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The Lichenologist 37(5): 445–462 (2005) 2005 The British Lichen Society
doi:10.1017/S0024282905014982 Printed in the United Kingdom
Isidia ontogeny and its effect on the CO
2
gas exchanges of the
epiphytic lichen Pseudevernia furfuracea (L.) Zopf
Mauro TRETIACH, Paola CRISAFULLI, Elena PITTAO, Simona RININO,
Enrica ROCCOTIELLO and Paolo MODENESI
Abstract: The development of isidia in thalli of Pseudevernia furfuracea from the Carnic Alps
(North-eastern Italy), and the effects of these structures on CO
2
gas exchanges were investigated. The
ontogenetic events were studied by comparison of sections stained with different histochemical tests
and SEM observations. A high cell turnover rate in both symbiotic partners is the first sign of isidium
development, followed by an increased aplanosporogenesis of algae and growth of neighbouring
medullary hyphae which become oriented upwards. Large nuclei and an intense cytoplasm activity
characterize the mycobiont cells. The surface of very young isidia shows an irregular structure of
spherical to ovoid protruding tips of perpendicular cortical hyphae, that are later organised in a
pseudomeristematic area similar to that observed in the apex of growing lobes. CO
2
gas exchange
measurements carried out in the laboratory confirmed the high metabolic activity of isidia. At optimal
water content and favourable light conditions, isolated isidia had rates of gross photosynthesis and
dark respiration that were twice those of non-isidiate lobes. Isolated isidia also had a very low CO
2
saturation point, probably because of their favourable surface/volume ratio, and a high light
saturation, probably linked to their high content of photosynthetic pigments. The different roles
played by isidia in the biology of Pseudevernia furfuracea, and particularly their rejuvenating effect on
aged lobes, are discussed, and the presence of thalloconidia is briefly mentioned.
Key words: chlorobiont, hydration, mycobiont, morphogenesis, ontogeny, photosynthesis, respir-
ation, thalloconidia, vegetative propagule
Introduction
One of the most striking features of the
lichen symbiosis is the production of a wide
spectrum of symbiotic vegetative diaspores
that permit the simultaneous dispersal of
both symbionts (Honegger 1998, 2001). In
this way the lichen symbiosis by-passes
the problem that any germinating sexual
ascospore encounters, i.e. to find a com-
patible photobiont partner to re-establish
the symbiotic phenotype (Poelt 1993;
Walser et al. 2001).
Soredia and isidia are the most frequent
type of diaspores produced by lichens
(Büdel & Scheidegger 1996). Corticated,
variable in size and shape, isidia are particu-
larly frequent in foliose and fruticose species
of Collemataceae,Parmeliaceae,Peltigeraceae,
Pyxinaceae,Stictaceae, and Umbilicariaceae
(Bowler & Rundel 1975), being rarer in
crustose species, although isidia occur
regularly in members of, for example,
Pertusariaceae (Dibben 1980). Soredia have
been extensively studied from many points
of view (Bailey & James 1979; Armstrong
1987, 1990, 1991; Raineri & Modenesi
1988; Stocker-Worgotter & Türk 1989,
1990; Tretiach & Carpanelli 1992; Marshall
1996; Lorentsson & Mattsson 1999; Zoller
et al. 2000; Nimis & Martellos 2003), but, in
contrast, our knowledge concerning isidia is
relatively poor. The most recent studies
have been mainly focused on their dispersal,
establishment and survival (Gilbert 1988;
Scheidegger et al. 1995a; Zoller et al. 2000),
M. Tretiach (corresponding author), P. Crisafulli
and E. Pittao: Dipartimento di Biologia, Università di
Trieste, Via L. Giorgieri 10, I-34127 Trieste, Italy.
S. Rinino, E. Roccotiello and P. Modenesi: DIP.
TE.RIS., Università di Genova, Corso Dogali 1/c,
I-16136 Genova, Italy.
or on development of the juvenile thallus
(Honegger 1987, 1993; Zoller et al. 2000).
The development of a dense cover of isidia,
however, causes many interesting modifica-
tions at the thallus level. The large increase
in surface area, for instance, modifies thallus
water absorption (Rikkinen 1997) and water
holding capacity (Jahns 1984), while the
darker colour typical of isidial outgrowths
probably influences the thermal operating-
environment (Kershaw 1983; Coxson et al.
1984).
The present paper analyses the results of a
multi-disciplinary investigation aimed at
clarifying two little known aspects of isidia:
the first stages of their ontogeny and the
influence they exert on the assimilative
capacity of the thallus. The locally common
fruticose lichen, Pseudevernia furfuracea (L.)
Zopf, was selected for the study because: (a)
it was readily available, (b) young lobes
devoid of isidia coexist on the same thallus
with old lobes completely covered with
isidia, and the two types of lobe can easily
be separated; (c) this species is often used
in ecophysiological (Türk 1983; Manrique
et al. 1993; Scheidegger et al. 1995b;
Vidergar-Gorjup et al. 2001; Kranner et al.
2003) and biomonitoring (Niewiadomska
et al. 1998; Bari et al. 2001; Vingiani et al.
2004) studies.
