Cladonia Grayi Research Articles

Modeling in yeast how rDNA introns slow growth and increase desiccation tolerance in lichens.

We connect ribosome biogenesis to desiccation tolerance in lichens, widespread symbioses between specialized fungi (mycobionts) and unicellular phototrophs. We test whether the introns present in the nuclear ribosomal DNA of lichen mycobionts contribute to their anhydrobiosis. Self-splicing introns are found in the rDNA of several eukaryotic microorganisms, but most introns populating lichen rDNA are unable to self-splice, being either catalytically impaired group I introns, or spliceosomal introns ectopically present in rDNA. Although the mycobiont’s splicing machinery removes all introns from rRNA, Northern analysis indicates delayed post-transcriptional removal during rRNA processing, suggesting interference with ribosome assembly. To study the effects of lichen introns in a model system, we used CRISPR to introduce a spliceosomal rDNA intron from the lichen fungus Cladonia grayi into all nuclear rDNA copies of Saccharomyces cerevisiae, which lacks rDNA introns. Three intron-bearing yeast mutants were constructed with the intron inserted either in the 18S rRNA genes, the 25S rRNA genes, or in both. The mutants removed the introns correctly but had half the rDNA genes of the wildtype, grew 4.4–6 times slower, and were 40–1700 times more desiccation tolerant depending on intron position and number. Intracellular trehalose, a disaccharide implicated in desiccation tolerance, was detected at low concentration. Our data suggest that the interference of the splicing machinery with ribosome assembly leads to fewer ribosomes and proteins and to slow growth and increased desiccation tolerance in the yeast mutants. The relevance of these findings for slow growth and desiccation tolerance in lichens is discussed.

Lichen 1. Solo fungal and algal partners

Lichens are slow-growing niche-constructing organisms that form a thallus via scripted symbiotic/mutualistic relationships between fungi, algae, and bacteria, and that are distributed across nearly all terrestrial ecosystems. Here we use quick-freeze deep-etch electron microscopy (QFDEEM) to probe the ultrastructure of a lichen-forming fungus, Cladonia grayi, and its partner alga, Asterochloris glomerata, grown separately (solo) in the laboratory. The solo fungus resembles its lichenized counterpart in: a) general cellular organization; b) the capacity to form lateral branches; c) the production of extracellular materials; and d) the generation of acellular hyphae that we call struts. It differs in carrying a fibrillar coat on its wall exterior and in often adopting two novel plasma-membrane configurations called pleated and pitted. The solo alga also resembles its lichenized counterpart in general cellular organization, but its algaenan-based wall carries a fibrillar coat not evident in the lichen, and its pyrenoid and plastoglobule endowments are less well developed. These findings are followed by four reports on the QFDEEM ultrastructure of four species of lichens, where we describe the differentiations that occur when the fungi and algae are living together in community with bacteria.

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata

BackgroundLichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.ResultsA) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting.ConclusionsThe surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi.

Peramine is a non-ribosomal peptide-derived pyrrolopyrazine (PPZ)-containing molecule with anti-insect properties. Peramine is known to be produced by fungi from genus Epichloë, which form mutualistic endophytic associations with cool-season grass hosts. Peramine biosynthesis has been proposed to require only the two-module non-ribosomal peptide synthetase (NRPS) peramine synthetase (PerA), which is encoded by the 8.3 kb gene perA, though this has not been conclusively proven. Until recently, both peramine and perA were thought to be exclusive to fungi of genus Epichloë; however, a putative perA homologue was recently identified in the genome of the insect-pathogenic fungus Metarhizium rileyi. We use a heterologous expression system and a hydrophilic interaction chromatography-based analysis method to confirm that PerA is the only pathway-specific protein required for peramine biosynthesis. The perA homologue from M. rileyi (MR_perA) is shown to encode a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë. Furthermore, perA is part of a larger seven-gene PPZ cluster in M. rileyi, Metarhizium majus and the stalked-cup lichen fungus Cladonia grayi. These PPZ genes encode proteins predicted to derivatize peramine into more complex PPZ metabolites, with the orphaned perA gene of Epichloë spp. representing an example of reductive evolution.

Records of terricolous lichens from páramos of southern Ecuador

Ecological studies of five páramos in Azuay and Loja provinces recorded one lichen species new to South America (Bryoria nitidula), five new to Ecuador (Cladonia halei, C. melanopoda, C. merochlorophaea, C. subreticulata, Diploschistes diacapsis), two new to mainland Ecuador (Cladonia grayi, C. pyxidata), and 20 new provincial records. Brief morphological descriptions, with remarks on distribution and ecology, are provided.

