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Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, Oregon 97331-2902
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ABSTRACT |
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Apothecia of Lobaria oregana and L. pulmonaria emerge in late spring and discharge ascospores throughout the year. Most populations have a few fertile thalli, although the proportion of fertile thalli usually is less than 25 percent. Ascospores fail to germinate in water or on water agar but do germinate on agar containing an adsorbant and either a sugar or the sugar-alcohol ribitol. It is postulated that the ascospores of these species contain an autoinhibitor that must be removed before germination. Widespread ascospore germination in the presence of an adsorbant in Peltigera aphthosa, P. membranacea, and Pseudocyphellaria anthraspis, as well as in Lobaria, suggest that this phenomenon might be widespread in the Peltigerales.
Key words: adsorbant, autoinhibitor, discharge, germination, lichens, Lobariaceae, Peltigerales, seasonality
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Both species fix atmospheric nitrogen, thereby contributing to the nitrogen economy of the forest (Pike 1978). Lobaria oregana is endemic to the Pacific Northwest, but L. pulmonaria occurs elsewhere as well. Both species are common and widespread in coastal forests of the Pacific Northwest. However, both species are threatened by the continuing destruction of the ancient forests they inhabit. Managers of federal lands (national forests, national parks, and Bureau of Land Management property) are required to provide protection for these species (USDA/USDI 1994). Protection of any species over years, decades or centuries requires knowledge of how the species maintains itself through time, that is, knowledge of its reproductive strategy.
Both L. oregana and L. pulmonaria reproduce sexually and asexually. Production of new thalli and their dissemination is primarily by means of minute, airborne, asexual propagules—lobules in the case of L. oregana and soredia in the case of L. pulmonaria (Jordan 1973, McCune and Geiser 1997). In addition to their asexual propagules, both species produce apothecia, the site of sexual reproduction. The apothecia are described as "infrequent", "occasional" or "uncommon" (Brodo et al 2001, Jordan 1973, McCune and Geiser 1997). However, it is not clear whether these terms refer to the proportion of populations that contain apothecia or to the frequency of apothecial thalli within populations. Similarly, it is not clear whether apothecia are seasonal and, if so, in which seasons they occur. This paper deals with these questions.
Lichen apothecia contain asci, which in turn contain ascospores. The ascospores are forcibly discharged into the air. Under suitable conditions, ascospores germinate to form a mycelium. Most reported attempts to germinate ascospores of species of Lobaria were unsuccessful (Crittenden et al 1995). An intriguing exception is the work of Lallemant (1977), who reported that ascospores of Lobaria pulmonaria germinated only in the presence of its phycobiont.
This paper deals with four aspects of sexual reproduction in L. oregana and L. pulmonaria: the seasonal occurrence of apothecia, the frequency of fertile thalli within and between populations, the seasonal occurrence of ascospore discharge and conditions required for ascospore germination in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To determine the frequency of apothecial thalli within and between populations, I visited 38 populations of Lobaria oregana and 32 populations of L. pulmonaria over a period of six years, January 1994 through December 1999, noting the presence or absence of fertile thalli among populations and among thalli within each population. The sites occupied by these populations ranged from near sea level along the Pacific Coast to above 1000 m on the west side of the Cascades, and between 43° and 45° north latitude. Most of the sites were visited once or twice; a few were sampled more frequently.
Sampling involved examining a large number of thalli at a site, recording both the total examined and the number that bore at least one apothecium. Sites with fewer than 50 thalli were not counted; where thalli were abundant, the count was stopped at 100. One sample, as the term is used here, consisted of a variable number of thalli that were counted at one site at one time. The thalli were examined in place on tree trunks from 50–250 cm above the ground.
To provide material for studies of spore discharge and germination, thalli were collected and taken to the laboratory, where they were allowed to dry at room temperature for up to one wk. An apothecium, removed from its thallus, was placed on a filter paper circle and dampened to induce discharge. For spore-discharge studies, ascospores were caught on a cover glass suspended on a ring (a 4 mm section, sliced from 19-mm-diam plastic pipe) ca 2 mm above the apothecium. The presence or absence of discharged ascospores could be monitored with a 40x dissecting microscope without disturbing the apparatus, but identification and photography of spores required a compound microscope. To prepare a slide for the compound microscope, a cover glass with adhering ascospores was removed and added, ascospore side down, to a drop of lactic acid containing aniline blue dye on a microscope slide.
A similar method was used to introduce ascospores to agar plates for germination studies. A small disk of filter paper was placed in the lid of an inverted 60 mm plastic Petri dish containing sterile medium; then an apothecium was added to the paper and the paper was wetted. Wetting the paper hydrated the apothecium causing it to discharge ascospores upward onto the medium. When ascospores were seen by looking through the agar with a dissecting microscope, the paper and apothecium were removed. With rare exceptions, an adequate number of spores were discharged within 24 h of wetting the apothecium.
