Experimental cultivation of lichens and lichen symbionts

Experimental cultivation of lichens and lichen symbionts

Elfie Stocker-Worgotter

Abstract: Culture experiments with lichens and lichen symbionts are helpful for giving answers to many open questions in different fields of modern lichenology. Cultures are especially required for investigations where the analysis of naturally grown thalli is inconclusive and more standardized research material is needed. In the first part a short review of the artificial resynthesis of the cyanobacterial Peltigera praetextata along with new results obtained by the culture of isidia are presented. Another series of experiments report a successful resynthesis of the photosymbiodeme Peltigera leucophlebia. Details of the thallus morphogenesis and the formation of cephalodia in culture are shown. The related Peltigera aphthosa was cultured from small thallus fragments. In this case, the alpine P. aphthosa forms solely the cephalodiate thallus. A cyanobacterial morphotype as known from other culture experiments and the related P. britannica is missing in culture. Further culture experiments are conducted with alpine species of the genus Cladorlia (e.g., C1. fimbriata, Cl. furcata). Results show a quick redifferentiation and regeneration of squamules and podetia both by soredia and fragments. The high capacity of thallus regeneration as shown for Cl. furcata seems to be abscnt in representatives of the Cladina group (c.g., Cl. portentosa, Cl. rangiferina).

Key words: lichen culture, resynthesis, tissue culture, Peltigeraceae, cyanobacterial lichens, photosymbiodemes, Peltigera leucophlebia, Peltigera aphthosa, Cladoniaceae, Cl. fimbriata, Cl. furcata.

Resume : Les experiences de culture avec les lichens et les symbiontes lichkniques permettent de rkpondre h plusieurs questions sans rCponse, dans differents champs de la IichCnologie moderne. Les cultures sont particulierement utiles dans les recherches oh l'analyse de thalles croissant en nature n'est pas concluante et oh du materiel de recherche plus standardis6 est nCcessaire. Dans la premikre partie l'auteur prCsentc une courte revue de la resynthbse artificielle du lichen cyanobacterien Peltigera praetextata ainsi que de nouveaux resultats obtenus avec la culture d'isidies. Un autre ensemble d'experiences font Ctat d'une resynthese reussie chez le photosymbiodeme Pelfigera leucophlebia. On prksente les dCtails de la morphogtnese et la formation de ckphalodies en culture. Le Peltigera aphthosa apparent6 a CtC cultiv6 a partir de petits fragments de thalle. Dans ce cas, le P. aphthosa alpin ne forme que des thalles ctphalodiCs. L'auteur n'a pas rkussi B cultiver un morphotype cyanobacterien tel que connu a partir de d'autres cultures expCrimentales ainsi que du P. britannica apparent&. D'autres cultures expCrimentales sont conduites avec des especes alpines du genre Cladonia (p. ex., Cl. fimbriata, Cl. furcata). On obtient une rapide redifferenciation et reg6nCration de squamules et de podeties a partir de soredies et de fragments. Cette importante capacitC de regentration telle que demontree chez le C1. furcata, semble absente chez les representants du groupe Cladina (p. ex., Cl. portentosa, Cl. rangiferina).

Mots elis : cultures de lichens, resynthbse, culture de tissus, Peltigeraceae, lichens cyanobact6riens, photosymbiodbmes, Peltigera leucophlebia, Peltigera aphthosa, Cladoniaceae, C1. fimbriata, Cl. furcata. [Traduit par la rtdaction]

Introduction microbiology and also to modern laboratory equipment; e.g.,

Experimental studies on lichens, especially culture experi- improved clean work benches allow manipulations under axenic conditions, and both adjustable culture chambers and

ments, have a long tradition of failure o r of very limited suc- phytotrons can be used to culture plants under various com-

cess (Ahmadjian 1959, 1962, 1973, 1993; Ahmadjian and binations of conditions. Heikkila 1970; Ahmadjian et al. 1980).