The lichens of the Cladonia pyxidata-chlorophaea complex in Belarus

Based on an examination of c. 500 herbarium specimens of the Cladonia pyxidata-chlorophaea complex, this study deals with their morphology, secondary chemistry, habitat requirements and distribution in Belarus. Eleven taxa have been accepted, of which C. conista, C. cryptochlorophaea, C. homosekikaica, C. merochlorophaea, C. monomorpha and C. novochlo- rophaea are new to the country. Cladonia grayi appears to be the commonest species in the country (c. 40% of the studied specimens), and C. pyxidata is uncommon in Belarus, known only from 10 localities. The chemotypes of the species and their frequency in Belarus are indicated.

High-throughput genome sequencing of lichenizing fungi to assess gene loss in the ammonium transporter/ammonia permease gene family

BackgroundHorizontal gene transfer has shaped the evolution of the ammonium transporter/ammonia permease gene family. Horizontal transfers of ammonium transporter/ammonia permease genes into the fungi include one transfer from archaea to the filamentous ascomycetes associated with the adaptive radiation of the leotiomyceta. The horizontally transferred gene has subsequently been lost in most of the group but has been selectively retained in lichenizing fungi. However, some groups of lichens appear to have secondarily lost the archaeal ammonium transporter. Definitive assessment of gene loss can only be made via whole genome sequencing.ResultsAmmonium transporter/ammonia permease gene sequences were recovered from the assembled genomes of eight lichenizing fungi in key clades including the Caliciales, the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacterium as a primary photobiont or lichens living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the lichens from the Acarosporomycetidae, Chaetotheriomycetes and Arthoniomycetes. In addition, the four ammonium transporter/ammonia permease genes from Cladonia grayi were shown to be functional by expressing the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source.ConclusionsGiven sufficient coverage, next-generation sequencing technology can definitively address the loss of a gene in a genome when using environmental DNA isolated from lichen thalli collected from their natural habitats. Lichen-forming fungi have been losing ammonium transporters/ammonia permease genes at a slower rate than the most closely related non-lichenized lineages. These horizontally transferred genes in the Cladonia grayi genome encode functional ammonium transporters/ammonia permeases.

Fungal and algal gene expression in early developmental stages of lichen-symbiosis

How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.

