I. CELLS AND CELL PRODUCTS
Any study of cells or cell-membranes in lichens should naturally include those of both symbionts, but the algae though modified have not been profoundly changed, and their response to the influences of the symbiotic environment has been already described in the discussion of lichen gonidia. The description of cells and their contents refers therefore mainly to the fungal tissues which form the framework of the plant; they have been transformed by symbiosis to lichenoid hyphae in some respects differing from, in others resembling, the fungal hyphae from which they are derived.
A. Cell-Membranes
a. Chitin. It was recognized by workers in the early years of the nineteenth century that the substance forming the cell-walls of fungal hyphae differed very markedly from the cellulose of the membranes in other groups of plants, the blue colouration with iodine and sulphuric acid so characteristic of cellulose being absent in most fungi. Various explanations were suggested; but it was always held that the doubtful substance was a cellulose containing something peculiar to fungi, this view being strengthened by the fact that, after long treatment with potash, a blue reaction was obtained. It was called fungus-cellulose by De Bary[740] in order to distinguish it from true cellulose.
It was not till a much later date that any exact work was done on the fungal cell, and that Gilson[741] by his researches was able to prove that the membranes of fungi contained probably no cellulose, or, “if cellulose were present, it was in a different condition from the cellulose of other plants.” Winterstein[742] followed with the results of his examination of fungus-cellulose: he found that it contained nitrogen and therefore differed very considerably from typical plant cellulose. Gilson[743] published a second paper dealing entirely with fungal tissues in which he also established the presence of nitrogen, and added that this nitrogenous compound resembled in various ways the chitin[744] of animal cells. He further discovered that by heating it with potash a substance was obtained that took a reddish-violet stain when treated with iodine and weak sulphuric acid. This substance, called by him mycosin, was proved later to be similar to chitosan[744], a product of chitin.
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.
c. Cellulose. Several workers claim to have found true cellulose in the cell-walls of the hyphal tissues of a few lichens; but the more careful analyses of Escombe[764], Wisselingh[762] and Wester[765] have disproved their results. The cell-walls of all the gonidia, however, are formed of cellulose, or according to Escombe of glauco-cellulose, except those of Peltigera in which Wester found neither cellulose nor chitin. Czapek[766] suggests that the blue reaction with iodine characteristic of the cell-walls in some apothecia, of the asci and of the hyphae in cortex or medulla in a few instances, may be due to the presence of carbohydrates of the nature of galactose. Moreau[767] in a recent paper terms the substance that gives a blue reaction with iodine at the tips of the asci “amyloid.” In Peltigera the ascus tip is occupied by such a plug of amyloid which at maturity is projected like a cork from the ascus and may be found on the surface of the hymenium.
B. Contents and Products of the Fungal Cells
a. Cell-substances. The cells of lichen hyphae contain protoplasm and nucleus with glucoses. It is doubtful if starch has been found in fungal hyphae; it is replaced, in some of the tissues at least, by glycogen, a carbohydrate (C₆H₁₀O₅) very close to, if not identical with, animal glycogen, a substance which is soluble in water and colours reddish-brown (wine-red) with iodine. Errera[768] first detected its presence in Ascomycetes where it is associated with the epiplasm of the cells, more especially of the asci, and he considered it to be physiologically homologous with starch. He included lichens, as Ascomycetes, in his survey of fungi and quotes, in support of his view that lichen hyphae also contain glycogen, a statement made by Schwendener[769] that “the contents of the ascogenous hyphae of Coenogonium Linkii stain a deep-brown with iodine.” Errera also instances the red-brown reaction with iodine, described by de Bary[770], as characteristic of the large spores of Ochrolechia (Lecanora) pallescens, while the germinating tubes of these spores become yellow with iodine like ordinary protoplasm. Glycogen has been, so far, found only in the cells of the reproductive system.
Iodine was found by Gautier[771] in the gonidia of Parmelia and Peltigera, i.e. both in bright-green and blue-green algae. The amount was scarcely calculable.
Herissey[772] claims to have established the presence of emulsin in a large series of lichens belonging to such widely separated genera as Cladonia, Cetraria, Evernia, Peltigera, Pertusaria, Parmelia, Ramalina, and Usnea. It is a ferment which acts upon amygdalin, though its presence has been proved in plants such as lichens where no amygdalin has been found[773]. Diastase was demonstrated in the cells of Roccella tinctoria, R. Montagnei and of Dendrographa leucophaea by Ronceray[774] who states that, in conjunction with air and ammonia, it forms orchil, the well-known colouring substance of these lichens. Diastatic ferments have also been determined[775] in Usnea florida, Physcia parietina, Parmelia perlata and Peltigera canina.
b. Calcium Oxalate. Oxalic acid (C₂H₂O₄) is an oxidation product of alcohol and of most carbohydrates and in combination is a frequent constituent of plant cells. Knop[776] held that it was formed in lichens by the reduction and splitting of lichen acids, though, as Zopf[777] has pointed out, these are generally insoluble. Hamlet and Plowright[778] demonstrated the presence of free oxalic acid in many families of fungi including Pezizae and Sphaeriae. The acid combines with calcium to form the oxalate (CaC₂0₄), which in the crystalline form is very common in lichens. In the higher plants the crystals are formed within the cell, but in lichens they are always deposited on the outer surface of the hyphal membranes, mainly of the medulla and the cortex.
