Lichen Chimeras: DNA Analysis Suggests That One Fungus Forms Twp Morphotypes DANIELE ARMALEO and PHILIPPE CLERC'

EXPERIMENTAL MYCOLOGY 15, l-10(1991)

Lichen Chimeras: DNA Analysis Suggests That One Fungus Forms Twp Morphotypes DANIELE ARMALEO AND PHILIPPE CLERC'

Department of Botany, Duke University, Durham, North Carolina 27706 Accepted for publication August 6, 1990

ARMALEO, D., AND CLERC, P. 1990. Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15, l-10. In most lichens, the symbiosis between one fungus (mycobiont) and one photosynthetic partner (photobiont) results in a uniform thallus whose morphology is distinctive for each combination of symbionts. In some lichens, two morphologically different thalli, one containing a green alga, the other a cyanobacterium, are joined together in a chimera called photosymbiodeme. The question whether the same or two different mycobionts are involved in the formation of the different chimera components (morphotypes) is relevant to lichen morphogenesis, physiology, and taxonomy, but has not been answered conclu- sively to date. We have developed nucleic acid extraction procedures suitable for lichens. Using Southern hybridization and the polymerase chain reaction we demonstrate the genetic near-identity of the mycobionts forming paired morphotypes in two different photosymbiodemes. o 1991 Academic Press, Inc. INDEX DESCRIPTORS: lichen DNA; symbiosis; photosymbiodemes; Sticta; Pseudocyphellaria; polymerase chain reaction.

One in five of all known fungi are lichen- lichen races (Culberson et al., 1988). Yet formers. Of the 13,500 lichen species major questions about the interactions known, 98% belong to the Ascomycotina, guiding the development of the symbiotic where they are distributed among 16 orders thallus remain unanswered. The system, in (Hawksworth, 1988). The biology of lichens nature or in culture, is ripe to be ap- is studied traditionally using field-collected proached with the tools of molecular biol- specimens. In the last decade, advances in ogy- cultural techniques, pioneered by Ahmad- In this study we apply molecular meth- jian (Ahmadjian and Jacobs, 1985), have ods to define the relatedness between improved the manipulation of lichens in the paired fungal components in photosymbio- laboratory under controlled conditions. It is demes. Photosymbiodemes (see Renner possible to dissociate and culture the sym- and Galloway, 1982; Ott, 1988) were first biotic partners separately and, in some analyzed in detail by Dughi (1936, 1937, cases, to reconstitute and grow lichens in 1944) in his studies on Dendriscocaulon. vitro from pure isolates of fungus and alga Whereas most lichens combine one type of (Yamamoto, 1987; Bubrick, 1988). In vitro fungus (mycobiont) and one type of photo- techniques have dramatically extended the synthetic partner (photobiont) in a morpho- range of observations on the early stages of logically homogeneous thallus, Dughi de- the symbiosis (see, for example, Ahmadjian scribed composite lichen thalli with two and Jacobs, 1985; Schuster et al., 1985; Ott, morphologically different structures (mor- 1987; Armaleo, 1990) and have been instru- photypes) attached together. In one the my- mental in demonstrating gene flow among cobiont was associated with a green alga, in the other with a cyanobacterium. The dif- i Systematisch Geobotanisches Institut der Univer- ferent morphotypes were also found as in- sitat Bern, Altenbergrain 21, CH-3013 Beme, Switzer- dependent thalli. The phenomenon, since land. observed in many genera of the Pelti-

