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
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12

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,
For personal use only.

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 o n lichens, especially culture experi-
ments, have a long tradition of failure o r of very limited suc-
cess (Ahmadjian 1959, 1962, 1973, 1993; Ahmadjian and
Heikkila 1970; Ahmadjian et al. 1980).
Recent advances in "lture techniques may be attributed
generally to progress in different fields of experimental
Received August 15, 1994.
E. Stocker-Worgotter. Institute of Plant Physiology,
University of Salzburg, Hellbrunner Str. 34, A-5020
Salzburg, Austria.
Can. J. Bot. 73(Suppl. 1): S579-S589 (1995). Printed in Canada I ImprimC au Canada
improved clean work benches allow manipulations under
axenic conditions, and both adjustable culture chambers and
phytotrons can b e used t o culture plants under various com-
binations of conditions.
In the opinion of Ahmadjian (1987), lichens are among
the most difficult organisms to work with experimentally.
Compared with other free-living fungi, mycobionts grow
much more slowly and have more complex nutrient require-
ments. This is not surprising since a great variety of fungi
are involved in the lichen symbioses and the polyols released
by green and blue-green photobionts are different. During
the last two decades, f o r certain species, (e.g., Endocalpon

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 pusil-
lum, Peltigera aphthosa, P. didactyla, P. leucophebia, P. prae-
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
textata, 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 ser-
ies of ex~erimentsare 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
For personal use only.
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 cul-
tures, 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) con-
taining 0.25 g KN031L and soil extract was prepared from
soil from the habitat of the lichen. LBM (+ additional sub-
stances) was sterile filtrated. The cultures were maintained
in a culture chamber at 20°C without light.
Mycobionts isolated from alpine Cladonia species (Cl. fur-
cata, Cl. jmbriata) were grown in liquid and solid LBM
containing 1 % Ribitol instead of glucose.
Can. J. Bot. Vol. 73 (Suppl. I ) , 1995
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. praetex-
tata) 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 frag-
ments 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 resyn-
thesis 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).
 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
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
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 photo-
bionts. 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 condi-
For personal use only.
tions 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 con-
tact with a compatible photobiont. Our first experiments con-
firmed 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 devel-
opment (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 pri-
mordia and from fused soredial clumps small lobules differ-
entiated (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 sub-
strate or emerged marginally from cyanobacterial lobules
formed by the lichen fungus with cyanobacteria. The fact that
in our cultures green primordia differentiated from cyano-
bacterial 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
 S582 Can. J. Bot. Vol. 73 (Suppl. I ) , 1995

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.
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
For personal use only.
 D4.2. Stocker-Worgotter

Figs. 8-13. Pelt~gerapraerextuta, 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.
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
For personal use only.
 Can. J . Bot. Vol. 73 (Suppl. I ) , 1995

Fig. 14. Artificial resynthesis of the photosymbiodeme Initially the first green lichenized stages were globules;
Peltigera leucophlebia. 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 cyanobac-
terial 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
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12

thalli with an average size of 2-3 mm were obtained after

6-8 months in culture. After 1 year the largest thalli mea-
sured 5 -7 mm.
Development of the green thallus
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
mixed primordia formed by
side of the cup where it had been attached to the substrate.
pure cyanobaclerial
......
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
For personal use only.

.....................................................................................
............................
..........
............ . . . . . . . . . . . . . . . .............
. . . . . . . . . . . . . .............. . ....
.......
-- .- ,.-. . , to be colonized by the cyanobacteria after the thalli had
511 ( r n l 2011 u l n 1 SO 11111 S&Gn
green tliallus primordla on a green thallus primordia differentiated small concavities (Fig. 14, step 7).
cyanobacterial prothalhls on acyanobacterial crust Microscopical examination showed that the Nostoc colo-
0green algae 0 cyanobacteria nies, 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 pre-
sented 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 differen-
tiated. The field cultures are always endangered to be over-
Dart of thallus
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 cyanobac-
teria entered the green thalli by watering after an incubation
green thalli with cephalodia cephalodium o n a green thallus
time of more or less 1 year. After an additional 2 months
14 and 18 month^ nld vertical section (12- 14 months) many cephalodia can be found within the
 D4.2.Stocker-Worgotter

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.
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
For personal use only.
 S586 Can. J. Bot. Vol. 73 (Suppl. I ) , 1

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.
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
For personal use only.