Calcium oxalate was first detected in lichens by Henri Braconnot[779], who extracted it by treating the powdered thallus of a number of species (Pertusaria communis, Diploschistes scruposus, etc.) with different reagents. The quantity present varies greatly in lichens: Zopf[780] found that it was abundant in all the species inhabiting limestone, and states that in such plants the more purely lichenic acids are relatively scarce. Errera[781] has calculated the amount of calcium oxalate in Lecanora esculenta, a desert lime-loving lichen, to be about 60 per cent. of the whole substance of the thallus. Euler[782] gives for the same lichen even a larger proportion, 66 per cent. of the dry weight. In Pertusaria communis, a corticolous species, the oxalate occurs as irregular crystalline masses in the medulla ([Fig. 116]) and has been calculated as 47 per cent. of the whole substance. Other crustaceous species such as Diploschistes scruposus, Haematomma coccineum, H. ventosum, Lecanora saxicola, Lecanora tartarea, etc., contain large amounts either in the form of octahedral crystals or as small granules.
Fig. 116. Pertusaria communis DC. Vertical section of thallus. a, cortex; b, gonidia; c, medulla; d, crystal of calcium oxalate. × ca. 100.
Rosendahl[783] has recently made observations as to the presence of the oxalate in the thallus of the brown Parmeliae. Of the fourteen species examined by him, eleven contained calcium oxalate as octahedral crystals or as small prisms, often piled up in thick irregular masses. Usually the crystals were located in the medullary part of the thallus, but in two species, Parmelia verruculifera and P. papulosa, they were abundant on the surface cells of the upper cortex.
c. Importance of Calcium Oxalate to the Lichen Plant. It is natural to conclude that a substance of frequent occurrence in any group of plants is of some biological significance, and suggestions have not been lacking as to the value of oxalic acid or of calcium oxalate in the economy of the lichen thallus. Oxalic acid is known to be one of the most efficient solvents of argillaceous earth and of iron oxides likely to be in the soil. These materials are also conveyed to the thallus as air-borne dust, and would thus, with the aid of the acid, be easily dissolved and absorbed. As a direct proof of this, Knop[784] has stated that lichen-ash always contains argillaceous earth. According to Kratzmann[785], aluminium, a product of clay, is stored up in various lichens. He proved the amount in the ash of Umbilicaria pustulata to be 4·46 per cent., in Usnea barbata 1·79 per cent., in U. longissima considerable quantities while in Roccella tinctoria it occurred in great abundance. It was also abundant in Diploschistes scruposus, 28·17 per cent.; it declined in Variolaria (Pertusaria) dealbata to 7·77 per cent., in Cladonia rangiferina to 1·76-2·12 per cent. and in Ramalina fraxinea to 1·8 per cent.
Calcium oxalate is directly advantageous to the thallus by virtue of the capacity of the crystals to reduce or prevent evaporation, as has been pointed out by Zukal[786]. A like service afforded by crystals to the leaves of the higher plants in desert lands has been described by Kerner[787]. These are frequently encrusted with lime crystals which allow the copious night dews to soak underneath them to the underlying cells, while during the day they impede, if they do not altogether check, evaporation.
Calcium oxalate crystals are insoluble in acetic acid, soluble in hydrochloric acid without evolution of gas; they deposit gypsum crystals in a solution of sulphuric acid.
C. Oil-Cells
a. Oil-Cells of Endolithic Lichens. Calcicolous immersed lichens are able to dissolve the lime of the substratum, and their hyphae penetrate more or less deeply into the rock. In some forms the entire thallus may thus be immersed, the fruits alone being visible on the surface of the stone. In two such species, Verrucaria calciseda and Petractis (Gyalecta) exanthematica, Steiner[788] detected peculiar sphaeroid or barrel-shaped cells that differed from the other hyphal cells of the thallus, not only in their form, but in their greenish-coloured contents. Similar cells were found by Zukal[789] in another immersed (endolithic) lichen, Verrucaria rupestris f. rosea. He describes them as roundish organs crowded on the hyphae and filled with a greenish shimmering protoplasm. He[790] found the same types of sphaeroid and other swollen cells in the immersed thallus of several calcicolous lichens and he finally determined the contents as fat in the form of oil. He found also that these fat-cells, though very frequent, were not constantly present even in the same species. His observations were confirmed by Hulth[791] for a number of allied crustaceous lichens that grow not only on limestone but on volcanic rocks. In them he found a like variety of fat-cells—intercalary or torulose cells, terminal sphaeroid cells and hyphae containing scattered oil-drops. Bachmann[792] followed with a study of the thallus of purely calcicolous lichens. The specialized oil-cells were fairly constant in the species he examined, and, as a rule, they were formed either in the tissues immediately below, or at some distance from, the gonidial zone. Fünfstück[793] has also published an account of various oil-cells in a large series of calcicolous lichens ([Fig. 117]).
Fig. 117. Lecidea immersa Ach. A, sphaeroid fat-cells from about 8 mm. below the surface × 550. B, oil-hyphae in process of emptying: a, sphaeroid cells containing oil; b, cells with oil-globules × 600 (after Fünfstück).