1 0147-5975191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved. 2 ARMALEO AND CLERC gerales, has been described with terms as used to address that prsble chimera (James and Henssen, 1976), symbiodeme from the genus chimeroid associations (Brodo and Rich- ardson, 1978), joined phycotypes (Tonsberg and Holtan-Hartwig, 1983), morphotypes (White and James, 19SS>, phyco- symbiodemes (Renner and Galloway, 1982), photosymbiodemes (Ott, 1988). In this paper, we will use “photosymbiodeme” for the dimorphic complex, “morphotype” for each component. The question whether the fungal partner is the same for each pair of morphotypes as major implications for lichen morphogenesis, physiology, and taxonomy. The prevailing assumption, based solely on his- tological observations of hyphal continuity, favors the involvement of a single fungal spec~rne~~ were ames, 1975; James and Henssen, do and Richardson, 1978; Renner way, 1982). However, as pointed out by W. I. Culberson (1977), there is no conclusive proof in support of that assumption Since frequent cases exist of taxonom- ically unrelated lichens that grow intimately attached to one another through hyphal com~enetration (Hawksworth, 1988), the orphological evidence alone leaves much ments are selected for space to the competing view that the different morphotypes are in fact formed by unrelated mycobionts. To resolve the ques- sts of the genetic origin of phae are needed. We report ere the results of two such tests on myco-

describe methods for the rapid extraction of undegraded RNA and of high- ecufar-weight DNA of purity suitable cloning. Reports on DNA extracted lichens are in the literature (Blum and evarov, 1986; Ahmadjian et d., 1987), effective procedures to rapidly purify nucleic acids suitable for molecular manip- ulations are lacking. Cloned DNA is used to define the genetic relationship between mycobionts in a photosymbiodeme of the ge- Pseudocyphellaria. The polymerase iately added to the g in reaction (PCR) (White et al., 1989) is owder is well sus MYCOBIONT DNA IN LICHEN PHOTOSYMBIODEMES 3 by gently shaking and inverting tube repeat- can be further purified by centrifugation edly by hand for 2-3 min (vortexing is through a CsCl cushion (Maniatis et al., avoided throughout the procedure to limit 1982) for uses not described in this paper]. shearing of high-molecular-weight DNA). Polysaccharide contaminants which inhibit The homogenate is centrifuged for 2 min in restriction enzymes are removed by chro- a microfuge and the supernatant is trans- matography through an Elutip minicolumn ferred with a pipet into a fresh microfuge (Schleicher and Schuell), as described be- tube and kept on ice. The pellet is resus- low. Method I requires about 4 h, including pended as above in 2 vol of guanidinium the Elutip step. buffer (after being first softened by stirring DNA extraction-Method ZZ. Dry, clean into it a small amount of buffer with a pipet thallus sections are fragmented with a blade tip). After 2 min of centrifugation, the su- or scissors into very small pieces (2-10 pernatant is pooled with the previous one. mm2), weighed, and transferred into a 1.5- A third extraction with 2 vol of guanidine ml microfuge tube. Routinely, method II is buffer is performed if the color intensity used with l-50 mg of material. Novozym- and opacity of the second supernatant have containing buffer is added [pH 5.8, 50 mM not decreased significantly relative to those Na citrate, 400 