Material and Methods
Lichen sampling
Thalli of Pseudevernia furfuracea were collected in
January and December 2003 from the lower branches of
isolated Larch (Larix decidua Mill.) trees, near Sauris di
Sotto (Carnic Alps, Italy), at 1500 m altitude for study-
ing isidia development (Figs 1–4) and gas exchange
measurements (Figs 7–10). Thalli were air-dried in the
forest, then transported immediately in plastic bags to
the laboratory, where they were cleaned of bark frag-
ments and debris under a dissecting microscope. The
negative reaction to C (sodium hypochlorite, 10%),
which is diagnostic for var. furfuracea (Culberson et al.
1977), was verified in 100 randomly selected samples;
less than 3% of the samples tested belonged to var.
ceratea (Ach.) D. Hawksw.
Further samples were collected in June 2004 for
quantifying the increase in external surface area due to
the presence of isidia (Fig. 5), and for studying water
loss processes (Fig. 6). Thirty randomly selected thalli
were collected with a short piece of the twig on which
they were growing, put in closed plastic boxes,
c.445·5 cm, without touching them, and trans-
ported to the laboratory for further processing.
Isidium ontogeny
Observations were carried out by SEM, bright field
and fluorescence light microscopy on small apical por-
tions of thalline lobe tips, c. 0·5–1 cm long. For SEM
dried thallus fragments were directly coated with a
200–220Å thick layer of gold in a sputter coater and
then examined in a Philips 515 SEM at 20 kV.
For bright field microscopy thalline fragments were
fixed in buffered formalin (Pearse 1985) for 24 h,
dehydrated in an ethanol series (30, 50, 70, 80, and
95%) and embedded in JB4 resin (Polysciences, Inc.
Warrington, UK). Sections 7–10 m thick were cut
with a glass knife on a Reichert OM2 ultramicrotome.
The following histochemical tests were performed on
the JB4-embedded sections: (1) periodic acid-Schiff
(PAS) for general water-insoluble polysaccharides
localization (Pearse 1985); (2) Toluidine Blue O
(TBO) 0·05% in acetate buffer pH 4·4 for 1 min as
metachromatic stain (for mechanism of TBO meta-
chromatic reaction, see Giordani et al. 2003). Another
series of unfixed and cryostatic sections was used to
show acid phosphatase activity according to the azo dye
method of Burstone (1958).
For fluorescence light microscopy, thalline fragments
were embedded in Technovit 7100 (Hereaeus Kulzer,
Wehrheim). Sections 7 m thick were mounted serially
and observed unstained to show autofluorescence or
treated with 0·001% DAPI (4#,6-diamidino-2-
phenylindole, Sigma-Aldrich, St Louis, USA) for
nuclear staining (Regan & Moffatt 1990). Observations
were made using an optical microscope Leitz Dialux
22EB equipped with an epi-illuminating condenser
mounted with types A (UV= 340–380 nm) and H2
(blue= 390–490 nm) fluorescence filter blocks. Photo-
micrographs were taken using Kodak Ektachrome
160T colour reversal film.
Control reactions for all histochemical methods were
carried out according to the methods suggested by the
respective authors.
Estimation of isidial density and increase in
external lobe surface area
Thirty-six isidiate lobes were selected randomly from
12 thalli stored in plastic boxes, and observed under a
stereomicroscope Wild M420 with a Leica DC300
camera connected to a computer. Entire and broken
isidia were counted on the computer monitor covered
with a plastic film, marking them with a felt-tip pen,
continuously adjusting the focus of the image. The dry
weight of each sample was measured after 24 h at
120(C, and its surface area was measured with a
LI3000A Leaf Area Meter (Licor Inc., Lincoln, Ne,
USA), using enlarged, calibrated photocopies. The
isidia were then removed. A randomly selected sub-
sample of 100 isidia was mounted in glycerine, observed
446 THE LICHENOLOGIST Vol. 37
with a transmitted light microscope at 100 magnifi-
cation and their length and basal diameter were
recorded. The isidium lateral surface area was approxi-
mated to that of a cylinder with the same basal
diameter. The surface area of each lobe was then
estimated as the sum of the two faces, plus the sum of
the lateral surface areas of the isidium population,
taking into account also fallen isidia.
Thallus water loss
Water loss was measured gravimetrically after soaking
silica dried thalli of similar weight (c. 0·22 g each) for
5 min in distilled water, blotting or shaking dry, and the
rate of weight change was followed by weighing at
increasing time intervals (every minute for the first
10 min, then every two minutes for half an hour, and
finally every 30 min until air dry). Between weighings,
samples were simultaneously placed on a latticework
placed on a laboratory bench in dim light. The
measurements were carried out at 27(C, 40%RH;
temperature and air humidity were monitored with an
Assmann aspired psychrometer. Two sets of thalli,
strongly isidiate, and weakly isidiate, were used; each
set consisted of two samples. At the end of experiments,
the thalli were dried to constant weight at 120(C.
CO
2
gas exchange
Treatment of samples
Three sets of samples were prepared: isidiate lobes
(IL), non-isidiate lobes (NIL), and isidia (I). The latter
were obtained by gently shaking plastic bags containing
air-dried thalli. The samples of all materials, c. 500 mg
each, were dried over silica gel for 24 h, sealed in Petri
dishes and kept at 32(C until use. Concomitant
chlorophyll fluorescence measurements demonstrated
the particularly high stability of light reactions of P.
furfuracea after 6 months of low temperature storage, in
good accordance with the results of Feige & Jensen
(1987).