The occurrence of oil-(or fat-)cells is not dependent on the presence of any particular alga as the gonidium of the lichen. Fünfstück[794] has described the immersed thallus of Opegrapha saxicola as one of those richest in fat-cells. The gonidia belong to the filamentous alga Trentepohlia umbrina and form a comparatively thin layer about 160µ thick near the upper surface; isolated algal branches may grow down to 350µ into the rock, while the fungal elements descend to 11·5 mm., and though the very lowest hyphae were without oil—as were those immediately beneath the gonidia—the interlying filaments, he found, were crowded with oil-cells. Sphaeroid terminal cells were not present.
Fünfstück[794] has re-examined the thallus of Petractis exanthematica, an almost wholly immersed lichen with a gelatinous gonidium, a species of Scytonema. The thallus is homoiomerous: the alga forms no special zone, it intermingles with the hyphae down to the very base of the thallus; the hyphae are extremely slender and at the base they measure only about 1µ in width. Oil-cells are abundant in the form of intercalary cells about 3-5µ in thickness. Nearer the surface sphaeroid cells are formed on short lateral outgrowths; they measure 14-16µ in diameter and occur in groups of 15 to 20. The superficial part of the thallus is a mere film; the hyphae composing it are slightly stouter and more thickly interwoven.
Bachmann[795] and Lang[796] have further described the anatomy of endolithic thalli especially with reference to oil-cells, and have supplemented the researches of previous workers. New methods of cutting the rock in thin slices and of dissolving away the lime enabled them to see the tissues in their relative positions. In these immersed lichens, as described by them and by previous writers, and more especially in calcicolous species, the gonidial zone of Protococcaceous algae lies near the surface of the rock, and is mingled with delicate, thin-walled hyphae which usually do not contain oil. The more deeply immersed layer is formed of a weft of equally thin-walled hyphae, some of the cells of which are swollen and filled with fat globules. These oil-cells may occur at intervals along the hyphae or they may form an almost continuous row. In addition, strands or bundles of hyphae ([Fig. 118]) containing few or many oil globules traverse the tissue, and true sphaeroid cells are generally present. These latter arise in great numbers on short lateral branchlets, usually near the tip of a filament and the groups of cells are not unlike bunches of grapes. Sometimes the oil-cells are massed together into a complex tissue. Hyphae from this layer pierce still deeper into the rock and constitute the rhizoidal portion of the thallus. They also produce sphaeroid oil-cells in great abundance ([Fig. 119]). In the immersed thallus of Sarcogyne (Biatorella) pruinosa Lang[797] estimated the gonidial zone as 175-200µ in thickness, while the colourless hyphae penetrated the rock to a depth of quite 15 mm.
Fig. 118. Biatorella (Sarcogyne) simplex Br. and Rostr. a, sphaeroid oil-cells; b, strand of oil-hyphae from 10-15 mm. below the surface. × 585 (after Lang).
Fig. 119. Biatorella pruinosa Mudd. a, complex of sphaeroid oil-cells from 10 mm. below the surface; b, hypha of sphaeroid cells also from inner part of the thallus. × 585 (after Lang).
b. Oil-cells of Epilithic Lichens. The general arrangement of the tissues and the occurrence and form of the oil-cells vary in the different species according to the nature of the substratum. This has been clearly demonstrated by Bachmann[798] in Aspicilia (Lecanora) calcarea, an almost exclusively calcareous lichen as the name implies. On limestone, he found sphaeroid cells formed in great abundance on the deeply penetrating rhizoidal hyphae ([Fig. 120]). On a non-calcareous brick substratum[799], a specimen had grown which of necessity was epilithic. The cortex and gonidial zone together were 40µ thick; immediately below there were hyphae with irregular cells free from oil; lower still there was formed a compact tissue of globose fat-cells. In this case the calcareous lichen still retained the capacity to form oil-cells on the non-calcareous impenetrable substance.
Fig. 120. Lecanora (Aspicilia) calcarea Sommerf. Early stage of sphaeroid cell formation × 175 (after Bachmann).
Very little oil is formed, as a rule, in the cells of siliceous crustaceous lichens which are almost wholly epilithic, but Bachmann found a tissue of oil-cells in the thallus of Lecanora caesiocinerea, from Labrador, on a granite composed of quartz, orthoclase and traces of mica. A thallus of the same species collected in the Tyrol, though of a thicker texture, contained no oil. Bachmann[799] suggests no explanation of the variation.
On granite, rhizoidal hyphae penetrate the rock to a slight extent between the different crystals, but only in connection with the mica[800] are typical sphaeroid cells formed.
More or less specialized oil-cells have been demonstrated by Fünfstück[801] in several superficial (epilithic) lichens which grow on a calcareous substratum, as for instance Lecanora (Placodium) decipiens, Lecanora crassa and other similar species. The oil in these lichens is usually restricted to more or less swollen or globose cells; but it may also be present in the ordinary hyphae as globules. Zukal[802] found that the smooth little round granules sprinkled over the thallus of the soil-lichens, Baeomyces roseus and B. rufus, contained in the hyphae typical sphaeroid oil-cells and that they were specially well developed in specimens from Alpine situations. In still another soil-lichen, Lecidea granulosa, shimmering green oil was found in short-celled torulose hyphae.