mM EDTA, 700 mM KCl, of the first. The pellet is discarded and the 1% B-mercaptoethanol, 10 mg/ml Novozym pooled supernatants are extracted at room 234 (Novo BioLabs); the last two compo- temperature by gentle agitation for l-2 min, nents are introduced just before use] and once with an equal volume of PC1 (phenol, the digestion is carried out at room temper- chloroform, isoamyl alcohol, 25:24: 1) and ature for 4 h. When using between 20 and 50 twice with an equal volume of CI (chloro- mg of tissue, 10 vol of buffer (w/v) are form, isoamyl alcohol 24:l). Between ex- added; 200 pl are used for any amount un- tractions, the sample is centrifuged for 3 der 20 mg. During the incubation, the frag- min and the upper aqueous phase is trans- ments settled on the bottom of the tube are ferred to a new tube. After the last extrac- occasionally stirred with a micropipet tip. tion, the sample is gently but thoroughly At the end of the incubation, proteinase K mixed with an equal volume of isopropanol from a 10 mg/ml stock is added (3 vl/lOO ~1 and kept at room temperature for 10 min. It of Novozym buffer), followed by 0.25 vol is then centrifuged for 10 min, the superna- of 10% Sarkosyl. The material dispersed on tant is poured off, the inverted tube is blot- the tube wall is pulse-pelleted and stirred ted on paper, and 0.5 ml of 70% ethanol are back into the buffer with a micropipet. The added to the pellet. The tube is centrifuged tube is incubated at 65°C for 10 min and for 2 min, the ethanol is discarded and the contents are mixed once more and centri- inverted tube is blotted on paper. Without fuged for 3 min. The greenish pellet (con- further drying, the pellet is gently resus- taining intact-looking algal cells amid obvi- pended in 200 ~1 of TE buffer (10 mM Tris, ously damaged hyphae) is discarded, the pH 7.5, 1 mM EDTA) with a minimum of supernatant is transferred to a new tube, pipetting. To aid resuspension and as a fur- shaken by hand for l-2 min with an equal ther insurance against nucleases, the tube is volume of PCI, heated at 65°C for 3 min, incubated at 65°C for 10 min, and pipetting shaken again briefly, and centrifuged for 3 is briefly repeated. To limit pipetting, after min. After extracting twice with an equal the incubation at 65°C the sample can be volume of CI at room temperature, the allowed to resuspend at 4°C overnight. In- aqueous upper phase is transferred to a new soluble material is then pelleted by centrif- tube and thoroughly mixed with 1.5 vol of ugation, and the supernatant is transferred isopropanol, and the mixture is centrifuged to a fresh tube and stored at -20°C. [RNA for 3 min. A further phase separation is ob- 4 ARMALEO AND CLERC tamed at this point, with a concentrated aqueous phase on the bottom. The upper phase is carefully withdrawn and discarded, and to the rest 3 vol of TE buffer and 5 p,g of yeast tRNA are added [the tRNA stock solution (10 mg/ml) is prepared by extracting commercially available yeast tRNA (Sigma) once with PC1 and twice precipitating it with ethanol]. ative to the final volume, 0.7 vol of iso- panol are added and the mixture is kept at room temperature for 5 min. The precip- itate is pelleted by centrifugation for 10 min, and the pellet is rinsed with 70% ethanol, resuspended without excessive drying p,l TE, and treated as described in I to ensure resuspension. The in- present are removed with an Elutip n. Method II requires about 6 h, in- bation with Novozym and

