Ascogenous hyphae in foliicolous species of Arthonia and allied genera

Mycol. Res. 105 (8) : 1007–1013 (August 2001). Printed in the United Kingdom.

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Ascogenous hyphae in foliicolous species of Arthonia and allied genera

Martin GRUBE1 and Robert LU$ CKING2 " Institut fuW r Botanik, Karl-Franzen-UniversitaW t Graz, Holteigasse 6, A-8010 Graz, Austria. # Lehrstuhl fuW r Pflanzensystematik, UniversitaW t Bayreuth, UniversitaW tsstrasse 30, D-95440 Bayreuth, Germany. E-mail : martin.grube!kfunigraz.ac.at Received 10 August 2000 ; accepted 26 February 2001.

Ascogenous hyphae of foliicolous members of the Arthoniaceae, including the genera Arthonia, Eremothecella, Amazonomyces, and Cryptothecia, were studied by epifluorescence microscopy. Two additional representatives of Opegrapha were included. The ascogenous hyphae in Arthonia, Eremothecella, Amazonomyces, and Opegrapha arise from a central region of the ascoma and form a dense and branched tree-like structure. The asci of Cryptothecia are not connected by such structures and arise singly. Differences of measurements, structures and branching patterns of ascogenous hyphae indicate that these are species-specific characters. Typical ascogones were not observed. The fusion of tips or croziers of ascogenous hyphae and haploid mycelium were seen in several species. The systematic significance of the observations is discussed.

INTRODUCTION Whether ascogones are formed prior to the development of other fruit body structures or afterwards distinguishes major groups of Ascomycota (Nannfeldt 1932), and these fungi were assigned to either the ascohymenial or the ascolocular type of development. While most orders of fungi were classified easily in that way, only the Arthoniales, or Arthoniomycetes sensu Eriksson (1999), remained controversial. Some representatives lack typical fruit body structures of other Ascomycota and because this group was thought to combine both ascohymenial and ascolocular characters, they were treated as a ‘ Zwischengruppe’ by Henssen & Jahns (1973) and Henssen & Thor (1994). Nevertheless, Tehler (1990) interpreted the ascomata of Arthoniales as ascolocular and introduced a detailed terminology to describe the diversity of fruit bodies in this order. The new terminology distinguished cases with single or several centres per ascoma or asci per locule. Arthonia radiata, the type species of Arthonia, has pluricarpocentral ascomata, as opposed to Opegrapha vulgata with monocarpocentral ascomata. Both species have a centrum with multiascal locules. The ‘centrum’ includes ascogenous hyphae, asci and sterile mycelium which occupy the locule within which the asci develop (Lutrell 1951). By anatomical studies of the ascomata, Tehler (1990) found that the individual centres in fruit bodies of the Arthoniales are separated by intrusions of interascal plectenchyma, i.e. by excipuloid strands. Consequently, differences in ascomatal characters between representatives of

the order were used in various phylogenetic approaches (e.g. Tehler 1990, Myllys et al. 1998). Although this works well for many genera with uniform ascomatal structure, considerable variation has been noticed in Arthonia and allied genera (Grube 1998). Albeit still insufficiently understood, the ascomatal heterogeneity may therefore be of use in a reclassification of the huge genus Arthonia with around 500 species. Foliicolous species in Arthonia and related genera are of special interest in this respect because the delimitation of these genera is based on subtle morphological differences (Lu$ cking 1995, Ferraro & Lu$ cking 1997, Lu$ cking et al. 1998). During investigations of the thallus structures of foliicolous lichens using epifluorescence microscopy (Grube & Lu$ cking 2000), we discovered the presence of distinctly stained ascogenous hyphae in the basal ascomatal layers of some species investigated, notably in Arthonia. As this could add to the systematic concept of this group, we have here studied the growth-patterns of ascogenous hyphae in correlation with the ascomatal construction of more foliicolous Arthoniales.

