Studies in the biology and physiology of lichens with special reference to Xanthoria parietina (L.) Th. Fr

The work described in this thesis had several main objects which are summarised as follows:
To study the carbohydrate movement from alga to fungus during photosynthesis in the lichen Xanthoria aureola (Ach.) Erichs. (since this work commenced the variety of Xanthoria parietina used in the experiments has been recognised as a separate species and is referred to by this new name). Previous research on this aspect of the physiology of lichens has been largely limited to Peltigera polydactyla. This lichen contains the blue-green alga Nostoc which produces glucose during photosynthesis that passes to the fungus where it is rapidly converted into mannitol. Xanthoria aureola was selected for the experiments in this investigation because it contains a green alga, Trebouxia, which is the commonest algal symbiont found in temperate lichens.
To study the carbohydrate movement between the symbionts of other lichens to determine the extent to which the pattern found in Peltigera polydactyla and Xanthoria aureola occurred in other species.
To study some general aspects of the carbohydrate metabolism of Xanthoria aureola such as the changes which occur during starvation, the uptake of carbohydrates and the variation in sugar alcohol content at various times of the year.
To study a number of ecological and taxonomic aspects of the biology of Xanthoria parietina sensu lato by transplanting thalli of different varieties between inland and sea shore habitats.
1. Carbohydrate movement from alga to fungus in Xanthoria aureola
a. Products of photosynthesis in the intact thallus.
Before investigating the role of the alga and fungus in the symbiosis, it was necessary to study photosynthesis in the complete thallus. When samples of this lichen were floated in the light on aqueous 14C sodium bicarbonate solutions, 14C was at first incorporated in substantial amounts into the sugar alcohol ribitol which had not previously been reported in lichens. After several hours 14C also began to accumulate in mannitol and a small amount was also found in arabitol. It was noted that less than 10% of the fixed 14C occurred in the ethanol-insoluble fraction over a 24 hour period, and in experiments where the redistribution of a pulse of 14C-ribitol was studied, it was found that this became largely converted into 14C-mannitol and 14C-arabitol over a 48 hour period.
b. Studies on the isolated components of the lichen
i. The alga. The alga of Xanthoria aureola, which is a species of Trebouxia, was successfully isolated and maintained in pure culture. Since there was the possibility that the behaviour of the alga in pure culture is different from that in the lichen, a technique was developed for isolating quantities of the alga direct from the thallus. This was done by gently homogenising thallus lobes and subjecting the homogenate to a regime of low speed differential centrifugation so as to produce a moderately pure suspension of living intact algal cells.
It was demonstrated that both the cultured and directly isolated alga produced 14C-ribitol and 14C-sucrose within the cells during photosynthesis on 14C-sodium bicarbonate solution. However when the alga had been in pure culture for some time, less 14C was incorporated into carbohydrates, especially ribitol, than when the alga was directly isolated from the thallus. Both kinds of algal preparation were found to release carbohydrate, mainly in the form of ribitol, into the medium. However they did this only slowly, and the cultured alga appeared to release substances less readily than the directly isolated alga.
ii. The fungus. This was also isolated into pure culture from fungal ascospores and was subsequently grown in liquid culture. The lichen fungus of Xanthoria aureola was found to grow on a range of sugars and sugar alcohols. It was able to convert 14C-glucose rapidly into 14C-mannitol. Under certain cultural conditions it was found also to contain arabitol, confirming that this is a fungal product.
c. Attempts to inhibit the movement of carbohydrates between the symbionts
If 12C-pentitols were present in the medium during photosynthesis by Xanthoria aureola on 14C-sodium bicarbonate solutions, then 14C-ribitol appeared in the medium and little 14C-mannitol was formed within the thallus. This was explained in terms of the 14C-ribitol produced by the alga being unable to compete with the high concentration (l or 2%) of non radioactive pentitols at the fungal uptake sites, so that the entry of 14C-ribitol into fungus was prevented and it therefore diffused out into the medium. It was only possible to obtain this inhibition with pentitols. Other sugars or sugar alcohols were unable to prevent carbohydrate movement, suggesting that the fungal uptake mechanisms had a high specificity for pentitols. These results suggested that carbon moved between the symbionts principally in the form of ribitol.
d. The rate of movement of carbohydrate between the symbionts
In Peltigera polydactyla it had been shown by earlier workers that 14C fixed by the alga moves to the fungal medulla within thirty minutes from the start of photosynthesis on 14C-sodium bicarbonate. In the type of inhibition experiment described above, at least 25% of the total fixed 14C was released from samples of Peltigera into the medium within three hours if it contained 12C-glucose. It was therefore concluded that movement of carbon between the symbionts was rapid. In analogous inhibition experiments carried out here using Xanthoria aureola, it took as long as twenty four hours for a similar proportion of the total fixed 14C to be released into the medium when it contained 12C-ribitol, and this suggested that the movement of carbohydrates between the symbionts in this species was much slower. Further experiments were devised in which the rate of transfer of a pulse of 14C from alga to fungus was estimated both in Peltigera polydactyla and Xanthoria aureola under identical conditions. These confirmed that movement of carbohydrate was much slower in the latter lichen.
