CHAPTER II
CONSTITUENTS OF THE LICHEN THALLUS
I. LICHEN GONIDIA
The thallus or vegetative body of lichens differs from that of other green plants in the sharp distinction both of form and colour between the assimilative cells and the colourless tissues, and in the relative positions these occupy within the thallus: in the greater number of lichen species the green chlorophyll cells are confined to a narrow zone or band some way beneath and parallel with the surface (Fig. 1); in a minority of genera they are distributed through the entire thallus (Fig. 2); but in all cases the tissues remain distinct. The green zone can be easily demonstrated in any of the larger lichens by scaling off the outer surface cells, or by making a vertical section through the thallus. The colourless cells penetrate to some extent among the green cells; they also form the whole of the cortical and medullary tissues.
These two different elements we now know to consist of two distinct organisms, a fungus and an alga. The green algal cells were at one time considered to be reproductive bodies, and were called “gonidia,” a term still in use though its significance has changed.
1. GONIDIA IN RELATION TO THE THALLUS
A. HISTORICAL ACCOUNT OF LICHEN GONIDIA
There have been few subjects of botanical investigation that have roused so much speculation and such prolonged controversy as the question of these constituents of the lichen plant. The green cells and the colourless filaments which together form the vegetative structure are so markedly dissimilar, that constant attempts have been made to explain the problem of their origin and function, and thereby to establish satisfactorily the relationship of lichens to other members of the Plant Kingdom.
In gelatinous lichens, represented by _Collema_, of which several species are common in damp places and grow on trees or walls or on the ground, the chains of green cells interspersed through the thallus have long been recognized as comparable with the filaments of _Nostoc_, a blue-green gelatinous alga, conspicuous in wet weather in the same localities as those inhabited by _Collema_. So among early systematists, we find Ventenat[152] classifying the few lichens with which he was acquainted under algae and hazarding the statement that a gelatinous lichen such as _Collema_ was only a _Nostoc_ changed in form. Some years later Cassini[153] in an account of _Nostoc_ expressed a somewhat similar view, though with a difference: he suggested that _Nostoc_ was but a monstrous form of _Collema_, his argument being that, as the latter bore the fruit, it was the normal and perfect condition of the plant. A few years later Agardh[154] claimed to have observed the metamorphosis of _Nostoc_ up to the fertile stage of a lichen, _Collema limosum_. But long before this date, Scopoli[155] had demonstrated a green colouring substance in non-gelatinous lichens by rubbing a crustaceous or leprose thallus between the fingers; and Persoon[156] made use of this green colour characteristic of lichen crusts to differentiate these plants from fungi. Sprengel[157] went a step further in exactly describing the green tissue as forming a definite layer below the upper cortex of foliaceous lichens.
The first clear description and delimitation of the different elements composing the lichen thallus was, however, given by Wallroth[158]. He drew attention to the great similarity between the colourless filaments of the lichen and the hyphae of fungi. The green globose cells in the chlorophyllaceous lichens he interpreted as brood-cells or gonidia, regarding them as organs of reproduction collected into a “stratum gonimon.” To the same author we owe the terms “homoiomerous” and “heteromerous,” which he coined to describe the arrangement of these green cells in the tissue of the thallus. In the former case the gonidia are distributed equally through the structure; in the latter they are confined to a distinct zone.
Wallroth’s terminology and his views of the function of the gonidia were accepted as the true explanation for many years, the opinion that they were solely reproductive bodies being entirely in accordance with the well-known part played by soredia in the propagation of lichens—and soredia always include one or more green cells.
B. GONIDIA CONTRASTED WITH ALGAE
In describing the gonidia of the Graphideae Wallroth[159] had pointed out their affinity with the filaments of _Chroolepus_ (_Trentepohlia_) _umbrina_. He considered these and other green algae when growing loose on the trunks of trees to be but “unfortunate brood-cells” which had become free and, though capable of growth and increase, were unable to form again a lichen plant.
Further observations on gonidia were made by E. Fries[160]: he found that the green cells escaped from the lichen matrix and produced new individuals; and also that the whole thallus in moist localities might become dissolved into the alga known as _Protococcus viridis_; but, he continues, “though these _Protococcus_ cells multiplied exceedingly, they never could rise again to the perfect lichen.” Kützing[161], in a later account of _Protococcus viridis_, also recognized its affinity with lichens; he stated that he could testify from observation that, according to the amount of moisture present, it would develop, either in excessive moisture to a filamentous alga, or in drier conditions “to lichens such as _Lecanora subfusca_ or _Xanthoria parietina_.”
A British botanist, G. H. K. Thwaites[162], at one time superintendent of the botanical garden at Peradeniya in Ceylon, published a notable paper on lichen gonidia in which he pointed out that as in _Collema_ the green constituents of the thallus resembled the chains of _Nostoc_, so in the non-gelatinous lichens, the green globose cells were comparable or identical with _Pleurococcus_, and Thwaites further observed that they increased by division within the lichen thallus. He insisted too that in no instance were gonidia reproductive organs: they were essential component parts of the vegetative body and necessary to the life of the plant. In a further paper on _Chroolepus ebeneus_ Ag., a plant consisting of slender dark-coloured felted filaments, he described these filaments as being composed of a central strand which closely resembled the alga _Chroolepus_, and of a surrounding sheath of dark-coloured cells (Fig. 3): “occasionally,” he writes, “the internal filament protrudes beyond the investing sheath, and may then be seen to consist of oblong cells containing the peculiar reddish oily-looking endochrome of _Chroolepus_.” Thwaites placed this puzzling plant in a new genus, _Cystocoleus_, at the same time pointing out its affinity with the lichen genus _Coenogonium_. The plant is now known as _Coenogonium ebeneum_. Thwaites was on the threshold of the discovery as to the true nature of the relationship between the central filament and the investing sheath, but he failed to take the next forward step.
Very shortly after, Von Flotow[163] published his views on some other lichen gonidia. He had come to the conclusion that the various species of the alga, _Gloeocapsa_, so frequently found in damp places, among mosses and lichens, were merely growth stages of the gonidia of _Ephebe pubescens_, and bore the same relation to _Ephebe_ as did _Lepra viridis_ (_Protococcus_) to _Parmelia_. The gonidium of _Ephebe_ is the gelatinous filamentous blue-green alga _Stigonema_ (Fig. 4), and the separate cells are not unlike those of _Gloeocapsa_. Flotow had also demonstrated that the same type of gonidium was enclosed in the cephalodia of _Stereocaulon_. Sachs[164], too, gave evidence as to the close connection between _Nostoc_ and _Collema_. He had observed numerous small clumps of the alga growing in proximity to equally abundant thalli of _Collema_, with every stage of development represented from one to the other. He found cases where the gelatinous coils of _Nostoc_ chains were penetrated by fine colourless filaments “as if invaded by a parasitic fungus.” Later these threads were seen to be attached to some cell of the _Nostoc_ trichome. Sachs concluded, however, from very careful examination at the time, that the colourless filaments were produced by the green cells. As growth proceeded, the coloured _Nostoc_ chains became massed towards the upper surface, while the colourless filaments tended to occupy the lower part of the thallus. He calculated that during the summer season the metamorphosis from _Nostoc_ to a fertile _Collema_ thallus took from three to four months. He judged that in favourable conditions the change would inevitably take place, though if there should be too great moisture no _Collema_ would be formed. His study of _Cladonia_ was less successful as he mistook some colonies of _Gloeocapsa_ for a growth condition of _Cladonia_ gonidia, an error corrected later by Itzigsohn[165].
But before this date Itzigsohn[166] had published a paper setting forth his views on thallus formation, which marked a distinct advance. He did not hazard any theory as to the origin of gonidia, but he had observed spermatia growing, much as did the cells of _Oscillaria_: by increase in length, and, by subsequent branching, filaments were formed which surrounded the green cells; these latter had meanwhile multiplied by repeated division till finally a complete thallus was built up, the filamentous tissue being derived from the spermatia, while the green layer came from the original gonidium. In contrasting the development with that of _Collema_, he represents _Nostoc_ as a sterile product of a lichen and, like the gonidia of other lichens, only able to form a lichen thallus when it encounters the fructifying spermatia.
Braxton Hicks[167], a London doctor, some time later, made experiments with _Chroococcus_ algae which grew in plenty on the bark of trees, and followed their development into a lichen thallus. He further claimed to have observed a _Chlorococcus_, which was associated with a _Cladonia_, divide and form a _Palmella_ stage.
C. CULTURE EXPERIMENTS WITH THE LICHEN THALLUS
It had been repeatedly stated that the gonidia might become independent of the thallus, but absolute proof was wanting until Speerschneider[168], who had turned his attention to the subject, made direct culture experiments and was able to follow the liberation of the green cells. He took a thinnish section of the thallus of _Hagenia_ (_Physcia_) _ciliaris_, and, by keeping it moist, he was able to observe that the gonidial cells increased by division; the moist condition at the same time caused the colourless filaments to die away. This method of investigation was to lead to further results. It was resorted to by Famintzin and Baranetzky[169] who made cultures of gonidia extracted from three different lichens, _Physcia_ (_Xanthoria_) _parietina_, _Evernia furfuracea_ and _Cladonia_ sp. They were able to observe the growth and division of the green cells and, in addition, the formation of zoospores. They recognized the development as entirely identical with that of the unicellular green alga, _Cystococcus humicola_ Naeg. Baranetzky[170] continued the experiments and made cultures of the blue-green gonidia of _Peltigera canina_ and of _Collema pulposum_. In both instances he succeeded in isolating them from the thallus and in growing them in moist air as separate organisms. He adds that “many forms reckoned as algae, may be considered as vegetating lichen gonidia such as _Cystococcus_, _Polycoccus_, _Nostoc_, etc.” Meanwhile Itzigsohn[171] had further demonstrated by similar culture experiments that the gonidia of _Peltigera canina_ corresponded with the algae known as _Gloeocapsa monococca_ Kütz., and as _Polycoccus punctiformis_ Kütz.
D. THEORIES AS TO THE ORIGIN OF GONIDIA
Though the relationship between the gonidia within the thallus and free-living algal organisms seemed to be proved beyond dispute, the manner in which gonidia first originated had not yet been discovered. Bayrhoffer[172] attacked this problem in a study of foliose and other lichens. According to his observations, certain colourless cells or filaments, belonging to the “gonimic” layer, grew in a downward direction and formed at their tips a faintly yellowish-green cell; it gradually enlarged and was at length thrown off as a free globose gonidium, which represented the female cell. Other filaments from the “lower fibrous layer” of the thallus at the same time grew upwards and from them were given off somewhat similar gonidia which functioned as male cells. His observations and deductions were fanciful, but it must be remembered that the attachment between hypha and alga in lichens is in many cases so close as to appear genetic, and also it often happens that as the gonidium multiplies it becomes free from the hypha.