Ascospore germination was attempted in three series of experiments. In the first, these media were used: 2.5% water agar (WA); 2.5% water agar plus 0.5% powdered charcoal (Sigma C-6289); 3% Knox gelatin [without agar]; cornmeal agar (CMA, BBL); Czapek Dox (CD, Sigma C-6095), and potato-dextrose agar (PDA, Difco).
In the second, the adsorbants 1% bovine serum albumin (BSA, Sigma A-7030) or 1% alpha-cyclodextrin (Sigma C-4642) were added to WA, CMA, PDA and CD. The BSA was filter sterilized and added to the melted and slightly cooled agar before pouring it into Petri dishes. Cyclodextrin was added to the medium before autoclaving.
In the third series, 1% sucrose, 1% glucose and 1% ribitol, a sugar alcohol, were added to WA, CMA, PDA and CD. Ascospores were shot onto the surface of the agar as described above, and the plates were incubated 7 d at 15–20 C.
One-half of a batch of plates (15 plates) was placed in a photographic changing bag, as soon as the apothecia were wet, and left one week; this was done to determine whether light is required for discharge and germination of spores. The other 15 plates were incubated in the light.
Percentage of ascospore germination was scored on the Petri dish using a 400x objective of a compound microscope and systematically selecting 100 spores, beginning in the top right field and continuing to the count of 100.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The among-population frequency of populations with one or more fertile thalli was 75% for Lobaria oregana and 84% for L. pulmonaria.
The within-population frequency of fertile thalli ranged from zero to more than 25% (Table I). The data are insufficient to determine whether there is a difference in these frequencies between the two species.
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In both species, apothecial color, far better than either apothecial size or shape, provided the best indicator of maturity. Any apothecium in which the hymenium was reddish brown (Vinaceous Rufous to Vandyke Red, Ridgeway 1912) could be expected to discharge ascospores. Paler tan apothecia were immature and those that were very dark brown or black either were exhausted or infected with a parasite and nonfunctional.
The effects of medium on ascospore germination are summarized in Table III. There was no germination on PDA or CD alone or with either of the adsorbants or sucrose, dextrose or ribitol alone (results not shown). No germination was observed on WA alone or with the adsorbants BSA or cyclodextrin. Equally, there was no germination on CMA alone. However, when either BSA or cyclodextrin were added to WA and CM, together with either of the sugars or ribitol, the percentage of germination in both L. oregana and L. pulmonaria increased dramtically. Increased germination was not observed in WA, to which one of the adsorbants was added in the absence of one of the sugars, whereas on CM, germination was greatly enhanced with the addition of either of the adsorbants in the absence of added sugar.
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At temperatures in the range of 15–18 C, germination began within three to four days and the proportion of germinated to ungerminated spores continued to increase several days thereafter.
The ascospores of Lobaria oregana (Figs. 1, 3) and L. pulmonaria (Figs. 2, 4) differ markedly in size and shape. The ascospores of L. oregana are acicular, straight or strongly curved, often more attenuate at one end, 42–73(–85) x 4.5–9.5 µm, whereas those of L. pulmonaria are fusiform-ellipsoidal, straight or curved, ends symmetrical, 23–29 x 6–10 µm (Jordan 1973). The majority of ascospores in both species are one-septate. The apparent occurrence of additional septa is an artifact attributable to the interface between guttules.
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Spores that are disintegrating under conditions that do not foster germination might produce a short unbranched tube that can be mistaken for a germ tube. Genuine germ tubes produce both branches and septa.
Bacterial contaminants routinely overwhelmed the plates shortly after germination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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noted that in lichens, in contrast to nonlichenized ascomycetes, the ascocarp is perennial and might function for several years; apothecia of the species of Lobaria studied here appear to persist and to discharge ascospores for about a year. Other published works, in which studies of spore discharge are accompanied by seasonal collection data, confirm for other lichens the presence of functional apothecia throughout the year (Clayden 1997, Ostrofsky and Denison 1980, Pyatt 1968). This remarkable obervation sharply contrasts with the behavior of most nonlichenized ascomycetes, in which apothecia and other ascomata are relatively short-lived and seasonal. In the case of pathogens, ascospore production usually is coordinated with the appearance of a vulnerable part of the host (Heald 1933). In the cases of saprobes, the morels for instance, the reason for seasonality is unclear. Nevertheless, most nonpathogenic, apothecial, nonlichenized ascomycetes also have relatively brief seasons of fruiting (Weber 2001). The ostropalean fungi might be exceptional; Sherwood (1977) noted the lack of evidence to support the contention that fruit bodies of Stictis and Schizoxylon are long-lived. During sampling, it seemed that the distribution of fertile thalli within the population was not random. Often the fertile thalli were grouped in one, or a small number, of areas within the population, sometimes limited to a single tree. However, the study was not designed to detect within-population spatial variability.
It is not known whether, or under what circumstances, Lobaria ascospores are able to produce new individuals. There are no reports that the mycelium that emanates from ascospores is able to establish a relationship with free, living algae. It might be that ascospores function in some other way to retain the benefits of meiotic recombination, perhaps by forming heterokaryons with established individuals, as Kyoviita (2000) proposed for ectomycorrhizal fungi.
