J. Hattori Bot. Lab. No. 76: 235- 248 (Oct. 1994)

LICHEN PROTEINS, SECONDARY PRODUCTS AND MORPHOLOGY: A REVIEW OF PROTEIN STUDIES IN WITH SPECIAL EMPHASIS ON

JAN-ERIC MATTSSON1

ABSTRACT. Almost 30 protein studies with different objectives for c. 50 lichens species are compared and the taxonomic relevance, in particular, and other aspects of the use of elec­ trophoretic techniques discussed. The results indicate large enzymatic variation both in vegetat­ ively and sexually propagating lichens, differences in enzyme patterns between lichenized and non-lichenized bionts but also, correlation between protein banding patterns and secondary product chemistry or spatial separation.

INTRODUCTION Electrophoretic studies of proteins and enzymes are still far behind those of other organisms and for taxonomic purposes very few investigations have been made. Reasons for this have been the technical problems with the methods and the uncertainty of how to interpret the electrophoretic data taxonomically. The reliability of earlier techniques such as starch gel or polyacrylamide gel electrophoresis have been questioned, especially when combined with simple methods of plant protein separation (Kershaw et al. 1983). Isoelectric focusing has been shown to be more reliable, and the development of a standardized method of isoelectric focusing for routine use, which could be used by others than chemists and physiologists, has been necessary to provide lichen taxonomists with access to protein data. Although such a method has been developed by Fahselt and co-workers it has rarely been used for taxonomic purposes. The objective of this paper is, from the viewpoint of the taxonomist, to briefly review most of the studies dealing with electrophoretic investigations of lichen proteins and to discuss some of the general problems when interpreting these results taxonom­ ically.

GENERAL PROBLEMS WITH LICHEN ELECTROPHORESIS One of the main problems for electrophoretical investigations of lichens has been the difficulty of breaking the cell walls in a process to yield a reasonable concentration of active protein. With low protein concentration some isozymes may not be detected

1 Dept. of Systematic Botany, Lund University, Ostra Vallgatan 18- 20, S-223 61 Lund, Sweden. Present address: Botanical Museum, Uppsala University, Villavligen 6, S-752 36 Uppsala, Sweden. 236 J. Hattori Bot . Lab. No. 76 I 9 9 4 and cause erroneous information on the variation between different samples. Thus, apparent homogeneity may actually be heterogeneity when methods are changed (Kershaw et al. 1983). Lack of detectable activity does not necessarily indicate absence of a certain enzyme. Alternative extraction methods would yield different kinds of proteins and no extraction method seems, so far, to be successful in extracting the same enzymes from all lichens (Hageman & Fahselt 1984). Some of the studies discussed here use mainly general protein stains, while others look at specific (particular) enzymes. In the former cases there is more problem comparing bands between thalli. There is a greater chance that bands looking the same are not the same. Another problem with lichen enzymes is to make sure that the appropriate enzyme really is stained. The staining methods are often developed for studies of enzymes of higher plants or animals and not for or lichen enzymes; for example esterase and carbonic anhydrase (CAN) have similar function and for C/adonia cristatella these enzymes have zymograms resembling each other (Fahselt 1985). It is possible that CAN in lichens may also have esterase activity just as some CAN isozymes in mammalian cells have (Tashian 1977). It is necessary therefore to be cautious when interpreting isozyme data for genetic or taxonomic purposes. A major problem is also attempts to characterize a species without investigating variation present in either species or populations before deciding on sampling regimes. For example, Mattsson ( 1991) had difficulties in evaluating enzyme data for taxonomic purposes because of the large intraspecific variation compared to the small sample. The sampling problem is a major one with many of the studies reviewed here.

FUNGAL AND ALGAL CONTRIBUTIONS TO THE ZYMOGRAMS The co-occurrence of at least two symbiotic organisms is also a complication. Some taxonomists have been uncertain how protein data from electrophoretic investi­ gations of such organisms as lichens should be interpreted taxonomically. Some bands of a zymogram could represent enzymes composed of mycobiont and photobiont enzymes or parts of them, although most bands may originate from enzymes produced by one or the other of the symbionts. The algae make up about 10- 15 % of the cellular volume of the lichen thallus (Ahmadjian & Paracer 1986) and will to some extent contribute to the enzyme banding patterns. If for taxonomic reasons one prefer to use mycobiont characters it is interesting to know to what extent the photobiont is responsible for the bands of a zymogram of a thallus preparation. For Cladonia cristate/la Fahselt (1985) showed a large similarity between isolated mycob­ iont and lichen zymograms and concluded that most of the detectable lichen protein could well have been produced by the mycobiont, in some cases under influence of the photobiont.