In the opinion of Ahmadjian (1987), lichens are among Recent advances in "lture techniques may be attributed the most difficult organisms to work with experimentally.

generally to progress in different fields of experimental Compared with other free-living fungi, mycobionts grow much more slowly and have more complex nutrient require-

Received August 15, 1994. ments. This is not surprising since a great variety of fungi

E. Stocker-Worgotter. Institute of Plant Physiology, are involved in the lichen symbioses and the polyols released

University of Salzburg, Hellbrunner Str. 34, A-5020 by green and blue-green photobionts are different. During Salzburg, Austria. the last two decades, fo r certain species, (e.g., Endocalpon

Can. J. Bot. 73(Suppl. 1): S579-S589 (1995). Printed in Canada I ImprimC au Canada

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pusillum, Cladonia cristatella) some of these problems have been overcome; e.g., isolation and culture techniques for photo- and myco-bionts and also methods for resynthesis and thallus tissue culture are well established. A growing number of lichens have been cultured under artificial conditions in the laboratory. A list of partially and completely successful resyntheses has been given by Ahmadjian (1993). A signifi- cant number of the cited species are terricolous and also saxi- colous lichen species.

. . . . . - . . . . During our culture experiments of the past 10 years, we

. . . . . found the most favourable culture objects to be lichens living

in nature on soil and sand substrates (e.g., Endocarpon pusillum, Peltigera aphthosa, P. didactyla, P. leucophebia, P. praetextata, alpine Cladonia species such as Cladoniajmbriata, C. furcata, and C1. gracilis, and tropical representatives from Brazil such as C1. clathrata, C1. substellata, and C1. verticillaris (Stocker-WorgMter et al. 1994).

The first part of my paper presents a short overview of our resynthesis experiments with P. praetextata. Then a new series of ex~eriments are described to show that the so-called wound isidia act both as initial stages of new lobes on a parent thallus and as vegetative diaspores. A report on a resynthesis of the photosymbiodeme P. leucophlebia is given along with results from tissue cultures conducted with the related P. aphthosa. The last part of my paper documents

I highlights from culture experiments with alpine species of the genus Cladonia and new data obtained from photo- and myco-biont cultures.

Materials and methods Fertile thalli of Peltigera praetextata Sommerf. ex Florke and P. leucophlebia (Nyl.) Gyelnik were collected in the Rottenstiner Valley, Carinthia (Austria) at 1300 m altitude. Specimens of P. aphthosa (without fruiting bodies) were collected in the Virgen Valley, eastern Tirol (Austria) at 1500 m elevation. A selected alpine species of the genus Cladonia, C1. jmbriata (L.) Fr. was also collected in the Rottensteiner Valley at an altitude of 1400 m. For the cultures, both fresh (1 -3 weeks old) or stored (refrigerated at -23°C) lichen material was used (Stocker-Worgotter et al. 1994).

Mycobionts Mycobionts were isolated from the following lichens (Pel- tigera leucophlebia, P. praetextata, Cladonia jmbriata, C1. furcata). Apothecia were cut off and first washed in phosphate-buffered saline (PBS) (+ Tween 80) after Bubrick and Galun (1986) and later on under running tap water for 30 min. Moist apothecia were fixed to the top cover of a Petri dish. Spores were discharged downwards in a Petri dish, containing sterile 2% BBM-agar (Bischoff and Bold 1963) and soil extract was prepared after Esser (1976). Mycelia (P. leucophlebia), grown from germinated spores were transferred to LBM (altered by Lallemant 1985) containing 0.25 g KN031L and soil extract was prepared from soil from the habitat of the lichen. LBM (+ additional substances) was sterile filtrated. The cultures were maintained in a culture chamber at 20°C without light.

Mycobionts isolated from alpine Cladonia species (Cl. furcata, Cl. jmbriata) were grown in liquid and solid LBM containing 1 % Ribitol instead of glucose.

Fig. 1. Tissue culture method (modified after Yamamoto 1990).

1. Original 111allus 2. Washing procedure 3. Homogenization cut into pieces (rnagnclic stirrer)

4. Filmtion wih 5. Sclcdon of 6. Incubation a sieve fragments on soil

Photobionts

Cyanobionts Small Nostoc colonies were isolated from thalli (P. praetextata) and cephalodia (P. leucophlebia, P. aphthosa). Purifi- cation and cultivation were conducted following the method of Boissikre et al. (1987), first on 2 % BBM-agar plates and later in liquid BBM.