Rosendahl’s[803] researches on the brown Parmeliae resulted in the unexpected discovery of specialized oil-cells situated in the cortices—upper and lower—of five species out of fourteen which he examined. In one of the species, P. papulosa, they also occurred in the cortex of the rhizoids. The oil-cells were thinner-walled and larger than the neighbouring cortical cells; they were clavate or ovate in form and sometimes formed irregular external processes. They were more or less completely filled with oil which coloured brown with osmic acid, left a fat stain on paper and, when extracted, burned with a shining reddish flame. These oil-cells were never formed in the medulla nor in the gonidial region.
c. Significance of Oil-formation. Zukal[804] regarded the oil stored in these specialized cells as a reserve product of service to the plant in the strain of fruit-formation, or in times of prolonged drought or deprivation of light. According to his observations fat was most freely formed in lichens when periods of luxuriant growth alternated with periods of starvation. He cites, as proof of his view, the frequent presence of empty sphaeroid cells, and the varying production of oil affected by the condition, habitat, etc. of the plant. Fünfstück[805], on the other hand, considers the oil of the sphaeroid and swollen cells as an excretion, representing the waste products of metabolism in the active tissue, but due chiefly to the presence of an excess of carbonic acid which, being set free by the action of the lichen acids on the carbonate of lime, forms the basis of fat-formation. He points out that the development of fat-cells is always greater in endolithic species in which the gonidial layer—the assimilating tissue—is extremely reduced. In epilithic lichens with a wide gonidial zone, the formation of oil is insignificant. He states further that if the oil were a direct product of assimilation, the cells in which it is stored would be found in contact with the gonidia, and that is rarely the case, the maximum of fat production being always at some distance.
Fünfstück tested the correctness of his views by a prolonged series of growth experiments: he removed the gonidial layer in an endolithic lichen, and found that fat storage continued for some time afterwards, its production being apparently independent of assimilative activity. The correctness of his deductions was further proved by observations on lichens from glacier stones. In such unfavourable conditions the gonidia were scanty or absent, having died off, but the hyphae persisted and formed oil. He[806] also placed in the dark two quick-growing calcicolous lichens, Verrucaria calciseda and Opegrapha saxicola. At the end of the experiment, he found that they had increased in size without using up the fat. Lang[807] also is inclined to reject Zukal’s theory, seeing that the fat is formed at a distance from the tissues—reproductive and others—in need of food supply. He agrees with Fünfstück that the oil is an excretion and represents a waste-product of the plant.
Considerable light is thrown on the subject of oil-formation by the results of recent researches on the nutrition of algae and fungi. Beijerinck[808] made comparative cultures of diatoms taken from the soil, and he found that so long as culture conditions were favourable, any fat that might be formed was at once assimilated. If, however, some adverse influence checked the growth of the organism while carbonic acid assimilation was in full vigour, fat was at once accumulated. The adverse influence in this case was the lack of nitrogen, and Beijerinck considers it an almost universal rule in plants and animals, that where there is absence of nitrogen, in a culture otherwise suitable, fat-oils will be massed in those cells which are capable of forming oil. He observed that in two of the cultures of diatoms the one which alone was supplied with nitrogen grew normally, while the other, deprived of nitrogen, formed quantities of oil-drops. Wehmer[809] records the same experience in his cultural study of Aspergillus. Sphaeroid fat-cells, similar to those described by Zukal in calcicolous lichens, were formed in the hyphae of a culture containing an overplus of calcium carbonate, and he judged, entirely on morphological grounds, that these were not of the nature of reserve-storage cells.
Stahel[810] has definitely established the same results in cultures of other filamentous fungi. In an artificial culture medium in which nitrogen was almost wholly absent, the cells of the mycelium seemed to be entirely occupied by oil-drops, and this fatty condition he considered to be a symptom of degeneration due to the lack of nitrogen. These experiments enable us to understand how the hyphae of calcicolous lichens, buried deep in the substratum, deprived of nitrogen and overweighted with carbonic acid, may suffer from fatty degeneration as shown by the formation of “sphaeroid-cells.” The connection between cause and effect is more obscure in the case of lichens growing on the surface of the soil, such as Baeomyces roseus, or of tree lichens such as the brown Parmeliae, but the same influence—lack of sufficient nitrogenous food—may be at work in those as well as in the endolithic species, though to a less marked extent.
It seems probable that the capacity to form oil- or fat-cells has become part of the inherited development of certain lichen species and persists through changes of habitat as exemplified in Lecanora calcarea[811].
In considering the question of the formation and the function of fat in plant cells, it must be remembered that the service rendered to the life of the organism by this substance is a very variable one. In the higher plants (in seeds, etc.) fat undoubtedly functions in the same way as starch and other carbohydrates as a reserve food. It is evidently not so in lichens, and in one of his early researches, Pfeffer[812] proved that similarly oil was only an excretion in the cells of hepatics. He grew various species in which oil-cells occurred in the dark and then tested the cell contents. He found that after three months of conditions in which the formation of new carbohydrates was excluded, the oil in the cells, instead of having served as reserve material, was entirely unchanged and must in that instance be regarded as an excretion.