In his _Mémoire sur les Lichens_, Tulasne[173] described the colourless filaments as being fungal in appearance. The green cells he recognized as organs of nutrition, and once and again in his paper he states that they arose directly by a sort of budding process from the medullary or cortical filaments, either laterally or at the apex. This apparently reasonable view of their origin was confirmed by other writers on the subject: by Speerschneider[174] in his account of the anatomy of _Usnea barbata_, by de Bary[175], and by Schwendener[176] in their earlier writings. But even while de Bary accepted the hyphal origin of the gonidia, he noted[177] that, accompanying _Opegrapha atra_ and other Graphideae, on the bark were to be found free _Chroolepus_ cells similar to the gonidia in the lichen thallus. He added that gonidia of certain other lichens in no way differed from _Protococcus_ cells; and as for the gelatinous lichens he declared that “either they were the perfect fruiting form of Nostocaceae and Chroococcaceae—hitherto looked on as algae—or that these same Nostocaceae and Chroococcaceae are algae which take the form of _Collema_, _Ephebe_, etc., when attacked by an ascomycetous fungus.”
All these investigators, and other lichenologists such as Nylander[178], still regarded the free-living organisms identified by them as similar to the green cells of the thallus, as only lichen gonidia escaped from the matrix and vegetating in an independent condition.
The old controversy has in recent years been unexpectedly reopened by Elfving[179] who has sought again to prove the genetic origin of the green cells. His method has been to examine a large series of lichens by making sections of the growing areas, and he claims to have observed in every case the hyphal origin of the gonidia: not only of _Cystococcus_ but also of _Trentepohlia_, _Stigonema_ and _Nostoc_. In the case of _Cystococcus_, the gonidium, he says, arises by the swelling of the terminal cell of the hypha to a globose form, and by the gradual transformation of the contents to a chlorophyll-green colour, with power of assimilation. In the case of filamentous gonidia such as _Trentepohlia_, the hyphal cells destined to become gonidia are intercalary. In _Peltigera_ the cells of the meristematic plectenchyma become transformed to blue-green _Nostoc_ cells.
A study was also made by him of the formation of cephalodia[180], the gonidia of which differ from those of the “host” thallus. In _Peltigera aphthosa_ he claims to have traced the development of these bodies to the branching and mingling of the external hairs which, in the end, form a ball of interwoven hyphae. The central cells of the ball are then gradually differentiated into _Nostoc_ cells, which increase to form the familiar chains. Elfving allows that the gonidia mainly increase by division within the thallus, and that they also may escape and live as free organisms. His views are unsupported by direct culture experiments which are the real proof of the composite nature of the thallus.
E. MICROGONIDIA
Another attempt to establish a genetic origin for lichen gonidia was made by Minks[181]. He had found in his examination of _Leptogium myochroum_ that the protoplasmic contents of the hyphae broke up into a regular series of globular corpuscles which had a greenish appearance. These minute bodies, called by him microgonidia, were, he states, at first few in number, but gradually they increased and were eventually set free by the mucilaginous degeneration of the cell wall. As free thalline gonidia, they increased in size and rapidly multiplied by division. Minks was at first enthusiastically supported by Müller[182] who had found from his own observations that microgonidia might be present in any of the lichen hyphae and in any part of the thallus, even in the germinating tube of the lichen spore, and was in that case most easily seen when the spores germinated within the ascus. He argued that as spores originated within the ascus, so microgonidia were developed within the hyphae. Minks’s theories were however not generally accepted and were at last wholly discredited by Zukal[183] who was able to prove that the greenish bodies were contracted portions of protoplasm in hyphae that suffered from a lowered supply of moisture, the green colour not being due to any colouring substance, but to light effect on the proteins—an outcome of special conditions in the vegetative life of the plant. Darbishire[184] criticized Minks’s whole work with great care and he has arrived at the conclusion that the microgonidium may be dismissed as a totally mistaken conception.
F. COMPOSITE NATURE OF THALLUS
Schwendener[185] meanwhile was engaged on his study of lichen anatomy. Though at first he adhered to the then accepted view of the genetic connection between hyphae and gonidia, his continued examination of the vegetative development led him to publish a short paper[186] in which he announced his opinion that the various blue-green and green gonidia were really algae and that the complete lichen in all cases represented a fungus living parasitically on an alga: in _Ephebe_, for example, the alga was a form of _Stigonema_, in the Collemaceae it was a species of _Nostoc_. In those lichens enclosing bright green cells, the gonidia were identical with _Cystococcus humicola_, while in _Graphideae_ the brightly coloured filamentous cells were those of _Chroolepus_ (_Trentepohlia_). This statement he repeated in an appendix to the larger work on lichens[187] and again in the following year[188] when he described more fully the different gonidial algae and the changes produced in their structure and habit by the action of the parasite: “though eventually the alga is destroyed,” he writes, “it is at first excited to more vigorous growth by contact with the fungus, and in the course of generations may become changed beyond recognition both in size and form.” In support of his theory of the composite constitution of the thallus, Schwendener pointed out the wide distribution and frequent occurrence in nature of the algae that become transformed to lichen gonidia. He claimed as further proof of the presence of two distinct organisms that, while the colourless filaments react in the same way as fungi on the application of iodine, the gonidia take the stain of algal membranes.
G. SYNTHETIC CULTURES
Schwendener’s “dual hypothesis,” as it was termed, excited great interest and no little controversy, the reasons for and against being debated with considerable heat. Rees[189] was the first who attempted to put the matter to the proof by making synthetic cultures. For this purpose he took spores from the apothecium of a _Collema_ and sowed them on pure cultures of _Nostoc_, and as a result obtained the formation of a lichen thallus, though he did not succeed in producing any fructification. He observed further that the hyphal filaments from the germinating spore died off when no _Nostoc_ was forthcoming.
Bornet[190] followed with his record of successful cultures. He selected for experiment the spores of _Physcia_ (_Xanthoria_) _parietina_ and was able to show that hyphae produced from the germinating spore adhered to the free-growing cells of _Protococcus[191] viridis_ and formed the early stages of a lichen thallus. Woronin[192] contributed his observations on the gonidia of _Parmelia_ (_Physcia_) _pulverulenta_ which he isolated from the thallus and cultivated in pure water. He confirmed the occurrence of cell division in the gonidia and also the formation of zoospores, these again forming new colonies of algae identical in all respects with the thalline gonidia. He was able to see the germinating tube from a lichen spore attach itself to a gonidium, though he failed in his attempts to induce further growth. In our own country Archer[193] welcomed the new views on lichens, and attempted cultures but with very little success. Further synthetic cultures were made by Bornet[194], Treub[195] and Borzi[196] with a series of lichen spores. They also were able to observe the first stages of the thallus. Borzi observed spores of _Physcia_ (_Xanthoria_) _parietina_ scattered among _Protococcus_ cells on the branch of a tree. The spores had germinated and the first branching hyphae had already begun to encircle the algae.
Additional evidence in favour of the theory of the independent origin of the colourless filaments and the green cells was furnished by Stahl’s[197] research on hymenial gonidia in _Endocarpon_ (Fig. 5). By making synthetic cultures of the mature spores with these bodies, he was able to observe not only the germination of the spores and the attachment of the filaments to the gonidia (Fig. 6), but also the gradual building up of a complete lichen thallus to the formation of perithecia and spores.
Some years later Bonnier[198] made an interesting series of synthetic cultures between the spores of lichens germinated in carefully sterilized conditions, and algae taken from the open (Figs. 7 and 8). Separate control cultures of spores and algae were carried on at the same time, with the result that in one case lichen hyphae alone, in the other algae were produced. The various lichen spores with which he experimented were sown in association with the following algae:
(1) PROTOCOCCUS.
Pure synthetic cultures of _Physcia_ (_Xanthoria_) _parietina_ were begun in August 1884 on fragments of bark. In October 1886 the thallus was several centimetres in diameter, and some of the lobes were fruited.
_Physcia stellaris_ was also grown on bark; in one case both thallus and apothecia were developed.
_Parmelia acetabulum_, another corticolous species, formed only a minute thallus about 5 mm. in diameter, but entirely identical with normally growing specimens.
(2) PLEUROCOCCUS.
_Lecanora_ (_Rinodina_) _sophodes_, sown on rock in 1883, reached in 1886 a diameter of 13 mm. with fully developed apothecia.
_Lecanora ferruginea_ and _L. subfusca_ after three years’ culture formed sterile thalli only.
_Lecanora coilocarpa_ in four years, and _L. caesio-rufa_ in three years formed very small thalli without fructification.
(3) TRENTEPOHLIA (Chroolepus).
_Opegrapha vulgata_ in two years had developed thallus and apothecia. The control culture of the spores formed, as in nature, a considerable felt of mycelium in the interstices of the bark, but no pycnidia or apothecia.
_Graphis elegans._ Only the beginning of a differentiated thallus was obtained with this species.
_Verrucaria muralis_ (?)[199] gave in less than a year a completely developed thallus.
Bonnier also attempted cultures with species of _Collema_ and _Ephebe_, but was unsuccessful in inducing the formation of a lichen plant.
H. HYMENIAL GONIDIA
Reference has already been made to the minute green cells which were originally described by Nylander[200] as occurring in the perithecia of a few Pyrenolichens as free gonidia, _i.e._ unentangled with lichen hyphae. Fuisting[201] found them in the perithecium of _Polyblastia_ (_Staurothele_) _catalepta_ at a very early stage of its development when the perithecial tissues were newly differentiated from those of the surrounding thallus. The gonidia enclosed in the perithecium differed in no wise from those of the thallus: they had become mechanically enclosed in the new tissue; and while those in the outer compact layers died off, those in the centre of the structure, where a hollow space arises, were subject to very active division, becoming smaller in the process and finally filling the cavity. Winter’s[202] researches on similar lichens confirmed Fuisting’s conclusions: he described them as similar to the thalline gonidia but lighter in colour and of smaller size, measuring frequently only 2·3 µ in diameter, though this size increased to about 7 µ when cultivated outside the perithecium.
Stahl[203] sufficiently demonstrated the importance of these gonidia in supplying the germinating spores with the necessary algae. They come to lie in vertical rows between the asci and, owing to pressure, assume an elongate form[204] (Figs. 5 and 6). They have been seen in very few lichens, in _Endocarpon_ and _Staurothele_, both rather small genera of Pyrenolichens, and, so far as is known, in two Discolichens, _Lecidea phylliscocarpa_ and _L. phyllocaris_, the latter recorded from Brazil by Wainio[205], and, on account of the inclusion of gonidia in the hymenium, placed by him in a section, _Gonothecium_.
I. NATURE OF ASSOCIATION BETWEEN ALGA AND FUNGUS
_a._ CONSORTIUM AND SYMBIOSIS. These cultures had established convincingly the composite nature of the lichen thallus, and Schwendener’s opinion, that the relationship between the two organisms was some varying degree of parasitism, was at first unhesitatingly accepted by most botanists. Reinke[206] was the first to point out the insufficiency of this view to explain the long continued healthy life of both constituents, a condition so different from all known instances of the disturbing or fatal parasitism of one individual on another. He recognized in the association a state of mutual growth and interdependence, that had resulted in the production of an entirely new type of plant, and he suggested _Consortium_ as a truer description of the connection between the fungus and the alga. This term had originally been coined by his friend Grisebach in a paper[206] describing the presence of actively growing _Nostoc_ algae in healthy _Gunnera_ stems; and Reinke compared that apparently harmless association with the similar phenomenon in the lichen thallus. The comparison was emphasized by him in a later paper[207] on the same subject, in which he ascribes to each “consort” its function in the composite plant, and declares that if such a mutual life of Alga and Ascomycete is to be regarded as one of parasitism, it must be considered as reciprocal parasitism; and he insists that “much more appropriate for this form of organic life is the conception and title of _Consortium_.” In a special work on lichens, Reinke[208] further elaborated his theory of the physiological activity and mutual service of the two organisms forming the consortium.