MATERIAL AND METHODS Entire ascomata or fragments where detached from the thallus using the edge of a broken razorblade. With forceps, ascomata where oriented upside down on a slide and a coverslip was placed on top. The dyes used were Calcofluor White and Cotton Blue, as 1 % aqueous solution and Diamino phenylindol dihydrochloride (DAPI), as 50 µg ml−" solution in PBS

Ascogenous hyphae in Arthoniaceae (phophate buffered saline) pH 7n4 (120 m NaCl, 7 m Na HPO , 3 m NaH PO , 2n7 m KCl). Eight µl of staining # % # % solution were added. After ca 5 min, excessive dye was washed using tap water, or 5 % KOH in the case of Calcofluor White. Light microscopy and epifluorescence microscopy were carried out using a Zeiss Axioskop microscope. For fluorescence detection of Calcofluor White we have used filter set 09 from Zeiss. Cryptothecia candida and Eremothecella macrocephala have very fragile ascomatal structures filled with crystals, but after treatment with potassium hydroxide (10 %), the structures become more compact and it is possible to retain integrity while removing the ascomata from the leaf surface. The following specimens were studied : Amazonomyces farkasiae : Costa Rica : Heredia province : La Selva Biological Station (O.T.S.), near Puerto Viejo de Sarapiquı! , 10m 26h N, 84m 03h W, 50–100 m, primary lowland rain forest, Jan.–Feb. 1999, R. LuW cking 99-87 (GZU). Arthonia accolens : loc. cit., R. LuW cking 99-95 (GZU). Arthonia aciniformis : loc. cit., R. LuW cking 99-90 (GZU). Arthonia cyanea : Costa Rica : San JoseT province : Braulio Carillo National Park, about 35 km NNE of San Jose! at the San Jose! –Limon highway, 83m 58h W, 10m 09h N, 700–800 m, primary forest, Sept. 1991, R. LuW cking 91-3126 (GZU). Arthonia leptosperma : Costa Rica : Puntarenas province : Monteverde Biological Reserve, 10 km NE of Santa Elena, 10m 16h N, 84m 46h W, 1600–1700 m, secondary forest close to entrance, foliicolous in understorey, Feb. 2000, R. LuW cking 00-291 (GZU). Arthonia mira : Brazil : Rio de Janeiro, Morro Canos da Carioca, 19. Aug. 1892, G.O. Malme 163 :1m (UPS). Arthonia palmulacea : Costa Rica : Corcovado National Park, about 130 km SSE of San Jose! , 50 km WSW of Golfito on the Osa Peninsula, 83m 35h W, 8m 28h N, 50–150 m, primary and secondary forest on steep trail from the ‘ Sirena ’ ranger station to Rio Claro, July 1992, R. LuW cking 92-3586 (GZU). Arthonia trilocularis : Costa Rica : Heredia province : La Selva Biological Station (O.T.S.), near Puerto Viejo de Sarapiquı! , 10m 26h N, 84m 03h W, 50–100 m, primary lowland rain forest, Jan.–Feb. 1999, R. LuW cking 99-92 (GZU). Cryptothecia candida : loc. cit., R. LuW cking 99-91 (GZU). Cryptothecia effusa : loc. cit., R. LuW cking 99-76 (GZU). Eremothecella calamicola : loc. cit., R. LuW cking 99-91 (GZU). Eremothecella macrocephala : Papua New Guinea : Madang prov. : Burbura logging site, ca. 35 km NNW of Madang, ca. 6 km along road from the coast, 50 m, 145m 38h E, 4m 48h S, virgin rainforest on low hills, 28 Aug. 1992, E. SeT rusiaux 13500-52 (LG). Eremothecella macrosperma : New Guinea : Monrobe, Boana, 1000–1300 m, 12. July 1940, Clemens (UPS). Eremothecella variratae : Sumatra : Oostkust v. Sumatra, Besitang, 30 m., 1926, B. Palm 59 p.p. (UPS). Opegrapha filicina : Costa Rica : Alajuela province, Arenal volcano forest reserve, 10m 28h N, 84m 40h W, 500 m, primary forest on steep trail to the waterfall of the Rio Fortuna, on leaves of dicotyledon, Dec. 1991, R. LuW cking 91-2156 (GZU). Opegrapha puiggiari : Costa Rica : Puntarenas province, Golfito village, Golfo Dulce, close to airstrip, 8m 39h N, 83m 12h W, 50 m, on leaves of Mangifera indica, Dec. 1991, R. LuW cking 91-2937 (GZU).