EEuiip columre. The volume of the nucleic id sample prepared by either method is rought to 400 ~1 with TE buffer, pH 7.5, NaCl in TE, pH 7.5, are y is performed as in- cturer, with all solu- y, the sample (in 0.2 onto the Elutip col- .2 M NaCVTE, and m were use eluted with 500 ~1 of 1.0 M NaWTE. Car- (5 pg) is added to the eluted sam- e nucleic acid is precipitated at perature was 60°C. - 80°C for 30 min in presence of 2 vol of 95% ethanol. After a lo-min centrifugation at 4°C the pellet is rinsed with 70% ethanol, dried, and gently suspended in 10-100 l.~l of TIE. The resulting DNA is a good substrate for restriction of PCR. screens. ~~nsty~ctiQ~ of genomic library. DNA Pol9meYase chain reaction. was extracted with method I from a large and well-decked specimen of P. murrayi (the blue-green morphotype). Particular care was taken to avoid contamination from the green morphotype. After partial digestion with 0.2 units of Mb01 for 45 min, 800 ng of DNA were electrophoresed through a 0.8% low-melting-point agarose gel. Frag- MYCOBIONT DNA IN LICHEN PHOTOSYMBIODEMES 5 the other 2.2 kb downstream (Vilgalys and also prepared with method I, each yielded Hester, 1990). Buffers and primer con- only a 2.2-kb fungal product. centrations were the standard ones described in bulletin 55645lo/88 from U.S. Biochemical Corporation, manufacturer of RESULTS AmpliTaq, the polymerase used. Template DNA (10 ng/lOO pl-reaction) was added in Nucleic Acid Extraction. 10 p,l of TE buffer, pH 8.0. Reaction cycles (30) were set to 1 min at 94°C 1 min at 50°C Method I yields l-3 pg DNA and l-6 pg and 3 min at 72°C with rapid transitions. RNA from 100 mg of tissue, depending on Samples were extracted once with 300 ~1 age and species. When extracted from fresh PCI, diluted with 1 vol of TE, and extracted material, both DNA and RNA are unde- twice with CI. After ethanol precipitation, graded, as judged from the sharp DNA and the amplified DNA (l-4 pg per reaction) rRNA bands observed in agarose gels. was suspended in 50 ~1 TE (controls con- Method I has been successfully applied to a taining only the yeast tRNA present as variety of different lichens and isolated my- carrier in our template DNA preparations cobionts (D. Armaleo, P. DePriest, and P. gave significant amplification of contami- Clerc, unpublished). Lichens with eukary- nating traces of yeast DNA only with otic photobionts yield a predominantly fun- amounts of tRNA 100-1000 times higher gal mixture of nucleic acids from both sym- than those used in our experiments; Paula bionts (the photobiont normally constitute DePriest, unpublished). The amplified 5-10% of the thallus). Cyanobacterial li- DNA (0.1-0.2 pg) was restricted and the chens also yield mostly fungal DNA. products separated by electrophoresis Method II yields 200-500 ng DNA/10 mg of through 3% agarose (NuSieve, FMC) in tissue. When extracted from fresh material, TBE buffer. The fragments from a Hue111 the DNA is only minimally degraded; RNA digest of plasmid PBR322 were the size is always degraded, due to nuclease con- markers. taminants in Novozym. Method II selec- The primer binding sites are highly con- tively enriches for fungal DNA. Under the served among eukaryotes (Vilgalys and conditions used here, Novozym mostly Hester, 1990). Since Sticta genomic DNA damages hyphae, leaving algal cells suffr- was prepared with method II, contamina- ciently intact so that they do not release tion with DNA from the eukaryotic photo- their DNA readily. We are currently testing biont was minimal (see Results), and the whether the greenish pellet discarded dur- predominant amplification product was ing preparation of fungal DNA with method fungal. Pseudocyphellaria genomic DNA II can be used as a source of enriched algal was prepared with method I, and PCR am- DNA. plification of the 25 S ribosomal repeat from With both methods, final purification is P. rufovirescens (the green morphotype) achieved by reverse phase chromatography yielded two fragments, 2.2 and 2.5 kb in through an Elutip mini-column (Schleicher length, the latter derived from algal DNA. and Schuell). The removed contaminants To allow digestion of just the fungal prod- inhibit restriction enzyme activity and are uct from P. rufovirescens, the two ampli- most likely polysaccharides from the hy- fied fragments were separated electropho- phal wall, much thicker in lichens than in retically and individually isolated from the most other fungi (Ahmadjian, 1982), and/or gel. The genomic DNAs from P. murrayi from the mucillaginous matrix permeating (the blue-green morphotype) and P. hook- many lichen thalli (Greenhalgh and Whit- eri (the blue-green control outgroup), both field, 1987). 6 ARMALEO AND CLERC