Green bionts Coccomyxa (P. leucophlebia) was isolated from carefully washed thallus fragments after the method of Ahmadjian (1973). Different species of Trebouxia were isolated from the alpine Cladonia specimens. For cultivation small fragments were macerated between two glass slides and groups of algal cells were transferred to BBM-agar (+ soil extract) in a Petri dish. Purified small groups or single Trebouxia cells were subcultured.

The photobiont and cyanobiont cultures were kept at 20 + 2"C, at a 14-h-1ight:lO-h-dark regime and a photon flux intensity of 60- 100 pE . m-2 . spl .

Resynthesis The first resynthesis experiments were conducted on agar plates but with very limited success (Stocker-Worgotter and Tiirk 1991). Improved conditions for resynthesis were obtained by the use of soil substrates. Soil from the lichen habitats was dried and sieved (mesh width of 2 mm). Then for each experiment 500 g soil was soaked with 300 mL twice- distilled water for 3-4 h. The moist soil was distributed in glass Petri dishes (100 x 15 mm) and steam-sterilized for 3 h on 2 successive days.

The substrates were first inoculated with the following photobionts (Nostoc: P. praetextata; Nostoc, Coccomyxa: P. leucophlebia) and incubated for 3 weeks. After this time span, spores or hyphal fragments from mycobiont cultures (obtained by homogenization; 30 s at 15 000 rpm from mycelia) were added. The culture conditions for the resynthesis cultures are described in Stocker-Worgotter and Tiirk (1991, 1994). The methods of micro- and macro-photography are found in Stocker-Worgotter and Tiirk (1990).

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D4.2. Stocker-Worgotter

Tissue culture method The tissue culture method (modified after Yamamoto 1990) is shown in Fig. 1. Well-developed thalli of P. aphthosa and C1. furcata were carefully washed (PBS + Tween 80; twice- distilled water) and then fragmented in an homogenizer at 15 000 rpm for 30 s. Segments (150-500 pm) were picked up by sterile blood lancets and then transferred to sterilized soil substrates in Petri dishes (100 x 20 mm).

Cultivation of soredia and isidia Soredia of C1. fimbriata were scratched off the podetia and collected in a small Petri dish. About 100-200 soredia were sown on sterile, moist soil in Petri dishes (100 x 15 mm). After 4 weeks of incubation, germinated and contamination-free soredia were selected and transferred to new sterilized soil substrates. The cultures were maintained under the same conditions as described above for the photobionts. At intervals of 6-7 weeks, the cultures were misted just around the developing soredia with sterile twice-distilled water.

Culture of isidia (P. praetextata) Isidia were isolated from cultivated thalli and therefore were free from contamination. Like the soredia they were sown on sterilized soil substrates in Petri dishes. The culture conditions were similar to the resynthesis cultures. Every 6 weeks the cultures were moistened with sterile distilled water by

I means of a micropipette.

Results

Peltigera praetextata According to Lallemant and Bernard (1977) and other authors (e.g., Scott 1964), some lichen spores (Lobaria, Peltigera) germinate but cannot develop into a mycelium without contact with a compatible photobiont. Our first experiments confirmed this reaction also for the mycobiont of P. praetextata. To get mycelia for the resynthesis experiment, germinated spores were inoculated onto agar plates containing pre- viously isolated and purified Nostoc colonies. In cultures with the cyanobiont, mycelia formed after 3 months. On agar the Nostoc chains were infected by the mycobiont but not lichenized.

Further experiments were conducted on sterilized soil substrate to attain a better regulation of the moisture content.

On soil, early resynthesis stages were formed after 3-4 months. By further growth, these associations showed a glo- bose and soredialike arrangement. In the next phase of development (5 -6 months), neighbouring soredial stages fused and differentiated into upraised lobelike primordia. Often very young lobules merged forming a larger primordial thallus lobe. After this fusing process prothalli of different growth forms and sizes were present. After 1 year in culture the primordial thalli had transformed into corticated lobes. After 20 months, the largest thalli measured 7 mm. The first well- developed rhizinae emerged after 2 years. At the margin of many cultivated thallus lobes isidia had been formed.