ELECTROPHORETIC STUDIES OF LICHENS Electrophoretic protein studies on lichens have mainly dealt with populations of distinct species (e.g. Hageman & Fahselt 1984, Fahselt 1991, 1992) or more or less J.-E. MATTSSON: Lichen proteins, secondary products and morphology 237

Table 1. Lichen species studied with proteins studied by electrophoretic methods. Nomenclature according to original publication. Species Topic Reference alvarensis Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria a/varensis Taxonomy: pop. sp. Mattsson 1991, 1993 Cetraria arenaria lsozyme var. : p., pop. Fahselt & Hageman 1983 Cetraria cucullata Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria ericetorum Taxonomy: g., sp. Karnefelt & Mattsson 1987 Cetraria halei Isozyme var. : pop. Fahselt 1988 Cetraria islandica Taxonomy: g., sp., ssp. Klirnefelt & Mattsson 1987 Cetraria juniperina Taxonomy: g., sp. Klirnefelt & Mattsson 1987 Cetraria juniperina Taxonomy: g., sp. Mattsson 1991, 1993 Cetraria pinastri Taxonomy: g., sp. Mattsson 1991, t 993 Cetraria tilesii Taxonomy: g., sp. Mattsson 1991, 1993 Cladonia cristatella Symbiosis. Fahselt 1985 C/adonia cristatella Morphological. var. Fahselt 1985 Cladonia cristatella Genetic var. in Fahselt t 987 C/adonia mitis Environmental effects Fahselt 1992 Cladonia rangiferina Methodology Fahselt 1980 Cladonia rangiferina Isozyme var. : sp. MacFarlane et al. 1983 Cladonia stel/aris Methodology Fahselt 1980 Cladonia stellaris lsozyme var. : sp. Kershaw et al. 1983 Cladonia unicialis Methodology Fahselt 1980 Coe/ocaulon acu/eatum Taxonomy: g., sp. Klirnefelt & Mattsson 1987 Coelocaulon muricatum Taxonomy: g., sp. Karnefelt & Mattsson 1987 Evernia mesomorpha Isozume var.: pop. Fahselt 1988 Dermatocarpon miniatum Methodology Fahselt 1980 Hypogymnia physodes Isozyme var. : pop. Fahselt 1988 Lasa//ia papulosa lsozyme: repr. strat. Hageman & Fahselt 1990b Parmelia caperata Symbiosis. Martin 1973 Parmelia conspersa Taxonomy: sp., ssp. Skult et al. 1986 Parmelia cumberlandia Methodology Fahselt 1980 Parmelia hypoleucina Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia hypotropa Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia omphalodes Taxonomy: sp., ssp. Skult et al. 1986 Parme/ia omphalodes Taxonomy: ssp., pop. Skult et al. 1990 Parmelia perforata Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia rigida Taxonomy: sp., ssp. Fahselt & Jancey 1977 Parmelia saxatilis Taxonomy: sp., ssp. Skult et al. 1986 Parmelia sulcata Taxonomy: sp., ssp. Skult et al. 1986 Peltigera canina Methodology Fahselt 1980 Peltigera rufescens Physiology. Brown & Kershaw 1985 Platismatia tuckermannii Isozyme var.: pop. Fahselt 1988 Ramalina cuspidata Taxonomy: sp., v., pop. Mattsson & Klirnefelt 1986 Ramalina siliguosa Taxonomy: sp., v., pop. Mattsson & Karnefelt 1986 Stereocaulon saxatile Taxonomy: sp., v. Fahselt 1991 Umbilicariaceae Taxonomy: sp., v., pop. Hageman & Fahselt 1992 238 J. Hattori Bot. Lab. No. 76 I 9 9 4