D. Lichen-Acids
a. Historical. The most distinctive and most universal of lichen products are the so-called lichen-acids, peculiar substances found so far only in lichens. They occur in the form of crystals or minute granules deposited in greater or less abundance as excretory bodies on the outer surface of the hyphal cells. Though usually so minute as scarcely to be recognized as crystals, yet in a fairly large series their form can be clearly seen with a high magnification. Many of them are colourless; others are a bright yellow, orange or red, and give the clear pure tone of colour characteristic of some of our most familiar lichens.
The first definite discovery of a lichen-acid was made towards the beginning of the nineteenth century and is due to the researches of C. H. Pfaff[813]. He was engaged in an examination of Cetraria islandica, the Iceland Moss, which in his time was held in high repute, not only as a food but as a tonic. He wished to determine the chemical properties of the bitter principle contained in it, which was so much prized by the Medical Faculty of the period, though the bitterness had to be removed to render palatable the nutritious substance of the thallus. He succeeded in isolating an acid which he tested and compared with other organic acids and found that it was a new substance, nearest in chemical properties to succinic acid. In a final note, he states that the new “lichen-acid,” as he named it, approached still nearer to boletic acid, a constituent of a fungus, though it was distinct from that substance also in several particulars. The name “cetrarin” was proposed, at a later date, by Herberger[814] who described it as a “subalkaloidal substance, slightly soluble in cold water to which it gives a bitter taste; soluble in hot water, but, on continued boiling, throwing down a brown powder which is slightly soluble in alcohol and readily soluble in ether.” Knop and Schnederman[815] found that Herberger’s “cetrarin” was a compound substance and contained besides other substances “cetraric acid” and lichesterinic acid. It has now been determined by Hesse[816] as fumarprotocetraric acid (C₆₂H₅₀O₃₅), a derivative of which is cetraric acid or triaethylprotocetraric acid with the formula C₅₄H₃₉O₂₄(OC₂H₅)₃ and not C₂₀H₁₈O₉ as had been supposed. Cetraric acid has not yet been isolated with certainty from any lichen[817].
After this first isolation of a definite chemical substance, further research was undertaken, and gradually a number of these peculiar acids were recognized, the lichens examined being chiefly those that were of real or supposed economic value either in medicine or in the arts. In late years a wider chemical study of lichen products has been vigorously carried on, and the results gained have been recently arranged and published in book form by Zopf[818]. Many of the statements on the subject included here are taken from that work. Zopf gives a description of all the acids that had been discovered up to the date of publication, and the methods employed for extracting each substance. The structural formulae, the various affinities, derivatives and properties of the acids, with their crystalline form, are set forth along with a list of the lichens examined and the acids peculiar to each species. In many instances outline figures of the crystals obtained by extraction are given. For a fuller treatment of the subject, the student is referred to the book itself, as only a general account can be attempted here.
b. Occurrence and Examination of Lichen-Acids. Acids have been found, with few exceptions, in all the lichens examined. They are sometimes brightly coloured and are then easily visible under the microscope. Generally their presence can only be determined by reagents. Over 140 different kinds have been recognized and their formulae determined, though many are still imperfectly known. As a rule related lichen species contain the same acids, though in not a few cases one species may contain several different kinds. In growing lichens, they form 1 to 8 per cent. of the dry weight, and as they are practically, while unchanged, insoluble in water, they are not liable to be washed out by rain, snow or floods. Their production seems to depend largely on the presence of oxygen, as they are always found in greatest abundance on the more freely aerated parts of the thallus, such as the soredial hyphae, the outer rind or the loose medullary filaments. They are also often deposited on the exposed disc of the apothecium, on the tips of the paraphyses, and on the wall lining the pycnidia. They are absent from the thallus of the Collemaceae, these being extremely gelatinous lichens in which there can be little contact of the hyphae with the atmosphere. No free acids, so far as is known, are contained in Sticta fuliginosa, but a compound substance, trimethylamin, is present in the thallus of that lichen. It has also been affirmed that acids do not occur in any Peltigera nor in two species of Nephromium, but Zopf[818] has extracted a substance peltigerin both from species of Peltigera and from the section Peltidea.
For purposes of careful examination freshly gathered lichens are most serviceable, as the acids alter in herbarium or stored specimens. It is well, when possible, to use a fairly large bulk of material, as the acids are often present in small quantities. The lichens should be dried at a temperature not above 40°C. for fear of changing the character of the contained substances, and they should then be finely powdered. When only a small quantity of material is available, it has been recommended that reagents should be applied and the effect watched under the microscope with a low power magnification. This method is also of great service in determining the exact position of the acids in the thallus.
In micro-chemical examination, Senft[819] deprecates the use of chloroform, ether, etc., seeing that their too rapid evaporation leaves either an amorphous or crystalline mass of material which does not lend itself to further examination. He recommends as more serviceable some oil solution, preferably “bone oil” (neat’s-foot oil), in which a section of the thallus should be broken up under a cover-glass and subjected to a process of slow heating; some days must elapse before the extraction is complete. The surplus oil is then to be drained off, the section further bruised and the substance examined.