Lallemant and Savoye (1985) reported that cutting the thalli of P. praetextata induced the formation of isidia and regeneration lobes. My studies agree with these authors in that lobes are formed from these isidioid structures, but not

in that their formation should be interpreted solely as a repair process of injured lobes. In my opinion, the main function of these isidia is the vegetative distribution of the lichen. My cultures showed that isidia also formed independently of any thallus wounding. In culture they differentiated on nearly all thalli that had reached a distinct stage of maturity (a minimal size of 1 cm and an age of more than 1 year).

My first investigations documented the differentiation of juvenile lobes from isidia at the margin of an adult thallus. The isidia surprisingly transformed first into soredial primordia and from fused soredial clumps small lobules differentiated (Figs. 2-7). This process occurred very rapidly, more rapidly than lobe development by resynthesis. Corti- cated lobes developed from isidia after 2-4 months, while larger lobes formed after 6 months.

In an additional experiment, the isidia were isolated from the parent thallus and sown onto a new soil substrate (Figs. 8 - 13). In this case, small juvenile thalli formed after 4 and 5 months (Figs. 11 and 12) and larger thalli formed after 8 months (Fig. 13).

From these studies, it became obvious that the isidia of P. praetextata serve both for the development of lobes directly on the parent thalli and as vegetative diaspores. Like the related P. didactyla, P. praetextata has two strategies of reproduction, sexually by spores and vegetatively by isidia.

Peltigera leucophlebia Mycobiont In our alpine valleys Peltigera aphthosa and P. leucophlebia are rarely found with fruiting bodies and often the few apothecia do not discharge enough spores. When they do, the contamination rate is very high.

When isolated without contamination (Fig. 2, step 3) the fungus grew very slowly; the average size of a mycelium was 0.5 -2 mm after 3 -4 months in culture. After 1 year, suffi- cient mycelial material was available for the resynthesis experiment.

The fungus, as spores or mycelia, was transferred to a sterilized soil substrate and inoculated on a mixture of Nostic and Coccomyxa cells that had been maintained in culture for almost 3 weeks (Fig. 14, steps 1-3).

The first lichenized stages were formed after 4 months (Fig. 14, step 4). Green initiation stages differentiated directly on a cyanobacterial layer now covering the soil substrate or emerged marginally from cyanobacterial lobules formed by the lichen fungus with cyanobacteria. The fact that in our cultures green primordia differentiated from cyanobacterial lobules or a cyanobacterial crust agrees very well with field observations of Brodo and Richardson (1978) with P. britannica and by Ott (1988) with P. verzosa.

In our cultures, four different types of primordia were found (Fig. 14, step 4): pure cyanobacterial primordia, pure green initiation stages, cyanobacterial lobules carrying green devel- opments, and also stages composed of a mixture of the fungus and the two photobionts. The composite lobules disintegrated very soon, during the following 2 months. Perhaps they may be interpreted as laboratory artefacts. Surprisingly, the cyanobacterial primordia also did not develop beyond a homeomerous thallus structure; they did not transform into heteromerous thalli as known from cyanobacterial Peltigera

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Figs. 2-7. Peltigera praetextnta, cultivation of isidia. Fig. 2. Young thallus primordia, developed from "wound" isidia on a thallus grown in laboratory culture. Scale bar = 1 mm. Figs. 3 and 4. Isidia differentiating into soredia-like agglomerations at the beginning of development (2 and 4 weeks). Scale bar = 1 mm. Fig. 5. New lobes growing at the margin of a cultivated thallus. Scale bar = 1 mm. Fig. 6. A more differentiated thallus lobe in contact with the parent thallus. Notice initiation stages of new isidia at the margin. Scale bar = 1 mm. Fig. 7. Fully developed thalli originating from isidia (formed by the cultivated parent thallus), 6 months in culture. Scale bar = 4 mm.

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Figs. 8-13. Pelt~gera praerextuta, culture of isolated isidia. Fig. 8. Wound isidia as formed by Peltigera praetexlata in nature. Scale bar = 1 mm. Fig. 9. Redifferentiation stage formed by isolated isidia grown on a cultivated thallus (notice also sorcdia-like stages), incubated for 2 months. Scale bar = 800 pm. Fig. 10. Juvenile thallus lobe, 3 months in culture. Scale bar = I mm. Fig. l I . More developed lobe, after 4 months. Scale bar = 1 mm. Fig. 12. Juvenilc thallus, just becoming branched (5 months). Scale bar = 5 mm. Fig. 13. Thallus lobes, developed from isidia after 8 months. Scale bar = 5 mm.