Table I. (Continued) Species Topic Reference Umbilicaria decussata Isozyme var. : pop. Fahselt 1989 Umbilicaria deusta Isozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria hirsuta lsozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria hyperborea Isozyme var. : pop. Fahselt 1989 Umbi/icaria mammulata lsozyme var.: sp. Hageman & Fahselt 1984 Umbilicaria mammulata Isozyme: Seasonal var. Hageman & Fahselt l 986b Umbilicaria mammulata Physiolog. var.: ind. Larson & Carey 1986 Umbilicaria mammulata Isozyme; repr. strat.: sp. Hageman & Fahselt 1990b Umbi/icaria muhlenbergii Isozyme; morph.: sp., pop. Hageman & Fahselt l 986a Umbilicaria muhlenbergii Isozyme; repr. strat. Hageman & Fahselt 1990b Umbilicaria vellea Physiolog. var.: ind. Larson & Carey 1986 Umbilicaria vellea Functional sexuality. Hageman & Fahselt l 990a Umbilicaria vellea Isozyme: repr. strat. Hageman & Fahselt l 990b Umbilicaria virginis lsozyme var. : pop. Fahselt 1989 Usnea subjioridana Isozyme var.: pop. Fahselt 1988 Xanthoria elegans Isozyme var.: morph, pop. Fahselt & Krol 1989 Abbreviations: g. = , ind. = individual, morph.= morphology, pop.= population, repr. strat. = reproductive strategies, sp. = species, ssp. = subspecies, v. = variety, var. = variation. closely related taxa from populations within a limited area (e.g. Fahselt & Jancey 1977, Mattsson & Karnefelt 1986, Skult et al. 1986, 1990, Karnefelt & Mattsson 1987). The number of species investigated so far is rather limited (Tab. 1). Electrophoretic studies with more or less taxonomic objectives have only been made in 7 genera (accepted in the broad sense); Cetraria, Cladonia, Parmelia, Ramal­ ina, Stereocaulon, Umbilicaria and Xanthoria. Some general observations can be made from these studies (Tab. 2). Most of the studies describe the enzymatic variation between or within populations or between or within morphotypes. It has been shown that in Umbilicaria, the similarity between two conspecific populations is greater than between populations belonging to different species (Hageman 1989, cf. Fahselt & Krol 1989), and that the similarity between conspecific lichens with different morphology belonging to the same population is greater than between specimens with similar morphology belonging to different populations. The low enzyme similarity between species of Umbilicaria, suggest that enzyme data may be most successfully used for intraspecific comparisons. However, this question should be further investigated in other groups of lichens. These research­ ers have also shown how the enzymatic similarity between different morphs of Cladonia and Xanthoria species are as large as these between conspecific populations of other lichens.

LICHENS AS ECOSYSTEMS The lichen thallus is an ecosystem of at least two individuals, and thus not J.-E. MATTSSON : Lichen proteins, secondary products and morphology 239

Table 2. Electrophoretic protein studies of lichens. Nomenclature according to original publication. Topic, author, species Objective, methods Results

METHOD DEVELOPMENT Fahselt 1980 Different techniques for disrupting Pulverization of acetone-washed Cladonia rangiferina, lichen cells were tried. General thallus fragments was effective. C. stel/aris, C. uncialis, protein staining. Isoelectric focusing on polyacryl­ Dermatocarpon miniatum, amide gels was found to be supe­ Parmelia cumber/andia, rior to electrophoresis. Peltigera canina Fahselt & Hageman 1983 To explore the range of isozymes Different activities of different Cetraria arenaria detectable in two stands of the enzymes were found. lichen.

STUDIES OF SYMBIOSIS Fahselt 1985 Comparison between enzyme bands Most of the lichen bands are of Cladonia cristatella of the lichen thallus and the isolated fungal origin. The origin of lichen P. caperata (Martin 1973) symbionts of Cladonia cristate/la. enzymes is discussed. The structure of Parmelia caperata invertase is discussed as well as the genetic variability within lichen populations. Martin 1973 Comparison of occurrence and Lichen invertase has other proper­ Parmelia caperata properties of invertases from thallus ties than the other invertases and and the isolated symbionts. is probably composed of subunits from both symbionts.

