O sis,
8:
33-40
(1993)
Con ibu ion o he s udy o
No mandina pulchella:
a cy ological app oach
Dona ella Ma es, Ma ia
P.
Fasulo and Alessand o B uni
Ins i u e o Bo any, Uni e si y o Fe a a, Co so Po a Ma e, 1-44100 Fe a a, I aly
Key
wo ds:
No mandina pulchella,
ul as uc u e, mycobion -pho obion in e ace.
Abs ac . A cy ological s udy was unde aken on
No mandina pulchella
(Bo .) Nyl. in o de o con-
ibu e o a be e knowledge o he s uc u e o he lichen and o acqui e use ul in o ma ion on i s
axonomic a angemen . Mic o- and submic oscopic obse a ions e ealed ha he allus is he e o-
ne ous, wi h a medulla y g issue* composed o a ne wo k o hyaline ungal h eads su ounding small
clumps o algal cells. These we e hick-walled and exhibi ed a single loba e chlo oplas in which a
conspicuous ne ame ic py enoid, small s a ch g anules and lipophilic plas oglobuli we e equen ly
p esen . The mycobion had hyphae wi h simple pe o a ed sep a wi h associa ed Wo onin bodies.
The ela ionship be ween he wo pa ne s was me ely o e y close p oximi y o hei cell walls and
in ol ed no ungal pene a ion in o alga1 cells. The p esence o Wo onin bodies, exclusi e o he
Ascomyce es
and o plas oglobuli, ha we e desc ibed only in he
Chlo ococcales
phycobion s in
ascolichens, indica es ha in all likelyhood
No mandina
is a membe o he ascolichens and no o
basidiolichens, as p e iously epo ed by some wo ke s.
Resumen.
Con ibucidn al es udio de
No mandina pulchella:
ap oximacidn ci ológica.
Pa a con i-
bui a un mejo conocimien o de la es uc u a de
No mandina pulchella
(Bo .) Nyl. y pa a ob ene
in o maciones ú iles pa a su co ec a colocación axonómica, se ha e np endido un es udio ci ológi-
co sob e el liquen. Obse aciones al mic oscopio Óp ico y elec ónico han e elado que el allo del
liquen es he e óme o, con un ejido nedula compues o po un en elazamien o de hi as hialinas de
hongos, que odean pequeños g upos de células algales. És as p esen aban una pa ed g uesa y un clo-
oplas ~ lobulado simple, en el cua1 ecuen e nen e es aban D esen es un oluminoso ~i enoide me a-
mi ico, pequeños de almidón y plas oglóbulos lipó ilos. El micobion e enia hi as con sep os
simples y co pusculos de Wo onin asociados. La elación en e 10s dos componen es consis ia en una
simple y densa ap oximación de sus pa edes, sin una pene ación del hongo en las células algales. La
p esencia de 10s co púsculos de Wo onin, exclusi os de 10s Ascomice es y de 10s plas oglóbulos, que
han sido desc i os s610 en 10s clo ococales icobion es de 10s ascolíquenes indica que, con oda p o-
babilidad,
No mandina
es un mie nb o de 10s ascolíquenes y no de 10s basidiolíquenes, como habia
sido p e iamen e a i mado po algunos au o es.
In oduc ion
No mandina pulchella
(Bo .) Nyl. is an omb ophy ic and hyg ophy ic small li-
chen, which is widely dis ibu ed in he No he n and Sou he n hemisphe es, in
locali ies wi h suboceanic condi ions. This species is one o he mos s iking
34
D.
MARES, M.P. FASULO
&
A. BRUNI
examples o <<hype epiphy ism)), g owing bo h on ocks o ba k and on he
hallus o o he epiphy es, chie ly li e wo s, mosses and e en o he lichens
(T e iach and Nimis 1988 and e e ences he ein). The hallus, whose colou may
a y om glaucous o pale g ey o g eenish-g ey, consis s o squamules (1-2 mm)
ha a e mo e o less ounded o kidney-shaped. The phycobion is
Nannochlo is
no mandinae
Tsch.-W. (Tsche mak-Woess 198 1). F ui ing s uc u es do no seem
o be p oduced by his lichen. In ac , he pe i hecia obse ed in he hallus by
some wo ke s (Ozenda and Clauzade 1970, Poel 1974) a e asc ibable o he in-
ading pa asi ic ungus
Sphae ulina chlo ococca
(Leigh .) R. San . The e o e,
he iden i y o he mycobion caused conside able axonomic p oblems. In pa -
icula , whe he
N. pulchella
is an ascolichen o a basidiolichen has emained an
open p oblem in published accoun s (Ozenda and Clauzade 1970, Poel 1974,
Wi h 1987). In ac ,
N. pulchella
was conside ed o a long ime as a membe
o basidiolichens (Henssen and Jahns 1973) because o he close mo phological
simila i y wi h
Co iscium i ide,
a lichen cu en ly ega ded as he impe ec
s age o he basidiomyce e
Omphalina hudsoniana
(Obe winkle 1970). On he
o he hand, he subsequen inding o lack o dolipo e sep a in
No mandina,
which a e ypical o
Basidiomyce es,
made i s a ibu ion o basidiolichens unli-
kely (Henssen 1976). Thus, he sys ema ic posi ion o
No mandina
is s ill o be
clea ly de ined.