Before the experiments IL and NIL samples, consist-
ing of 12 to 16 lobes taken from different thalli, were
re-hydrated for 18 h in the dark in a closed chamber
filled with wet paper, then immersed in distilled water
for 30–40 sec, and then gently hand centrifuged. The
lobes were then placed in a Petri dish, covered with
a thin mesh to avoid the curling of wetted lobes, and
used in the CO
2
gas exchange measurements. If neces-
sary, in order to maintain hydration, the material was
gently sprayed with distilled water after each series of
measurements.
A different protocol was adopted for the samples of
isidia (I). These were also kept for 18 h in a humid
chamber, but then positioned with a soft brush on a
disc of commercial horticultural fleece (‘‘non-woven
fabric’’), stretched on a plastic ring of c. 10 cm diam.
The disc was laid for 120 min on a layer of Whatman
no. 1 filter paper soaked in distilled water, in a Petri
dish, which was placed in a yet larger petri dish lined
with wet paper. Half an hour before starting the CO
2
gas exchange measurements, samples were exposed to a
photosynthetic photon fluence rate (PPFR) measured
over the waveband 400–700 nm of 100 mol photons
m
2
s
1
, weighed after removing excess water on the
fleece disc with absorbing paper, and then used in
the experiments. Thallus relative water content (RWC)
was expressed as a percentage of dry weight (i.e.
RWC%= [(wet weightdry weight)/dry weight]
100). Following this protocol, hydration of isidia was
constantly around 150%, but only two hydration cycles
per day could be carried out, with a loss of c. 3–4% of
the isidia per cycle.
Water content, CO
2
, and light response curves
The CO
2
exchange rates of the three sets of samples
(IL, NIL, I) were characterized by building up response
curves to water content, CO
2
, and light, at a thallus
temperature of 171(C. Maximum net photosynthe-
sis (Ph
n
) and dark respiration (R
d
) at different CO
2
concentrations and light conditions were calculated as
the average of three to six values measured at optimal
thallus water content. Gross photosynthesis (Ph
g
) was
calculated as the sum of Ph
n
and R
d
.Attheendofthe
CO
2
gas exchange experiments, samples were dried to
constant weight over silica gel, and divided into two
parts, one for the estimation of their photosynthetic
pigments (see below), and the other for measuring their
dry weight at 120(C for 24 h, and then put over silica
gel for 2 h.
The CO
2
exchange rates were measured in a closed
system with a LICOR-6250 infrared gas analyzer
(IRGA) (Licor Inc., Lincoln, Ne, USA; estimated
precision 0·2–0·3 ppm CO
2
; cuvette volume
4000 cm
3
; internal volume 130 cm
3
; maximum flow
rate 1000–1100 mol s
1
), connected to a data-logger.
During the experiments, a natural light spectrum
generated by a halogen incandescent lamp was provided
by a Fl-400 fiber illuminator (Walz, Effeltrich,
Germany) and the PPFR was measured using a Licor
Quantum meter model LI 185A inserted within the
cuvette at the same position as the lichen thalli.
Chlorophylls and carotenoids
Samples of IL, NIL (n=10), and I (n= 16), each
c. 50 g, were dried over silica gel for 24 h, then repeat-
edly washed with 5 ml aliquots of pure spectrophoto-
metric grade acetone, until the solution did not turn
yellow after the addition of a drop of conc. KOH, to
remove the lichen substances (Brown & Hooker 1977).
Approximately, 8–10 washings were necessary for IL
and NIL, and 20–22 washings for I. Further samples,
consisting of seven single isidiate lobes, were cut in half
along their axis into two portions, c. 13–22 mg in
weight, and from one half isidia carefully removed
under a dissecting microscope; both groups of samples,
with and without isidia, were washed as above.
The samples were homogenized with a small aliquot
of polyvinyl polypyrrolidone in 15 ml DMSO for 50 mg
of material under dim, green light, the tube left
overnight at room temperature in the darkness, and
then centrifuged at 5000 rpm. The absorbance of
supernatant was measured with a spectrophotometer
Perkin-Elmer mod. 554. Estimation of chlorophylls and
total carotenoids was carried out using the equations of
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 447
448 THE LICHENOLOGIST Vol. 37
Pfeifhofer et al. (2002), and the ratio OD
435
/OD
415
was
calculated according to Ronen & Galun (1984).
Statistics
Statistics were carried out with the program Excel97
(Microsoft) and Statistica 6.0 (StatSoft). Polynomial
curves were obtained with the program Curvefit.
Results
Isidium ontogeny
Three zones can be recognised in a
growing lobe of Pseudevernia furfuracea: the
pseudomeristematic apical tip, covered by
an amorphous epicortex (Rikkinen 1997)
darkened by melanin pigments; the differen-
tiation zone (or elongation zone sensu
Honegger 1993), where the epicortex pro-
gressively deteriorates; and the fully differ-
entiated zone, where the epicortex is
completely absent. In the latter zone the
upper cortex consists of two morphologically
and histochemically different layers: the
outer layer, formed by anticlinally oriented
hyphae with free, swollen, PAS positive,
TBO negative tips, and a cementing, PAS
positive and TBO metachromatic matrix at
their base, and the inner layer, consisting in
a scleroplectenchyma of hyphae congluti-
nated by a PAS positive, TBO metachro-
matic matrix (Figs 1B, 2B, 3A & B).