PHYSIOLOGICAL STUDIES Kershaw et al. 1983 To characterize the potential range Good level of isozyme homogeneity C/adonia stellaris of enzyme polymorphism in two between the two morphotypes contrasting morphotypes of Cladonia studied. ste/laris from sun and shade locations. Larson & Carey 1986 Study of intraindividual enzymatic Large thalli were polymorphic. Umbilicaria mammulata polymorphism. Umbilicaria ve/lea MacFarlane et al. 1983 To characterize the potential range Good level of isozyme homogeneity C/adonia rangiferina of enzyme polymorphism in between the two morphotypes two contrasting morphotypes of studied but large differences C/adonia rangiferina from sun between tips and bases of podetia. and shade locations.

POPULATION, VARIATION, REPRODUCTION AND DISPERSAL Brown & Kershaw 1985 Differences i proteins and enzymes Co-variation in gas exchange Peltigera rufescens between three physiologically dif­ and protein banding patterns ferent populations. between the populations indicates adaptations to the environment. Fahselt 1987 To examine mycobiont cultures Some loci in the mycobiont must Cladonia cristate/la derived from single ascospores have undergone meiotic recom­ from one apothecium. bination. 240 J. Hattori Bot. Lab. No. 76 I 9 9 4

Table 2. (Continued) Topic, author, species Objective, methods Results

Fahselt 1988 Evaluation whether some enzyme Esterase and alcaline phosphatase Cetraria halei, Evernia systems are variable in many seems to be most variable. Both mesomorpha, Hvpogymnia , lichens and to study enzyme sorediate and fertile lichens physodes, Platismatia variability among unrelated showed great enzyme variability. tuckermannii, Usnea species growing together. subfioridana Fahselt 1989 Comparison of enzyme variability Stands of the fertile species had Umbilicaria decussata , between apotheciate and sterile the greatest variability, suggesting U. hyperborea , U. virginis species. that regular sexual reproduction contributes to greater diversity. Hageman & Fahselt 1984 To determine whether there was Each site had a distinct set of Umbilicaria mammulata intraspecific variability of isozymes, protein characters and close collec­ intersite differences related to dis­ tion sites had greater similarity tance and to compare the contri­ of enzyme forms. Esterase, 6- bution of different enzyme systems phosphogluconate dehydrogenase to the elucidation of group structure. and alcaline phosphatase were most variable. Hageman & Fahselt 1990a To address the question of whether Sexual recombination is probably Umbilicaria vellea the sexual life cycle might function responsible for some variability in in U. veilea. U. vellea and the ascospores play some role in the reproduction and the dispersal. Hageman & Fahselt 1990b To investigate and interpret the There may be considerable genetic Lasa/ia papulosa, patterns of variability in one group variability both in apotheciate and Umbilicaria deusta, of lichens in relation the reproduc­ strictly vegetatively reproducing U. hirsuta, U. mammulata, tion and dispersal potential of each lichen species, and sterile lichen U. muhlenbergii, U. vellea species. species do not have inherently less evolutionary potential.

SEASONAL VARIATION Hageman & Fahselt 1986b Study of seasonal variation in one Variation in some but not all Umbilicaria mammulata population. enzymes studied. Skult et al. 1990 Specimens of three subspecies from The subspecies had different en­ Parmelia omphalodes different populations were compared zymatic and protein characteristics. for seasonal variation of isoenzymes.

TAXONOMIC STUDIES Fahselt 1986 Comparison between enzymes and No significant similarities found. Cladonia cristatel/a morphological characters. Fahselt 1991 To determine whether multiple The enzymatic variation between Stereocau/on saxatile enzyme forms indicated a genetic the two forms was similar to that distinction between two forms and if between conspecific populations a distinction was observed to assess rather than populations between whether it was similar in magnitude two different species. to that between separate species. Fahselt & Jancey 1977 To develop a general technique to Support of taxonomic separation Parmelia hypoleucina, observe total banding patterns in based on secondary chemistry. P. hypotropa, P. perforata, lichens. To determine whether such Parmelia rigida bands constitute taxonomically use­ ful information. J.-E. MATTSSON : Lichen proteins, secondary products and morphology 241