Frank[209] suggested the term _Homobium_ as appropriate, but it is faulty inasmuch as it expresses a relationship of complete interdependence, and it has been proved that the fungus partly, and the alga entirely, have the power of free growth.
A wider currency was given to this view of a mutually advantageous growth by de Bary[210]. He followed Reinke in refusing to accept as satisfactory the theory of simple parasitism, and adduced the evident healthy life of the algal cells—the alleged victims of the fungus—as incompatible with the parasitic condition. He proposed the happily descriptive designation of a _Symbiosis_ or conjoint life which was mostly though not always, nor in equal degree, beneficial to each of the partners or symbionts.
_b._ DIFFERENT FORMS OF ASSOCIATION. The type of association between the two symbionts varies in different lichens. Bornet[211], in describing the development of the thallus in certain members of the Collemaceae, found that though as a rule the two elements of the thallus, as in some species of _Collema_ itself, persisted intact side by side, there was in other members of the genus an occasional parasitism: short branches from the main hyphae applied themselves by their tips to some cell of the _Nostoc_ chain (Fig. 9). The cell thus seized upon began to increase in size, and the plasma became granular and gathered at the side furthest away from the point of attachment. Finally the contents were used up, and nothing was left but an empty membrane adhering to the fungus hypha. In another species the hypha penetrated the cell. These instances of parasitism are most readily seen towards the edge of the thallus where growth is more active; towards the centre the attached cells have become absorbed, and only the shortened broken chains attest their disappearance. The other cells of the chains remain uninjured.
In _Synalissa_, a small shrubby gelatinous genus, the hypha, as described by Bornet and by Hedlund[212], pierces the outer wall of the gelatinous alga (_Gloeocapsa_) and swells inside to a somewhat globose haustorium which rests in a depression of the plasma (Fig. 10). The alga, though evidently undamaged, is excited to a division which takes place on a plane that passes through the haustorium; the two daughter-cells then separate, and in so doing free themselves from the hypha.
Hedlund followed the process of association between the two organisms in the lichens _Micarea_ (_Biatorina_) _prasina_ and _M. denigrata_ (_Biatorina synothea_), crustaceous species which inhabit trunks of trees or palings. In these the alga, one of the Chlorophyceae, has assumed the character of a _Gloeocapsa_ but on cultivation it was found to belong to the genus _Gloeocystis_. The cells are globose and rather small; they increase by the division of the contents into two or at most four portions which become rounded off and covered with a membrane before they become free from the mother-cell. The lichen hypha, on contact with any one of the green cells, bores through the outer membrane and swells within to a haustorium, as in the gonidia of _Synalissa_.
Penetrating haustoria were demonstrated by Peirce[213] in his study of the gonidia of _Ramalina reticulata_. In the first stage the tip of a hypha had pierced the outer wall of the alga, causing the protoplasm to contract away from the point of contact (Fig. 11). More advanced stages showed the extension of the haustorium into the centre of the cell, and, finally, the complete disappearance of the contents. In many cases it was found that penetration equally with clasping of the alga by the filament sets up an irritation which induces cell-division, and the alga, as in _Synalissa_, thus becomes free from the fungus. Hue[214] has recorded instances of penetration in an Antarctic species, _Physcia puncticulata_. It is easy, he says, to see the tips of the hyphae pierce the sheath of the gonidium and penetrate to the nucleus.
Lindau[215] has described the association between fungus and alga in _Pertusaria_ and other crustaceous forms as one of contact only (Fig. 12). He found that the cell-membrane of the two adhering organisms was unbroken. Occasionally the algal cell showed a slight indentation, but was otherwise unchanged. The hyphal branch was somewhat swollen at the tip where it touched the alga, and the wall was slightly thinner. The attachment between the two cells was so close, however, that pressure on the cover-glass failed to separate them.
Generally the hypha simply surrounds the gonidium with clasping branches. Many algae also lie free in the gonidial zone, and Peirce[216] claims that these are larger, more deeply coloured and in every way healthier looking than those in the grasp of the fungus. He ignores, however, the case of the soredial algae which though very closely invested by the fungus are yet entirely healthy, since on their future increase depends in many cases the reproduction of new individual lichens.
In a recent study of a crustaceous sandstone lichen, “_Caloplaca pyracea_,” Claassen[217] has sought to prove a case of pure parasitism. The rock was at first covered with the green cells of _Cystococcus_ sp. Later there appeared greyish-white patches on the green, representing the invasion of the lichen fungus. These patches increased centrifugally, leaving in time a bare patch in the centre of growth which was again colonized by the green alga. The lichen fruited abundantly, but wherever it encroached the green cells were more or less destroyed. The true explanation seems to be that the green cells were absorbed into the lichen thallus, though enough of them persisted to start new colonies on any bare piece of the stone. In the same way large patches of _Trentepohlia aurea_ have been observed to be gradually invaded by the dark coloured hyphae of _Coenogonium ebeneum_. In time the whole of the alga is absorbed and nothing is to be seen but the dark felted lichen. The free alga as such disappears, but it is hardly correct to describe the process as one of destruction.
This algal genus _Trentepohlia_ (_Chroolepus_) forms the gonidia of the Graphideae, Roccelleae, etc. It is a filamentous aerial alga which increases by apical growth. In the Graphideae, many of which grow on trees beneath the outer bark (hypophloeodal), the association between the two symbionts may be of the simplest character, but was considered by Frank[218] to be of an advanced type. According to his observations and to those of Lindau[219], the fungal hyphae penetrate first between the cells of the periderm. The alga, frequently _Trentepohlia umbrina_, tends to grow down into any cracks of the surface. It goes more deeply in when preceded by the hyphae. In some species both organisms maintain their independent growth, though each shows increased vigour when it comes into contact with the other. In some instances the cells of the alga are clasped by the fungus which causes the disintegration of the filament. The cells lose their bright yellow or reddish colour and are changed in appearance to greenish lichen gonidia; but no penetration by haustoria has ever been observed in _Trentepohlia_.
Bachmann’s[220] study of a similar gonidium in a calcicolous species of _Opegrapha_ confirms Frank’s results. The algae had pierced not only between the looser lime granules but also through a crystal of calcium carbonate, and occupied nests scooped out in the rock by means of acid formed and excreted by their filaments. When association took place with the fungus, the algal cells were more restricted to a gonidial zone; but some of the cells, having been pushed aside by the hyphae, had started new centres of gonidia. On contact with the hyphae there was a tendency to bud out in a yeast-like growth.
In the thallus of the Roccelleae, the algal filament, also a _Trentepohlia_, is broken up into separate cells, but in the Coenogoniaceae, whether the gonidium be a _Cladophora_ as in _Racodium_, or a _Trentepohlia_ as in _Coenogonium_, the filaments remain intact and are invested more or less closely by the hyphae.
A somewhat different type of association takes place between alga and fungus in _Strigula complanata_, an epiphyllous lichen more or less common in tropical regions. Cunningham[221], who found it near Calcutta, described the algal constituent and placed it in a new genus, _Mycoidea_ (_Cephaleuros_). It forms small plate-like expansions on the surface of the leaf, and also penetrates below the cuticle, burrowing between that and the epidermal cells; occasionally, as observed by Cunningham, rhizoid-like growths pierce deeper into the tissue—into and below the epidermal layer. Very frequently, in the wet season, a fungus takes possession of the alga and slender colourless hyphae creep along its surface by the side of the cell rows, sending out branches which grow downwards. Marshall Ward[222] described the same lichen from Ceylon. He states that the alga may be attacked at any stage, and if it is in a very young condition it is killed by the fungus; at a more advanced period of growth it continues to develop as an integral part of the lichen thallus, but with more frequently divided and smaller cells. Vaughan Jennings[223] observed _Strigula complanata_ in New Zealand associated with a closely allied chroolepoid alga _Phycopeltis expansa_. He also noted the growth of the fungus over the alga breaking up the plates of tissue and separating the cells which, from yellow, change to a green colour and become rounded off (Fig. 13). The mature lichen, a white thallus dotted with black fruits, contrasts strikingly with the yellow membranous alga. Lichen formation usually begins near the edge of the leaf and the margin of the thallus itself is marked by a green zone showing where the fungus has recently come into contact with the alga.
More recently Hans Fitting[224] has described “_Mycoidea parasitica_” as it occurs on evergreen leaves in Java. The alga, a species of _Cephaleuros_, though at first an epiphyte, becomes partially parasitic at maturity. It penetrates below the cuticle to the outer epidermal cells and may even reach the tissue below. When it is joined by the lichen fungus, both constituents grow together to form the lichen. Fitting adds that the leaf is evidently but little injured. In this lichen the alga in the grip of the fungus loses its independence and may be killed off: it is an instance of something like intermittent parasitism.
J. RECENT VIEWS ON SYMBIOSIS AND PARASITISM
No hyphal penetration of the bright-green algal cell by means of haustoria had been observed by the earlier workers, Bornet[225], Bonnier[226] and others, though they followed Schwendener[227] in regarding the relationship as one of host and parasite. Lindau, also, after long study accepted parasitism as the only adequate explanation of the associated growth, though he never found the fungus actually preying on the alga.
In recent years interest in the subject has been revived by the researches of Elenkin[228], a Russian botanist who claims to have established a case for parasitism or rather “endosaprophytism.” He has demonstrated by means of staining reagents the presence in the thallus of large numbers of dead algal cells. A few empty membranes are to be found in the cortex and in the gonidial zone, but the larger proportion occur below the gonidial zone and partly in the medulla. He describes the lower layer as a “necral” or “hyponecral” zone, and he considers that the hyphae draw their nourishment chiefly from dead algal material. The fungus must therefore be regarded in this case as a saprophyte rather than a parasite. The algae, he considers, may have perished from want of sufficient light and air or they may have been destroyed by an enzyme produced by the fungus. The latter he thinks is the more probable, as dead cells are frequently present among the living algae of the gonidial zone. To the action of the enzyme he also attributes the angular deformed appearance of many gonidia and the paler colour and gradual disintegration of their contents which are finally used up as endosaprophytic nourishment by the fungus. Dead algal cells were more easily seen, he tells us, in crustaceous lichens associated with “_Pleurococcus_” or “_Cystococcus_”; they were much less frequent in the larger foliose or fruticose lichens. Dead cells of _Trentepohlia_ were also difficult to find.
In a second paper Elenkin records one clear instance of a haustorium entering an algal cell, and says he found some evidence of hyphal branches penetrating otherwise uninjured gonidia, round holes being visible in their outer wall, but he holds that it is the cell-wall of the alga that is mainly dissolved by the ferment and then used as food by the hyphae.
No allowance has been made by Elenkin for the normal wasting common to all organic beings: the lichen fungus is continually being renewed, especially in the cortical structures, and the alga must also be subject to change. He[229] claims, nevertheless, that his observations have proved that the one symbiont is always preying on the other, either as a parasite or as a saprophyte. He has likened the conception of symbiosis to that of a balance between two organisms, “a moveable equilibrium of the symbionts.” If, he says, we could conceive a state where the conditions of life would be equally favourable for both partners there would be true mutualism, but in practice one only is favoured and gains the upper hand, using its advantage to prey on the other. Unless the balance is redressed, the complete destruction of the weaker is certain, and is followed in time by the death of the stronger. The fungus being the dominant partner, the balance, he considers, is tipped in its favour.