The earliest developmental stages of an
isidium, according to our observations, can
be observed only in the fully differ-
entiated zone. The first event consists in the
increased aplanosporogenesis of a small
clump of algae, and is immediately followed
by the proliferation of the neighbouring
medullary hyphae, that become oriented
upwards (Figs 1A & B, 2A). At this stage the
photobiont cells are small in size, the small-
est occurring towards the upper cortex, and
have a reduced plastidial pigmentation due
to a lower chlorophyll content (Fig. 2D).
The hyphae surrounding these algae have
thin walls, intensely stained cytoplasm and
large nuclei, perfectly visible in DAPI
stained sections (Fig. 2B & C). The intense
metabolism of both myco- and photobiont is
shown by the pronounced activity of acid
phosphatase enzymes (Fig. 1G), associated
with dephosphorilative processes, which
are presumably involved in active intercellu-
lar movements of glucids (Gahan 1984).
Further algal proliferation forms a sort of
wedge penetrating into the upper cortex
(Figs 1B–D, 2A & B). Only at this stage the
conglutinated cortical cells in the immediate
proximity of the primordium show an
increased, anti- and periclinally oriented
division, becoming easily recognisable for
their large lumen and strongly stained
nucleus (Figs 1C & D, 2B). Also the com-
position of their wall is progressively modi-
fied, because both metachromatic reaction
to TBO and DAPI blue fluorescence are
weakened.
The growth of cortical cells, further
algal divisions and the constant intrusion of
medullary hyphae carrying the algal cells
upwards to produce a small protuberance
above the thallus surface (Fig. 1C–E). At
this stage the protruding tips of the cortical
cells bordering the young isidium appear
thicker and almost brown-black due to
deposition of dark brown pigments. These
pigments turn dark green when stained by
the ferrous ions technique, have a positive
green reaction with TBO, are bleached only
by strong oxidising agents and therefore can
be identified as melanins (Pearse 1985). The
F. 1. Light micrographs of transverse sections showing developmental stages of isidium formation in Pseudevernia
furfuracea. A, first stage, algal cells and medullary hyphae (arrow) penetrate the upper cortex, TBO test;
B, aplanosporogenesis of algae and oriented tip growth of neighbouring medullary hyphae in an initial stage of
development, the arrow points to a wall darkening due to early melanin deposition in the cortical cell walls, TBO
test; C–E, formation of an isidial primordium, subsequent anti- and periclinally oriented growth of cortical cells,
intrusion of medullary hyphae and algal division elevate a protuberance above the thallus surface, cortical cells
bordering an isidial primordium show darkening and thickening at their protruding tips, forming a brown-black cap
above the protuberance, TBO test; F, cortex, algal layer and medulla differentiated in a young isidium, TBO test;
G, Burstone test showing the strong reactivity for acid phosphatases of algal and cortical cells next to protuberance
of a young isidium. Scales: A= 10 m;B&F=30m; C=20 m;D&E=25m.
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 449
surface of the emerging structure is similar
to that of the pseudomeristematic zone at
the apex of growing lobes, being formed by
the spherical to ovoid protruding tips of
perpendicularly arranged hyphae (Figs 1E &
F, 2F, 3C). Internally, the young isidium is
completely filled by algae (Fig. 2E). When it
attains a height of c. 200–250 m, however,
a well-developed medulla is already differen-
tiated, and is connected to the medulla of
the lichen thallus (Figs 1F, 3D).
Mature isidia have a simple or branched
cylindrical body and are often constricted at
their bases (Fig. 3A). Scars left by detached
isidia may be frequently seen in untouched
or carefully handled thalli. Recent scars can
be easily recognized because they expose
photobiont cells and medulla. Older scars
are covered by a newly formed cortical layer,
and therefore are recognized as shallow
depressions on the thallus surface (Fig. 3B).
Further cortical structures of P. furfuracea,
which are present on both faces of the
dorsiventral thallus (Fig. 4A–C, F), are
superficially similar to young, developing
isidia. Almost spherical in shape (30–50 m
diam.) (Fig. 4A–F), they can be recognized
by their intense dark brown, verging upon
F. 2. Fluorescence light micrographs of transverse sections showing early developmental stages of isidium
formation in Pseudevernia furfuracea. A–C, DAPI staining, UV-A excitation light; D–F, autofluorescence, blue
excitation light. Scales: A–D= 10 m;E&F=20m.
450 THE LICHENOLOGIST Vol. 37
F. 3. SEM micrographs of surface features in Pseudevernia furfuracea. A, simple and branched mature isidia
occurring in a non-epicorticate thalline area, note the roughness of mature cortical surface due to minute crevices
separating protruding hyphal tips; B, scars left by detachment of isidia, the arrow points to recent scars exposing
photobiont cells, older scars are visible as depressions in the thallus surface covered by a newly formed cortical layer;
C, top of an isidial primordium, the surface shows an irregular structure of spherical to ovoid protruding tips of
anticlinally oriented cortical hyphae; D, transverse section through a mature isidium showing the continuum of
medullary spaces between isidium and lobe. E–F: surface of a lobe after the mechanical removal of isidia. Scales:
A, B, D & F= 100 m; C=10 m; E =200 m.