In his con ex , a cy ological in es iga ion was unde aken on his lichen, o-
cusing he a en ion on he o ganiza ion o he hallus, he s uc u al cha ac e is-
ics o bo h myco-and pho obion and he na u e o he ungal-alga1 con ac .
The pu pose o his s udy was o con ibu e o a be e knowledge o he s uc-
u e o he lichen, and o acqui e use ul in o ma ion o i s axonomic a ange-
men .
Ma e ials and me hods
No mandina pulchella
(Bo .) Nyl., g own on
F ullania dila a a
(L.)
Dum., was
collec ed on
Que cus pubescens
L.
a Opicina (250 m) in he Ka s Region (T ies e,
I aly). Mic o- and submic oscopic obse a ions we e ca ied ou on hallus
pieces soaked o 12 h in ap-wa e in a he mos a ically con olled incuba o a
25
+
1°C unde con inuous ligh (50 pE.m2.S-1 a he su ace).
Mic oscopic examina ions we e pe o med wi h a Zeiss Axiopho pho omi-
c oscope equipped wi h con en ional o luo escen a achmen s
(BP
436,
FT
460,
LP 470 il e se ). Fo ansmission elec on mic oscopy (TEM), he specimens
we e ixed in 3% glu a aldehyde in 0.1 M phospha e bu e , pH 6.8, a 4°C o 3
hou s, insed o se e a1 hou s in bu e and pos ixed wi h 1
%
Os04 in he same
bu e . A e dehyd a ion in a g aded e hanol se ies, hey we e embedded in Epon-
A aldi e esin, cu wi h a LKB Ul a ome I11 and s ained wi h u anyl ace a e and
lead ci a e. Obse a ions we e made wi h a Zeiss EM 109 elec on mic oscope
a 80 kV. Scanning elec on mic oscopy (SEM) was ca ied ou on glu a aldehy-
NORMANDINA
PULCHELLA:
A CYTOLOGICAL APPROACH
35
de ixed hallus pieces ha we e b ie ly pos ixed wi h 1% Os04, apidly dehy-
d a ed in ace one, c i ica1 poin d ied and gold coa ed, as p e iously desc ibed
(Ma es and Fasulo 1990). Obse a ions we e made wi h a scanning elec on mi-
c oscope Camb idge S e eoscan 360 a an accele a ing ol age o 20 kV.
Resul s and discussion
As obse ed by he ligh mic oscope, he hallus o
N.
pulchella
has he same
mo phological cha ac e is ics p e iously desc ibed by Henssen (1976). In pa i-
cula , i is he e ome ous wi h an uppe pseudopa enchyma ous o e anescen co -
ex, whe eas a dis inc lowe co ex is absen . The medulla y cc issue,, o he li-
chen is composed o a ne wo k o hyaline ungal h eads, which su ound small
clumps o alga1 cells (Fig. 1 .I). These la e ha e no well de ined dis ibu ion pa -
e n in he hallus and a e p esen in wo di e en o ms, ha p obably conespond
o wo di e en s ages o hei li e cycle. Mo e p ecisely, he pho obion popula-
ion consis s o bo h la ge and hick- walled ma u e cells, and smalle young mi-
ospo es, which can be ee in he hallus o s ill enshea ed by he mo he cell wall.
Figu a
1.1
Ligh mic og aph o he hallus o
N.
pulchella
showing a ne wo k o hya-
line mycobion cells en eloping small g oups o alga1 cells.
(x
400).
1.2 Fluo escence
mic oscope aspec o he lichen showing he pho obion popula ion wi h a well de i-
ned ed luo escence which is mo e in ense in he smalle mi ospo es (a ows).