Elenkin’s conclusions are not borne out by the long continued and healthy life of the lichen. There is no record of either symbiont having succumbed to the other, and the alga, when set free, is unchanged and able to resume its normal development. Without the alga the fungus cannot form the ascigerous fruit. Is that because as a parasite within the lichen it has degenerated past recovery, or has it become so adapted to symbiosis that in saprophytic conditions it fails to develop?
Another Russian lichenologist, U. N. Danilov[230], records results which would seem to support the theory of parasitism. He found that from the clasping hyphae minute haustoria were produced, which penetrate the algal cell-wall, and branch when within the outer membrane, thus forming a delicate network over the plasma; secondary haustoria arising from this network protrude into the interior and rob the cell-contents. He observed gonidia filled with well-developed hyphae and these, after having exhausted one cell, travel onwards to others. Some gonidia under the influence of the fungus had become deformed and were finally killed. As a proof of this latter statement he adduces the presence in the thallus of some gonidia containing shrivelled protoplasm, of others entirely empty. He considers, as further evidence in favour of parasitism, the finding of empty membranes as well as of colourless gonidia filled with the hyphal network. This description hardly tallies with the usual healthy appearance of the gonidial zone in the normal thallus, and it has been suggested that where the fungus filled the algal cell, it was as a saprophyte preying on dead material.
The gradual perishing of algal cells in time by natural decay and their subsequent absorption by the fungus is undisputed. It is open to question whether the varying results recorded by these workers have any further significance.
These observations of Elenkin and Danilov have been proved to be erroneous by Paulson and Somerville Hastings[231]. They examined the thalli of several lichens (_Xanthoria parietina_, _Cladonia_ sp., etc.) collected in early spring when vegetative growth in these plants was found to be at its highest activity. They found an abundant increase of gonidia within the thallus, which they regarded as sporulation of the algae, and the most careful methods of staining failed to reveal any case of penetration of the gonidia by the hyphae.
Nienburg[232] has published some recent observations on the association of the symbionts. In the wide cortex of a _Pertusaria_ he found not only the densely compact hyphae, but also isolated gonidia. In front of these latter there was a small hollow cavity and, behind, parallel hyphae rich in contents. These gonidia had originated from the normal gonidial zone. They were moved upward by special hyphae called by Nienburg “push-hyphae.” After their transportation, the algae at once divide and the products of division pass to a resting stage and become the centre of a new thalline growth. A somewhat similar process was noted towards the apex of _Evernia furfuracea_. Radial hyphae pushed up the cortex, leaving a hollow space over the gonidial zone. Into the space isolated algae were thrust by “push-hyphae.” In this lichen he also observed the penetration of the algal cell by haustoria of the fungus. He considers that the alga reaps advantage but also suffers harm, and he proposes the term _helotism_ to express the relationship.
An instructive case of the true parasitism of a fungus on an alga has been described by Zukal[233] in the case of _Endomyces scytonemata_ which he calls a “half-lichen.” The mature fungus formed small swellings on the filaments of the _Scytonema_ and, when examined, the hyphae were seen to have attacked the alga, penetrating the outer gelatinous sheath and then using up the contents of the green cells. It is only after the alga has been destroyed and absorbed, that asci are formed by the fungus. Zukal contrasts the development of this fungus with the symbiotic growth of the two constituents in _Ephebe_ where both grow together for an indefinite time.
Mere associated growth however even between a fungus and an alga does not constitute a lichen. An instance of such growth is described by Sutherland[234] in an account of marine microfungi. One of these, a species of _Mycosphaerella_, was found on _Pelvetia canaliculata_, and Sutherland claims that as no apparent injury was done to the alga, it was a case of symbiosis and that there was formed a new type of lichen. The mycelium, always intercellular, pervaded the whole host-plant, and the fungal fruits were invariably formed on the algal receptacles close to the oogonia. Their position there is, of course, due to the greater food supply at that region. Both fungus and alga fruited freely. A closer analogy could have been found by the writer in the smut fungus which grows with the host-cereal until fruiting time; or with the mycorrhiza of _Calluna_ which also pervades every part of the host-plant without causing any injury. In the true lichen, the alga, though constituting an important part of the vegetative body, takes no part in reproduction, except by division and increase of the vegetative cells within the thallus. The fruiting bodies are always of a modified fungal nature.
2. PHYSIOLOGY OF THE SYMBIONTS
The occurrence of isolated cases of parasitism—the fungus preying on the alga—in any case leaves the general problem unsolved. The whole question turns on the physiological activity and requirements of the two component elements of the thallus. From what sources do they each procure the materials essential to them as living organisms? It is chiefly a question of nutrition.
A. NUTRITION OF ALGAE
_a._ CHARACTER OF ALGAL CELLS. Gonidia are chlorophyll-containing bodies and assimilate carbon-dioxide from the atmosphere by photosynthesis as do the chlorophyll cells of other plants. They also require water and mineral salts which, in a free condition, they absorb from their immediate surroundings, but which, in the lichen thallus, they must obtain from the fungal hyphae. If the nutriment supplied to them in their inclosed position be greater or even equal to what the cells could procure as free-living algae, then they undoubtedly gain rather than lose by their association with the fungus, and are not to be considered merely as victims of parasitism.
_b._ SUPPLY OF NITROGEN. Important contributions on the subject of algal nutrition have been made by Beyerinck[235] and Artari[236]. The former conducted a series of culture experiments with green algae, including the gonidia of _Physcia_ (_Xanthoria_) _parietina_. He successfully isolated the lichen gonidia and, at first, attempted to grow them on gelatine with an infusion of the Elm bark from which he had taken the lichen. Growth was very slow and very feeble until he added to the culture-medium a solution of malt-extract which contains peptones and sugar. Very soon he obtained an active development of the gonidia, and they multiplied rapidly by division[237] as in the lichen thallus. This proved to him conclusively the great advantage to the algae of an abundant supply of nitrogen.
Artari in his work has demonstrated that there are two different physiological races of green algae: (1) those that absorb peptones—which he designates peptone-algae—and (2) those that do not so absorb peptones. He tested the cells of _Cystococcus humicola_ taken from the thallus of _Physcia parietina_, and found that they belonged to the peptone group and were therefore dependent on a sufficiency of nitrogenous material to attain their normal vigorous growth. It was also discovered by Artari that the one race can be made by cultivation to pass over to the other: that ordinary algae can be educated to live on peptones, and peptone-algae to do without.
We learn further from Beyerinck’s researches that Ascomycetes, the group of fungi from which the hyphae of most lichens are derived, are what he terms ammonia-sugar fungi; that is to say, the hyphae can abstract nitrogen from ammonia salts and, with the addition of sugar, can form peptones. The lichen peptone-algae are thus evidently, by their contact with such fungi, in a favourable position for securing the nitrogenous food supply most suited to their requirements. In their deep-seated layers, they are to a large extent deprived of light, but it has been proved by Artari[238] in a series of culture experiments extending over a long period, that the gonidia of _Xanthoria parietina_ remain green in the dark under very varied conditions of nutriment, though the colour is distinctly fainter.
Recently Treboux[239] has revised the work done by Artari and Beyerinck in reference to _Cystococcus humicola_. He denies that two physiological races are represented in this alga, the lichen gonidia, in regard to the nitrogen that they absorb, behaving exactly as do the free-living forms of the species. He finds that the gonidium is not a peptone-carbohydrate organism in the sense that it requires nitrogen in the form of peptones, inorganic ammonia salts being a more acceptable food supply. Treboux concludes that his results favour the view that the gonidia are in an unfavourable situation for receiving the kind of nitrogenous compound most advantageous to them, that they are therefore in a sense “victims” of parasitism, though he qualifies the condition as being a lichen-parasitism or helotism. This view does not accord with Chodat’s[240] results: in his cultures of gonidia he observed that with glycocoll or peptone, which are nearly equivalent, they developed four times better than with potassium nitrate as their nitrogenous food, and he concluded that they assimilated nitrogen better from bodies allied to peptides.
_c._ EFFECT OF SYMBIOSIS ON THE ALGA. Treboux’s observations however convinced him that the alga leads but a meagre existence within the thallus. Cell-division—the expression of active vitality—was, he held, of rare occurrence in the slowly growing lichen-plant, and zoospore formation in entire abeyance. He contrasts this sluggish increase[241] with the rapid multiplication of the free-living algal cells which cover whole tree-trunks with their descendants in a comparatively short time. These latter cells, he finds, are indeed rather smaller, being generally the products of recent division, but mixed with them are numbers of larger resting cells, comparable in size with the lichen gonidia. He states further, that the gonidia are less brightly green and, as he judges, less healthy, though in soredial formation or in the open they at once regain both colour and power of division. Treboux had entirely failed to observe the sporulation which is so abundant at certain seasons.
Their quick recovery seems also a strong argument in favour of the absolutely normal condition of metabolism within the gonidial cell; and the paler appearance of the chlorophyll is doubtless associated with the acquisition of carbohydrates from other sources than by photosynthesis. There is a wide difference between any degree of unfavourable life-conditions and parasitism however slight, even though the balance of gain is on the side of the fungus. It is not too fanciful to conclude that the demand for nitrogen on the part of the alga has influenced its peculiar association with the fungus. In the thallus of hypophloeodal lichens it has been proved indeed that the alga _Trentepohlia_ with apical growth is an active agent in the symbiotic union. _Cystococcus_ and other green algal cells are stationary, but they are doubtless equally ready for—as many of them are equally benefited by—the association. Keeble[242] has pointed out in the case of _Convoluta roscoffensis_ that nitrogen-hunger induces the green algae to combine forces with an animal organism, though the benefit to them is only temporary and though they are finally sacrificed. The lichen gonidia, on the contrary, persist for a long time, probably far beyond their normal period of existence as free algae.
Examples of algal association with other plants might be cited here: of _Nostoc_ in the roots of _Cycas_ and in the cells of _Anthoceros_, and of _Anabæna_ in the leaf-cells of _Azolla_, but in these instances it is generally held that the alga secures only shelter. It was by comparing the lichen-association with the harmless invasion of _Gunnera_ cells by _Nostoc_ that Reinke[243] arrived at his conception of “consortism.”
_d._ SUPPLY OF CARBON. Carbon, the essential constituent of all organic life, is partly drawn from the carbon-dioxide of the air, and assimilated by the green cells; it is also partly contributed by the fungus as a product of its metabolism. A proof of this is afforded by Dufrenoy[244]: he found a _Parmelia_ growing closely round pine needles and even sending suckers into the stomata. He covered the lichen with a black cloth and after seven weeks found that the gonidia had remained very green. That growth had not been checked was evidenced by an unusual development of soredia and of spermogonia. Dufrenoy describes the condition as a parasitism of the algae on the fungus which in turn was drawing nourishment from the pine needles.