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 451
F. 4. Light and SEM micrographs of thalloconidia in Pseudevernia furfuracea. A, unstained section of the lower
cortex with three thalloconidia, the cutting plane omitted their base; B, thalloconidium on the upper cortex, the
base forming a continuum with the cortical cells, PAS test; C, cross-section of lobe with young isidium (arrow
marked ‘1’), epi- and hypothalline thalloconidia (arrows marked ‘2’), and pycnidium (arrow marked ‘3’), TBO test;
D, cross section of thalloconidium, TBO test; the arrows point to deposition of melanins;E&F,SEMmicrographs
of thalloconidia: external surface (E), and cross section (F). Scales: A= 50 m;B&D=25m; C=250 m;
E=10m; F=100 m.
452 THE LICHENOLOGIST Vol. 37
black pigmentation. These structures are
completely devoid of algae and are derived
from the proliferation of the cortical external
layer, to which they remain connected by a
very short strand of hyphae (Fig. 4B & C, E
& F), but forming a clear basal constriction.
The outgrowth, however, is not accom-
panied by the intrusion of medullary hyphae
as during isidium ontogeny. Their external
surface (Fig. 4) consists of strongly ad-
pressed, dematiaceous, more or less iso-
diametric cells, c. 3–4 m diam., whereas in
the internal portion wide intercellular spaces
divide somewhat larger cells (c. 6–8 m
diam.), characterized by scarcely pigmented
walls, in which deposits of polyphenolic pig-
ments (precursors of melanins), turning
green in TBO, are present (Fig. 4D). The
cytoplasm of the inner cells is less dense than
in the hyphae of a growing isidium (compare
Figs 1A–G, 2A–F with Fig. 4A–F) and they
have larger vacuoles and histochemical tests
for lipophilic substances show the presence
of numerous lipidic bodies.
Estimation of isidium density and
increase in external lobe surface
Isidium density ranged between 24 and
111 isidia mm
2
, with a mean value of
5518 isidia mm
2
. Broken isidia were
observed on all lobes, although their fre-
quency was rather variable, ranging from 1
to 13%. The density of isidia and frequency
of broken isidia were not statistically corre-
lated. Isidia were morphologically rather
heterogeneous, and therefore estimates of
their individual external surface ranged be-
tween 0·017 and 0·146 mm
2
, with a mean
value of 0·0630·027 mm
2
, and a basal
thickness of 8216 m(n=100). The fre-
quency distribution of homogenous classes
of 0·011 mm
2
each is shown in Figure 5.
The increase in external lobe surface
caused by the development of isidia was
estimated to range between 107 and 211%.
This noteworthy figure, obtained with a
minimum increase in weight, c. 14–63%,
is possibly still higher in deformed, over-
isidiate lobes, which were purposely
excluded from the present investigation.
Thallus water loss
Isidiate and non-isidiate thalli clearly dif-
fered in water storage capacity and speed of
water loss (Fig. 6). Isidiate thalli reached a
maximum RWC of 240–245%, against 150–
160% for non-isidiate thalli, and retained
water more efficaciously. This can probably
be explained by the increased external capil-
lary system of isidiate thalli, consisting of the
open spaces among isidia (Rikkinen 1997),
and the greater quantity of polysaccharides
and other hygroscopic materials covering, as
shown before, the external surface of isidia.
CO
2
gas exchanges
The response curves of CO
2
gas
exchanges of I, NIL, and IL to thallus water
content, CO
2
concentration, and light inten-
sity are presented in Figures 7–9, respect-
ively. Compared to NIL and IL, data for I
were exceptionally homogeneous, only small
differences being observed among duplicates
of the same material. In contrast, striking
differences were observed between the three
different materials, particularly in Ph
n
and
its dependence on thallus water content (Fig.
7). Depression of Ph
n
regularly occurred at
high thallus water contents (>150%) in NIL
but was only slight in IL and absent in I, with
an optimum at c. 100–120% RWC (see also
Scheidegger et al. 1995b). In contrast, no
significant difference was detected in R
d
,
which remained relatively constant (from
F. 5. Size distribution (by surface area) in 100 ran-
domly selected isidia of Pseudevernia furfuracea (size
classes increment by 0·011 mm
2
).
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 453
0·7 to 1·0 mg CO
2
g
1
h
1
) within a broad
range of RWC values. It must be emphasised
that the I desiccation curves started from a
maximum RWC of 150%, and therefore
ignore what happens at higher values,
although a considerable depression of CO
2
gas exchange similar to that observed in NIL
seems to be unlikely.
The differences in Ph
n
(and Ph
g
) noted in
Figure 7, with I>NIL>IL, are confirmed by
data in Figure 8 where further differences
include the Ph
g
saturation value to external
CO
2
concentration, that was >600 ppm in I,
350 ppm in NIL, and only c. 270 ppm in IL.
The estimated CO
2
compensation point was
lowest in I (9 ppm CO
2
), and highest in NIL
(40 ppm CO
2
) (Table 1).
The three materials also showed a differ-
ent reaction to increasing light (Fig. 9),
although the difference between IL and NIL
F. 6. Variation over time of thallus relative water content (A), and rate of water loss in non-isidiate (B) and
isidiate (C) thalli of Pseudevernia furfuracea under the same environmental conditions. Duplicate data for isidiate
(,) and non isidiate (,d) thalli.