Southern Analysis of gests from other photosy Paired Morphotypes. forming species of the sam knig~tiil~ivido~usca~ P. In the Pseudocyphellaria species ana- the probes did not hybridiz lyzed here, the cyanobacterial thallus lobes very weakly to correspond to P. murrayi, the ones with the those seen w green alga to P. rufovirescens. The two rufovirescens pair (not s morphotypes are readily distinguished by stringency employed in t their color and can be cleanly separated un- and wasbes ensures that t der a dissecting microscope. DNA was ex- tern generated by the probes is tracted with method I from the individual very similar, if not identical, g morphotypes (each from a separate thallus) quences a and digested with PstI or with EcoRI. The igests were probed according to the PC’R Analysis of Paired ~~r~~~ method of Southern (as described by Mani- In the photosymbiodeme from t atis et al., 1983, using three 32P-labeled sin- Sticta, the cyanobacterial t e-copy clones from a fungal genomic li- classified as S. dufourii, th y derived from the cyanobacterial mor- green alga as S. canariensis. type (Fig. 1). If P. murrayi (the blue- ite thallus, the two mo green morphotype) and P. rufovirescens (the green morphotype) share the same fungus, the probes derived from one of the members of the pair will highlight fragments identical in size and sequence in par- each mo~hotype allel digests from both morphotypes. The sections of the same thallus) an results shown in Fig. 1 conform to that ex- pectation. The converse is also true: used under the same conditions on genomic di-

ba action (White et al., 1989). DNA was restricted with fo nition enzymes and the fragme