Artari[245] has proved that lichen gonidia can obtain carbohydrates from the substratum as well as by photosynthesis. He cultivated the gonidia of _Xanthoria parietina_ and _Placodium murorum_ on media which contained organic substances as well as mineral salts, while depriving them of atmospheric carbon-dioxide and in some cases of light also. The gonidia not only grew well but, even in the dark, they remained normally green, a phenomenon coinciding with Etard and Bouilhac’s[246] experience in growing _Nostoc_ in the dark: with suitable culture media the alga retained its colour. _Nostoc_ also grows in the dark in the rhizome of _Gunnera_. Radais’[247] experiments with _Chlorella vulgaris_ confirmed these results. On certain organic media growth and cell-division were as rapid in the dark as in the light, and chlorophyll was formed. The colour was at first yellowish and the full green arrived slowly, especially on sugar media, but in ten days it was uniform and normal.
When making further experiments with the alga, _Stichococcus bacillaris_, Artari[248] found that it also grew well on an organic medium and that grape sugar was the most valuable carbonaceous food supply. Chodat[249] also found that sugar or glucose was a desirable ingredient of culture media.
Treboux[250], in his work on organic acids, has also proved by experimental cultures with a large series of algae, including the gonidia of _Peltigera_, that these green plants in the absence of light and in pure cultures would grow and form carbohydrates if the culture medium contained a small percentage of organic acids. The acids he employed were combined with potassium and were thus rendered neutral or slightly alkaline; acetate of potash proved to be the most advantageous compound of any that was tested. Amino-acids and ammonia salts were added to provide the necessary nitrogen. Oxalic acid and other organic acids of varying composition are peculiarly abundant in lichen tissues and may be a source of carbon supply. Marshall Ward[251] has found calcium carbonate crystals in the lower air-containing tissues of _Strigula complanata_.
Treboux finally concluded from his researches that just as fungi can extract carbohydrates from many sources, so algae can secure their carbon supply in a variety of ways. He affirms that the metabolic activity of the alga in these cultural conditions is entirely normal, and the various cell-contents are formed as in the light. Whether, in this case, starch is formed directly from the acids or through a series of combinations has not been determined. Uhlir[252], with electric lighting, made successful cultures of _Nostoc_ isolated from Collemaceae on silicic acid, proving thereby that these gonidia do not require a rich nutriment. A certain definite humidity was however essential, and bacteria were never eliminated as they are associated with the gelatinous membranes of Nostocaceae.
_e._ NUTRITION WITHIN THE SYMBIOTIC PLANT. Culture experiments bearing more directly on the nutrition of lichens as a whole were carried out by F. Tobler[253]. He proved that the gonidia had undoubtedly drawn on the calcium oxalate secreted by the hyphae for their supply of carbon. In a culture medium of poplar-bark gelatine he grew hyphae of _Xanthoria parietina_, and noted an abundant deposit of oxalate crystals on their cell-walls. A piece of the lichen thallus including both symbionts and grown on a similar medium formed no crystals, and microscopic examination showed that crystals were likewise absent from the hyphae of the thallus that had grown normally on the tree, the inference being that the gonidia used them up as quickly as they were deposited. It must be remembered in this connection, however, that Zopf[254] has stated that where lichen acids are freely formed as, for instance, in _Xanthoria parietina_, there is always less formation and deposit of calcium oxalate crystals, which may partly account for their absence in the normal thallus so rich in parietin.
Tobler next introduced lichen gonidia into a culture medium in which the isolated hyphal constituent of a thallus had been previously cultivated, and placed the culture in the dark. In these circumstances he found that the gonidia were able to thrive but formed no colour: they were obtaining their carbohydrates, he decided, not from photosynthesis, but from the excretory products such as calcium oxalate that had been deposited in the culture medium by the lichen hyphae. We may conclude with more or less certainty that the loss of carbohydrates, due to the partial deprivation of light and air suffered by the alga owing to its position in the lichen thallus, is more than compensated by a physiological symbiosis with the fungus[255]. It has indeed been proved that in the absence of free carbon-dioxide, algae may utilize the half-bound CO₂ of carbonates, chiefly those of calcium and magnesium, dissolved in water.
_f._ AFFINITIES OF LICHEN GONIDIA. Chodat[256] has, in recent years, made cultures of lichen gonidia with a view to discovering their relation to free-living algae and to testing at the same time their source of carbon supply. He has come to the conclusion that lichen gonidia are probably in no instance the normal _Protococcus viridis_: they differ from that alga in the possession of a pyrenoid and in their reproduction by zoospores when free.
Careful cultures were made of different _Cladonia_ gonidia which were morphologically indistinguishable, and which varied in size from 10 to 16µ in diameter, though smaller ones were always present. He recognized them to be species of _Cystococcus_: they have a pyrenoid[257] in the centre and a disc-like chromatophore more or less starred at the edge. These gonidia grew well on agar, still better on agar-glucose, but best of all with an addition of peptone to the culture. There was invariably at first a slight difference in form and colour in the mass between the gonidia of one species and those of another, but as growth continued they became alike.
In testing for carbon supply, he found that gonidia grew slowly without sugar (glucose), and that, as sources of carbon, organic acids could not entirely replace glucose though, in the dark, the gonidia used them to some extent; the colony supplied with potassium nitrate, and grown in the dark, had reached a diameter of only 2 mm. in three months. With glucose, it measured 5 mm. in three weeks, while in three months it formed large culture patches.
A further experiment was made to test their absorption of peptones by artificial cultures carried out both in the light and the dark. The gonidia grew poorly in all combinations of organic nitrogen compounds. When combined with glucose, growth was at once more vigorous though only half as much in the dark as in the light, the difference in this respect being especially noticeable in the gonidia from _Cladonia pyxidata_. He concludes that as gonidia in these cultures are saprophytic, so in the lichen thallus also they are probably more or less saprophytic, obtaining not only their nitrogen in organic form but also, when possible, their carbon material as glucose or galactose from the hyphal symbiont which in turn is saprophytic on humus, etc.
B. NUTRITION OF FUNGI
Fungi being without chlorophyll are always indebted to other organisms for their supply of carbohydrates. There has never therefore been any question as to the advantage accruing to the hyphal constituent in the composite thallus. The gonidia, as various workers have proved, have also a marked preference for organized nourishment, and, in addition, they obtain carbon by photosynthesis. Chodat[258] considers that probably they are thus able to assimilate carbon-dioxide in excess, a distinct advantage to the hyphae. In some instances the living gonidium is invaded and the contents used up by the fungus and any dead gonidia are likewise utilized for food supply. It is also taken for granted that the fungus takes advantage of the presence of humus whether in the substratum or in aerial dust. In such slow growing organisms, there is not any large demand for nourishment on the part of the hyphae: for many lichens it seems to be mere subsistence with a minimum of growth from year to year.
C. SYMBIOSIS OF OTHER PLANTS
The conception of an advantageous symbiosis of fungi with other plants has become familiar to us in Orchids and in the mycorhizal formation on the roots of trees, shrubs, etc. Fungal hyphae are also frequent inhabitants of the rhizoids of hepatics though, according to Gargeaune[259], the benefit to the hepatic host-plant is doubtful.
An association of fungus and green plant of great interest and bearing directly on the question of mutual advantage has been described by Servettaz[260]. In his study of mosses, he was able to confirm Bonnier’s[261] account of lichen hyphae growing over such plants as _Vaucheria_ and the protonema of mosses, which is undoubtedly hurtful; but he also found an association of a moss with one of the lower fungi, _Streptothrix_ or _Oospora_, which was distinctly advantageous. In separate cultivation the fungus developed compact masses and grew well in peptone agar broth.
Cultures of the moss, _Phascum cuspidatum_, were also made from the spores on a glucose medium. The specimens in association with the fungus were fully grown in two months, while the control cultures, without any admixture of the fungus, had not developed beyond the protonema stage. Servettaz draws attention to the proved fact that, in certain instances, plants benefit when provided with substances similar to their own decay products, and he considers that the fungus, in addition to its normal gaseous products, has elaborated such substances, as acid products, from the glucose medium to the great advantage of the moss plant.
A symbiotic association of _Nostoc_ with another alga, described by Wettstein[262], is also of interest. The blue-green cells were lodged in the pyriform outgrowths of the siphoneous alga, _Botrydium pyriforme_ Kütz., which the author of the paper places in a new genus, _Geosiphon_. The sheltering _Nostoc symbioticum_ fills all of the host left vacant by the plasma, and when the season of decay sets in, it forms resting spores which migrate into the rhizoids of the host, so that both plants regenerate together.
Wettstein has compared this symbiotic association with that of lichens, and finds the analogy all the more striking in that the membrane of his new alga had become chitinous, which he thinks may be due to organic nutrition.
II. LICHEN HYPHAE
A. ORIGIN OF HYPHAE
Lichen hyphae form the ground tissue of the thallus apart from the gonidia or algal cells. They are septate branched filaments of single cell rows and are colourless or may be tinged by pigments or lichen acids to some shade of yellow, brown or black. They are of fungal nature, and are produced by the mature lichen spore.
The germination of the spore was probably first observed by Meyer[263]. His account of the actual process is somewhat vague, and he misinterpreted the subsequent development into thallus and fruit entirely for want of the necessary magnification; but that he did succeed in germinating the spores is unquestionable. He cultivated them on a smooth surface and they grew into a “dendritic formation”—a true hypothallus. Many years later the development of hyphae from lichen spores was observed by Holle[264] who saw and figured the process unmistakably in _Borrera_ (_Physcia_) _ciliaris_.
A series of spore cultures was undertaken by Tulasne[265] with the twofold object of discovering the exact origin of hyphae and gonidia and of their relationship to each other. The results of his classical experiment with the spores of _Verrucaria muralis_—as interpreted by him—were accepted by the lichenologists of that time as conclusive evidence of the genetic origin of the gonidia within the thallus.
The spores of the lichen in large numbers had been sown by Tulasne in early spring on the smooth polished surface of a piece of limestone, and were covered with a watch-glass to protect them from dust, etc. At irregular intervals they were moistened with water, and from time to time a few spores were abstracted from the culture and examined microscopically. Tulasne observed that the spore did not increase or change in volume in the process of germination, but that gradually the contents passed out into the growing hyphae, till finally a thin membrane only was left and still persisted after two months (Fig. 14). For a considerable time there was no septation; at length cross-divisions were formed, at first close to the spore, and then later in the branches. The hyphae meanwhile increased in dimension, the cells becoming rounder and somewhat wider, though always more slender than the spore which had given rise to them. In time a felted tissue was formed with here and there certain cells, filled with green colouring matter, similar to the gonidia of the lichen and thus the early stages at least of a new thallus were observed. The green cells, we now know, must have gained entrance to the culture from the air, or they may have been introduced with the water.
B. DEVELOPMENT OF LICHENOID HYPHAE
Lichen hyphae are usually thick-walled, thus differing from those of fungi generally, in which the membranes, as a rule, remain comparatively thin. This character was adduced by the so-called “autonomous” school as a proof of the fundamental distinction between the hyphal elements of the two groups of plants. It can, however, easily be observed that, in the early stages of germination, the lichen hyphae, as they issue from the spore, are thin-walled and exactly comparable with those of fungi. Growth is apical, and septation and branching arise exactly as in fungi, and, in certain circumstances, anastomosis takes place between converging filaments. But if algae are present in the culture the peculiar lichen characteristics very soon appear.