Table 2. (Continued) Topic, author, species Objective, methods Results

Fahselt & Krol 1989 Comparison between enzymes, No substantial biochemical differ­ Xanthoria e/egans secondary metabolites, morphology ences between the morphologically and populations. distinct forms was found. Hageman & Fahselt 1987a Comparison between morphology, No significant similarities between Umbilicaria muhlenbergii populations and enzyme bands. enzymes and morphology but similarities between individuals from the same population. Hageman & Fahselt 1992 To assess relationships among six Similarities between conspecific Lasallia papulosa, umbilicate lichen species based on populations are of a different order Umbilicaria deusta, enzyme characters, and to relate of magnitude than those between U. hirsuta, U. mammulata, enzyme data to currently used species. Electrophoretic analysis U. muh/enbergii, U. vellea classifications of the Umbilicariaceae. constitutes means of assessing systematic relationships. Kiirnefelt & Mattsson 1987 Comparison between protein bands Similarities between morphological Cetraria cucu/lata, and species/subspecies. species and banding patterns. C. ericetorum, C. islandica , C. juniperina , Coelocaulon aculeatum, C. muricatum Mattsson & Kiimefelt 1986 Comparison between protein bands Weak support for taxonomic re­ Ramalina cuspidata, and secondary product chemistry. cognition of chemotypes. R. siliquosa Mattsson 1991, 1993 To study variation in populations No significant variation between Cetraria alvarensis, of Cetraria a/varensis in Europe two morphs of C. a/varensis was C. juniperina, C. pinastri, and to compare them with popu­ found. Specimens of different C. tiles ii ( Vulpicida lation of C. tilesii from North species from the same continent tubulosus, V. juniperinus, America. showed higher similarity than V. pinastri, V. tilesii) specimens of C. pinastri from different continents. Skult et al. 1986 Comparison of banding patterns of Support of earlier taxonomic Parmelia conspersa, total protein and isozymes of species separation based on morphology P. omphalodes, P. saxatilis, and subspecies. and secondary chemistry. P. sulcata comparable with an individual plant or an individual fungus. This is a complication not only for lichen taxonomists but also for scientists in other fields of lichen biology when designing experiments and evaluating results. A lichen should never be presumed to act like an individual, although in many cases it really does. Most lichen species show a wide morphological, physiological and biochemical variation which may be caused by the symbiosis. In the lichenized state, the mycobiont and the photobiont form a thallus which the mycobiont is unable to produce by itself. The formation of the thallus is also influenced by external factors. The morphology of thalli of a lichen species may therefore be quite variable. Thus, the lichen taxonomist has to be aware of the possibility for similar mycological genotypes to form morphologically different thalli and that similarity in morphology does not automatically indicate close phylogenetic relationship. 242 J. Hattori Bot. Lab. No. 76 l 9 9 4

The importance of the interaction between the symbionts and the environment has until recently mainly been studied by ecophysiologists. The physiological responses of lichens exhibit great plasticity; lichens growing in similar habitats often show similar physiological responses even if they belong to different genera, and specimens of the same species show different responses when growing in ecologically different sites. On the other hand, lichens of different species, with different morphology and from different habitats sometimes have similarities in their physiological response (cf. Kershaw 1985). To some extent a lichen is comparable to individuals of other organisms but the former may also often exhibit features of a micro-ecological unit. The character set of a lichen includes attributes of both symbionts, influenced by the symbiosis and the environment.

DIFFERENCES BETWEEN THE LICHEN AND ITS ISOLATED SYMBIONTS Secondary metabolites, such as depsides, depsidones, and other products of the acetate-polymalonate pathway, are usually produced only in the lichenized thalli and often not by the isolated symbionts, although mycobionts are able to produce them alone under certain conditions. These products are depositeC: extracellularly and although their function in many cases is uncertain, the cost of production is so high that it is reasonable to assume that the secondary metabolites in some way are beneficial to the lichen. Primary metabolites, such as lichen enzymes, may also be produced only in the lichenized state and deposited outside the cell walls. Harley and Smith (1956) found extracellular surface carbohydrase activity in Peltigera polydactyla. Exogenously suppl­ ied sucrose was converted to monosaccharides which were taken up by the mycobiont. Martin (1973) compared the occurrence and the properties of invertase isolated from the lichen thallus of Parmelia caperata with those of the invertases isolated from each of the symbionts grown in pure culture. Five forms of invertases were studied. Two from the photobiont in pure culture, one of these were intracellular and the other one was recovered from the growth medium. Two invertases were intracellular mycob­ iont enzymes and one was the lichen thallus invertase. The latter was studied both in vivo and in vitro. The optimal pH range of the P. caperata lichen thallus invertase was as wide as or wider than the invertases of the isolated symbionts (Fig. I.). The heat stability of the thallus invertase was greater than for the invertases of the symbionts (Fig. 2), especially the in vivo air dry thallus invertase which was more thermally stable, in fact up to 100°C. The invertases of the isolated symbionts were also more sensitive to inhibitors than the lichen thallus invertase. Thus, the lichen enzyme may be more adaptive than the corresponding enzymes in the isolated symbionts. Martin (1973) also suggested, and this idea has been further developed by Fahselt (1985) with support of molecular weight and substrate specificity data, that the lichen thallus invertase is composed of the entire mycobiont protein and one half of the extracellular photobiont protein compo­ nent. J.-E. MATTSSON: Lichen proteins, secondary products and morphology 243

