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Summary• Attenuated total reflectance (ATR) spectra of plant leaves display complex absorption features related to organic constituents of leaf surfaces. The spectra can be recorded rapidly, both in the field and in the laboratory, without special sample preparation.• This paper explores sources of ATR spectral variation in leaves, including compositional, positional and temporal variations. Interspecific variations are also examined, including the use of ATR spectra as a tool for species identification.• Positional spectral variations generally reflected the abundance of cutin and the epicuticular wax thickness and composition. For example, leaves exposed to full sunlight commonly showed more prominent cutin-and wax-related absorption features compared with shaded leaves. Adaxial vs. abaxial leaf surfaces displayed spectral variations reflecting differences in trichome abundance and wax composition. Mature vs. young leaves showed changes in absorption band position and intensity related to cutin, polysaccharide, and possibly amorphous silica development on and near the leaf surfaces.• Provided that similar samples are compared (e.g. adaxial surfaces of mature, sun-exposed leaves) same-species individuals display practically identical ATR spectra. Using spectral matching procedures to analyze an ATR database containing 117 individuals, including 32 different tree species, 83% of the individuals were correctly identified.

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