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Fig. 14. Artificial resynthesis of the photosymbiodeme Peltigera leucophlebia.

Development of the green thallus

mixed primordia formed by pure cyanobaclerial

......

..................................................................................... ............................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ ......... ............. ... ........... -- .- ,.-. . , 511 ( r n l 2011 uln 1 SO 11111 S&Gn

green tliallus primordla on a green thallus primordia cyanobacterial prothalhls on acyanobacterial crust

0 green algae 0 cyanobacteria

Dart of thallus

green thalli with cephalodia cephalodium on a green thallus 14 and 18 month^ nld vertical section

Initially the first green lichenized stages were globules; later on the globules differentiated into more flattened and fingerlike primordia (Fig. 14, step 5). The young prothalli are products of merged neighbouring stages. By fusing, small joint lobules of different size developed (Fig. 14, steps 5 and 6). Prothallus formation occurred after a similar pattern already found in former studies with the cyanobacterial P. didactyla and P. praetextata and also with lichens representing other growth forms as shown by Schuster (1985).

By further growth and differentiation the juvenile thalli became more cup- and shell-shaped (Fig. 14, step 6).

Finally, our cultures were dominated by well-developed green thalli arising from a cyanobacterial layer. Corticated thalli with an average size of 2-3 mm were obtained after 6-8 months in culture. After 1 year the largest thalli measured 5 -7 mm.

After 14 months of cultivation (Fig. 14, steps 7 and 8), the first cyanobacterial colonies were found on the upper side (within the cup of the green thalli). Most of the cyanobacteria reached the thallus surface by watering and entered at that side of the cup where it had been attached to the substrate. After the next desiccation of the cultures, Nostoc colonies were preferentially found in slight depressions of the thallus surface. Such microforms of the thalli are very fitted to house a second symbiotic partner. The green thalli seemed to be colonized by the cyanobacteria after the thalli had differentiated small concavities (Fig. 14, step 7).

Microscopical examination showed that the Nostoc colonies, beforethey enter the cup, had been almost infected by the fungus outside. Mycobiont-free Nostoc chains were rarely present. In laboratory culture only the fungus-infected Nostoc became attached and rapidly differentiated into cephalodia. Details of the formation of cephalodia are presented in Stocker-Worgotter and Tiirk (1994).

After 1.5 years in culture most of the obtained green thalli carried well-developed cephalodia. It was found that after the green thalli had a representative number of cephalodia their growth accelerated.

Culture of fragments of Peltigera aphthosa In both cases (laboratory cultures, field cultures in the flowerpots) the de novo regeneration of the fragments into primordia occurred very slowly. As found in all former studies, the fragments first disintegrated into soredialike agglomerations and afterwards new primordia were formed.

After 3-4 months small thallus primordia had differentiated. The field cultures are always endangered to be over- grown by mosses (Figs. 15 and 16). After 5-6 months in both field and laboratory cultures small prothalli were present (Figs. 16 and 17).

Generally, the sequence of development was very similar with the stages formed by resynthesis as shown before with Peltigera leucophlebia. After 8 months, the stages in the field were further developed than the thalli in the laboratory (Figs. 18 and 19). In both cases the young lobes are first found without cephalodia.

As reported before with P. leucophlebia, the cyanobacteria entered the green thalli by watering after an incubation time of more or less 1 year. After an additional 2 months (12- 14 months) many cephalodia can be found within the

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Figs. 15-21. Tissue fragment culture of Pelrigera aphthosa. Fig. 15. P. aphthosa fragments in field culture at the natural habitat of the lichen (flowerpot cultures). Scale bar = 200 pm. Fig. 16. Thallusprimordium, differentiated from a fragment in the field. Scale bar = 500 pm. Fig. 17. Primordia, grown on a cyanobacterial crust in the laboratory. Scale bar = 300 pm. Fig. 18. Juvenile thallus grown in field culture, 8 months old. Scale bar = 1.25 mm. Fig. 19. Thallus formed after the same time span in the laboratory (8 months). Scale bar = 500 pm. Fig. 20. Detail of thallus grown in the laboratory. Cephalodium, just releasing hormogonia. Scale bar = 500 pm. Fig. 21. Cultivated thallus with cephalodia after 1 year. Scale bar = 3 mm.