2. Studies of carbohydrate movement in other lichens
a. Lichens containing blue-green algae
Three lichens were examined, two contained Nostoc (Sticta fuliginosa, Lobaria scrobiculata) and the third, Rivularia (Lichina pygmaea). They all showed similarities with Peltigera polydactyla in that glucose appeared to move between the symbionts and this was converted into mannitol by the fungal part of the thallus. There was evidence that the movement of carbohydrate from alga to fungus was rapid in the species containing Nostoc but slower in Lichina pygmaea.
b. Other lichens containing green algae
Lichens containing four genera of green algae (including Trebouxia) were studied and it was remarkable that in each case 14C initially accumulated in sugar alcohols, during photosynthesis. The compounds ribitol, erythritol and sorbitol appeared to be the respective photosynthetic products of the algae Trebouxia (and Coccomyxa), Trentepohlia and Myrmecia. These sugar alcohols were converted into mannitol by the fungal part of the lichen and to a lesser extent into arabitol and volemitol in certain species.
In the lichens containing Trebouxia that were studied (Parmelia saxatilis, Lecanora conizaeoides, Umbilicaria pustulata, Lobaria laetevirens, Lobaria pulmonaria) movement between the symbionts appeared to occur at a similar rate to that found in Xanthoria aureola and little arabitol was formed. In the lichens containing Coccomyxa (Solorina saccata, Peltigera aphthosa) the movement of carbohydrates was more rapid and a much larger proportion of the fixed carbon was found in arabitol after twenty four hours photosynthesis. Similarly the transfer of carbohydrate from alga to fungus was faster in Dermatocarpon miniatum, which contains Myrmecia, than in Xanthoria aureola and this lichen also differed in that substantial amounts of volemitol accumulated in the thallus. However the slowest rate of movement of carbohydrates between the symbionts was observed in those lichens containing Trentepohlia (Roccella fuciformis, R. phycopsis, Lecanactis stenhamarii, Gyalecta cupularis).
c. Lichens symbiotic with more than one alga
Three lichens were examined in which a green alga was symbiotic in the greater part of the thallus but where there are limited areas within the thallus (or outgrowths from it) where the fungus is symbiotic with a blue-green alga such as Nostoc. These areas, called cephalodia, have usually been assumed to be important in the nitrogen metabolism of the thallus. The pattern of carbohydrate movement between the fungus and its two symbionts was studied to try and find out if it was the fungus or alga which controlled the pattern of carbohydrate transfer in the thallus.
It was possible to excise the cephalodia of one species (Lobaria amplissima) and in this case the pattern of carbohydrate movement from alga to fungus in the cephalodia was closely similar to that described for Peltigera polydactyla which also contains Nostoc. In the rest of the thallus, which contained Trebouxia, it was similar to that found in other lichens containing this alga. When samples of the other two lichens (Solorina saccata, Peltigera aphthosa) in which the cephalodia could not be excised, were placed in the light on solutions of 14C-sodium bicarbonate to which had been added 12C-ribitol or 12C-glucose, fixed 14C appeared in the medium. This was either in the form of 14C-ribitol or 14C-glucose depending on which sugar had been used. If a mixture of the two sugars was placed in the medium, then both 14C-glucose and 14C-ribitol were found therein. Thus some 14C-glucose appeared to be moving from the cephalodia to the fungal part of the thallus and this indicates that they may contribute to the carbohydrate nutrition of the thallus, particularly in Solorina saccata, where they are quite large in proportion to the rest of the thallus.
These results suggest that the type of alga symbiotic in the thallus has an important role in dictating the pattern of carbohydrate movement in the thallus. This was confirmed by studies on the directly isolated algae and by an investigation (described in Appendix A) into four species of the genus Lobaria which contain closely related fungi symbiotic with either Nostoc or Trebouxia. The two patterns of carbohydrate transference observed were there- fore described as the 'Nostoc' and 'Trebouxia' patterns.
3. Absorption and loss of carbohydrates
Samples of Xanthoria aureola were found to absorb both pentitols and mannitol rapidly at 18°C which is in agreement with other studies on the uptake of substance from solution by lichens. On starvation in the dark at the same temperature, the content of pentitols (initially about 3% of the dry weight) in Xanthoria aureola fell rapidly and these compounds were not detectable after four days. By contrast, there was at first a rise in mannitol content (initially also about 3% dry weight), presumably due to conversion of pentitols into mannitol, followed by a slow fall. Mannitol was detectable in samples starved under these conditions for fourteen days. Some measurements were made on the seasonal variation in the total sugar alcohol content of this lichen and the results suggested that the sugar alcohol content fell in winter but rose rapidly in early spring.
4. The Transplantation of lichen thalli
Xanthoria parietina sensu lato now conprises two species, Xanthoria parietina and Xanthoria aureola. The former is separated into two varieties, var parietina and var ectanea. Lichen thalli were transplanted between Devon and Berkshire to gain further information about the exact taxonomic status of the species and varieties. The material of Xanthoria aureola from Berkshire was planted on the rocks near Slapton in Devon whilst the material from that habitat, Xanthoria parietina var ectanea. was transplanted to the habitat of Xanthoria aureola at Wytham in Berkshire. No method for transplanting saxicolous lichens had apparently been published, so one was developed using resin glue. This proved successful as the thalli survived and grew.
On preliminary evidence it was found that Xanthoria parietina and Xanthoria aureola were distinct species. However on transplanting X. parietina var ectanea to Berkshire, the lobe width increased so that it could not be distinguished from X. parietina var parietina.
The experiments also suggest that the amount of the yellow pigment, parietin, in these lichens is largely controlled by the environment. It was found that on transplanting thalli between the two habitats, the parietin content changed to that of control transplants within six months. It has been suggested that the amount of parietin developed is related to the light intensity. The Devon transplants had some four times more parietin than those from Berkshire. Since all the thalli grew in unshaded habitats it seems that light may not be the only factor responsible for the development of this pigment.