fo active., ·~ ~ ~ 0 ~~ !Cl 0 0 • o ;0 • i • 0 0 A 0 60 o. ~• 0 0 0 A ~ • 0 20 • ~ 0 • • 0 • 0 • • 2.5 3 4 5 6 7 8pH Fig. 1. Activity of the invertases of P. caperata as a function of pH (after Martin 1973). Legend in Fig. 2.

VARIATION IN ENZYMATIC ACTIVITY CAUSED BY ENVIRONMENTAL FACTORS Seasonal variability in some enzyme systems which occur in lichens (Hageman & Fahselt 1986b, Skult et al. 1990) is another complication. For taxonomic purposes it is necessary to make comparable collections at the same time of the year. The adaptations of lichens to different climatic conditions may also influence the banding patterns just as these adaptations change the physiological properties of the lichens ( cf. Kershaw 1986). To ensure comparable results in enzyme studies, as is the case in ecophysiolog­ ical studies, the material collected for taxonomic purposes should not only be collected at the same time of the year but also under the same microclimatic conditions. To avoid changes in the seasonal physiology of the lichens all material should be kept under similar conditions during transport and storage. Fahselt (1992) has also showed how external factors such as geothermal outgass­ ings result in enzymatic differences between Cladonia stands comparable to those between geographically separated conspecific populations. This suggested that factors, 244 J. Hattori Bot. Lab. No. 76 I 9 9 4

% activity 10 0 0 0 0

60 • • Trebouxia (P. caperata) cell invertase 0 Trebouxia (P. caperata) medium invertase • ~ P. caperata mycobiont A-form invertase A. P. caperata mycobiont B-form invertase • 0 P. caperata thallus invertase

20 •

5 10 20 30min Fig. 2. Change in activity of the invertases of P. caperata at 55 °C as a func­ tion of time (after Martin 1973). such as temperature, pH and soil chemistry may affect enzyme forms in lichens. The large enzymatic variation in populations of sorediate lichens, also demonstrated by Fahselt (1988), is also important to the taxonomist.

POLYMORPHIC THALL! WITH RESPECT TO lsOZYME PHENOTYPES Larson and Carey ( 1986) reported that large thalli of Umbilicaria mammulata and U. vel/ea are polymorphic with respect to the isozyme phenotypes. This is also a challenge for the taxonomist. They discussed seven possible explanations: 1) The fusion of intact thalli during colonization; 2) the invasion of intact lichens by fungal spores, algal cells or intact soredia; 3) the accumulation of somatic mutations; 4) heterokary­ osis; 5) parasexual cycle in which separate nuclei fuse following heterokaryosis; 6) age-dependant developmental processes, and 7) Microclimatic or nutritional factors within the thalli which could select for differences in expressed genotype. The possibility for lichens to grow for at least 100 years makes it possible for several somatic mutations to be maintained in different parts of one thallus (Hageman J.-E. MATTSSON: Lichen proteins, secondary products and morphology 245

& Fahselt 1990b). Ott (1987a, 1987b) showed how a single thallus may be derived from several different propagules. This can also explain the large genetic variation among lichens.