Recent literature on lichens—217

Abbott, B. F. M. 2009. Checklist of the Lichens and Lichenicolous Fungi of Greece. Bibliotheca Lichenologica No. 103. J. Cramer in der Gebruder Borntraeger Verlagsbuchhandlung. Berlin and Stuttgart. 368 pages. [Exhaustive catalog of Greek lichens based on both literature records and unpublished specimens. Includes extensive listings of synonyms and Greek place names.] Abonyi, D. O., M. U. Adikwu, C. O. Esimone & E. C. Ibezim. 2009. Plants as sources of antiviral agents. African Journal of Biotechnology 8(17): 3989–3994. [Lichens are mentioned as one of the many plant sources.] Ahti, T. 2009. A tribute to John Walter Thomson (1913–2009). Lichenologist 41(6): 561–563. [Short article remembering his life and work, especially on arctic lichens.] Allen, A. & B. Hilton. 2010. [Review] C. W. Smith, A. Aptroot, B. J. Coppins, A. Fletcher, O. L. Gilbert, P. W. James & P. A. Wolseley (eds.). The Lichens of Great Britain and Ireland. The British Lichen Society, Botany Department, Natural History Museum, London. x + 1046 pages. 2009. Lichenologist 42(1): 123–125. Alstrup, V., J. Kocourkova, M. Kukwa, J. Motiejūnaitė, W. Von Brackel & A. Suija. 2009. The lichens and lichenicolous fungi of South Greenland. Folia Cryptogamica Estonica 46: 1–24. [Notes on 744 lichens, 156 lichenicolous and 6 allied fungi. Many species new to Greenland. New: Naetrocymbe kentrospora (Branth) Alstrup comb. nov.] ——— & U. Sochting. 2009. Galloea cladoniicola, a new hyphomycetous lichenicolous fungus from Denmark. Graphis Scripta 21(2): 33–35. [New: Galloea cladoniicola gen. et sp. nov.] Aptroot, A. 2009. A revision of the lichen genus Stirtonia. Lichenologist 41(6): 615–625. [Treatment of 13 species; key. Stirtonia neotropica (Netherlands Antilles), S. schummii (Seychelles) and S. curvata Aptroot spp. nov. (Indonesia).] ———. 2009. Diversity and endemism in the pyrenocarpous lichen families Pyrenulaceae and Trypetheliaceae in the Malesian flora region. Blumea 54(1–3): 145–147. [‘‘Species numbers in both families seem highest in Australia and Papua New Guinea, but some potentially rich regions are much undercollected. The numbers of local endemics are also highest in these areas, at least in the Pyrenulaceae, and remarkably high for lichens.’’] Armaleo, D. & S. May. 2009. Sizing the fungal and algal genomes of the lichen Cladonia grayi through quantitative PCR. Symbiosis 49(1): 43–51. [Authors report first genome size for a lichen, 28.6 Mb (mycobiont) and 106.7 Mb (Asterochloris photobiont). DNA was obtained from culture of soredia.] Armstrong, R. A. & S. N. Smith. 2009. Carbohydrates in the hypothallus and aerolae of the crustose lichen Rhizocarpon geographicum (L.) DC. Symbiosis 42(2): 95–100. [Lichen material from Wales. Authors report 8 carbohydrates present. The results suggest that the ‘‘ ... hypothallus has a lower demand for carbohydrates than the areolae or there is limited transport ...’’ and the ‘‘ ... increased trehalose in the non-lichenised hypothallus may be an adaptation to withstand stress and desiccation ....’’] Asplund, J. & Y. Gauslaa. 2010. The gastropod Arion fuscus prefers cyanobacterial to green algal parts of the tripartite lichen Nephroma arcticum due to low chemical defense. Lichenologist 42(1): 113–117. [Gastropods in experiments preferred cephalodia which lack secondary lichen metabolites. This feeding behavior reduces N concentration 1 Author e-mail: regan@unomaha.edu The cumulative database for this series is available in searchable form on the World Wide Web at http:// www.nhm.uio.no/botanisk/bot-mus/lav/sok_rll.htm. I owe special thanks to Bill Buck for providing copies of papers by other authors, which were otherwise unavailable to me, and to the cooperating authors who send reprints or electronic versions of their works to me for inclusion in this series. DOI: 10.1639/0007-2745-113.2.390

Sizing the fungal and algal genomes of the lichen Cladonia grayi through quantitative PCR

Using a method based on quantitative PCR, we determined that the nuclear genome sizes for the mycobiont and photobiont of the lichen Cladonia grayi are 28.6 Mb and 106.7 Mb, respectively. This is the first genome size determination for lichens, and suggests that between 20,000 and 25,000 genes function in C. grayi. The mycobiont genome size is near the middle of the range observed within the Pezizomycota, the subphylum containing all known ascomycete lichen fungi. The genome size of the photobiont (the green alga Asterochloris sp.) is near the lower end of its class, the Trebouxiophyceae. Genomes in this size range can be sequenced at relatively low cost with current pyrosequencing-based methods. The genome sizing method requires very small amounts of precisely quantified DNA and should be applicable to any lichen whose symbionts can be reliably isolated from one another. Since the symbionts used in this project were isolated from soredia, the lichen’s vegetative propagules, we also describe a method for the establishment of axenic symbiont cultures from large numbers of soredia.

Growth dynamics in Cladonia grayi

The erect secondary thallus (podetium) of the lichen Cladonia grayi develops in distinct stages that can be traced to the development of its fungal meristem tissue. In early ontogeny, while the contiguous meristem grows unilaterally both outward and vertically, the gross morphology of the podetium resembles other cup-forming species. In later ontogeny, when the divided meristem grows vertically, the podetium forms a subtly articulated spiral. The spiral growth form is most apparent in branching proliferations that grow along the cup margin. The meristem tissue at the apex of these proliferations is curved, maturing sequentially in a spiral pattern. The proliferations curve in a spiral around the vertical axis of the podetium. Apothecia at the apex of proliferations curve in a similar manner. Evidence for spiral growth along the cup margin can also be inferred from the sequential development of pycnidia that emerge from it. Similar to early land plants, the growth dynamics of C. grayi may represent an evolutionary advance from an orthotropous growth pattern to a branched, photosynthetic thallus.