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Figs. 22-27. Cladonia firnbriata, cultivation of soredia. Fig. 22. Thalli of CI. fimbriata from the natural environment. Scale bar = 4 rnm. Fig. 23. "Germinated" soredia after 2 weeks of incubation. Scale bar = 200 pm. Fig. 24. Merged soredia, differentiating into squamule primordia. Scale bar = 300 pm. Fig. 25. Primordium, just developing into a squamule. Scale bar = 500 pm. Fig. 26. Juvenile podetium, growing up from well-defined squamules (4, 5 months). Scale bar = 1 mm. Fig. 27. More differentiated podetium forming a juvenile scyphus. Scale bar = 1 mm.

young green thalli (Figs. 20 and 21). Figure 20 shows Nostoc Cladonia fimbriata filaments (hormogonia) that are released by a developing In the first investigation I tried to cultivate CL. j5tnbriata f i

cephalodiurn. soredia. Initial growth reactions were apparent after 2 we rorn :eks

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of incubation (Fig. 23). In the next step of development neighbouring soredia merged forming an undifferentiated algal-fungal association (Fig. 23). In the following 2 months the basal tissue clumps (Schuster 1985; Ott 1987) differentiated first into presquamules (Fig. 24). After 3 months of cultivation the whole surface of the soil substratum was covered by corticated squamules (Fig. 25), the thallus horizontalis of Cladonia 5mbriata.

After a time span of 4 months some of the lobelike squamules began to differentiate podetia. Initiation of podetia formation was recognized by small reddish brown spots (primordia of generative tissue; Jahns 1993) on different parts of the squamule. From these areas podetia began to grow up. Figures 26 and 27 show different stages of podetium development. After half a year of development the podetia had reached an average height of 3-5 mm. They resembled in all characteristics the very juvenile podetia well known from populations of Cladonia jtnbriata in nature.

A very interesting object for tissue culture (Yamamoto method) is Cladoniafircata. We found that nearly all parts of the vegetative and generative thallus have the capacity to regenerate the whole plant.

First regeneration stages of tissue fragments of Cl. furcara , developed after 3 weeks of incubation, which in comparison

with the investigated Peltigera species was very rapid. Pre- squamules were formed on different parts of the lichen explant. In these areas the growth of the fungus took place in synchronization with the increase of the algal colony available for integration. On certain parts of the tissues the symbiotic partners were separated through homogenization and so sometimes the mycobiont without photobionts grew into mycelia, not getting further contact with photobiont cells.

The basal squamules differentiated after the same developmental sequence as observed with Cladoniajmbriata (see above) or Cl. cristatella (Ahmadjian and Jacobs 1983). First presquamule and finally (after a cultivation time of 3-4 months) corticated squamules with a pigmented basis were formed.

After an incubation time of 4-5 months a high number of the horizontal thalli (ca. 60%) initiated podetia formation. The differentiation of the podetia also began with the brown coloured spots (as described above) and continued with the formation of the podetium stem. As already found by Ander- son and Ahmadjian (1962) the stem was first formed by the lichen fungus alone, consisting of a bundle of hyphal cords that were tightly cemented together. Then in a second step algal colonies grew up the fungal stems until these were covered by the irregular algal layer very typical for young Cladonia fircara podetia from the natural habitat. Further- more, it was found that podetia branches were formed at a very early stage of stem differentiation, generally after a cultivation time of 5 months. Under favourable conditions the growth of the podetia occurred very fast, sometimes 2 -3 mm within 1 month. A high growth rate during the development of the podetia was also reported by Anderson and Ahmadjian (1962) and Jahns (1993). Fully differentiated juvenile thalli (with podetia of 4-5 mm in height) carrying initials of fruit bodies of Cladonia fircata had developed after a time span of 7 - 8 months. Although the thalli have not reached the size of the adult lichens they show all characteristics (including

the small lobules that had grown out of the podetial stems) of young Cladonia furcata thalli as they are found in the natural environment.