Pst I ECORI FIG. 1. Southern blot autoradiographs showing the correspondence between restriction fragments highlighted by random fungal probes in parallel genomic sis pair, whereas t digests of the green (g) and blue-green (bg) morpho- revealed different types of the Pseudocyphellaria photosymbiodeme. Shown are the patterns obtained from PstI and EcoRI digestions. The labeled probes are three fungal DNA clones (14, 25, 43) chosen at random from a genomic library of the blue-green morphotype. Two probes were hybridized together to the parallel g and bg digests (14 and 25 to the PstI digests, 25, and 43 to the EccaRI digests). The numbers indicate the sizes, in kb, of the highlighted fungal DNA fragments. MYCOBIONT DNA IN LICHEN PHOTOSYMBIODEMES 7

g bg o A g bg o g bg o B 9 bg 0 tern and is not involved here (see also Ma- terials and Methods). This single intermor- photype heterogeneity does not change the conclusion, supported by the hybridization experiments and by the rest of the PCR data, that the murraylrufovirescens mycobionts have largely identical genomes. The point is exemplified by the data listed in

Hpa I, Table 1 which place at the lower end of the FIG. 2. (A) Ethidium bromide-stained agarose gel variability spectrum the restriction frag- with HpaII and Hue111 digests of the 2.2-kb rDNA ment length polymorphism observed within segment amplified by PCR from Sticta DNA. The each morphotype pair. symbols g and bg represent the paired SGcta green and blue-green morphotypes, respectively, and o repre- sents the Sticta outgroup. The fainter bands inter- DISCUSSION spersed through the predominant pattern in the g lanes are due most likely to background amplification of re- Nucleic Acid Extraction sidual algal rDNA. The numbers indicate the size range of the fragments, in bp. Arrows mark differences Method I yields high-molecular-weight between the outgroup and the photosymbiodeme. (B) DNA and RNA, and is best used with fresh Ethidium bromide-stained agarose gel with Hue111 starting material, which contains largely and NczI digests of the 2.2-kb rDNA segment ampli- undegraded DNA and RNA (progressive tied by PCR from Pseudocyphelluria DNA. Symbols, degradation of nucleic acid, most rapidly of other than referring to the Pseudocyphekzria morphotypes and outgroup, are as in (A). Asterisks mark the RNA, occurs in stored specimens, whether heterogeneity mentioned in the text. See also Table 1. dried in a herbarium or frozen at ambient moisture). The development of method II was based on the capacity of Novozym to We used PCR as an independent test of generate protoplasts from isolated lichen the results of the hybridization experiments mycobionts (Ahmadjian et al., 1987), and performed with Pseudocyphellaria. The ex- on some of the digestion parameters de- perimental design was the same as with scribed for other fungi (Stephen and Nasim, Sticta. The 2.2-kb ribosomal DNA segment 1981; Hamlyn et al., 1981; Chadegani et al., was amplified using genomic DNA from P. 1989). Method II is appropriate when the murrayi, from P. rufovirescens (the paired amount of tissue is too small to be ground morphotypes were sampled from separate efficiently (we used 25 mg of S. dufourii and thalli), and from a different species, P. 5 mg of S. canariensis). hookeri, used as control outgroup. Restric- tion patterns obtained with four-base rec- The One MycobiontlTwo ognition enzymes were compared on agar- Photobiont Hypothesis ose gels. Figure 2B shows the NciI and HaeIII digests, which confirm the conclu- Taxonomic implications. Our data ex- sion of the hybridization experiments and clude the possibility that phylogenetically fully parallel the Sticta data. unrelated fungi form paired morphotypes in A single Hue111 site differentiates one the photosymbiodemes analyzed. In fact, Pseudocyphellaria morphotype from the the results indicate that the mycobionts are other and is present only in a subset of the genetically very similar within each photo- repeats amplified from the green compo- symbiodeme. The high level of DNA relat- nent (the resulting bands are marked by * in edness shown here confirms the conclusion the g lane of Fig. 2B). Algal contamination of the anatomical studies which suggested would generate a clearly recognizable pat- hyphal continuity between the members of 8 ARMALEQ AND CLERC a pair in a variety of photosymbiodemes taxonomic status of each mo (James and Henssen, 1976; Brodo and Ri- within the framework of tbe pres chardson, 1978). Therefore, the combined The examples shown indicate t evidence strongly suggests (a) that paired patterns obtained from D mycobionts within a photosymbiodeme PCR can rapidly convey should be assigned to the same species, and information. Even withi (b) that the phenomenon is likely to be of relatively conserved widespread among photosymbiodemes in ences increase with taxonomic dist general. (Table 1). Our qualitative corn~a~§o~s wtt However, the debate about what consti- congeneric outgroups t tutes a lichen species is often far from set- bridization or PCR fi tled (Hawksworth, 1988, and references bined with the almos therein). White and James (1988), for in- observed polymorphism between paired stance, argue that morphotypes within a morpbotypes, strongly support their con- photosymbiodeme should be considered specificity. Near-identity was members of different species, even if even when the morphotypes we formed by the same mycobiont. Their pro- from separate thalli, as was posal contradicts the one fungus/one lichen Pseudocyphellaria. Detailed q rule set by the International Code of Botan- definitions of relatedness vs ~Ql~c~~a~ vari- ical Nomenclature (Greuter et al., 1988) ation in lichen mycobionts are ~ey~~~ the and, in our opinion, the biology of photo- scope of this study and will need the inciiu- symbiodemes. At the core of the lichen sion of additional taxa and d symbiosis lies the nutritional interaction be- Evolutionary impii tween a heterotroph and an autotroph. The ies have shown that paired morphotypes of photosymbiodemes, tal factors appear to favor when viewed as nutritional adaptations of or the other morphotyp the same fungus “feeding” on two different James and Henssen, 1976; photobionts, should not be considered dif- ologically, therefore, the associat~~r~ of a ferent species. By careful assessment of the single mycobiont with two p~~t~b~~~t§ as individual cases (Hawksworth, 1988) it divergent as a prokaryote and a ~~kary~te should be possible to define the appropriate y increasing the abil-