Previous studies of ascospore germination in Lobaria and other Peltigerales found little or no germination with media that induced germination in many other lichens (Ahmadjian 1989, Crittenden et al 1995). However, Lallemont (1977) and Scott (1959) observed ascospore germination in Lobaria and Peltigera, respectively, when the spores were accompanied by the appropriate mycobiont. It now seems likely that mycobionts served both as adsorbants and as a source of sugar or ribitol.
Preliminary studies of germination in Peltigera aphthosa, P. membranacea and Pseudocyphellaria anthraspis, using the same media and conditions described here, yielded high rates of germination, suggesting that the need for an adsorbant is widespread in the Peltigerales (Denison unpubl).
The fact that Lobaria ascospores are stimulated to germinate by adsorbants suggests that the spores contain at discharge a self-inhibitor that must be removed before they can germinate. This phenomenon is known about the spores of other fungi (Mako et al 1976, Stone et al 1994) and about the seeds of flowering plants (Mayer and Poljakoff-Mayber 1989, Evanari 1949). Typically, the self-inhibitor is a dormancy mechanism, delaying germination until environmental conditions are favorable.
In Lobaria the apothecia are the site of sexual reproduction. In Lobaria, as in many lichens, multiplication and dissemination appear to be accomplished primarily, if not exclusively, by asexual means. This suggests that the primary function of apothecia in these species is meiotic recombination and that the widespread occurrence of apothecia argues for the importance of meiotic recombination for the survival of the species. Other lichens, such as some species of Xanthoria and Physcia, have abundant apothecia and no asexual propagules; ascospores are the agents of multiplication and dissemination in these lichens (Chrismas 1980).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication November 18, 2002.
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LITERATURE CITED |
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Ahmadjian V., 1989 Studies on the isolation and synthesis of bionts of the cyanolichen Peltigera canina (Peltigeraceae). Pl Sys Evol 165:29-38
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Chrismas M., 1980 Ascospore discharge and germination in Xanthoria parietina. Lichenologist 12:403-406
Clayden IR., 1997 Seasonal variation in ascospore discharge in Rhizocarpon lecanorinum. Lichenologist 29:495-499
Crittenden PD, David JC, Hawksworth DL, Campbell FS., 1995 Attempted isolation and success in culturing of a broad spectrum of lichen-forming and lichenicolous fungi. New Phytol 130:267-297
Evanari M., 1949 Germination inhibitors. Bot Rev 15:153-194
Heald FD., 1933 Manual of plant disease. New York: McGraw-Hill. 953 p
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Kytoviita M-M., 2000 Do symbiotic fungi refresh themselves by incorporating their own or closely related spores into existing mycelium?. Oikos 90:606-608
Lallemant R., 1977 Obtention de cultures pures des mycosymbiontes du Lobaria laetevirens (Lightfoot) Zahlbr. et du Lobaria pulmonaria (L.) Hoffm.: le role des gonidies. Rev Bryol Lichenol 43:303-308
Mako V, Staples RC, Yanev Z, Granados RR., 1976 Self-inhibitors of fungal spore germination. In: Weber DJ, Hess WM, eds. The fungal spore: form and function. New York: Wiley-Interscience. p 73–98
Mayer AM, Poljakoff-Mayber A., 1989 The germination of seeds. Oxford: Pergamon Press. 270 p
McCune B, Geiser L., 1997 Macrolichens of the Pacific Northwest. Corvallis: Oregon State University Press. 236 p
Ostrofsky A, Denison WC., 1980 Ascospore discharge and germination in Xanthoria polycarpa. Mycologia 72:1171-1179
Pike L., 1978 The importance of epiphytic lichens in mineral cycling. Bryologist 81:247-257
———, Rydell R, Denison W., 1977 A 400-year-old Douglas fir tree and its epiphytes: biomass, surface area, and their distributions. Can J For Res 7:680-699
Pyatt FB., 1968 An investigation into conditions influencing ascospore discharge and germination in lichens. Rev Bryol Lichenol 36:323-329
Ridgeway R., 1912 Color standards and color nomenclature. Washington, D.C.: Published by the author. 43 p + 53 pl
Scott GD., 1959 Observations on spore discharge and germination in Peltigera praetexta (Flk.) Vain. Lichenologist 1:109-111
Sherwood MA., 1977 The ostropalean fungi. Mycotaxon 5:1-277
Stone JK, Pinkerton JN, Johnston KB., 1994 Axenic culture of Anisogramma anomala: evidence for the self inhibition of ascospore germination and colony growth. Mycologia 86:674-683
USDA/USDI. 1994 Forest plan (Record of decision: standards and guidelines). USDA-Forest Service and USDI-Bureau of Land Management. April 1994. 74 p
Weber NS., 2001 Musings on mushrooming. McIlvainea 15:63-76
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