Our presented results (Stocker-Worgotter and Tiirk 1994) show that soredia and tissue fragments of two selected Cladonia species develop or regenerate very quickly into crowded associations of basal squamules carrying podetia with initials of fruiting bodies. It remains still open if fiutifi- cation can be obtained in in vitro cultures.

Discussion

Culture of cyanobacterial and green Peltigera species In agreement with Scott (1964) we found that mycelia of P. praetextata and other cyanobacterial Peltigera species only developed when the fungus came into direct contact with the cyanobionts or an extract of them. These findings indicate that the Pelrigera fungi, probably during their life in symbiosis with Nostoc and (or) Coccotnyxa had adapted very specialized nutrient requirements.

Later culture experiments with mycobionts of cyanobacterial and green Peltigera revealed the possibility to grow these fungi without photobionts, but in media containing substances available for the mycobionts in symbiosis such as nitrogenous compounds, ribitol or glucose, and soil extract. In contrast to Stone and Denison' we found that our tested Peltigera mycobionts grow without adsorbants by using an enriched medium. In the improved media no inhibition of germination and growth was observed. The question whether growth in nature is accelerated by phytohormones released from associated bacteria (as found in symbioses between higher plants and bacteria; Werner 1992) is almost open. The possibility that nitrogenous compounds play an essential role for the growth of Peltigera fungi was discussed by Critten- den et al. (1994).

It became clear that the slow growth of Peltigera fungi had handicapped former manipulations with these lichens in the laboratory, e.g., resynthesis experiments conducted with P. canina by Ahmadjian (1989). Part of the culture problems with Peltigera have been overcome by using our method of long-term cultivation on soil substrate. With this method an artificial resynthesis of thalli of the cyanobacterial P. prae- rextata and also with the photosymbiodeme P. leucophlebia was achieved. Soil substrate was also used with success for the cultivation of isidia of P. praetextata. Thallus primordia and young thalli were formed more rapidly than by resynthesis.

Former investigations to culture discs cut from thalli of P. praerexrata were conducted by Scott (1960). Fragments of P. praetextata were studied by- Yoshimura and ~ a m a m o t o (1991) using their tissue culture method. On agar plates young thallus primordia were obtained after 1 year. As the water content of agar plates could not be regulated, the experiment was stopped, before thallus differentiation had been completed.

Photosymbiodemes or lichens representing a tripartite symbiosis are of great interest for culture experiments. Studies

' Stone, J . , and Denison, W. 1994. Regulation and germination of lichen ascospores by endogenous inhibitors. Fifth International Mycological Congress. August 14 - 2 1, Vancouver, B.C. Poster communication.

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on three-membered lichens allow to show how the morphogenesis of one mycobiont can be influenced by different not related photobionts, in the case of P. leucophlebia cyanobacteria and green algae.

In agreement to field studies of Brodo and Richardson (1978) and Ott (1988) it was found that the green thallus primordia of P. leucophlebia differentiated on a cyanobacterial crust covering the soil substrate or even on cyanobacterial lobules (homeomerous thalli formed by the lichen fungus and cyanobacteria). The differentiation of mixed primordia (composed of fungus, cyanobacteria, and green algae) occurred probably as a reaction to light. Field studies of other photosymbiodemes conducted by Poelt (1986) revealed that different intensities of sunlight obviously influenced the morphogenesis of the fungus and determined whether the cyanobacterial or the green morphotype was differentiated. The green morphotype dominated under higher light intensities, whereas the cyanobacterial morphotype was found growing under lower light intensities. Between the green and cyanobacterial phototypes mixed morphotypes were present. In our cultures, these mixed stages were first formed, but disintegrated before a mixed morphotype could develop. In the soil cultures only the green stages survived growing into mussel-shaped thalli. A heteromerous thallus was formed by the P. leucophlebia mycobiont only with the green photobiont; the capacity to form also heteromerous thalli with cyanobacteria (like the thalli of cyanobacterial Peltigera species) was missing. Field investigations of Tonsberg and Holtan-Hartwig (1983) confirmed the absence of a cyanobacterial morphotype of P. leucophlebia in nature. In our resynthesis experiment we found that both photobionts cyanobacteria and green algae are lichenized, but stages with cyanobacteria rested incomplete or disintegrated.

