Raman spectroscopy of pigments and oxalates in situ within epilithic lichens: Acarospora from the Antarctic and Mediterranean

Abstract

Fourier Transform laser Raman spectroscopy was used to generate diagnostic spectra for pigments and biodegradative calcium oxalate in situ in two yellow-pigmented species of the lichen genus Acarospora from contrasting sites in the Antarctic and the Mediterranean. This non-intrusive technique was used to identify the photoprotective pigments rhizocarpic acid and β-carotene by their unique Raman spectral fingerprints. The use of low energy near-IR excitation at 1064 nm eliminated interference from autofluorescence of photosynthetic pigments. The insensitivity of the technique to water permitted the use of field-fresh material. The dominant yellow pigment, rhizocarpic acid, gave a diagnostic pattern of corroborative bands at wavenumbers (ν) 1596, 1665, 1620 and 1000 cm⁻¹. It was possible to discriminate between hydration states of calcium oxalate; the monohydrate (whewellite) featured a ν(CO) stretching band at 1493 cm⁻¹ whereas the dihydrate (weddellite) had a contrasting ν(CO) stretching band at 1476 cm⁻¹. Fourier Transform deconvolution and intensity measurements were used to obtain relative quantitative data for rhizocarpic acid by using its ν(CO) and ν(CONH) amide modes, for carotenoid pigment by its ν(C = C) band at 1520 cm⁻¹ and for calcium oxalates by their ν(CO) bands. ν(CO), ν(CONH) and ν(C = C) are the vibrational stretching modes of the carbonyl C = O, protein amide 1 and alkenyl C = C moieties, respectively, in the pigments and metabolic products of the Acarospora lichens. The ability to determine the precise (20 μm spot diameter) spatial distribution of these key functional molecules in field-fresh thallus profiles and variegations has great potential for understanding the survival strategies of lichens, which receive high insolation, including elevated levels of UV-B, under extremes of desiccation and temperature in hot and cold desert habitats.