REPRODUCTION AND DISPERSAL Sexually reproducing lichens seem to have a larger enzymatic variability compared to vegetatively reproducing lichens but the differences are small (Fahselt 1988, Hage­ man & Fahselt 1990b). Sterile Umbilicaria mammu/ata, that produces thalloconidia and sorediate U hirsuta showed particularly higher diversity values than the apothec­ iate species (Hageman & Fahselt 1990b). Studies of other usually sterile species show similar results, especially sorediate species such as Parmelia hypotropa and P. hypoleucina (Fahselt & Jancey 1977). Hypogymnia physodes and Usnea subfioridana (Fahselt 1988) show large enzymatic variation, but other rarely apotheciate species such as Cetraria arenaria (Fahselt & Hageman 1983 ), C. tilesii ( Vulpicida tilesii) and C. alvarensis ( V. tubulosus ), (Mattsson 1991, 1993) are enzymatically variable. These findings suggest that there may be considerable genetic variability in not only apotheciate species but also strictly vegetatively reproducing lichen species. Therefore, each population or species must be sampled carefully before it can be characterized enzymatically.

TAXONOMIC STUDIES The total number of taxonomic studies in this field is low, therefore, one should not make sweeping generalizations about enzyme data being correlated with morphology, secondary chemistry etc. Anyhow, the studies reviewed here show correlations which urge further studies on the genetic vs. the environmental impact on lichens. In a study which primarily had method development of lichen electrophoresis as an objective, Fahselt and Jancey ( 1977) found support in protein evidence for recognition of members of the Parme/ia perforata group at species level based on secondary product chemistry. Skult et al. ( 1986) presented isozyme data from the Parme/ia omphalodes group, which also supported earlier proposed taxonomic recognition based on secondary product chemistry and morphology. Mattsson and Kiirnefelt ( 1986) published a study of proteins in the Ramalina siliquosa group which only gave weak support to the suggested taxonomy based on secondary product chemistry. Later, Kiirnefelt and Mattsson ( 1987) compared six Cetraria species and found similarities between the accepted morphological species and the protein banding patterns, but only weak support for recognition of intraspecific (morphological) taxa. Skult et al. ( 1990) however found enzymatic differences between the subspecies of Parme/ia omphalodes. In my own studies of the vulpinic acid containing Cetraria ( Vulpicida) species I found no support for a taxonomic recognition of the terete and fiat morphs of C. 246 J. Hattori Bot. Lab. No. 76 I 9 9 4 alvarensis ( V. tubulosus) when only the most typical specimens of each morph were analyzed (Mattsson 1991, 1993). The first study on generic comparison within a family was recently published (Hageman & Fahselt 1992). The results are only partly in concordance with proposed classifications mainly based on morphological characteristics. The number of studies are to small for general conclusions and many of the more or less taxonomic studies are not based on enzymes but "proteins" without reference to what they might be, and therefore with less chance of knowing if they are concordant or not. This is the major drawback of taxonomic studies based on just "proteins". The correlation between protein and enzyme banding patterns and geographic distance, secondary product chemistry and morphology differ between the species studied so far but can be of similar magnitude.

DISCUSSION It seems to be easier to find support by protein data or isozyme data for recognition on species level, if the taxonomy proposed is based on qualitative differences in secondary product chemistry rather than on sometimes minor but often discrete, morphological characters. Is secondary product chemistry, within a mainly morpholog­ ically defined species, better for taxonomic recognition than morphological characters? Morphological characters are subject to environmental influence to a large extent and the response to this influence is quite variable as manifested in the great plasticity of many of these characters. Secondary products may be essential for the maintenance of the symbiosis in the lichen thallus and the production of these substances may also be genetically controlled. If this is true, the chemical variation within a species will also be rather small which is the case among many lichens. Thus, differences in secondary product chemistry may be strong indications for taxonomic recognition. The large enzymatic variation in several vegetatively propagating lichens is of importance to the taxonomist. Similar patterns may also occur in other characters. This variation may explain why so many sorediate species are distributed over large areas and are common in quite different habitats. They are not only propagating efficiently, they also have sufficient genetic variability to occupy a range of habitats. Thus, it could be questioned if they should be regarded as evolutionary dead ends as proposed by Tehler (1982). Evolutionary potential may remain if genetic variability is maintained by means other than sexuality.

ACKNOWLEDGEMENTS This study was supported by a grant from the K. A. Wallenberg Foundation. I thank Dr. R. Moberg for letting me use the facilities of the Herbarium in Uppsala and Dr. L. Tibell and M. Wedin for constructive criticism of the manuscript. Finally I also give my sincere thanks to two referees for their constructive help. J.-E. MATTSSON: Lichen proteins, secondary products and morphology 247

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