(x
250).
36
D.
MARES, M.P. FASULO &A.
BRUNI
Figu a
2.
Fine s uc u e o
N.
pulchella
associa ion showing nume ous mycobion hy-
phae (my) adjacen o a phycobion cell (ph) in which a single loba e chlo oplas wi h
a cen al me ame ic py enoid may be obse ed. The physical con ac be ween he sym-
bion s consis s o a simple ela ionship o wall- o-wall p oximi y. Ba
=
1
Fm.
When obse ed unde
UV
ligh , all he algae exhibi a well de ined ed luo es-
cence which is mo e in ense in he younge ones. (Fig. 1.2).
A TEM, he cells o he phycobion ha e a a he da kly s ained wall wi h a
a iable hickness acco ding o he s age in li e cycle. Howe e , he alga1 wall is
always hicke han ha o he ungal pa ne . The cen e o he cell is occupied
by
a
single loba e chlo oplas wi h a conspicuous py enoid ha ing a me ame ic
s uc u e (Fig. 2). Small s a ch g anules and nume ous plas oglobuli a e p esen
close o he hylakoid memb anes (Fig.
3).
The mycobion shows hyphae wi h mul ilaye ed walls, some imes in il a ed
by elec on-dense g anula p ecipi a es unce ain in na u e. The sep a a e always
simple s uc u es, ypically pe o a ed, ha dly e e linea , bu o en wa ped, and
equen ly associa ed wi h mic obodies and Wo onin bodies. The la e o ganules
a e sphe ical (abou 0.2 ym in diame e ), bounded by a single uni memb ane and
con ain a homogeneous elec on-opaque ma ix. Wo onin bodies appea singly in
NORMANDINA
PULCHELLA:
A CYTOLOGICAL APPROACH
37
Figu a
3.
In he phycobion , he chlo oplas ma ix p esen s some small s a ch g a-
nules (a owheads) close o he hylacoid memb anes and many plas oglobuli (pg)
pe iphe ally dis ibu ed. Ba
=
1
pm.
Figu a
4.
Hyphal sep um wi h a single cen al po e ha ing Wo onin bodies (a ows)
associa ed. Ba
=
1
pm.

38
D.
MARES, M.P. FASULO
&
A.
BRUNI
he po e o as a pai a bo h sides o he sep a (Fig.
4),
Dolipo e sep a o clamp
connec ions, ypical o he
Basidiomyce es,
we e ne e obse ed.
As i no mally happens in he squamulose o ms o lichens (Honegge 1985),
each phycobion appea s o be enci cled by a ious mycobion hyphae (Figs. 2,5).
The physical connec ion be ween he wo symbion s is me ely he close p oximi y
o hei cell wall wi hou he o ma ion o ungal haus o ia. This was es ablished
by obse a ions on 30 se ial lichen sec ions, om h ee esin blocks, and by
SEM
specimens.
In ou opinion, he s uc u al aspec s obse ed in
N.
pulchella
can u nish some
s uc u al de ails use ul o a be e knowledge o his in e es ing species o li-
chen. As iewed, in ac , he physical con ac be ween he symbion s, consis s o
a simple ela ionship o wall- o-wall p oximi y wi h no ungal pene a ion in o he
alga1 cells. Acco ding o Galun e al. (1970), he p e en ion o he hyphae om
en e ing in o he phycobion could be ela ed o he p esence in he alga o a e y
hick wall.
Ano he in e es ing inding in ou samples is he cons an p esence in he my-
cobion o simple sep a. This aspec was al eady desc ibed by Henssen (1976)
Figu a
5.
SEM
mic og aph o an alga1 cell con ac ed by nume ous adhe ing
mycobion hyphae. Ba
=
1
pm.
NORMANDINA
PULCHELLA:
A CYTOLOGICAL APPROACH 39
which, jus on his basis, asc ibed
N.
pulchella o ascolichens. Ou inding o
Wo onin bodies associa ed wi h he simple po es subs an ia es his a ilia ion,
because hese s uc u es a e conside ed o be exclusi e o he Ascomyce es (Moo e
1965, Becke e al. 1974, Vannini and Ma es 1975, Wi h ow and Ahmadjian
1983). Thus, we hink ha he mycobion o
N.
pulchella is he s e ile s age o
an Ascomyce e. A suppo o his hypo hesis seems o be o e ed by he p esen-
ce o plas oglobuli in he phycobion . In ac , in o he lichens ha ing Chlo ococcales
as pho obion s, hese plas idial inclusions we e ound only when he algae a e in
symbiosis wi h Ascomyce es, whe eas he same phycobion s do no show plas-
oglobuli when he symbiosis is wi h Basidiomyce es (Pe eling and Galun 1976).