454 THE LICHENOLOGIST Vol. 37
was less evident. The highest rate (c. 9·6 mg
CO
2
g
1
h
1
at saturation) was again
recorded in I, being approximately double
that of IL. However, when expressed on
a chlorophyll abasis (data from Table 2),
this difference was considerably reduced,
although Ph
n
still remained slightly higher in
I than in IL and NIL. Isidia also had higher
quantities of total carotenoids (Table 2), in
accordance with the fact that in this material
we observed a particularly high content of
lichen substances. It is noteworthy that NIL
and I had practically identical Chl
a
/Chl
b
(3·570·07 against 3·570·11), and
OD
435
/OD
415
ratios (1·280·04 against
1·300·01), which were statistically higher
than in IL (3·200·11, and 1·190·05,
respectively).
F. 7. Dark respiration (negative values) and net photosynthesis (positive values) of Pseudevernia furfuracea
at 171(C, 270 mol m
2
s
1
PPFR, and 360 ppm CO
2
. A, isidia (O,:) and isidiate lobes (,); B,
non-isidiate lobes (,d). Two samples (open vs closed symbols) were used for each type of material. Means (n=3)
are plotted SD except where it is exceeded by the symbol.
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 455
In all experiments the lowest rates of both
Ph
n
and Ph
g
were always measured in IL, an
apparent contradiction with the observa-
tion that I had impressively high rates. The
importance of isidium development for
increasing the CO
2
gain of mature lobes is
evident from Fig. 10, in which two desicca-
tion curves of the same IL sample, before
and after the mechanical removal of isidia,
are compared. Whereas R
d
did not change
significantly, Ph
n
drastically decreased to
54%, with a concomitant reduction of c. 28%
in both chlorophyll aand total carotenoids
(Table 2). It must be noted that the mech-
anical removal of isidia, carried out with
tweezers used under a stereomicroscope at
high magnification, was less traumatic than
expected, because SEM observations re-
vealed that the cortical surfaces were still
relatively intact, with very few medullary
hyphae emerging from the broken cortex
(Fig. 3E & F).
Discussion
Surprisingly, morphogenesis of isidia has
attracted little attention, probably because
these structures have long been interpreted
as simple cortical outgrowths (‘hernies’, see
Ozenda 1963). Except for a very few ana-
tomical studies (Rosendahl 1907; Moreau &
Moreau 1919; Dughi 1933; Honegger 1987;
Ott et al. 1993), most work deals with the
external morphology of these structures
from a systematic point of view (Du Rietz
1922, Beltman 1978). Little is known about
the first stages of isidium development,
and the factors that trigger their phenotypic
F.8. CO
2
dependence curves of gross photosynthesis
(mg CO
2
g
1
h
1
) in isidia (O,:), and isidiate (,
) and non-isidiate (,d)lobesofPseudevernia
furfuracea, at optimal water content, 171(C, and
350 mol m
2
s
1
PPFR. Two samples (open vs
closed symbols) were used for each type of material.
Means (n=6) are plotted SD except where it is
exceeded by the symbol.
F. 9. Light dependence curves of net photosynthesis
in isidia (O,:), isidiate (,), and non-isidiate (,
d)lobesofPseudevernia furfuracea, at optimal water
content, 171(C, and 360 ppm CO
2
. A, expressed
on the basis of dry weight (mg CO
2
g
1
h
1
); B,
expressed on basis of chlorophyll acontent (mg CO
2
mg
1
chl
a
h
1
). Two replicates (open vs closed sym-
bols) were used for each material. Means (n=3) are
plotted SD except where exceeded by the symbol.
456 THE LICHENOLOGIST Vol. 37
expression are still unknown. It is worth
noting that in Pseudevernia furfuracea isidia
start to develop only after the disappearance
of the melanin-darkened epicortex. The
presence of a true epicortex in this species
had been excluded by Hale (1973), but it
was later correctly recognized by Rikkinen
(1997), who also showed that the develop-
ment from a smooth continuous epicortex to
the rough surface topography typical of old
P. furfuracea lobes seems to involve transi-
tional stages in which the epicortex first
achieves a pored structure and then later
deteriorates. Our observations fully confirm
these results. Rikkinen (1995: 239) also sug-
gested that in P. furfuracea an initial increase
in algal proliferation could be triggered by a
local increase in irradiance within a light-
limited algal layer. These observations are
potentially important for explaining the first
stages of isidium development. The progres-
sive disappearance of the brown melanin-
pigmented epicortex could both cause an
increase in the intensity and quality of
light reaching the photobiont layer, with an
enrichment of the blue component, since
T 1. Main significant physiological parameters inferred from Figs 7–9 relative to isidiate lobes (IL), non-isidiate lobes
(NIL), and isidia (I) of Pseudevernia furfuracea
Isidiate lobes Non-isidiate lobes Isidia
Optimal RWC (%) 110–250 100–150 >150
CO
2
compensation point (ppm) 22 40 9
CO
2
saturation point (ppm) 270 350 >600
Light compensation point (mol photons m
2
s
1
)26 15 27
Light saturation point (mol photons m
2
s
1
) 300 500 900
T 2. Photosynthetic pigments content (gmg
1
dry weight; C
(x+c)
=total carotenoids; Chl
a
,Chl
b
=chlorophyll a,b)
in non-isidiate and isidiate lobes, and isidia of Pseudevernia furfuracea, and per cent variation recorded in isidiate lobes
after the mechanical removal of isidia
n
Non isidiate
lobes
10
Isidiate lobes
10
Isidia
16
After isidia
removal
7
Wilcoxon
matched pairs
test
P-level
C
(x+c)
0·590·07† 0·49 0·06 0·850·05 27% 11 0·018*
Chl
a
2·030·10 1·70 0·26 2·900·17 29% 14 0·018*
Chl
a
/Chl
b
3·570·07 3·20 0·11 3·570·11 5% 7 0·091
OD
435
/OD
415
1·280·04 1·19 0·05 1·300·01 5% 5 0·028
*Statistically significant differences (last column) (P<0.02).