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 rorn
cephalodiurn. soredia. Initial growth reactions were apparent after 2 we:eks
 D4.2. Stocker-Worgotter
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
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
parts of the squamule. From these areas podetia began to
grow up. Figures 26 and 27 show different stages of podet-
ium 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
For personal use only.
, 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 avail-
able for integration. On certain parts of the tissues the sym-
biotic 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 devel-
opmental 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 cyanobac-
terial and green Peltigera revealed the possibility to grow
these fungi without photobionts, but in media containing sub-
stances 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 resyn-
thesis.
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 sym-
biosis 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.

on three-membered lichens allow to show how the morpho-
genesis of one mycobiont can be influenced by different not
related photobionts, in the case of P. leucophlebia cyanobac-
teria 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 cyanobac-
terial crust covering the soil substrate or even on cyanobac-
terial lobules (homeomerous thalli formed by the lichen
fungus and cyanobacteria). The differentiation of mixed pri-
mordia (composed of fungus, cyanobacteria, and green algae)
occurred probably as a reaction to light. Field studies of
other photosymbiodemes conducted by Poelt (1986) revealed
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
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 intensi-
ties, 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 photo-
For personal use only.
biont; 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 cyano-
bacterial 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 cepha-
lodia 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 hormo-
gonia, 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. aph-
thosa 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, leu-
cophlebia. On soil cultures only green thalli with cephalodia
were formed. Cyanobacterial thalli were not differentiated.
Other tissue fragment cultures of P. aphthosa have been
Can. J. 601. Val. 73 (Suppl. I ) , 1995
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 cyano-
bacterial 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 mor-
photype 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 experi-
ments should be able to define the genetic relationships
between cyanobacterial, cephalodiate, and very spectacular
Peltigera species forming two morphotypes like P. britan-
nica. 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).
References
Ahmadjian, V. 1959. A contribution towards lichen synthesis.
Mycologia, 51: 56-60.
Ahmadjian, V. 1962. Investigation on lichen synthesis. Am.
J. Bot. 49: 277-283.
Ahmadjian, V. 1966. Artificial reestablishment of the lichen
 D4.2. Stocker-Worgotter
Cladonia cristatella. Science (Washington, D.C.), 151:
199-201.
Ahmadjian, V. 1973. Methods of isolating lichen symbionts and
thalli. In The lichens. Edited by V. Ahmadjian and M.E.
Hale. Academic Press, New York. pp. 653-659.
Ahmadjian, V. 1987. Laboratory culture of lichens and lichen
symbionts. Proceedings of the Symposium on Tissue Culture
of Lichen and Bryophyte, April 23, Kyoto. Edited by Nippon
Paint, Osaka. pp. 1- 13.
Ahmadjian, V. 1989. Studies on the isolation and resynthesis of
bionts of the cyanolichen Peltigera canina (Peltigeraceae).
Plant Syst. Evol. 165: 29-39.
Ahmadjian, V. 1993. The lichen symbiosis. John Wiley & Sons,
New York.
Can. J. Bot. Downloaded from www.nrcresearchpress.com by Entomology on 09/24/12
Ahmadjian, V., and Heikkila, H. 1970. The culture and
resynthesis of Endocarpon pusillurn and Staurothele clopima.
Lichenologist, 4: 259 -267.
Ahmadjian, V., and Jacobs, J.B. 1981. Relationship between
fungus and alga in the lichen Cladonia cristatella Tuck.
Nature (London), 289: 169- 172.
Ahmadjian, V., and Jacobs, J.B. 1983. Algal fungal
relationships in lichens: recognition, synthesis and
development. In Algal symbiosis. Edited by J.L. Goff.
Cambridge University Press, Cambridge. pp. 147- 172.