Growth Dynamics in Cladonia grayi

The erect secondary thallus (podetium) of the lichen Cladonia grayi develops in distinct stages that can be traced to the development of its fungal meristem tissue. In early ontogeny, while the contiguous meristem grows unilaterally both outward and vertically, the gross morphology of the podetium resembles other cup-forming species. In later ontog- eny, when the divided meristem grows vertically, the podetium forms a subtly articulated spiral. The spiral growth form is most apparent in branching prolifer- ations that grow along the cup margin. The meristem tissue at the apex of these proliferations is curved, maturing sequentially in a spiral pattern. The prolif- erations curve in a spiral around the vertical axis of the podetium. Apothecia at the apex of proliferations curve in a similar manner. Evidence for spiral growth along the cup margin can also be inferred from the sequential development of pycnidia that emerge from it. Similar to early land plants, the growth dy- namics of C. grayi may represent an evolutionary ad- vance from an orthotropous growth pattern to a branched, photosynthetic thallus.

Induction of a complete secondary-product pathway in a cultured lichen fungus

Experimental studies on the secondary metabolism characteristic of lichens have been impeded by the slow growth of the fungi and by the inconsistent results of many attempts to induce the pathways in the fungi isolated from their photosynthetic partners. In the present study, a lichen-specific secondary pathway was consistently induced in a lichen fungus ( Cladonia grayi) grown in the absence of the alga. The depside (4- O-demethylsphaerophorin) and two depsidones (grayanic and 4- O-demethylgrayanic acids) found in the natural lichen began to accumulate a few days after the transfer of lightly fragmented mycelia from liquid to solid medium. Induction was enhanced on drier substrates and was correlated with the proliferation of aerial hyphae, where the major product (grayanic acid) accumulated in extracellular patches visible by fluorescence microscopy. The time course was analyzed by quantitative high-performance liquid chromatography of extracts from small cultures grown on nylon filters. Induction was rapid in view of the slow growth of the fungus, and secondary productivity was comparable to that of some nonlichen fungi. These results confirm that the alga is not needed for catalysis in lichen depside and depsidone biosynthesis and suggest instead that the characteristic secondary metabolism of natural lichen is linked to their aerial habit of growth.

Chemistry and chorology of the Cladonia chlorophaea-pyxidata complex (Lichenes, Cladoniaceae) in Italy

Summary 252 samples of lichens of the Cladonia chlorophaea—pyxidata complex collected in Italy were analyzed by thin layer chromatography. Six chemospecies occur in Italy; they are: Cladonia chlorophaea s.str. (80.9% of the samples), throughout the Peninsula, Cladonia grayi (4.0%) restricted to mountain areas on acid substrate in the North, Cladonia merochlorophaea (5.5%) and Cladonia cryptochlorophaea (2.4%), most frequent in Central Italy, at the western side of the peninsula, Cladonia conoidea (5.5%) most frequent in Central and Southern Italy, in lowlands and within mediterranean vegetation, Cladonia perlomera (1.6%), very rare throughout the country. Five of the six chemospecies are new to Italy; one (C. perlomera) is new to Europe. The higher frequency of C. chlorophaea s.str. is probably due to the fact that this is the only chemospecies within the complex that grows on limestone soils.

Cladonia chlorophaea sens. lat. in Southeastern Australia

The distribution of species within the Cladonia chlorophaea group in southeastern Australia has been determined from a chemical examination of 148 specimens. The commonest species is Cladonia merochlorophaea Asah.; Cladonia grayii Merr. ex Sandst. does not appear to occur in the area studied. Cladonia homosekikaica Nuno is reported for the first time from Australia. The Cladonia chlorophaea group contains several morphologically similar species which are differentiated on the basis of their constituent lichen acids. Five species of this group occur in Australia viz. C. chlorophaea (Flirke ex Sommerf.) Spreng. (fumarprotocetraric acid); C. cryptochlorophaea Asah. (cryptochlorophaeic acid ? fumarprotocetraric acid with traces of paludosic acid); C. merochlorophaea Asah. (merochlorophaeic and 4-O-methyl- cryptochlorophaeic acids ? fumarprotocetraric acid with traces of homosekikaic and cryp- tochlorophaeic acids); C. conoidea Ahti (atranorin and fumarprotocetraric acid) and C. homosekikaica Nuno, with homosekikaic and fumarprotocetraric acid. A recent chemical examination of 148 specimens of C. chlorophaea sens. lat. collected in southeastern Australia (excluding Tasmania) gave the results shown in Table 1. The specimens were collected from 31 locations as shown in Fig. 1. The altitudes of the collection sites ranged from 2200 m (Mt. Kosciusko, N.S.W.) to 50 m (Gladesville, N.S.W.). No collections were made from north and west of the 500 mm isohyet; C. chlorophaea was not found in this dryer area (Rogers & Lange 1972). Acetone extracts of the specimens were examined by thin-layer chromatography (Culberson 1972) and the separated com- pounds were detected with sulphuric acid (Culberson 1972) and MBTH (Archer 1978). Grayanic acid, for use as a reference compound, was extracted from Gymnoderma mela- carpum (Wilson) Yoshimura (Chester & Elix 1980). The results in Table 1 show that the most frequently collected species was C. merochloro- phaea; 58% of the specimens contained fumarprotocetraric acid compared to 89% in North America (Bowler 1972); C. merochlorophaea var. novochlorophaea Sipman (Sipman 1973) was not found. C. chlorophaea sens. str. formed 30% of the specimens examined and a further 13% were C. cryptochlorophaea; 75% of these contained fumarprotocetraric acid compared to 82% in North America. The less common species were C. conoidea and C. homosekikaica; the latter species is reported from Japan (Nuno 1975). The apparent absence of C. grayi from southeastern Australia is in marked contrast to the distribution of the species within the C. chlorophaea group reported from the Northern Hemisphere (Nourish & Oliver 1976), particularly from North Carolina (Kristinsson 1971) and the southern Appalachians (Dey 1978) where it was the most common species found. Cladonia merochlorophaea, the most common species in southeastern Australia, occurs relatively infrequently in North America. Cladonia grayi is not reported from South Aus- tralia (Filson & Rogers 1979) or Tasmania (Wetmore 1963) but is reported from South America (Dey 1978) and New Zealand (Martin 1958). However, a recent chemical ex-