TABLE 1 RFLPs Observed in a 2.2-kb rDNA Segment Derived from Photosymbiodeme Mycobionts”

No. of Compared groups difference@ Enzymes S. dufourii vs S. canariensis 0 P. marrayi vs P. rufovirescens 2 Sticta pair vs outgroup 5 Pseudocyphellaria pair vs outgroup 11 Sticta vs Pseudocyphellaria” 13

n The same rDNA segment was amplified by PCR from each group and restricted, and digests were compared after electrophoresis. For every comparison, the number combines ah the band differences observed with each of two enzymes. b A difference is scored whenever a band is not shared among all the compared taxa, regardless of the possible kinds of mutations involved. ’ Both genera belong to one family, the Stictaceae (James and Renssen, 1975). MYCOBIONT DNA IN LICHEN PHOTOSYMBIODEMES 9 ity of one species (the photosymbiodeme) Carolina Biotechnology Center and by a grant from the to adapt to different environments. Photo- Swiss National Science Foundation. symbiodemes amplify and diversify the lichen’s basic evolutionary strategy of sym- REFERENCES biosis, and are part of a remarkable array of AHMADJIAN, V. 1982. Algahfungal symbioses. In complex myco- and photobiont combina- Progress in Phycological Research (F. E. Round tions (Hawksworth, 1988). Solorina, Pla- and D. J. Chapman, Eds.), Vol. 1, pp. 179-233. Elsevier, Amsterdam/New York. copsis, and Stereocaulon are genera bear- AHMADJIAN, V., CHADEGANI, M., KORIEM, A. M., ing mostly a green alga, with cyanobacteria AND PARACER, S. 1987. DNA and protoplast isola- restricted to small nodules (cephalodia) on tion from lichens and lichen symbionts. Lichen or within the thallus; in Peltigera venosa Physiol. Biochem. 2: l-11. the mycobiont associates with the green AHMADJIAN, V., AND JACOBS, J. B. 1985. Artificial re-establishment of lichens IV. Comparison be- alga Coccomyxa and two cyanobacteria, tween natural and synthetic thalli of Usnea strigosa. Nostoc and Scitonema (Ott, 1988). Den- Lichenologist 17: 149-165. driscocaulon, Lobaria, Sticta, and ARMALEO, D. 1990. Experimental microbiology of li- Pseudocyphellaria show varying degrees of chens: Mycelia fragmentation, a novel growth morphological divergence between paired chamber, and the origins of thallus differentiation. morphotypes (good color photographs of Symbiosis, in press. BLUM, 0. B., AND KASHEVAROV, G. P. 1986. DNA photosymbiodemes are in Brodo and Rich- homologies as proof of the legitimacy of the estab- ardson, 1978.) lishment of the lichen genus Lasallia M&at (Umbili- Experimental implications. Photosym- cariaceae). Dok. Akad. Nauk UrkSSR Ser. B biodemes provide a challenge to under- 1986(12): 61-64 (in Russian). stand at the molecular level how different BRODO, I. M., AND RICHARDSON, D. H. S. 1978. Chimeroid associations in the genus Peltigera. Li- developmental pathways can result from chenologist 10: 157-170. the interactions of one mycobiont with two BUBRICK, P. 1988. Methods for cultivating lichens and radically different photobionts. In this re- isolated bionts. In Handbook of Lichenology (M. gard, it will be interesting to investigate Galun, Ed.), Vol. III, pp. 127-138. CRC Press, Boca whether small genomic changes of the kind Raton, FL. CHADEGANI, M., BRINK, J. J., SHEHATA, A., AND seen in Pseudocyphellaria may reproduc- AHMADJIAN, V. 1989. Optimization of protoplast ibly develop during morphotype differenti- formation, regeneration, and viability in Microspo- ation. Photosymbiodemes should not be re- rum gypseum. Mycopathology 107(l): 33-50. garded as isolated evolutionary curiosities, CULBERSON, W. L. 1977. Lichenology: Progress and but as natural experiments on the basic problems. Bryologist 80: 22%230. mechanisms of lichen symbiosis and mor- CULBERSON, C. F., CULBERSON, W. L., AND JOHNSON, A. 1988. Gene flow in lichens. Amer. J. phogenesis. Bot. 75: 1135-1139. DUGHI, R. 1936. Etude comparee du Dendriscocaulon ACKNOWLEDGMENTS bolacinum Nyl. et de la cephalodie fruticuleuse du We thank Chicita F. Culberson, William L. Culber- Ricasolia amplissima (Stop.) Leight. Bull. Sot. Bot. son, Stephen Johnston, and Anita Johnson for their Fr. 83: 671-693. encouragement, suggestions, criticisms, and frequent DUGHI, R. 1937. Une cephalodie libre lichenogene: Le material support; Allan Green (University of Waikato, Dendriscocaulon bolacinum Nyl. Bull. Sot. Bot. Fr. New Zealand) for collecting the Pseudocyphellariu, 84: 430-437. and Peter James (British Museum) for sending the DUGHI, R. 1944. Sur les relations, la position systtm- Sticta specimens; Rytas Vilgalys for many discus- atique et l’extension du genre Dendriscocaulon. sions, for suggesting the appropriate PCR strategy, Ann. Fat. Sci. Marseille 16: 147-157. and for donating the primers; Bruce D. Kohorn for FEINBERG, A. P., AND VOGELSTEIN, B. 1983. A tech- making his laboratory widely available to us; and nique for radiolabeling DNA restriction endonu- Paula DePriest for the PCR controls. The work was clease fragments to high specific activity. Anal. Bio- conducted in C. F. Culbersons’ laboratory and was them. 132: 6-13. supported by a grant (ARIG882134) from the North GREENHALGH, G. N., AND WHITFIELD, A. 1987. 10 ARMALEO AND CLERC