Bonnier[266], who made a large series of synthetic cultures, distinguishes three types of growth in lichenoid hyphae (Fig. 15):
1. Clasping filaments, repeatedly branched, which attach and surround the algae.
2. Filaments with rather short swollen cells which ultimately form the hyphal tissues of cortex and medulla.
3. Searching filaments which elongate towards the periphery and go to the encounter of new algae.
In five days after germination of the spores, the clasping hyphae had laid hold of the algae which meanwhile had increased by division; the swollen cells had begun to branch out and ten days later a differentiation of tissue was already apparent. The searching filaments had increased in number and length, and anastomosis between them had taken place when no further algae were encountered. The cell-walls of the swollen hyphae and their branches had begun to thicken and to become united to form a kind of cellular tissue or “paraplectenchyma[267].” At a later date, about a month after the sowing of the spores, there was a definite cellular cortex formed over the thallus. The hyphal cells are uninucleate, though in the medulla they may be 1-2-nucleate.
The hyphae in close contact with the gonidia remain thin-walled, and have been termed by Wainio[268] “meristematic.” They furnish the growing elements of the lichen either apical or intercalary. In most genera the organs of fructification take rise from them, or in their immediate neighbourhood, and isidia and soredia also originate from these gonidial hyphae.
As the filaments pass from the gonidial zone to other layers, the cell-walls become thicker with a consequent reduction of the cell-lumen, very noticeable in the pith, but carried to its furthest extent in the “decomposed” cortex where the cells in the degenerate tissue often become reduced to disconnected streaks indicating the cell-lumen, and the outer cortical layer is merely a continuous mass of mucilage.
All lichen tissues arise from the branching and septation of the hyphae, the septa always forming at right angles to the long axis of the filaments. There is no instance of longitudinal cell-division except in the spores of certain genera (_Collema_, _Urceolaria_, _Polyblastia_, etc.). The branching of the hypha is dichotomous or lateral, and very irregular. Frequent septation and coherent growth result in the formation of plectenchyma.
C. CULTURE OF HYPHAE WITHOUT GONIDIA
Artificial cultures had demonstrated the germination of lichen spores, with the formation of hyphae, and from synthetic cultures of fungus and alga complete lichen plants had been produced. To Möller[269] we owe the first cultures of a thalline body from the fungus alone, both from spermatia and from ascospores. The germination of the spermatia has a direct bearing on their function as spores or as sexual organs and is described in a later chapter.
The ascospores of _Lecanora subfusca_ were caught in a drop of water on a slide as they were ejaculated from the ascus, and, on the following day, a very fine germinating tube was seen to have pierced the exospore. The hypha became slightly thicker, and branching began on the third day. If in water alone the culture soon died off, but in a nutrient solution growth slowly continued. The hyphae branched out in all directions from the spore as a centre and formed an orbicular expansion which in fourteen days had reached a size of ·1 mm. in diameter. After three weeks’ growth it was large enough to be visible without a lens; the mycelial threads were more crowded, and certain terminal hyphae had branched upwards in an aerial tuft, this development taking place from the centre outwards. Möller marked this stage as the transition from a mere protothallus to a thallus formation. In three months a diameter of 1·5-2 mm. was reached; a transverse section gave a thickness of ·86 mm. and from the under side loose hyphae branched downwards and attached the thallus, when it had been transferred to a solid substratum such as cork. Above these rhizoidal hyphae, a stratum of rather loose mycelium represented the medulla, and, surmounting that, a cortical layer in which the hyphae were very closely compacted. Delicate terminal branches rose into the air over the whole surface, very similar in character to hypothallic hyphae at the margin of the thallus.
_Lecanora subfusca_ has a rather small simple spore; it emitted germinating tubes from each end, and a septum across the middle of the spore appeared after germination had taken place. Another experiment was with a much larger muriform spore measuring 80 µ in length and 20 µ in thickness. On germination about 20 tubes were formed, some of them rising into the air at once, the others encircling the spore, so that the thallus took form immediately; growth in this case also was centrifugal. In three months a diameter of 6 mm. was reached with a thickness of 1 to 2 mm. and showing a differentiation into medulla and cortex. The hyphae did not increase in width, but frequently globose or ovate swellings arose in or at the ends, a character which recurs in the natural growth of hyphae both of lichens and of Ascomycetes. These swellings depend on the nutrition.
_Pertusaria communis_ possesses a very large simple spore, but it is multinucleate and germinates with about 100 tubes which reach their ultimate width of 3 to 4 µ before they emerge from the exospore. The hyphae encircle the spore, and an opaque thalline growth is quickly formed from which rise terminal hyphal branches. In ten weeks the differentiation into medulla and cortex was reached, and in five months the hyphal thallus measured 4 mm. in diameter and 1 to 2 mm. in thickness.
Möller instituted a comparison between the thalli he obtained from the spores and those from the spermatia of another crustaceous lichen, _Buellia punctiformis_ (_B. myriocarpa_). After germination had taken place the hyphae from the spermatia grew at first more quickly than those from the ascospores, but as soon as thallus formation began the latter caught up and, in eight weeks, both thalli were of equal size.
Another comparative culture with the spermatia and ascospores of _Opegrapha subsiderella_ gave similar results: the spores of that species are elongate-fusiform and 6-to 8-septate; germination took place from the end cells in two to three days after sowing. The germinating hyphae corresponded exactly with those from the spermatia and growth was equally slow in both. The middle cells of the spores may also produce germinating tubes, but never more than about five were observed from any one spore. A browning of the cortical layer was especially apparent in the hyphal culture from another lichen, _Graphis scripta_: a clear brown colour gradually changing to black appeared about the same period in all the cultures.
The hyphae from the spores of _Arthonia_ developed quickest of all: the hyphae were very slender, but in three to four months the growth had reached a diameter of 8 mm. In this plant there was the usual outgrowth of delicate hyphae from the surface; no definite cortical layer appeared, but only a very narrow line of more closely interwoven somewhat darker hyphae. Frequently, from the surface of the original thallus, excrescences arose which were the beginnings of further thalli.
Tobler[270] experimenting with _Xanthoria parietina_ gained very similar results. The spores were grown in malt extract for ten days, then transferred to gelatine. In three to five weeks there was formed an orbicular mycelial felt about 3 mm. in diameter and 2 mm. thick. The mycelium was frequently brownish even in healthy cultures, but the aerial hyphae which, at first, rose above the surface were always colourless. After these latter disappeared a distinct brownish tinge of the thallus was visible. In seven months it had increased in size to 15 mm. in length, 7 mm. in width and 3 mm. thick with a differentiation into three layers: a lower rather dense tissue representing the pith, above that a layer of loose hyphae where the gonidial zone would normally find place, and above that a second compact tissue, or outer cortex, from which arose the aerial hyphae. The culture could not be prolonged more than eight months.
D. CONTINUITY OF PROTOPLASM IN HYPHAL CELLS
Wahrlich[271] demonstrated that continuity of protoplasm was as constant between the cells of fungi as it has been proved to be between the cells of the higher plants. His researches included the hyphae of the lichens, _Cladonia fimbriata_ and _Physcia_ (_Xanthoria_) _parietina_.
Baur[272] and Darbishire[273] found independently that an open connection existed between the cells of the carpogonial structures in the lichens they examined. The subject as regards the thalline hyphae was again taken up by Kienitz-Gerloff[274] who obtained his best results in the hypothecial tissue of _Peltigera canina_ and _P. polydactyla_. Most of the cross septa showed one central protoplasmic strand traversing the wall from cell to cell, but in some instances there were as many as four to six pits in the walls. The thickening of the cell-walls is uneven and projects variously into the cavity of the cell. Meyer’s[275] work was equally conclusive: all the cells of an individual hypha, he found, are in protoplasmic connection; and in plectenchymatous tissue the side walls are frequently perforated. Cell-fusions due to anastomosis are frequent in lichen hyphae, and the wall at or near the point of fusion is also traversed by a thread of protoplasm, though such connections are regarded as adventitious. Fusions with plasma connections are numerous in the matted hairs on the upper surface of _Peltigera canina_ and they also occur between the hyphae forming the rhizoids of that lichen. The work of Salter[276] may also be noted. He claimed that his researches tended to show complete anatomical union between all the tissues of the lichen plant, not only between the hyphae of the various tissues but also between hyphae and gonidia.
III. LICHEN ALGAE
A. TYPES OF ALGAE
The algal constituents of the lichen thallus belong to the two classes, Myxophyceae, generally termed blue-green algae, and Chlorophyceae which are coloured bright-green or yellow-green. Most of them are land forms, and, in a free condition, they inhabit moist or shady situations, tree-trunks, walls, etc. They multiply by division or by sporulation within the thallus; zoospores are never formed except in open cultivation. The determination of the genera and species to which the lichen algae severally belong is often uncertain, but their distribution within the lichen kingdom is as follows:
_a._ MYXOPHYCEAE ASSOCIATED WITH PHYCOLICHENS. The blue-green algae are characterized by the colour of their pigments which persists in the gonidial condition giving various tints to the component lichens, and by the gelatinous sheath in which most of them are enclosed. This sheath, both in the lichen gonidia[277] and in free-living forms, imbibes and retains moisture to a remarkable extent and the thallus containing blue-green algae profits by its power of storing moisture. Myxophyceae form the gonidia of the gelatinous lichens as well as of some other non-gelatinous genera. Several families are represented[278]:
Fam. CHROOCOCCACEAE. This family includes unicellular algae with thick gelatinous sheaths. They increase normally by division, and colonies arise by the cohesion of the cells. Several genera form gonidia:
1. CHROOCOCCUS Naeg. Solitary or forming small colonies of 2-4-8 cells (Fig. 16) generally surrounded by firm gelatinous colourless sheaths in definite layers (lamellate). _Chroococcus_ is considered by some lichenologists to form the gonidium of _Cora_, a genus of Hymenolichens.
2. MICROCYSTIS Kütz. Globose or subglobose cells forming large colonies surrounded by a common gelatinous layer (gonidia of _Coriscium_).
3. GLOEOCAPSA Kütz. (including _Xanthocapsa_). Globose cells with a lamellate gelatinous wall, forming colonies enclosed in a common sheath (Fig. 17); the inner integument is often coloured red or orange. These two genera form the gonidia in the family Pyrenopsidaceae. _Gloeocapsa polydermatica_ Kütz. has been identified as a lichen gonidium.
Fam. NOSTOCACEAE. Filamentous algae unbranched and without base or apex.
NOSTOC Vauch. Composed of flexuous trichomes, with intercalary heterocysts (colourless cells) (Fig. 18). Dense gelatinous colonies of definite form are built up by cohesion. In some lichens the trichomes retain their chain-like appearance, in others they are more or less broken up and massed together, with disappearance of the gelatinous sheath (as in _Peltigera_); colour mostly dark blue-green.
_Nostoc_ occurs in a few or all of the genera of Pyrenidiaceae, Collemaceae, Pannariaceae, Peltigeraceae and Stictaceae, and _N. sphaericum_ Vauch. (_N. lichenoides_ Kütz.) has been determined as the lichen gonidium. When the chains are broken up it has been wrongly classified as another alga, _Polycoccus punctiformis_.