Acids in bulk should be extracted by ether, acetone, chloroform, benzole, petrol-ether and lignoin or by carbon bisulphide. Such solvents as alcohols, acetates and alkali solutions should not be used as they tend to split up or to alter the constitution of the acids. For the same reason, the use of chloroform is to a certain extent undesirable as it contains a percentage of alcohol. Ether and acetone, or a mixture of both, are the most efficient solvents, and all acids can be extracted by their use, if the material is left to soak a sufficient length of time, either in the cold or warmed. It is however advisable to follow with a second solvent in case any other acid should be present in the tissues. Concentrated sulphuric acid dissolves out all acids but often induces colour changes in the process.
All known lichen-acids form crystals, though the crystalline form may alter with the solution used. After filtering and distilling, the residue will be found to contain a mixture of these crystals along with other substances, which may be removed by washing, etc.
c. Character of Acids. Many lichen-acids are more or less bitter to the taste; they are usually of an acid nature though certain of the substances are neutral, such as zeorin, a constituent of various Lecanoraceae, Physciaceae and Cladoniaceae, stictaurin, originally obtained from Sticta aurata, leiphemin, from Haematomma coccineum, and others.
A large proportion are esters or alkyl salts formed by the union of an alcohol and an acid; these are insoluble in alkaline carbonates. It is considered probable that the fungus generates the acid, while the alcohol arises in the metabolic processes in the alga. It has indeed been proved that the alcohol, erythrit, is formed in at least two algae, Protococcus vulgaris and Trentepohlia jolithus; and the lichen-acid, erythrin (C₂₀H₂₂O₁₀), obtained from species of Roccella in which the alga is Trentepohlia, is, according to Hesse, the erythrit ester of lecanoric acid (C₁₆H₁₄O₇), a very frequent constituent of lichen thalli. It is certain that the interaction of both symbionts is necessary for acid production. This was strikingly demonstrated by Tobler[820] in his cultural study of the lichen thallus. He succeeded in growing, to a limited extent, the hyphal part of the thallus of Xanthoria parietina on artificial media; but the filaments remained persistently colourless until he added green algal cells to the culture. Almost immediately thereafter the characteristic yellow colour appeared, proving the presence of parietin, formerly known as chrysophanic acid. Tobler’s observation may easily be verified in plants from natural habitats. A depauperate form of Placodium citrinum consisting mainly of a hypothallus of felted hyphae, with minute scattered granules containing algae, was tested with potash, and only the hyphae immediately covering the algal granules took the stain; the hypothallus gave no reaction.
It has been suggested[821] that when a decrease of albumenoids takes place, the quantity of lichen-acid increases, so that the excreted substance should be regarded as a sort of waste product of the living plant, “rather than as a product of deassimilation.” The subject is not yet wholly understood.
d. Causes of Variation in Quantity and Quality of Lichen-Acids. Though it has been proved that lichen-acids are formed freely all the year round on any soil or in any region, it happens occasionally that they are almost or entirely lacking in growing plants. Schwarz[822] found this to be the case in certain plants of Lecanora tartarea, and he suggests that the gyrophoric acid contained in the outer cortex of that lichen had been broken up by the ammonia of the atmosphere into carbonic acid and orcin which is soluble in water, and would thus be washed away by rain. It has also been shown by Schwendener[823] and others that the outer layers of the older thallus in many lichens slowly perish, first breaking up and then peeling off; the denuded areas would therefore have lost, for some time at least, their particular acids. Fünfstück[824] considers that the difference in the presence and amount of acid in the same species of lichen may be due very often to variation in the chemical character of the substratum, and this view tallies with the results noted by Heber Howe[825] in his study of American Ramalinae. He observed that, though all showed a pale-yellow reaction with potash, those growing on mineral substrata gave a more pronouncedly yellow colour.
M. C. Knowles[826] found that in Ramalina scopulorum the colour reaction to potash varied extremely, being more rapid and more intense, the more the plants were subject to the influence of the sea-spray.
Lichen-acids are peculiarly abundant in soredia, and as, in some species, the thallus forms these outgrowths, or even becomes leprose more freely in damp weather, the amount of acids produced may depend on the amount of moisture in the atmosphere.
Their formation is also strongly influenced by light, as is well shown by the varying intensity of colour in some yellow thalli. Placodium elegans, always a brightly coloured lichen, changes from yellow to sealing-wax red in situations exposed to the full blaze of the sun. Haematomma ventosum,though greenish-yellow in lowland situations is intensely yellow in the high Alps. The same variation of colour is characteristic of Rhizocarpon geographicum which is a bright citron-yellow at high altitudes, and becomes more greenish in hue as it nears the plains. The familiar foliose lichen Xanthoria parietina is a brilliant orange-yellow in sunny situations, but grey-green in the shade, and then yielding only minute quantities of parietin. West[827] and others have noted its more luxuriant growth and brighter colour when it grows in positions where nitrogenous food is plentiful, such as the roofs of farm-buildings, which are supplied with manure-laden dust, and boulders by the sea-shore frequented by birds.
e. Distribution of Acids. Some acids, so far as is known, are only to be found in one or at most in very few lichens, as for instance cuspidatic acid which is present in Ramalina cuspidata, and scopuloric acid, a constituent of Ramalina scopulorum, the acids having been held to distinguish by their reactions the one plant from the other.