Ahmadjian, V., Russell, L.A., and Hildreth, K.C. 1980.
Artificial reestablishment of lichens 1. Morphological
interactions between the phycobionts of different lichens and
For personal use only.
the mycobionts Cladonia cristatella and Lecanora
chrysoleuca. My cologia, 72: 73 - 89.
Anderson, K.A., and Ahmadjian, V. 1962. Investigation on the
development of lichen structures in laboratory controlled
cultures. Sven. Bot. Tidskr. 56: 501 -506.
Bischoff, H.W., and Bold, H.C. 1963. Some soil algae from
enchanted rocks and related species. University of Texas
Publications No. 6318. Phycol. Stud. 4: 1-95.
Boissibre, J.C., Boissibre, M.C., Champion-Arnaud, P.,
Lallemant, R., and Wagner, J. 1987. Le cycle des Nostoc
des genres Peltigera et Collema en cultures in vitro et dans
le thalle lichknique. Can. J. Bot. 65: 1468-1477.
Brodo, I.M., and Richardson, D.H.S. 1978. Chimeroid
associations in the genus Peltigera. Lichenologist, 10:
157 - 170.
Crittenden, P.D., Kalucka, I., and Oliver, E. 1994. Does
nitrogen supply limit the growth of lichens. Cryptogam. Bot.
4: 143-155.
Esser, K. 1976. Kryptogamen: Blaualgen, Algen, Pilze,
Flechten. Springer-Verlag, Berlin.
Holtan-Hartwig, J. 1993. The lichen genus Peltigera, exclusive
of the P. canina group, in Norway. Sommerfeltia, 15: 1-77.
Jahns, H.M. 1993. Culture experiments with lichens. Plant Syst.
Evol. 187: 145- 174.
Kinoshita, Y. 1993. The production of lichen substances for
pharmaceutical use by lichen tissue culture. Kyoto
University, Japan. pp. 1-77.
Lallemant, R. 1985. Le dCveloppement en cultures pures in vitro
des mycosymbiotes des lichens. Can. J. Bot. 63: 681 -722.
S589
Lallemant, R., and Bernard, T. 1977. Obtension de cultures
pures des mycosymbiontes du Lobaria laetevirens (Lightf.)
Zahlbr. et du Lobaria pulmonaria (L.) Hoffm., le role des
gonidies. Rev. Bryol. Lichenol. 43: 303 -308.
Lallemant, R., and Savoye, D. 1985. Lectins and
morphogenesis: facts and outlooks. In Lichen physiology and
cell biology. Edited by D.H. Brown. Plenum Press, New
York. pp. 335-350.
Ott, S. 1987. Sexual reproduction and developmental adaptations
in Xanthoria parietina. Nord. J . Bot. 7: 219-228.
Ott, S. 1988. Photosymbiodemes and their development in
Peltigera venosa. Lichenologist, 20: 361 -368.
Poelt, J. 1986. Morphologie der Flechten. Fortschritte und
Probleme. Ber. Dtsch. Bot. Ges. 99: 3-29.
Rai, A.N. 1988. Nitrogen metabolism. In Handbook of
lichenology. Vol. I. Edited by M. Galun. CRC Press, Boca
Raton, Fla. pp. 201 -237.
Schuster, G. 1985. Die Jugendentwicklung von F1echten.-Ein
Indikator fiir Klimabedingungen und Umweltbelastung. Bibl.
Lichenol. 20: 1-206.
Scott, G.D. 1960. Studies of the lichen symbiosis. 1. New
Phytol. 59: 374-381.
Scott, G.D. 1964. Studies of the lichen symbiosis. 2. Ascospore
germination in the genus Peltigera. Z. Allg. Mikrobiol. 4:
326-336.
Stocker-Worgotter, E., and Tiirk, R. 1990. Thallus formation of
the cyanobacterial lichen Peltigera didactyla from soredia
under laboratory conditions. Bot. Acta, 103: 315 -321.
Stocker-Worgotter, E., and Tiirk, R. 1991. Artificial resynthesis
of thalli of the cyanobacterial lichen Peltigera praetextata
under laboratory conditions. Lichenologist, 23: 127- 138.
Stocker-Worgotter, E., and Tiirk, R. 1994. Artificial resynthesis
of the photosymbiodeme Peltigera leucophlebia under
laboratory conditions. Cryptogam. Bot. 4: 300-308.
Stocker-Worgotter, E., Xavier-Filho, L., and Tiirk, R. 1994.
Laboratory culture of lichen fragments of two selected
tropical Cladoniaceae (Cladonia verticillaris, Cl. substellata)
from the Northeast Coast of Brazil. Cryptogam. Bot. 4:
309-313.
Thomas, E. 1939. ~ b e dier Biologie von Flechtenbildnern.
Beitr. zur Kryptogamenflora der Schweiz, 9: 1-206.
Tonsberg, T., and Holtan-Hartwig, J. 1983. Phycotype pairs in
Nephroma, Peltigera and Lobaria in Norway. Nord. J. Bot.
3: 681 -688.
Werner, D. 1992. Symbiosis of plants and microbes. Chapman
& Hall, London.
Yamamoto, Y. ,l990. Studies of cell aggregates and the
production of natural pigments in plant cell culture.
Yoshimura, I., and Yamamoto, Y. 1991. Development of
Peltigera praetextata lichen thalli in culture. Symbiosis, 11:
109- 117.
Yoshimura, I., Kurokawa, T., Yamamoto, Y., and Kinoshita, Y.
1993. Development of lichen thalli in vitro. Bryologist, 96:
412-421.