RESULTS Ascogenous hyphae were recognized by the presence of asci at the branch-tips and by the presence of croziers (except in Arthonia leptosperma). In one species, A. aciniformis, we

1008 observed two nuclei per cell in the ascogenous hyphae and one nucleus in each vegetative hypha in the ascomata. Nuclei were otherwise difficult to observe using DAPI staining and are presumed to degrade rather quickly in dried herbarium specimens. All ascogenous hyphae differ from vegetative hyphae in their larger size and the brighter coloured cell walls when stained with Calcofluor White. Calcofluor White (excitation at 437 nm, emission at 490 nm) has a high affinity to linear polysaccharides with certain properties (Wood 1980) : β-conformation at the C1, the presence of a CH OH# 6-moiety, and an equatorial arrangement of hydroxyl groups at the C2 and C4 positions. Such polysaccharides apparently occur in larger amounts in the walls of ascogenous hyphae. Ascogenous hyphae in several foliicolous Arthoniales are arranged in a plane in the basal layer of the ascomata and can be visualized in preparations of complete ascomata (Figs 1–6). In Arthonia leptosperma, the tiny asci are hardly distinguished in whole ascomatal mounts, and asci are not formed at the periphery of the ascogenous hyphae (Fig. 1). In A. accolens asci are distinct and also formed at the periphery (Fig. 2) ; this was also observed in A. palmulacea, in which the central part is less stained (Fig. 3). In other species, algal colonies may be present below the ascomata, for example in Arthonia trilocularis and Eremothecella macrocephala (Figs 4–5). Lichenicolous hyphae which are present and distinct below the ascomata do not disturb or interrupt the branching pattern of ascogenous hyphae (Fig. 6, E. variratae). The basal arrangement of ascogenous hyphae has been observed also in Amazonomyces farkasiae, Eremothecella calamicola, and in other investigated Arthonia and Opegrapha species. In Crypthothecia candida and C. effusa, the ascogenous hyphae are not conspicuous and developed only below the individual asci. The situation in Mazosia requires further studies and we were unable to distinguish ascogenous hyphae in Enterographa species. Likewise, ascogenous hyphae were inapparent in other foliicolous lichens with flat ascomata ; e.g. Byssolecania, were a dense basal layer of sterile mycelium occurs below the layers with ascogenous hyphae. The ascogenous hyphae of the species studied form strongly branched hyphal systems. In several Arthonia spp., in Amazonomyces farkasiae, and in Eremothecella calamicola, they originate from a central region of the ascomata and radiate towards the periphery (Fig. 7, Arthonia aciniformis). The radiate pattern is less distinct in other species, for example in the lichenicolous A. cinnabarinula (Fig. 8) or in A. palmulacea (Fig. 9). Whereas asci and ascogenous hyphae are more or less regularly spaced in these species, Eremothecella macrocephala has ascogenous hyphae which may grow in bundles (Fig. 10), something also seen in Amazonomyces farkasiae. In Opegrapha filicina, long stretches of primary ascogenous hyphae, from which side branches emerge, grow with the extending lirellate ascomata (Fig. 11) ; this is also seen in O. puiggarii. Frequently, more than one long primary ascogenous hypha forms in parallel. In Arthonia trilocularis, a green fluorescent pigment interfered with the Calcofluor White stain. KOH can be used used to remove this pigment, after which the ascomata are more translucent. Asci with ascospores can clearly be recognized (Fig. 12). The asci arise directly as side-branches of

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Figs 1–6. Entire ascomata stained with Calcofluor White seen from the lower side. Fig. 1. Arthonia leptosperma. Fig. 2. A. accolens. Fig. 3. A. palmulacea. Fig. 4. A. trilocularis. Fig. 5. Eremothecella macrocephala. Fig. 6. E. variratae. Bars l 100 µm.