Morphological and Chemical Correlations in the Cladonia chlorophaea Complex

Measurements of granules from within the podetial cups of different chemical variants of the Cladonia chlorophaea complex in North Carolina show clear correlation between granule size and certain chemical characters. The results support the view that the replacement of cryptochlorophaeic acid by grayanic acid in this group is taxonomically more significant than the presence or absence of fumarprotocetraric acid. In the species tested, the soredia of C. conista are the smallest, those of C. perlomera and C. grayi are intermediate, and those of C. cryptochlorophaea are the largest. The Cladonia chlorophaea group was treated by Sandstede (1931) as including two species-C. chlorophaea (Flirke) Spreng., containing fumarprotocetraric acid and consequently distinguished by a bitter taste, and C. grayi Merr. ex Sandst., lacking fumarprotocetraric acid and consequently with a mild taste. Asahina (1940) discovered grayanic acid, cryptochlorophaeic acid, and merochlorophaeic acid in this group and divided the complex into four species: Cladonia grayi, C. cryptochlorophaea Asah., and C. merochlo?rophaea Asah. are characterized by the presence of grayanic acid, cryptochlorophaeic acid, and merochlorophaeic acid, respectively, while C. chlorophaea is considered to have fumarprotocetraric acid only. Within each of the first three species, the presence or absence of fumarprotocetraric acid is generally ignored from taxonomic considerations, but Asahina treated such fumarprotocetraric acid variants as forms. Although these four species are the most common ones of the chlorophaea group, many other variants have been recorded (Ahti, 1966; Culberson & Kristinsson, 1969). The morphologic variability has generally been considered to be the same within all the chemically different races in the complex. Ahti (1966), however, indicated some morphological differences between some of the plants called C. merochlorophaea and those of the other chemical species, and Krog (1968) claimed that the rangiformic acid variant in Alaska has a characteristic morphology. Wetherbee (1969) has demonstrated substrate specificity in some of the chemical species in Michigan. The present paper deals with the morphology and distribution of the chemical variants of the C. chlorophaea group in North Carolina. Since a preliminary survey indicated that there was a significant difference in the size of the soredia in the podetial cups between some of the chemical variants, a large-scale survey was made. Other 1 This study was supported by grant GB-6041X from the National Science Foundation. I thank Dr. William Louis Culberson for his interest in this work and for linguistic corrections and Dr. Chicita F. Culberson for her advice on the identification of the lichen substances. 2 Department of Botany, Duke University, Durham, North Carolina 27706. Present address: NMttlirugripasafnit Q Akureyri, P.O. Box 580, Akureyri, Iceland. This content downloaded from 157.55.39.153 on Mon, 19 Sep 2016 04:46:55 UTC All use subject to http://about.jstor.org/terms 14 THE BRYOLOGIST [Volume 74 100 Cladonia grayi PD+ s ---C. grayi PD?. C. cryptochlorophaea PD+ . .... .. ........ C. cryptochlorophaea PDo/ .... C)' I 0 100 200 300p LLI N A