Others of these peculiar products are abundant and widely distributed. Usninic acid, one of the commonest, has been determined in some 70 species belonging to widely diverse genera, and atranorin, a substance first discovered in Lecanora atra, has been found again many times; Zopf gives a list of about 73 species or varieties from which it has been extracted. Another widely distributed acid is salazinic acid which has been found by Lettau[828] in a very large number of lichens.
E. Chemical Grouping of Lichen-Acids
Most of these acids have been provisionally arranged by Zopf in groups under the two great organic series: I. The Fat series; and II. The Benzole or Aromatic series.
I. LICHEN-ACIDS OF THE FAT SERIES
Group 1. Colourless substances soluble in alkali, the solution not coloured by iron chloride. Exs. protolichesterinic acid (C₁₉H₃₄O₄) obtained from species of Cetraria, and roccellic acid (C₁₇H₃₂O₄) from species of Roccella, from Lecanora tartarea, etc.
Group 2. Neutral colourless substances insoluble in alkalies, but soluble in alcohol, the solution not coloured by iron chloride. Exs. zeorin (C₅₂H₈₈O₄), a product of widely diverse lichens, such as Lecanora (Zeora) sulphurea, Haematomma coccineum, Physcia caesia, Cladonia deformis, etc. and barbatin (C₉H₁₄O), a product of Usnea barbata.
Group 3. Brightly coloured acids, yellow, orange or red, all derivatives of pulvinic acid (C₁₈H₁₂O₅), a laboratory compound which has not been found in nature. The group includes among others vulpinic acid (C₁₉H₁₄O₅) from the brilliant yellow Evernia (Letharia) vulpina, stictaurin (C₃₆H₂₂O₉) deposited in orange-red crystals on the hyphae of Sticta aurata, and rhizocarpic acid (C₂₆H₂₀O₆) chiefly obtained from Rhizocarpon geographicum: it crystallizes out in slender citron-yellow prisms.
Group 4. Only one acid, usninic (C₁₈H₁₆O₇), a derivative of acetylacetic acid, is placed in this group. It is of very wide-spread occurrence, having been found in at least 70 species belonging to very different genera and families of crustaceous shrubby and leafy lichens. Zopf himself isolated it from 48 species.
Group 5. The thiophaninic acid (C₁₂H₆O₉) group representing only a small number. They are all sulphur-yellow in colour and soluble in alcohol, the solution becoming blackish-green or dirty blue on the addition of iron chloride, with one exception, that of subauriferin obtained from the yellow-coloured medulla of Parmelia subaurifera which stains faintly wine-red in an iron solution. Thiophaninic acid, which gives its name to this group, occurs in Pertusaria lutescens and P. Wulfenii, both of which are yellowish crustaceous lichens growing mostly on the trunks of trees.
II. LICHEN-ACIDS OF THE BENZOLE SERIES
The larger number of lichen-acids belong to this series, of which 94 at least are already known. They are divided into two subseries: I. Orcine derivatives, and II. Anthracene derivatives.
Subseries I. Orcine Derivatives
Zopf specially insists that the grouping of this series must be regarded as only a provisional arrangement of the many lichen-acids that are included therein. All of them are split up into orcine and carbonic acid by ammonia and other alkalies. On exposure to air, the ammoniacal or alkaline solution changes gradually into orceine, the colouring principle and chief constituent of commercial orchil. Orcine is not found free in nature. The orcine subseries includes five groups:
Group 1. The substances in this group form, with hypochlorite of lime (“CaCl”), red-coloured compounds which yield, on splitting, orsellinic acid. Zopf enumerates seven acids as belonging to this group, among which is lecanoric acid (C₁₆H₁₄O₇), found in many different lichens, e.g. Roccella tinctoria, Lecanora tartarea, etc.: whenever there is a differentiated pith and cortex it occurs in the pith alone. Erythrin (C₂₀H₂₂O₁₀), a constituent of the British marine lichen Roccella fuciformis, also belongs to this orsellinic group.
Group 2. Substances which also form red products with CaCl, but do not break up into orsellinic acid. Among the most noteworthy are olivetoric acid (C₂₁H₂₆O₇), a constituent of Evernia furfuracea, perlatic acid (C₂₈H₃₀O₁₀) and glabratic acid (C₂₄H₂₆O₁₁), which are obtained from species of Parmelia.
Group 3. Contains three acids of somewhat restricted occurrence. They do not form red products with CaCl, and they yield on splitting everninic acid. They are: evernic acid (C₁₇H₁₆O₇), found in Evernia prunastri var. vulgaris, ramalic acid (C₁₇H₁₆O₇) in Ramalina pollinaria, and umbilicaric acid (C₂₅H₂₂O₁₁) in species of Gyrophora.
Group 4. The numerous acids of this group are not easily soluble and have a very bitter taste. They are not coloured by CaCl; when extracted with concentrated sulphuric acid, the solution obtained is reddish-yellow or deep red. Among the most frequent are fumarprotocetraric acid (C₆₂H₅₀O₃₅), the bitter principle of Cetraria islandica, Cladonia rangiferina, etc., psoromic acid (C₂₀H₁₄O₉), obtained from Alectoria implexa, Lecanora varia, Cladonia pyxidata and many other lichens, and salazinic acid (C₁₉H₁₄O₁₀), recorded by Zopf as occurring in Stereocaulon salazinum and in several Parmeliae, but now found by Lettau[829] to be very wide-spread. He used micro-chemical methods and detected its presence in 72 species from twelve different families. The distribution of the acid in the thallus varies considerably.