the ascogenous hyphae (Figs 12–14), but not at the tips of main branches. Usually, no more than one side branch is formed from each ascogenous cell which develops into an ascus. In these cases, the branching point is below the apical septum in the hooked part of the branching cell. In A. leptosperma (Fig. 14) we also observed more than one ascus per ascogenous hyphal cell. In this species, the asci branch in a more or less vertical direction, whereas in other species (e.g. Eremothecella calamicola and Arthonia palmulacea) the stalks of the asci branch more or less horizontally, and then bend upwards.

In young ascomata, a central single plexus of a few cells can be discerned from which the hyphae branch, whereas in mature ascomata (Fig. 7) the central part is often less conspicuously stained, somewhat disorganized, or filled with irregularly branching hyphae (Fig. 3). Ascogenous hyphae in the ‘ diffuse ’ centre have poorly stained cell walls or are degraded. Differences were seen in the branching patterns and density of ascogenous hyphae. For example, Arthonia cinnabarinula (Fig. 13) develops numerous short side branches whereas these are less distinct in A. leptosperma (Fig. 14). More sinuous

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Figs 7–12. Branching patterns of ascogenous hyphae. Fig. 7. Arthonia aciniformis. Fig. 8. A. cinnabarinula. Fig. 9. A. palmulacea. Fig. 10. Eremothecella macrocephala Fig. 11. Opegrapha filicina. Fig. 12. Arthonia trilocularis. Bars l 25 µm.

branches are seen in Eremothecella macrocephala (Fig. 10) and Amazonomyces farkasiae. These contrast with the straight branches of Arthonia palmulacea (Fig. 9). In E. calamicola, the hyphae may form relatively dense webs, as do some Arthonia species (e.g. A. accolens, A. cyanea, A. mira, A. leptosperma, A. trilocularis), while others seem to have rather spaced ascogenous hyphae (e.g. A. palmulacea and A. aciniformis). Croziers are formed in most species, with Arthonia leptosperma as the only exception (Fig. 14). The thickness of ascogenous hyphae varies between 2–5 µm, those of

Amazonomyces farkasiae were the thickest at 4–5 µm. The tips of the ascogenous hyphae can be rounded, but in some instances acute tips are present in the vicinity of vegetative hyphae. We did not see whether these acute tips contact or penetrate adjacent haploid hyphae. It is not yet fully clear how the ascogeneous hyphae originate. In mature ascomata of Arthonia palmulacea (Fig. 3), after a primary ‘ flush ’ of radiating ascogenous hyphae, new ascogenous hyphae form in central parts of the ascomata (Fig. 15). The initial stages consist of a few cells from which asci

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Figs 13–17. Details of ascogenous hyphae. Fig. 13. Arthonia cinnabarinula. Fig. 14. A. leptosperma. Fig. 15. A. palmulacea. Fig. 16. Eremothecella macrocephala. Fig. 17. A. aciniformis. Bars l 10 µm.

may already arise. No typical ascogones are formed, and the ascogenous hyphae probably arise from somatic cells. However, the haploid hyphae which have contact to the initials of ascogenous hyphae are often in poor condition (Fig. 16), the details of the underlying processes therefore remain to be studied in detail. In various species, we occasionally observed contacts between ascogenous hyphae with the haploid hyphae at the periphery of the ascomata : e.g. in Arthonia aciniformis (Fig. 17), A. palmulacea, A. trilocularis, Eremothecella macrosperma,

E.variratae, and E. macrocephala (Fig. 16). Haploid hyphae can either project from a crozier or the tips of the ascogenous hyphae have contact to haploid hyphae. DISCUSSION According to the classic textbook pattern (e.g. Mu$ ller & Lo$ ffler 1982), ascogenous hyphae can form by the transmission of a nucleus to an ascogone or by the fusion of undifferenciated vegetative hyphae (somatogamy). Except for fragmentary