The e o e, on he basis o ou esul s, we a e led o belie e ha
N.
pulchella
is a membe o ascolichens.
Acknowledgemen s
The au ho s a e indeb ed o P o . P.L. Nimis, T ies e, who sen lichen speci nens. They a e likely g a-
e ul o he s a o he Elec on Mic oscopy Cen e o Fe a a Uni e si y o he use o he SEM ins-
umen a ion. This wo k was suppo ed by g an s om Consiglio Nazionale delle Rice che (CNR)
and Minis e o dell'uni e si l e della Rice ca Scien i ica e Tecnologica (MURST) o I aly.
Re e ences
Becke , A., Hea h, I.B.
&
McLaughlin, D.J. 1974. An a las o ungal ul as uc u e. Longman,
London.
Galun,
M.,
Pa an, N.
&
Ben-Shaul, Y. 1970. The ungus-alga associa ion
in
he Lecano aceae:
An ul as uc u a1 s udy. New Phy ol. 69: 599-603.
Henssen, A. 1976. S udies in he de elopmen al mo phology o lichenized Ascomyce es.
In: B own, D.H., Hawskwo h, D.L.
&
Bailey, R.H. (eds.), Lichenology: P og ess and
P oblems. Sys ema ics Ass., Academic P ess, London and New Yo k, Vol. VI11
PP.
107-138.
Henssen, A.
&
Jahns, H.M. 1973. Lichenes. Eine Ein üh ung in die Flech enkunde. Thieme,
S u ga .
Honegge , R. 1985. Fine s uc u e o di e en ypes o symbio ic ela ionship in Lichens.
In: B own, D.H. (ed.), Lichen physiology and cell biology. Plenum P ess, New Yo k
and London.
Ma es, D.
&
Fasulo, M.P. 1990. Ul as uc u a1 al e a ions in
Epide mophy on~occosum
and
T ichophy on men ag ophy es
exposed
in i o
o p o oanemonin. Cy obios
61: 89-95.
Moo e, R.T. 1965. The Ul as uc u e o ungal cells. In: Ainswo h, G.C.
&
Sussman, A.S.
(eds.), The ungi. Academic P ess, London and New Yo k, Vol. I pp. 95-1 18.
Obe winkle , F. 1970. Die Ga ungen de Basidiolichenen. Vo . bo . Ges. (D sch. bo . Ges.)
N.
F.
4:
139-169.
Ozenda,
P.
&
Clauzade,
G.
1970. Les lichens. Masson and Cie (eds.), Pa is.
40
D.
MARES,
M.P.
FASULO &A.
BRUNI
Pe eling, E. 1969. Elek onenop ische Un e suchungen an Flech en.
11.
Die Feins uk u
on T ebouxia-Phycobion en. Plan a 87: 69-85.
Pe eling, E.
&
Galun, M. 1976. Elec on-mic oscopical s udies on he phycobion
Coccomyxa
Schmidle. New Phy ol. 77: 713-718.
Poel ,
J.
1974. Bes i nmungsschlüsse1 eu opaische Flech en. C ame ,
J.
(ed.), Vaduz.
T e iach,
M.
&
Nimis, P.L. 1988. Lichenological s udies in NE-I aly.
11.
The dis ibu ion o
No mandinapulchella
(Bo .) Nyl. Go ania, A i Museo F iul. S o ia Na . 10: 133-144.
Tsche nak-Woess, E. 1981.
Zu
Kenn is de Phycobion en on
Loba ia lini a
und
No mandina
pulchella.
No a Hedwigia 35: 63-73.
Vannini, G.L.
&
Ma es, D. 1975. Fine s uc u al cha ac e iza ion o mic obodies and
Wo onin bodies in
T ichophy on men ag ophy es.
Expe ien ia 3 1
:
949-95 1.
Wi h, V. 1987. Die Flech en Baden-Wü embe gs. Ulme , E. and Co. (eds.), S u ga .
Wi h ow, K
.
&
Ahmadjian, V. 1983. The ul as uc u e o lichens. VII.
Chiodec on san-
guineum.
Mycologia 75: 337-339.
Manuscp i ecei ed on Decembe
1991