Evaluation of different protocols for anatomical studies in Parmeliaceae (lichenized Ascomycota)

Anatomical information on most lichen species, including Parmeliaceae species, is scarce and superficial. This is partially due to the technical difficulties found during the preparation of samples and sections suitable for optical microscopy analysis. There is a lack of pictures of anatomical sections as well as detailed anatomical protocols related to the sample processing made by specialized plant anatomists in literature. This work aimed to look for a standardized histological technique and to develop a protocol for histological studies of foliose Parmeliaceae under light microscopy. Four common pantropical species of Parmeliaceae, abundant in the Brazilian cerrado vegetation, were processed in several ways in relation to fixing and inclusion media, sectioning thicknesses, and staining, including both fresh and 2-year-old herbarium specimens. The best technique found for anatomical studies of Parmeliaceae under light microscopy is represented by 2 or 3 microm thick sections of fresh or herbarium samples, desiccated and fixed in FAA 50, infiltrated and embedded in glycol-methacrylate resin (Leica), and stained by Toluidine Blue or Methylene Blue.