Ascogenous hyphae in Arthoniaceae branching structures, the resulting filiform branching system of ascogenous hyphae from which the asci develop has hardly been studied in situ previously, partly as ascogenous hyphae grow in three dimensions in most species and because they are often not easy to distinguish from sterile hyphae by transmission microscopy. Previously, ascogenous hyphae stained with Calcofluor White were visualized in lichenicolous Arthonia species by Grube & Matzer (1997). In that study, the variable thicknesses of the walls of the ascogenous hyphae were used to distinguish closely related species. The ascogenous cells were seen at variable levels in the ascomata, indicating a three-dimensional growth in the developing fruit body. This is different in the comparatively plane ascomata of foliicolous Arthonia species where no basal hypothecium of vegetative tissue is apparent, and the ascogenous hyphae grow in a basal layer. Looking at the lower surface of the ascomata, the entire branching system of ascogenous hyphae can be studied. The results show that most species possess ascogenous hyphae of the crozier type, the commonest in ascomycetes ; only Caliciales exhibit a chain-type of development (Schmidt 1970). Previously, not many illustrations focus on the structure of ascogenous hyphae. Long stretches of unbranched ascogenous hyphae found in Daldinia (Ingold 1954) are clearly different from those in Arthoniales. The former have a unique septation : only the tips of the ascogenous hyphae and the young asci are filled with dense protoplasm and each unbranched ascogenous hypha represents a single cell ; septa are only formed towards the asci. The ascogenous hyphae in Arthoniales are always septate. Romero & Minter (1988) observed short stretches of septate ascogenous hyphae in fragmentary preparations of hymenia from Pleurostoma ootheca and Diatrype disciformis. In the latter, many asci arise from one ascogenous cell – similar to Arthonia leptosperma, but in a higher number. Large voluminous ascogones as found in certain other nonlichenized ascomycetes (Mu$ ller & Lo$ ffler 1982) are not present, and we have not observed typical trichogynes, although they were reported by Henssen & Jahns (1973) for non-foliicolous taxa. Some individual hyphae emerging at the surface of the ascomata, however, may represent such organs. If trichogynes are present, their individual function still needs to be clarified, especially as Moreau (1928) suggested that trichogynes could be functionless appendices of ascogones. It is still not clear whether the ascogenous hyphae originate directly from single somatic hyphae, from the fusion of dikaryotic hyphae, or from fertilization by spermatia. Beside those which produce large and complex macroconidia (Se! rusiaux 1992, Lu$ cking 1995, Grube et al. 1995), conidiomata producing microconidia are common in foliicolous Arthonia and Eremothecella species. Such conidiomata often occur in proximity to ascomata, or the ascomata may emerge from microconidiomata. It would be surprising if the microconidia would did not take part in the sexual processes as their thin walls suggest an ephemeral rather than a diasporic role. The contacts between ascogenous hyphae and haploid mycelium in the fruit bodies was surprising and we have not found any reference to this phenomenon in ascomycetes, however, fusion between dikaryotic and monokaryotic hyphae

