Chapter II: Constituents of the Lichen Thallus (2)

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.