†Mean values 1 SD.
F. 10. Dark respiration (negative values) and net
photosynthesis (positive values) in isidiate lobes of
Pseudevernia furfuracea at 171(C, 270 mol m
2
s
1
PPFR, and 360 ppm CO
2
, before (d) and after
(:) mechanical removal of isidia (see Fig. 3E–F); n=3.
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 457
melanins, having a broad monotonic absorp-
tion curve that decreases from the UV region
(Turkovskii & Yurlova 2002), would absorb
more in the blue than in the red region.
These factors suggest that phytochrome or
more probably cryptochrome-mediated pro-
cesses are triggered in the algae, with a
cascade series of events leading to the
well-coordinated morphogenetic process
described earlier. In the lichen sym-
biosis phytochrome-mediated processes
had already been hypothesized (but not
demonstrated) by several authors (Avalos &
Vicente 1985, 1986; Giles 1970), for
example to explain the formation of para-
soredia in the foliose lichen Pseudoparmelia
caperata (L.) Hale (Raineri & Modenesi
1986). On the other hand, the presence of
cryptochrome-mediated processes has been
neglected so far, although the role of this
molecule as a morphogenetic regulator is
well known in green algae (Suetsugu &
Wada 2003), and also in fungi (Suzuki et al.
1977; Ross 1985; Galland & Senger 2001).
The importance of events in the algal layer
for the process of isidium formation was
recognized by Nilson-Kajanus (1903, 1911),
who however attributed the proliferation
of algae to an excess in humidity. More
recently, Ott et al. (1993) recognized the
proliferation of photobiont cells as a key
event, but only in Parmotrema crinitum
(Ach.) M. Choisy, and not in P. furfuracea.
In the latter species they described an in-
creased division rate of the cortical layer as a
primary event, which only later was followed
by an increase in algal division beneath the
growing cortex. The medulla only partici-
pated after isidia had already emerged from
the cortex in the final differentiation stage,
when hyphae of the medulla then grew
towards the base of the protuberance (Ott
et al. 1993: 67). In our opinion, this series of
events is not confirmed by the limited icon-
ography reported. In contrast, the impor-
tance played by medullary hyphae in isidium
formation can be inferred from Jahns
(1984), who holds that in Parmelia saxatilis
(L.) Ach. isidia appear not in corticated
areas, but in the crevices of the pseudo-
cyphellae, i.e. where there is no cortex, and
medullary hyphae are directly exposed (see,
on this point, also Beltman 1978).
In P. furfuracea there are also cortical
outgrowths derived from proliferation of
cortical hyphae, and which are devoid of
algae (Fig. 4). Their morphology is quite
different from that of very young isidia, and
therefore they cannot be confused with
them. It might be argued that they are
formed by a parasite, but the reactivity to
histochemical tests of their cellular com-
ponents is identical to that of neighbouring
tissues, and they form an evident continuum
with the upper layer of the cortex. Alterna-
tively they might be galls induced by the
infection of a fungus, whose hyphae have not
been demonstrated by the dozens of staining
techniques used in this and other concurrent
studies (Giordani et al. 2004) or they might
be thalloconidia. We strongly support the
latter hypothesis, because they are practi-
cally identical to the thalloconidia described
by Hestmark (1990) in Umbilicaria esculenta
and U. mammulata (loc. cit. figs 9, 13,
respectively). As in our species, the vegeta-
tive mycobiont propagules of the two
Umbilicaria species are derived by direct
proliferation of cortical scleroplectenchyma-
tous tissue, the phenomenon being limited
to the lower cortex, although in Umbilicaria
species their frequency is considerably
higher (Hestmark 1992). Interestingly,
thalloconidia have also been reported from
some crustose members of the Parmeliaceae
(e.g. Protoparmelia) (Poelt & Obermayer
1990), but they were not known to occur in
foliose or fruticose members of the family. If
confirmed by further experimental work, the
presence of thalloconidia in P. furfuracea
would represent an important novelty in our
knowledge of the reproduction strategy of
this species, which relies strongly on isidium
dispersal (see Nienburg 1919).
Our experimental data demonstrate that
the growth of isidia strongly modifies the
relationships of the thallus with its en-
vironment, particularly CO
2
assimilation
economy, as already suggested by Smith
(1921). The high surface/volume ratio typi-
cal of isidia, the proximity of their algal
cells to the cortex, and the high content of
458 THE LICHENOLOGIST Vol. 37
photosynthetic pigments (Table 2) are prob-
ably the basis of the high carboxylation
efficiency and very low CO
2
compensation
point (9 ppm CO
2
) (Fig. 8 and Table 1).
