Escombe[745] analysed the hyphal membranes of Cetraria and found that they consisted mainly of a body called by him lichenin and of a paragalactan. From Peltigera he extracted a substance with physical properties agreeing fairly well with those of chitosan, though analysis did not give percentages reconcilable with that substance; the yield however was very small. No lichenin was detected.
Van Wisselingh[746] examined the hyphae of lichens as well as of fungi and experimented with a considerable number of both types of plants. He succeeded in proving the presence of chitin in the higher fungi (Basidiomycetes and Ascomycetes) and in lichens with one or two exceptions (Cladonia and Cetraria). Though in some the quantity found was exceedingly small, in others, such as Peltigera, the walls of the hyphae were extremely chitinous. More recently Wester[747] has gone into the question as regards lichens, and he has practically confirmed all the results previously obtained by Wisselingh. In some species, as for instance in Cladonia rangiferina, Cl. squamosa, Cl. gracilis, Ramalina calicaris, Solorina crocea and others, he found that chitin existed in large quantities, while in Evernia prunastri, Usnea florida, U. articulata, Sticta damaecornis and Parmelia saxatilis very little was present. The variation in the amount present may be very great even in the species of one genus: none for instance has been detected in Cetraria islandica nor in C. nivalis while it is abundant in other Cetrariae. There is also considerable variation in quantity in different individuals of the same species, and even in different parts of the thallus of one lichen. Factors such as habitat, age of the plant, etc., may, however, account to a considerable extent for the differences in the results obtained.
b. Lichenin and Allied Carbohydrates. It has been proved, as already stated, that chitin is present in the hyphal cell-walls of all the lichens examined except in those of Cetraria islandica (Iceland Moss), C. nivalis and, according to Wester[747], in those of Bryopogon (Alectoriae). In these lichens another substance of purely carbohydrate nature is the chief constituent of the cell-walls which swell up when soaked in water to a colourless gelatinous substance.
Berzelius[748] first drew attention to the peculiar qualities of this lichen product, and, recognizing its resemblance in many respects to ordinary starch, he called it “lichen-starch” or “moss-starch.” More exact observations were made later by Guérin-Varry[749] who described its properties and showed by his experiments that it contained no admixture of either starch or gum. He adopted the name lichenin for this organic soluble part of Iceland Moss. An analysis of lichenin was made by Mulder[750] who detected in addition to lichenin, which coloured yellow with iodine, small quantities of a blue-colouring substance which could be dissolved out from the lichenin and which he considered to be true starch. Berg[751] also demonstrated the compound nature of lichenin: he isolated two isomerous substances with the formula C₆H₁₀O₅. The name “isolichenin” was given to the second blue-colouring substance by Beilstein[752] in 1881.
More recently Escombe[753] has chemically analysed the cell-wall of Cetraria islandica: after the elimination of fat, oil, colouring matter and bitter constituents he found that there remained the compound lichenin, an anhydride of galactose with the formula C₆H₁₀O₅, which, as stated above, consists of two substances lichenin and isolichenin[754]; the latter is soluble in cold water and gives a blue reaction with iodine, lichenin is only soluble in hot water and is not coloured blue. Both are derivatives of galactose, a sugar found in a great number of organic tissues and substances, among others in gums.
Lichenin has also been obtained by Lacour[755] from Lecanora esculenta, an edible desert lichen supposed to be the manna of the Israelites. Wisselingh[756] tested the hymenium of thirteen different lichens for lichenin. He found it in the walls of the ascus of all those he examined except Graphis. Everniin, a constituent of Evernia prunastri, was isolated and described by Stüde[757]. It is soluble in water and, though considered by Czapek[758] to be identical with lichenin, it differs, according to Ulander[759], in being dextro-rotatory to polarized light; lichenin on the contrary is optically inactive. Escombe[753] also obtained a substance from Evernia which he considered to be comparable with chitosan. Usnein which has been extracted[756] from Usnea barbata may also be identical with lichenin, but that has not yet been established. Ulander[759] examined chemically the cell-walls of a fairly large number of lichens. Cetraria islandica, C. aculeata and Usnea barbata, designated as the “Cetraria group,” contained soluble mucilage-forming substances similar to lichenin. A second “Cladonia group” which included Cl. rangiferina with the variety alpestris, Stereocaulon paschale and Peltigera aphthosa yielded almost none. After the soluble carbohydrates were removed by hot water, the insoluble substances were hydrolysed and the “Cetraria group” was found to contain abundant d-glucose with small quantities of d-mannose and d-galactose; the “Cladonia group,” abundant d-mannose and d-galactose with but little d-glucose. Hydrolysis was easier and quicker with the former group than with the latter.
Besides these, which rank as hexosans, Ulander found small quantities of pentosans and methyl pentosans. All these substances which are such important constituents of the hyphal membranes of lichens are classed by Ulander as hemicelluloses of the same nature as mannan, galactan and dextran, or as substances between hemicellulose and the glucoses represented by lichenin, everniin, etc. They are doubtless reserve stores of food material, and they are chiefly located in the cell-walls of the medullary hyphae which are often so thick as almost to obliterate the lumen of the cells. Ulander made no test for chitin in his researches.
Ulander’s results have been confirmed by those obtained by K. Müller[760]. In Cladonia rangiferina, Müller found that the cell-membranes of the hyphae contained, as hemicelluloses, pentosans in small quantities and galactan, but no lichenin and very little chitin. In Evernia prunastri hemicelluloses formed the chief constituents of the thallus, and from it he was able to isolate galactan soluble in weak hot acid, and everniin soluble in hot water, the latter with the formula C₇H₁₅O₆, a result differing from that obtained by Stüde[761] who has given it as C₉H₁₄O₇; chitin was also present in small quantities. In Ramalina fraxinea, the soluble part of the thallus (in hot water) differed from everniin and might probably be lichenin. Cetraria islandica was also analysed and yielded various hemicelluloses, chiefly dextran and galactan, with less pentosan. No chitin has ever been found in this lichen. In testing minute quantities of material for chitin, Wisselingh[762] heated the tissue in potash to 160° C. The potash was then gradually replaced by glycerine and distilled water; the precipitate was placed on a slide and the preparation stained under the microscope by potassium-iodide-iodine and weak sulphuric acid. Chitin, if present, would have been changed by the potash to mycosin which gives a violet colour with the staining solution.
It has been stated by Schellenberg[763] that these lichen membranes may become lignified. He obtained a red reaction with phloroglucine test for lignin in Cetraria islandica and Cladonia furcata. Further research is required.