Thallus tip structure and matrix development in Cold Spring Harbor Laboratory, Cold Spring Har- Bryoria fiscescens. Lichenologist 19(3): 295-305. bor, NY. GREUTER, W., BURDET, H. M., CHALONER, W. G., OTT, S. 1987. Sexual reproduction and ~eve!o~rn~~~l DEMOIJLIN, V., GROLLE, R., HAWKSWORTH, D. L., adaptations in Xantoria parietina. Nerd. .T. Bat. 7: NICOLSON, D. H., SILVA, P. C., STAFLEU, F. A., 219-228. AND Voss, E. 6. 1988. International Code of Bo- Orr, S. 1988. Photosymbiodemes and their develop- tanical Nomenclatwre. Koeltz Scientific, Koenig- ment in Peltigeru venosa. Lichenologist 20(4): 361- stein 368. HAMEYN, P. F., BRADSHAW, R. E., MELLON, F. M., RENNER, B.,AN~GALLQwA~, I). 9. 1982. PRycosym- SANTIAGO,~. M., WILSON, J.M., AND PEBERDY, biodemes in Pseudocyphellaria in lhjew Zealand. J. F. 1981. Efficient protoplast isolation from fungi Mycotaxon S(1): 197-231. using commercial enzymes. Enzyme Microb. Tech- SCHUSTER,G.,OTT,S.,ANIPJAPINS.H. 1985. Ar- nol. 3: 321-325. tificial cultures of lichens in the nat I environ- HAWKSWORTH, D. L. 1988. The variety of fungal- ment. Lichenologist 17: 247-253. algal symbioses, their evolutionary significance, and STEPHEN, E.R., ANDNASIM, A. i the nature of lichens. Bot. J. Linn. Sot. 96: 3-20. protoplasts in different yeasts by HILL, J. E., MYERS, A. M., KOERNER, T. J., AND 8. Microbial. 27: 550-553. TZAGOLOFF, A. 1986. Yeast/E. coli shuttle vectors [email protected]., and HOLTAN-HARTWIG? J. 1983. Phy- with multiple unique restriction sites. Yeast 2: 163- cotype pairs in Nephroma, Peltigera and Lobaria in Norway. Nord. J. Bot. 3: 681438. 167. VILGALYS, R., APID HESTER, M. 1990. Rapid genetic JAHNS, H. M. 1987. New trends in developmental identification and mapping of enzymatically ampli- morphology of the thallus and their influence on fied ribosomal DNA from several Cryprococcus concepts of lichen symbiosis and systematic% Bib/. species. J. Bacterioi. 172(x): 4238-4246. Lichenol. 25: 17-33. WHITE, F. J., APEDJAMES, P. W. 1988. Studies on the JAMES, P. W. 1975. Lichen chimeras. Rep. Brit. Mus. genus Nephroma. II: The southern temperatespe- Nat. Hist. 191274: 37-43. cies. Lichenologist Z@(2): 103-166. JAMES, P. W., AND HENSSEN, A. 1976.The morpho- WHITE, T.J., !+RWHEIM. N., AldIP &?LICH, H. A. logical and taxonomic significance of cephalodia. In 1989. The polymerase chain reaction. rr”ren& Genet. Lichenology: Progress and Problems (D. H. S(6): 185-189. Brown, D. L. Hawksworth, and R. H. Bailey, YAMAMOTO, Y. 1987. Tissue cultures of lichens. In Eds.), pp. 27-77. Academic Press, London/New Proceedings of Symposium on Tissue Cultwe ofLi- York. then and Bryophyte, pp. 14-25. Nippon Paint Co., MANXATIS, T., FRITSCH, E. F., AND SAMBROOK, J. Ltd., 19-17 Ikedanaka-machi, Neyagawa, 572. Ja- 1982. Molecular Cloning: A Laboratory Manual. pan.