Contribution to the study of Normandina pulchella : a cytological approach

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. 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