Fam. SCYTONEMACEAE. Trichomes of single-cell rows, differentiated into base and apex. Pseudo-branching arises at right angles to the main filament.
SCYTONEMA Ag. Pseudo-branches piercing the sheath and passing out as twin filaments (Fig. 19); colour, golden-brown. This alga occurs in genera of Pyrenidiaceae, Ephebaceae, Pannariaceae, Heppiaceae, in _Petractis_ a genus of Gyalectaceae, and in _Dictyonema_ one of the Hymenolichens.
Fam. STIGONEMACEAE. Trichomes of several-cell rows with base and apex; colour, golden-brown.
STIGONEMA Ag. Stouter than _Scytonema_, with transverse and vertical division of the cells, and generally copious branching (Fig. 20). This alga occurs only in a few genera of Ephebaceae. _S. panniforme_ Kirchn. (_Sirosiphon pulvinatus_ Bréb.) has been determined as forming the gonidium.
Fam. RIVULARIACEAE. Trichomes with a heterocyst at the base and tapering upwards, enclosed in mucilage (Fig. 21).
RIVULARIA Thuret. In tufts fixed at the base and forming roundish gelatinous colonies; colour, blue-green. The gonidium of Lichinaceae has been identified as _R. nitida_ Ag.
Algae belonging to one or other of these genera of Myxophyceae also combine with the hyphae of Archilichens to form cephalodia[279] and Krempelhuber[280] has recorded and figured a blue-green alga, probably _Gloeocapsa_, in _Baeomyces paeminosus_ from the South Sea Islands. They also form the gonidia in a few species and genera of such families as Stictaceae and Peltigeraceae.
_b._ CHLOROPHYCEAE ASSOCIATED WITH ARCHILICHENS. The lichens of this group are by far the most numerous both in genera and species, though fewer algal families are represented.
Fam. PROTOCOCCACEAE. Consisting of globular single cells, aggregated in loose colonies, dividing variously.
1. PROTOCOCCUS VIRIDIS Ag. (_Pleurococcus vulgaris_ Menegh., _Cystococcushumicola_ Naeg.). Cells dividing into 2, 4 or 8 daughter-cells and not separating readily; in excessive moisture forming short filaments. The cells contain parietal chloroplasts, and, according to Chodat[281], are without a pyrenoid (Fig. 22). This alga, and allied species, forms the familiar green coating of tree-trunks, walls etc., and, in lichenological literature, are quoted as the gonidia of most of the crustaceous foliose and fruticose lichens. Chodat[281], who has recently made comparative artificial cultures of algae, throws doubt on the identity of many such gonidia. He lays great emphasis on the presence or absence of a pyrenoid in algal cells. West, on the contrary, considers the pyrenoid as an inconstant character. Chodat insists that the gonidia that contain pyrenoids belong to another genus, _Cystococcus_ Chod. (_non_ Naeg.), a pyrenoid-containing alga, which, in addition to multiplying by division of the cells, also forms spores and zoospores when cultivated. He further records the results of his cultures of gonidia, and finds that those taken from closely related lichens, such as different species of _Cladonia_, though they are alike morphologically, yet show constant variations in the culture colonies. These, he holds, are sufficient to indicate difference of race if not of species and he designates the algae, according to the lichen in which they occur, as _Cystococcus Cladoniae pyxidatae_, _C. Cladoniae fimbriatae_, etc.
Meanwhile Paulson and Somerville Hastings[282] by their careful research on the growing thallus have thrown considerable light on the identity of the Protococcaceous lichen gonidium. They selected such well-known lichens as _Xanthoria parietina, Cladonia_ spp. and others, which they collected during the spring months, February to April, the period of most active growth. Many of the gonidia, they found, were in a stage of reproduction, that showed a simultaneous rounding off of the gonidium contents into globose bodies varying in number up to 32. Chodat had figured this method of “sporulation” in his cultures of the lichen gonidium both in _Chlorella_ Beij. and in _Cystococcus_ Chod. (Fig. 23). It has now been abundantly proved that this form of increase is of frequent occurrence in the thallus itself. _Chlorella_ has been suggested as probably the alga forming these gonidia and recently West has signified his acquiescence in this view[283].
2. CHLORELLA Beij. Occurring frequently on damp ground, bark of trees, etc., dividing into numerous daughter-cells, probably reduced zoogonidia (Fig. 23).
Chodat distinguishes between _Cystococcus_ and _Chlorella_ in that _Cystococcus_ may form zoospores (though rarely), _Chlorella_ only aplanospores. He found three gonidial species, _Chlorella lichina_ in _Cladonia rangiferina, Ch. viscosa_ and _Ch. Cladoniae_ in other _Cladonia_ spp.
3. COCCOBOTRYS Chod. The cells of this new algal genus are smaller than those of _Cystococcus_ or _Protococcus_ and have no pyrenoid. They were isolated by Chodat from the thallus of _Verrucaria nigrescens_ (Fig. 24), and, as they have thick membranes, they adhere in a continuous layer or thallus. Chodat also claims to have isolated a species of _Coccobotrys_ from _Dermatocarpon miniatum_, a foliose Pyrenolichen.
4. COCCOMYXA Schmidle. Cells ellipsoid, also without a pyrenoid. Two species were obtained by Chodat from the thallus of _Solorinae_ and are recorded as _Coccomyxa Solorinae croceae_ and _C. Solorinae saccatae_.
_Coccomyxa subellipsoidea_ is given[284] as the gonidium of the primitive lichen _Botrydina vulgaris_ (Fig. 25). The cells are surrounded by a common gelatinous sheath.
5. DIPLOSPHAERA Bial.[285] _D. Chodati_ was taken from the thallus of _Lecanora tartarea_ and successfully cultivated. It resembles _Protococcus_, but has smaller cells and grows more rapidly; it is evidently closely allied to that genus, if not merely a form of it.
6. UROCOCCUS Kütz. Cells more or less globose, rather large, and coloured with a red-brown pigment, with the cell-wall thick and lamellate, forming elongate strands of cells (Fig. 26). Recorded by Hue[286] in the cephalodium of _Lepolichen coccophorus_, a Chilian lichen.
Fam. TETRASPORACEAE. Cells in groups of 2 or 4 surrounded by a gelatinous sheath.
1. PALMELLA Lyngb. Cells globose, oblong or ellipsoid, grouped without order in a formless mucilage (Fig. 27). Among lichens associated with _Palmella_ are the Epigloeaceae and Chrysothricaceae.
2. GLOEOCYSTIS Naeg. Cells oblong or globose with a lamellate sheath forming small colonies; colour, red-brown (Fig. 28). This alga along with _Urococcus_ was found by Hue in the cephalodia of _Lepolichen coccophora_, but whereas _Gloeocystis_ frequently occupies the cephalodium alone, _Urococcus_ is always accompanied by _Scytonema_, the normal gonidium of the cephalodium.
Fam. TRENTEPOHLIACEAE. Filamentous and branched, the filaments short and creeping or long and forming tufts and felts or cushions; colour, brownish-yellow or reddish-orange.
TRENTEPOHLIA Born. Branching alternate; cells filled with red or orange oil; no pyrenoids (Fig. 29). A large number of lichens are associated with this genus: Pyrenulaceae, Arthoniaceae, Graphidaceae, Roccellaceae, Thelotremaceae, Gyalectaceae and Coenogoniaceae, etc., in whole or in part. Two species have been determined, _T. umbrina_ Born., the gonidium of the Graphidaceae, and _T. aurea_ which is associated with the only European _Coenogonium, C. ebeneum_ (Fig. 3). Deckenbach[287] claimed that he had proved by cultures that _T. umbrina_ was a growth stage of _T. aurea._
Fam. CLADOPHORACEAE. Filamentous, variously and copiously branched, the cells rather large and multinucleate.
CLADOPHORA Kütz. Filaments branching, of one-cell rows, attached at the base; colour, bright or dark green; mostly aquatic and marine (Fig. 30). Only one lichen, _Racodium rupestre_, a member of the Coenogoniaceae, is associated with _Cladophora_. It is a British lichen, and is always sterile.
Fam. MYCOIDEACEAE. Epiphytic algae consisting of thin discs which are composed of radiating filaments.
1. MYCOIDEA Cunningh. (Cephaleuros Kunze). In _Mycoidea parasitica_ the filaments of the disc are partly erect and partly decumbent, reddish to green (Fig. 31). It forms the gonidium of the parasitic lichen, _Strigula complanata_, which was studied by Marshall Ward in Ceylon[288]. Zahlbruckner gives _Phyllactidium_ as an alternative gonidium of Strigulaceae.
2. PHYCOPELTIS Millard. Disc a stratum one-cell thick, bearing seta, adnate to the lower surface of the leaf, yellow-green in colour. _Phycopeltis_ (Fig. 32) has been identified as the gonidium of _Strigula complanata_ in New Zealand and of _Mazosia_ (Chiodectonaceae), a leaf lichen from tropical America.
There is some confusion as to the genera of algae that form the gonidia of these epiphyllous lichens. _Phyllactidium_ given by Zahlbruckner as the gonidium of all the Strigulaceae (except _Strigula_ in part) is classified by de Toni[289] as probably synonymous with _Phycopeltis_ Millard, and as differing from _Mycoidea parasitica_ in the mode of growth.
Fam. PRASIOLACEAE. Thallus filamentous, often expanded into broad sheets by the fusion of the filaments in one plane.
PRASIOLA Ag. Thallus filamentous, of one-to many-cell rows, or widely expanded (Fig. 33). The gonidium of Mastoidiaceae (Pyrenocarpeae).
B. CHANGES INDUCED IN THE ALGA
_a_. MYXOPHYCEAE. Though, as a general rule, the alga is less affected by its altered life-conditions than the fungus, yet in many instances it becomes considerably modified in appearance. In species of the genus _Pyrenopsis_—small gelatinous lichens—the alga is a _Gloeocapsa_ very similar to _G. magma_. In the open it forms small colonies of blue-green cells surrounded by a gelatinous sheath which is coloured red with gloeocapsin. As a gonidium lying towards or on the outside of the granules composing the thallus, the red sheath of the cells is practically unchanged, so that the resemblance to _Gloeocapsa_ is unmistakable. In the inner parts of the thallus, the colonies are somewhat broken up by the hyphae and the sheaths are not only less evident but much more faintly coloured. In _Synalissa_, a minute shrubby lichen which has the same algal constituent, the tissue of the thallus is more highly evolved, and in it the red colour can barely be seen and then only towards the outside; at the centre it disappears entirely. The long chaplets of _Nostoc_ cells persist almost unchanged in the thallus of the Collemaceae, but in heteromerous genera such as _Pannaria_ and _Peltigera_ they are broken up, or they are coiled together and packed into restricted areas or zones. The altered alga has been frequently described as _Polycoccus punctiformis_. A similar modification occurs in many cephalodia, so that the true affinity of the alga, in most instances, can only be ascertained after free cultivation.