From our results we conclude that the formation of cephalodia was mainly regulated by ecological factors. Fungus- infected Nostoc entered the green thallus cups after watering the cultures or under very moist conditions and colonized small concavities of the green thallus. The growth of the cephalodia was closely connected with the release of hormogonia, occurring only under very moist conditions. The differentiation of the cephalodiate cortex occurred during the next desiccation phase. Finally the growth of the cephalodia may be reduced by a very limited nutrient supply on the green thallus and also by the great number of Nostoc cells transforming from photoactive cells into heterocysts.

The question which internal factors may increase the number of heterocysts within the cephalodia is still open. A regulation by poyols, e.g., Ribitol is suggested by Rai (1988) but not proved experimentally. Rai (1988) indicated that the greatest part of the heterocysts is localized in the center of the cephalodia, whereas the photosynthetic active and coloured cells are found directly below the cephalodiate cortex.

Additional investigations showed that the related P. aphthosa can be cultivated by fragments both in the laboratory and also in the field. In this case the development of the green thallus primordia occurred after a very similar sequence and also the morphogenetic features of the developmental stages resemble strongly the thallus formation obtained with P, leucophlebia. On soil cultures only green thalli with cephalodia were formed. Cyanobacterial thalli were not differentiated.

Other tissue fragment cultures of P. aphthosa have been

conducted by Kinoshita (1993) using agar plates. Depending on the moisture content and light intensities on the agar plates, the cyanobacterial morphotype of P. aphthosa was formed. This result is astonishing, as the cyanobacterial morphotype seems not to exist in nature. On the other hand this experiment shows that the mycobiont of P. aphthosa has the potential or morphogenetic capacity to form a cyanobacterial thallus when grown in a medium of high moisture content. This result agrees with field observations of Holtan- Hartwig (1993) who reported that the cyanobacterial morphotype of the related photosymbiodeme P. britannica is preferentially found in humid and shady habitats.

The features of species of the genus Peltigera are confus- ing for taxonomists. In the future, molecular genetic experiments should be able to define the genetic relationships between cyanobacterial, cephalodiate, and very spectacular Peltigera species forming two morphotypes like P. britannica. Furthermore, by improving culturing, we hope to pro- vide molecular biologists with more standardized mycobiont material of these fascinating symbiotic systems.

In conclusion our results with selected Cladonia species show that soredia and also tissue fragments developed into primordia and young thalli (thallus squamules with podetia) more rapidly than by resynthesis. Thalli cultivated on soil were almost identical with thalli grown in nature. Burnt clay, showing nearly similar properties as soil was tested for several Cladonia species by Jahns (1993).

Former resynthesis experiments with species of the genus Cladonia (Cl. pyxidata, the North American C1. cristatella) were conducted by Thomas (1939) and by Ahmadjian and co-workers (Ahmadjian 1966; Ahmadjian et al. 1980; Ahmadjian and Jacobs 1981, 1983). Yoshimura et al. (1993) succeeded in culturing thallus squamules from fragments of C1. hurnilis on agar plates.

In comparison with the resynthesis cultures our soredia and tissue-fragment cultures revealed a very similar sequence of developmental stages and also the morphogenesis of the primordia and thallus squamules occurred more or less after the same scheme already described for foliose lichens.

A surprising result of other unpublished experiments was that species of the Cladina group (e.g., C1. rangiferina, C1. portentosa) did not show the excellent capacity of thallus regeneration found with C1. furcata.

Acknowledgements

In particular I thank Roman Tiirk for valuable discussions and his support in the cultivation project. Furthermore I am very grateful to the Fonds zur Forderung der wissenschaft- lichen Forschung for financing the project 9171-BIO. Many thanks to Roberto Zorer for help with the computer drawings. This investigation was also supported by the dsterreichische Akademie der Wissenschaften (Apart-stipendium).

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Experimental cultivation of lichens and lichen symbionts