Bull. Natn. Sci. Mus., Tokyo, Ser. B, 31(4), pp. 127–131, December 22, 2005

Lobule Formation from Isidia in Parmotrema tinctorum
Yoshiaki Kon1 and Hiroyuki Kashiwadani2
1Kohoku Senior High School,4–14–30 Nishiayase, Adachiku, Tokyo, 120–0014 Japan
E-mail: yoshkon@xw.catv.ne.jp
2
Department of Botany, National Science Museum,4–1–1 Amakubo, Tsukuba, 305–0005 Japan
E-mail: hkashiwa@kahaku.go.jp

Abstract Vegetative diaspores (isidia) of Parmotrema tinctorum were kept in nylon bags. Thesenylon bags were stapled on the trunks of Cryptomeria japonica. Morphological changes of isidia
were observed every three months after stapling in the natural field. Several protuberances wereformed on the isidia six months after stapling. Isidia with protuberances at this stage were anchored by thin bundles of hyphae to the nylon bags. These protuberances developed into dorsiventral lobules with rhizines 12 months after stapling and the lobules were stratified as seen in the natural thalli of P. tinctorum. Lecanoric acid and atranorin were produced in the newly developed lobules.
Key words : Isidia, lobules formation, Parmotrema tinctorum.

Introduction
In contrast to the sexual reproduction of themycobiont which requires re-lichenization with asuitable photobiont, the asexual reproduction bysoredia, isidia or schizidia are regarded as ideal
adaptations to lichens which are composed offungal and algal partners. Unfortunately, however, we have only a limited knowledge on the development of these vegetative propagules into
thalli either in the field or under culture. Scheidegger (1995) reported early development ofisidioid soredia in Lobaria pulmonaria andSchuster et al. (1985) also reported early growthand development of soredia for Hypogymnia
physodes and Physcia tenella in the natural environment. Stocker-Wörgötter & Türk (1988) also
succeeded producing young thalli from sorediaof Peltigera didactyla. However, development oftrue isidia has been observed only in Peltigera
praetextata, a cyanobacterial lichen (StockerWörgötter & Türk, 1995).
Parmotrema tinctorum (Nyl.) Hale is a common foliose lichen containing Trebouxia (a greenalga) as the photobiont. It is widely distributed intemperate and tropical areas in the world, being
easily recognized by the e-ciliate rounded lobes
with dense isidia and by the distinct C red reaction in the medulla. It has the potential for two
divergent strategies of reproduction, i.e. sexual,
through ascospores ejection, and vegetative by
the symbiotic propagules, isidia. However, fertile
specimens are found only rarely in luxuriant populations and they are absent in most localities inJapan. Therefore, dispersal by ascospores may
play only limited role in reproduction in mostpopulations. In contrast, isidia are regularly
found even in small thalli in small populations
and may play major role in dispersal.
In the present paper, the successful development of juvenile lobules from isidia will be reported for Parmotrema tinctorum. The structureof both young and mature isidia was also observed.
Materials and Methods
Isidia used for the present study were collectedfrom Parmotrema tinctorum growing on thetrunks of trees at Mt. Kiyosumi, Chiba Prefec-

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Yoshiaki Kon and Hiroyuki KashiwadaniFig. 1.

Nylon bag stapled on the trunks of Cryptomeria japonica. Scale10 mm.

ture. They were cut off with a knife from thalliand kept in bags about 55 cm in size whichwere made of 150 m m meshed nylon. Each bag
contained about 30 cylindrical or branched isidia(ca. 100–150 m m in length). These nylon bags
were stapled on the trunks of Cryptomeria japonica D. Don growing near by the collecting site
(Fig. 1). Developmental stages of isidia were investigated by light microscopy and scanningelectron microscopy every three months after stapling. For light microscopic observation, sections
were made by using a freezing microtome andwashed with acetone solution in order to remove
the secondary substances such as lecanoric acidand atranorin. Naturally dried specimens werecemented on specimen-holders, sputter coatedwith about 100 Å gold, and examined at 15 kV ina JEOL JSM-5410LV scanning electron microscope. Lobules newly formed on the isidia werechemically tested by the standardized thin-layer
chromatographic method (Culberson, 1972).
Results and Discussion
Formation of isidia
In the initial stage, isidia appear to be small
verrucae on the upper surface in P. tinctorum. In

this stage (Fig. 2B), the verrucae are filled withalgal cells, which are mostly the same as those of
the medulla in size and have cortex extended
from the upper cortex of the thallus. As isidia
grow a little further (Fig. 2C) and become shortcylindrical projections, algal cells increase innumber. It is noteworthy that algal cells formed
inside of the isidial projections are smaller in size
comparing those derived from or in the thallus.
In the later stages, these projections become distinct cylindrical isidia (Fig. 2D), which can berecognized even with the naked eye. In this stage,algal cells are concentrically distributed below
the cortex and medullary hyphae of the thallus
penetrate into the central part of isidia (Fig. 2A).
However, no distinct stratification is observed.
Formation of juvenile thalli from isidia
Nylon bags staled on the trunks of Cryptomeria japonica contained about 30 isidia in various
stages of development. However they were mostly cylindrical or coralloid. In the present study,
cylindrical isidia were mainly observed. Cylindrical isidia were about 75 m m in diameter and
100–200 m m long (Fig. 3A).
The cut end of isidia was exposed when thenylon bags were stapled (Fig. 3A). Three months