Being young, rich in actively growing
pseudomeristematic tissue and algal cells,
isidia have relatively high R
d
and, above all,
spectacularly high Ph
n
, although at 17(C
(the temperature chosen for our experi-
ments) it is only 50% of the maximum,
which occurs at c.5(C (Türk 1983). The
high rates of CO
2
assimilation by isidia on
the one hand, and the low rates by isidiate
lobes on the other, are only superficially a
contradiction: the quantity of isidia pro-
duced on the surface of old lobes is not
enough to equal the Ph
n
rates typical of
young, non-isidiate lobes. However, the Ph
n
of isidiate lobes would be considerably lower
if isidia were absent (see Fig. 10). From this
point of view, isidium production might be
considered a process by which old lobes
are rejuvenated. This is exactly the opposite
to what happens when soredia develop,
because in the parmelioid lichens investi-
gated so far, sorediate lobes have lower R
d
and Ph
g
, than esorediate lobes due to a lower
chlorophyll content (Tretiach & Carpanelli
1992).
The formation of isidia has two further
effects that positively influence CO
2
gain:
the reduction of Ph
n
inhibition at high thal-
lus water contents (Fig. 7), and a retarded
dehydration (Fig. 6). Both are caused by the
fact that the formation of a thick layer of
isidia increases the water holding capacity
of the thallus by storing large quantities of
water in the capillary spaces between the
bases of isidia. The tops of isidia tend to
remain free of liquid water, because they
are often raised above the level of the water
film eventually present (Scheidegger et al.
1995b), and have positive water potentials
(Rikkinen 1997). Consequently, the CO
2
assimilation of the most active parts of the
thallus, the isidia, is not limited by the
increased CO
2
diffusion resistance caused
by the presence of liquid water (Green et al.
1994; Lange et al. 1998, 1999); on the
contrary their hydration is guaranteed by the
water present at their base. On the other
hand, the smooth surfaces of non-isidiate
lobes are covered by a thin water film at high
RWC, and this strongly affects the CO
2
gas
exchange (Scheidegger et al. 1995b). Thus,
whereas non-isidiate lobes suffer the typical
Ph
n
inhibition at high RWC, isidiate lobes
and isidia maintain relatively constant rates
in a wide range of RWCs (Table 1).
The capillary forces and the high content
of mucopolysaccharides probably also
explain the lower rate of thallus dehydration
of isidiate thalli (Fig. 6C). These observa-
tions agree with those of Valladares et al.
(1993), who demonstrated that isidiate
Lasallia pustulata has a higher water storage
capacity than other anatomically similar
species of the same genus devoid of isidia.
From this point of view, it might be of some
interest to study the behaviour of isidiate and
non-isidiate morphotypes exposed to humid
air currents, because the development of the
small, three-dimensional irregular structures
might cause a strong increase of condensa-
tion phenomena, as observed by Rundel
(1974) in Ramalina menziesii Taylor, and by
Lange et al. (1990) in Teloschistes capensis
(L.f.) Vain. Also in our case any difference
could be explained by morphological fea-
tures only, because preliminary measure-
ments indicate that the two morphotypes
have similar water potentials (data not
shown). Of course, the increase in water
condensation capacity might be off-set by a
higher evaporative loss due to the thermal
increase caused by melanin-darkening of the
epicortex, a factor not influencing the results
of the water loss experiments of Fig. 6,
which were carried out in dim light.
It is noteworthy that although isidia are
very efficient in increasing CO
2
gain in older
lobes, isidiate lichens are less frequent than
sorediate ones. The lichen flora of Italy, for
instance, has only 85 isidiate species (3·7%),
whereas sorediate species are four times
more frequent (15·1%) (data derived from
Nimis 2003). Similar results were given by
Bowler & Rundel (1975) in their com-
prehensive study of the reproductive strate-
gies in lichens. These observations can be
explained by two non-mutually exclusive
hypotheses. Either isidia are less effective in
2005 Isidia and CO
2
exchange in Pseudevernia furfuracea—Tretiach et al. 459
securing the establishment of new thalli,
probably because they are less efficient in
dispersal and in the first stages of fixation to
the substratum (Jahns 1984; Kärnefelt 1990;
Scheidegger et al. 1995a; Zoller et al. 2000)
and/or the morphogenetic events leading to
the formation of isidia are more complex,
being under the control of a larger number
of genes. The latter hypothesis seems to be
the less supportable: isidia are known from
many unrelated taxa, and isidiate species
often have close relatives without isidia
(Bowler & Rundel 1975). The capacity to
produce isidia can apparently be acquired
and lost quite easily, probably because it
requires only minor changes in the chain of
events that controls thallus growth.
We thank Lucia Muggia (Trieste), and Laura Carletti
(Siena) for assistance in the field, Antonio Corallo
(Genova) and Tito Ubaldini (Trieste) for help with
SEM photographs, and Laurence Baruffo (Trieste) for
assistance in the laboratory. This study was carried
our in the framework of the COFIN 2002 project
‘‘Sviluppo di metodologie per il monitoraggio biologico
dell’inquinamento atmosferico da metalli in traccia
nelle aree urbane ed industriali italiane’’, co-ordinated
by Roberto Bargagli (Siena).
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Accepted for publication 9May 2005
462 THE LICHENOLOGIST Vol. 37