1012 is known in basidiomycetes (Ku$ es 2000). Whether this is a character specific to ascomata of Arthoniales still needs to be studied. It is also unclear whether the occasionally formed acute tips of ascogenous hyphae actively contact haploid hyphae and fuse with them at a later stage, whether haploid hyphae branch off from dikaryotic ascogenous hyphae, or whether these are actively contacted by haploid hyphae. More knowledge on sexual processes in Arthonia would be interesting in this context. Henssen & Jahns (1973) stated that morphogenesis in Arthoniales starts with the development of a ‘ generative tissue ’ in which ascogones are then formed. As is typical of ascolocular fungi, ‘ generative tissue ’ and ascogones form repeatedly in the ascostroma of Arthoniales. These authors also observed that asci arise in groups in Arthonia and Arthothelium, whereas those in Stirtonia and Cryptothecia are single, although Tehler (1990) describes uniascal locules in Arthothelium. We have not completed studies on Arthothelium, but can confirm that the single asci of foliicolous Cryptothecia spp. are apparently not connected by a net of ascogenous hyphae. The foliicolous C. candida and C. effusa have asci which are aggregated in thallus patches and superficially similar to those in Amazonomyces. However, A. farkasiae, originally described as a Cryptothecia (Lu$ cking 1995) and related to the species now called Amazonomyces sprucei, possesses ascogenous hyphae which originate from a single central point in the ascomata. Thus, our results confirm the position of this species as separate from foliicolous Cryptothecia spp. The same is true for Eremothecella macrocephala, originally described in Stirtonia (Santesson 1952). The ascogenous hyphae of foliicolous Arthonia and Eremothecella species branch in a regular pattern without any apparent subdivision of the ascomata towards the periphery ; the whole ascoma originally represents a single centre. In agreement with this observation, we have not seen any intrusions of interascal plectenchyma in these species by transmission light microscopy. The monocarpocentral ascomata in foliicolous Arthonia species contrast with pluricarpocentral ascomata described in A. radiata (Tehler 1990). This raises the question whether the genus Arthonia should be split according to these ascomatal characters. The foliicolous Arthonia species are apparently not closely related to Arthonia s.str. because they usually lack a well-developed epithecioid layer with predominantly vertical hyphae, except in a few species such as Eremothecella and to some extent A. palmulacea (Lu$ cking 1995). The foliicolous representatives are more similar anatomically to members of subgen. Coniangium, which formerly included species with 1-septate ascospores and red pigments (e.g. Arthonia spadicea, A. vinosa). However, it has not yet been determined whether these species have similarly organized ascogenous hyphae. In non-foliicolous species, these grow in three dimensions and their branching pattern can only be studied by threedimensional reconstruction from serial sections. Another character in common with the foliicolous Arthonia species is that species in Coniangium do not have excipular strands and thus are not composed of several centres. The differences in the arrangement of ascogeneous hyphae and asci in Arthonia and its allies appear to be species-specific. However, some of the phenomena observed might also reflect

M. Grube and R. Lu$ cking different ontogenetic stages and a general adaptation to the foliicolous habit : since leaves are usually shed early, lichens growing on them with a rapid reproduction and formation of numerous diaspores will be selected for. In certain non-related foliicolous lichens, such as Echinoplaca (Ostropales : Gomphillaceae) or Byssolecania (Lecanorales : Pilocarpaceae), the flat ascomata produce mature asci and ascospores from a very early stage and then grow laterally continuing to produce ascospores. A similar phenomenon can be seen in Arthonia : while A. leptosperma still has not developed asci in the most peripheral parts of the ascomata, in A. palmulacea the original centre of the ascomata is already degenerated while new ascogeneous hyphae are being formed locally. The peripheral ascogeneous hyphae of foliicolous Arthoniaceae grow radially like a prothallus, and the production of asci is slightly retarded, so that from the periphery to the centre of the ascomata different ontogenetic stages are evident. In Pulvinodecton (Henssen & Thor 1998) the repeatedly prolifering ascomata might represent a situation where the central part of the initial ascomata develops into an excipuloid ‘ tissue ’, while ascogenous hyphae continue to grow in the prolifering centres. This pattern could differ between Arthonia species. However, if initials of ascogenous hyphae, which are formed in a central region after a primary radiating growth of ascogenous hyphae, as observed in A. palmulacea, are distant, and if a later ontogenetic stage involves the pigmentation of ascomatal plectenchyma, this could also be recognized as a secondarily pluricarpocentral ascoma. Foliicolous Arthoniales are clearly interesting subjects for the study of branching patterns, growth and ontogeny in ascogenous hyphae. Now we are trying to find more examples where ascogenous hyphae can be studied using fluorescence microscopy. This is not limited to Arthoniales, as we already found a comparable situation in the lichenicolous Hemigrapha tenellula, a member of Dothideales (Grube & Lu$ cking, unpubl.). A C K N O W L E D G E M E N TS We thank Niki Hoffmann (Graz) for comments on the text and Peter Kosnik (Graz) for photoprocessing.

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