Group 5. This is called the atranorin group from one of the most important members. They are colourless substances and, like the preceding group, are not affected by CaCl, but when split they form bodies that colour a more or less deep red with that reagent. Atranorin (C₁₉H₁₈O₈) is one of the most widely spread of all lichen-acids; it occurs in Lecanoraceae, Parmeliaceae, Physciaceae and Lecideaceae. Barbatinic acid (C₁₉H₂₀O₇), another member, is found in Usnea ceratina, Alectoria ochroleuca and in a variety of Rhizocarpon geographicum. A very large number of acids more or less fully studied belong to this group.
Subseries II. Anthracene Derivatives
The constituents of this subseries are derived from the carbohydrate anthracene, and are characterized by their brilliant colours, yellow, red, brown, red-brown or violet-brown. So far, only ten different kinds have been isolated and studied. Parietin[830] (C₁₆H₁₂O₅), one of the best known, has been extracted from Xanthoria parietina, Placodium murorum and several other bright-yellow lichens; solorinic acid (C₁₅H₁₄O₅) occurs in orange-red crystals on the hyphae of the pith and under surface of Solorina crocea; nephromin (C₁₆H₁₂O₆) is found in the yellow medulla of Nephromium lusitanicum; rhodocladonic acid (C₁₂H₈O₆ or C₁₄H₁₀O₇) is the red substance in the apothecia of the red-fruited Cladoniae.
There are, in addition, a short series of coloured substances which are of uncertain position. They are imperfectly known and are of rare occurrence. An acid containing nitrogen has been extracted from Roccella fuciformis, and named picroroccellin[831] (C₂₇H₂₉N₃O₅). It crystallizes in comparatively large prisms, has an exceedingly bitter taste, and is very sparingly soluble. It is the only lichen-acid in which nitrogen has been detected.
One acid at least, belonging to the Fat series, vulpinic acid, which gives the greenish-yellow colour to Letharia vulpina, has been prepared synthetically by Volkard[832].
F. Chemical Reagents as Tests for Lichens
The employment of chemical reagents as colour tests in the determination of lichen species was recommended by Nylander[833] in a paper published by him in 1866. Many acids had already been extracted and examined, and as they were proved to be constant in the different species where they occurred, he perceived their systematic importance. As an example of the new tests, he cited the use of hypochlorite of lime, a solution of which, applied directly to the thallus of species of Roccella, produced a bright-red “erythrinic” reaction. Caustic potash was also found to be of service in demonstrating the presence of parietin in lichens by a beautiful purple stain. Many lichenologists eagerly adopted the new method, as a sure and ready means of distinguishing doubtful species; but others have rejected the tests as unnecessary and not always to be relied on, seeing that the acids are not always produced in sufficient abundance to give the desired reaction, and that they tend to alter in time.
The reagents most commonly in use are caustic potash, generally indicated by K; hypochlorite of calcium or bleaching powder by CaCl; and a solution of iodine by I. The sign + signifies a colour reaction, while- indicates that no change has followed the application of the test solution. Double signs ⁺₊ or any similar variation indicate the upper or lower parts of the thallus affected by the reagent. In some instances the reaction only follows after the employment of two reagents represented thus: K(CaCl)+. In such a case the potash breaks up the particular acid and compounds are formed which become red, orange, etc., on the subsequent application of hypochlorite of lime.
As an instance of the value of chemical tests, Zopf cites the reaction of hypochlorite of lime on the thallus of four different species of Gyrophora, the “tripe de roche”:—
| Gyrophora torrefacta | CaCl ⁻₊. |
| ” polyrhiza CaCl | ⁺₊. |
| ” proboscidea CaCl | ⁺₋. |
| ” erosa CaCl | ⁻₋. |
It must however be borne in mind that these species are well differentiated and can be recognized, without difficulty, by their morphological characters. Experienced systematists like Weddell refuse to accept the tests unless they are supported by true morphological distinctions, as the reactions are not sufficiently constant.
G. Chemical Reactions in Nature
Similar colour changes may often be observed in nature. The acids of the exposed thallus cortex are not unfrequently split up by the gradual action of the ammonia in the atmosphere, one of the compounds thus set free being at the same time coloured by the alkali. Thus salazinic acid, a constituent of several of our native Parmeliae, is broken up into carbonic acid and salazininic acid, the latter taking a red colour. Fumarprotocetraric acid is acted on somewhat similarly, and the red colour may be seen in Cetraria at the base of the thallus where contact with soil containing ammonia has affected the outer cortex of the plant. The same results are produced still more effectively when the lichen comes into contact with animal excrement.
Gummy exudations from trees which are more or less ammoniacal may also act on the thallus and form red-coloured products on contact with the acids present. Lecanora (Aspicilia) cinerea is so easily affected by alkalies that a thin section left exposed may become red in time owing to the ammonia in the atmosphere.