Chapter II: Constituents of the Lichen Thallus (1)

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_.