral fluorescence intensity than females, indicating sexual dimorphism. In contrast, Rheohyla miotympanum exhibited weaker dorsal but pronounced ventral fluorescence, without significant sexual differences. Our findings demonstrate that standardized image analysis provides a robust framework for detecting and comparing biofluorescence patterns, particularly when spectrophotometric equipment is unavailable. These results suggest that ventral biofluorescence may serve communicative or sexual signaling functions in hylid frogs and underscore the need for methodological consistency and behavioral integration in future biofluorescence research.