Lobule formation from isidia in Parmotrema tinctorum

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Fig. 2. Inner structure of isidia of Parmotrema tinctorum. A: Cross-section of mature isidia formed on the thalliof P. tinctorum. Algae are distributed concentrically under the cortex of the isidia and hyphae are situated in
the center of isidia. B–D: Longitudinal section of young and mature isidia of P. tinctorum. B: Juvenile isidia.
C: Young isidia. D: Mature isidia. (A–C scales50 m m, D scale100 m m).

after stapling, it was covered by mycobiont hyphae which seemed to adhere each other withgelatinous substances (Fig. 3B). In the present
study, isidia of P. tinctorum did not seem to be
fixed on 150 m m nylon mesh.
Six months after stapling, a clear morphological changes of isidia were observed. In this stage,
each isidium produces several verrucae or protuberances 20–30 m m broad and 50–60 m m long(Fig. 3C). There is no distinct boundary between
isidia and verrucae or protuberances and cortex
of isidia is spontaneously joined to those of verrucae or protuberances. On some of them, bundles of hyphae tightly anchoring to the substratum (nylon fiber) were observed (Fig. 3C). They
are comparable with anchoring hyphae reported
in the early stages of development of isidioidsoredia of Lobaria pulmonaria by Scheidegger(1995).
Twelve months after stapling, protuberancesdeveloped into dorsiventral lobules, which werepale gray and 0.2–0.35 mm broad and 0.2–0.3 mm long (Fig. 3D).
The cortex was often disintegrating at thebasal parts of lobules as well as on the isidia(Fig. 3D). In this stage distinct rhizines were observed on the lower surface of the lobules (Fig.
3E). At present, however, it is uncertain whether
these rhizines have derived directly from anchoring hyphae mentioned above or not. It should be
noted here that lobules in this stage were clearlystratified, upper and lower cortices, algal layer
and medulla being recognized (Fig. 3F), being
quite similar to thalli of P. tinctorum in nature,

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Yoshiaki Kon and Hiroyuki KashiwadaniFig. 3. Scanning electron micrography of developmental stages of lobule formation observed in the field and alobule. A: Cylindrical isidium removed from a thallus. B: Isidium three months after stapling. The cut end(arrow) is covered with hyphae. C: Verrucae and protuberances formed on the isidia six months after stapling, showing bundles of hyphae anchoring to the substratum (arrow). D: Lobules formed on the isidia 12months after stapling. E: Lobules 12 months after stapling, showing rhizines (arrow) on the lower surface. F:Cross-section of lobules formed on isidia 12 months after stapling. Stratified composition is similar to that oforiginal thalli of P. tinctorum, though the medulla is rather thin. Scale100 m m.

though the medulla was rather thin. In addition,
secondary products of newly formed lobules
were tested by the TLC method and lecanoricacid and atranorin, constant components in P.

tinctorum, were demonstrated on the chromatograms.
Stocker-Wörgötter & Türk (1995) observedthat isidia transformed into soredial primordial atLobule formation from isidia in Parmotrema tinctorum

first and then small lobules were differentiatedfrom fused soredial clumps in Peltigera praetextata. In P. tinctorum, however, such developmental process was not observed in any stages andverrucae or protuberances were formed directly
on isidia and they developed into small lobules.
When Scheidegger (1995) investigated early development of isidioid soredia of Lobaria pulmonaria, he reported that apical and lateral
pseudomeristematic growth zones were formed
six months after transplantation and then thegrowth zones further differentiated into small
lobes which have a stratified thallus as seen in foliose lichens. In P. tinctorum, however, verrucae
or protuberances were formed in the early stages
and they directly developed into lobules. Thus,verrucae or protuberances can be considered tobe equivalent to pseudomeristems observed byScheidegger (1995). It is noteworthy, on the otherhand, these verrucae or protuberances resemble
very much those observed on the periphery of
the transplanted fragments of P. tinctorum (Konet al., 2003).
Isidia of P. tinctorum are cylindrical and algalcells are concentrically distributed inside of thecortex and protuberances formed on isidia seemto have a similar inner morphology as isidia. Incontrast, lobules developed from isidia are clearly stratified as mention above, though the medulla is rather thin (Fig. 3F). Therefore, dispositionof algal cells and fungal hyphae should bechanged, while dorsiventral lobules are developing, although such process has not been observedin the present study.
It is now obvious that isidia removed from thethallus commonly produce many protuberancesand juvenile lobules in six to twelve months after

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detaching from the thallus. As having been suspected by most lichenologists, thus, isidia can beconsidered to play a major and important role indispersal in P. tinctorum. Since regeneration ofisidia has never been observed while they adhere
onto the thallus, formation of verrucae or protuberances on isidia observed through the present
study seems to be triggered by detaching fromthe thallus.
Acknowledgements
We express our sincere thanks to Dr. S.
Kurokawa, Botanical Gardens of Toyama for his
valuable suggestion and criticism.
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