Bornet[290] has described in _Coccocarpia molybdaea_ the change that the alga _Scytonema_ undergoes as the thallus develops: in very young fronds the filaments of _Scytonema_ are unchanged and are merely enclosed between layers of hyphae. At a later stage, with increase of the thallus in thickness, the algal filaments are broken up, their covering sheath disappears, and the cells become rounded and isolated. _Petractis_ (_Gyalecta_) _exanthematica_ has also a _Scytonema_ as gonidium, and equally exact observations have been made by Fünfstück[291] on the way it is transformed by symbiosis: with the exception of a very thin superficial layer, the thallus is immersed in the rock and is permeated by the alga to its lowest limits, 3 to 4 mm. below the surface, _Petractis_ being a homoiomerous lichen. The _Scytonema_ trichomes embedded in the rock become narrower, and the sheath, which in the epilithic part of the thallus is 4µ wide, disappears almost entirely. The green colour of the cells fades and septation is less frequent and less regular. The filaments in that condition are very like oil-hyphae and can only be distinguished as algal by staining reagents such as alkanna. They never seem to be in contact with the fungal elements: there is no visible appearance of parasitism nor even of consortism.
_b._ CHLOROPHYCEAE. As a rule the green-celled gonidium such as _Protococcus_ is not changed in form though the colour may be less vivid, but in certain lichens there do occur modifications in its appearance. In _Micarea_ (_Biatorina_) _prasina_, Hedlund[292] noted that the gonidium was a minute alga possessing a gelatinous sheath similar to that of a _Gloeocapsa_. He isolated the alga, made artificial cultures and found that, in the altered conditions, it gradually increased in size, threw off the gelatinous sheath and developed into normal _Protococcus_ cells, measuring 7 to 10µ in diameter. The gelatinous sheath was thus proved to be merely a biological variation, probably of value to the lichen owing to its capacity to imbibe and retain moisture. Zukal[293] also made cultures of this alga, but wrongly concluded it was a _Gloeocystis_.
Moebius[294] has described the transformation from algae to lichen gonidia in a species epiphytic on Orchids in Porto Rico. He had observed that most of the leaves were inhabited by a membranaceous alga, _Phyllactidium_, and that constantly associated with it were small scraps of a lichen thallus containing isolated globose gonidia. The cells of the alga, under the influence of the invading fungus, were, in this case, formed into isolated round bodies which divided into four, each daughter-cell becoming surrounded by a membrane and being capable, in turn, of further division.
Frank[295] followed the change from a free alga to a gonidium in _Chroolepus_ (_Trentepohlia_) _umbrinum_, as shown in the hypophloeodal thalli of the Graphideae. The alga itself is frequent on beech bark, where it forms wide-spreading brownish-red incrustations consisting of short chains occasionally branched. The individual cells have thick laminated membranes and vary in width from 20 µ to 37 µ. The free alga constantly tends to penetrate below the cortical layers of the tree on which it grows, and the immersed cells become not only longer and of a thinner texture, but the characteristic red colour so entirely disappears, that the growing penetrating apical cell may be light green or almost colourless. As a lichen gonidium the alga undergoes even more drastic changes: the red oily granules gradually vanish and the cells become chlorophyll-green or, if any retain a bright colour, they are orange or yellow. The branching of the chains is more regular, the cells more elongate and narrower; usually they are about 13 to 21 µ long and 8 µ wide, or even less. Deeper down in the periderm, the chains become disintegrated into separate units. Another notable alteration takes place in the cell-membrane which becomes thin and delicate. It has, however, been observed that if these algal cells reach the surface, owing to peeling of the bark, etc., they resume the appearance of a normal _Trentepohlia_.
In certain cases where two kinds of algae were supposed to be present in some lichens, it has been proved that one species only is represented, the difference in their form being caused by mechanical pressure of the surrounding hyphae, as in _Endocarpon_ and _Staurothele_ where the hymenial gonidia are cylindrical in form and much smaller than those of the thallus. They were on this account classified by Stahl[296] under a separate algal genus, _Stichococcus_, but they are now known to be growth forms of _Protococcus_, the alga that is normally present in the thallus. Similar variations were found by Neubner[297] in the gonidia of the Caliciaceae, but, by culture experiments with the gonidia apart from the hyphae, he succeeded in demonstrating transition forms in all stages between the “_Pleurococcus_” cells and those of “_Stichococcus_,” though the characters acquired by the latter are transmitted to following generations. The transformation from spherical to cylindrical algal cells had been also noted by Krabbe[298] in the young podetia of some species of _Cladonia_, the change in form being due to the continued pressure in one direction of the parallel hyphae.
Isolated algal cells have been observed within the cortex of various lichens. They are carried thither by the hyphae from the gonidial zone in the process of cortical formation, but they soon die off as in that position they are deprived of a sufficiency of air and of moisture. Forssell[299] found _Xanthocapsa_ cells embedded in the hymenium of _Omphalaria Heppii_. They were similar to those of the thallus, but they were not associated with hyphae and had undergone less change than the thalline algae.
C. CONSTANCY OF ALGAL CONSTITUENTS
Lichen hyphae of one family or genus, as a rule, combine with the same species of alga, and the continuity of genera and species is maintained. There are, however, related lichens that differ chiefly or only in the characters of the gonidia. Among such closely allied genera or sections of genera may be cited _Sticta_ with bright-green algae and the section _Stictina_ with blue-green; _Peltidea_ similarly related to _Peltigera_ and _Nephroma_ to _Nephromium_. In the genus _Solorina_, some of the species possess bright-green, others blue-green algae, while in one, _S. crocea_[300], there is an upper layer of small bright-green gonidia that project in irregular pyramids into the upper cortex; while below these there stretches a more or less interrupted band of blue-green _Nostoc_ cells. The two layers are usually separated by strands of hyphae, but occasionally they come into close contact, and the hyphal filaments pass from one zone to the other. In this genus cephalodia containing blue-green _Nostoc_ are characteristic of all the “bright-green” species. Harmand[301] has recorded the presence of two different types of gonidia in _Lecanora atra_ f. _subgrumosa_; one of them, the normal _Protococcus_ alga of the species, the other, pale-blue-green cells of _Nostoc_ affinity.
Forssell[302] states that in _Lecanora_ (_Psoroma_) _hypnorum_, the normal bright-green gonidia of some of the squamules may be replaced by _Nostoc_. In that case they are regarded as cephalodia, though in structure they exactly resemble the squamules of _Pannaria pezizoides_, and Forssell considers that there is sufficient evidence of the identity of the hyphal constituent in these two lichens, the alga alone being different.
It may be that in Archilichens with a marked capacity to form a second symbiotic union with blue-green algae, a tendency to revert to a primitive condition is evident—a condition which has persisted wholly in _Peltigera_ with its _Nostoc_ zone, but is manifested only by cephalodia formation in the _Peltidea_ section of the genus. In this connection, however, we must bear in mind Forssell’s view that it is the Archilichens that are the more primitive[303].
The alien blue-green algae with their gelatinous sheaths are adapted to the absorption and retention of moisture, and, in this way, they doubtless render important service to the lichens that harbour them in cephalodia.
D. DISPLACEMENT OF ALGAE WITHIN THE THALLUS
_a._ NORMAL DISPLACEMENT. Lindau[304] has contrasted the advancing apical growth of the creeping alga _Trentepohlia_ with the stationary condition of the unicellular species that multiply by repeated division or by sporulation, and thus form more or less dense zones and groups of gonidia in most lichens. The fungus in the latter case pushes its way among the algae and breaks up the compact masses by a shoving movement, thus letting in light and air. The growing hypha usually applies itself closely round an algal cell, and secondary branches arise which in time encircle it in a network of short cells. In the thallus of _Variolaria_[305] the hyphae from the lower tissues, termed push-hyphae by Nienburg[306], push their way into the algal groups and filaments composed of short cells come to lie closely round the individual gonidia. Continued growth is centrifugal, and the algae are carried outward with the extension of the hyphae (Fig. 12). Cell-division is more active at the periphery, that being the area of vigorous growth, and the algal cells are, in consequence, generally smaller in that region than those further back, the latter having entered more or less into a resting condition, or, as is more probable, these smaller cells are aplanospores not fully mature.
_b._ LOCAL DISPLACEMENT. Specimens of _Parmelia physodes_ were found several times by Bitter, the grey-green surface of which was marbled with whitish lines, caused by the absence of gonidia under these lighter-coloured areas. The thallus was otherwise healthy as was manifested by the freely fruiting condition: no explanation of the phenomenon was forthcoming. Bitter compared the condition with the appearance of lighter areas on the thallus of _Parmelia obscurata_.
Something of the same nature was observed on the thallus of a _Peltigera_ collected by F. T. Brooks near Cambridge. The marking took the form of a series of concentric circles, starting from several centres. The darker lines were found on examination to contain the normal blue-green algal zone, while the colour had faded from the lighter parts. The cause of the difference in colouration was not apparent.
E. NON-GONIDIAL ORGANISMS ASSOCIATED WITH LICHEN HYPHAE
Bonnier[307] made a series of cultures with lichen spores and green cells other than those that form lichen gonidia. In one instance he substituted _Protococcus botryoide_s for the normal gonidia of _Parmelia_ (_Xanthoria_) _parietina_; in another of his cultures he replaced _Protococcus viridis_ by the filamentous alga _Trentepohlia abietina_. In both cases the hyphae attached themselves to the green cells and a certain stage of thallus formation was reached, though growth ceased fairly early. Another experiment made with the large filaments of _Vaucheria sessilis_ met with the same amount of success (Fig. 34). The germinating hyphae attached themselves to the alga and grew all round it, but there was no advance to tissue formation.
Cultures were also made with the protonema of mosses. Either spores of mosses and lichens were germinated together, or lichen spores were sown in close proximity to fully formed protonemata. The developing hyphae seized on the moss cells and formed a network of branching anastomosing filaments along the whole length of the protonema without, however, penetrating the cells. If suitable algae were encountered, proper thallus formation commenced, and Bonnier considers that the hyphae receive stimulus and nourishment from the protonema sufficient to tide them over a considerable period, perhaps until the algal symbiont is met. An interesting variation was noted in connection with the cultures of _Mnium hornum_[308]. If the protonema were of the usual vigorous type, the whole length was encased by the hyphal network; but if it were delicate and slender, the protoplasm collected in the cell that was touched by hyphae and formed a sort of swollen thick-walled bud (Fig. 35). This new body persisted when the rest of the filament and the hyphae had disappeared, and, in favourable conditions, grew again to form a moss plant.
F. PARASITISM OF ALGAE ON LICHENS
A curious instance of undoubted parasitism by an alga, not as in _Strigula_ on one of the higher plants, but on a lichen thallus, is recorded by Forssell[309]. A group of _Protococcus_-like cells established on the thallus of _Peltigera_ had found their way into the tissue, the underlying cortical cells having degenerated. The blue-green cells of the normal gonidial layer had died off before their advance but no zone was formed by the invading algae; they simply withdrew nourishment and gave seemingly no return. The phenomenon is somewhat isolated and accidental but illustrates the capacity of the alga to absorb food supply from lichen hyphae.
An instance of epiphytic growth has also been recorded by Zahlbruckner[310]. He found an alga, _Trentepohlia abietina_, covering the thallus of a Brazilian lichen, _Parmelia isidiophora_, and growing so profusely as to obscure the isidiose character towards the centre of the thallus. There was no genetic connection of the alga with the lichen as the former was not that of the lichen gonidium. Lichen thalli are indeed very frequently the habitat of green algae, though their occurrence may be and probably is accidental.