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Leprocaulon beechingii is described as new to science based on collections from exposed rock outcrops in the southern Appalachian Mountains in eastern North America. Taxonomic placement in Leprocaulon, and delimitation from other members of the genus with usnic acid, is supported by molecular phylogenetic analyses of ITS and mtSSU sequence data. The species is readily recognized by its occurrence on non-calcareous rocks, normandinoides-type placodioid thallus, and the production of usnic acid and zeorin.

Molecular phylogenetic analyses revealed a high diversity of unrecognized species of Lecanora s.l. in Brazil, with many near-cryptic species that require a combined analysis of morphology, secondary metabolites and molecular sequences for accurate delimitation. In this study, a new species of Lecanora is described which is morphologically close to L. achroa and L. helva. The new species was collected in the Guadalupe environmental protection area, located in the municipality of Barra de Sirinhaem, a mangrove region in the state of Pernambuco in the Brazilian Northeast. Lecanora parachroa sp. nov. agrees with L. achroa in morphological and anatomical features but differs in secondary chemistry, lacking usnic acid and 2′-O-methylperlatolic acid. Molecular data using the ITS barcoding marker suggest that commonly identified species in this group, including L. helva and L. leprosa, are collective taxa, each including several lineages.

Agnello, G., M. Catinon, S. Ayrault, J. Asta, M. Tissut & P. Ravanel. 2010. A comparative study of atmospheric deposits and lichen populations in a protected alpine area in the Grenoble region (France). International Journal of Environment and Health 4(2–3): 235–249. [‘‘Concurrently, the lichen flora of Seiglieres was studied from 1970 to 2007, showing a high biodiversity as well as important changes occurring in its composition, with a marked increase in the nitrophilous species, possibly due to higher amounts of NOx, P, Mg and S wafted over in the atmosphere.’’] Alstrup, V., A. Aptroot, P. K. Divakar, S. LaGreca & L. Tibell. 2010. Lichens from Tanzania and Kenya III. Macrolichens and calicioid lichens. Cryptogamie, Mycologie 31(3): 333–351. [Report of 156 species, including ‘‘ ... 28 species are new for Tanzania and 2 for Kenya. New for Africa are Hypotrachyna novella, H. physcioides, Melanelia panniformis, Physcidia squamulosa, and Xanthoparmelia microspora.’’] Aragon, G., R. Lopez & I. Martinez. 2010. Effects of Mediterranean dehesa management on epiphytic lichens. Science of the Total Environment 409(1): 116–122. [Study of Spanish holm oak in open woodlands (dehesas) in Spain. ‘‘Total species richness and cover exhibited considerable variation among management regimes. Both parameters tended to decrease with the intensity of management, abandoned dehesas maintaining a higher number of species than more intensively managed habitats. Lichen composition also significantly differed among the four regimes. Nitrophytic species were clearly associated with more intensive management regimes (farming or livestock management), whereas non-nitrophytic species favored abandoned dehesas.’’] Archer, A. W. 2010. The Rt. Rev. James Hannington, D. D., F. L S., F. R. G. S., collector, missionary and martyr 1847– 1885. British Lichen Society Bulletin 107: 18–19. [Notes on specimens at Kew from East Africa including a list of 11 new taxa described by Muller based on his collections.] Backorova, M., M. Backor, J. Mikes, R. Jendželovský & P. Fedorocko. 2011. Variable responses of different human cancer cells to the lichen compounds parietin, atranorin, usnic acid and gyrophoric acid. Toxicology in Vitro 25(1): 37–44. [Tests on nine human cancer cell lines. ‘‘However, in comparison with parietin and gyrophoric acid, the suppression of viability and cell proliferation by usnic acid or atranorin was found to be more efficient at equitoxic doses and correlated more strongly with an increased number of floating cells or a higher apoptotic index. This study has confirmed a differential sensitivity of cancer cell lines to lichen secondary metabolites.’’] Bajpai, R., S. Nayaka & D. K. Upreti. 2010. Calcium accumulation in lichens of Bhimbetka rock shelter — World Heritage Zone, India. British Lichen Society Bulletin 107: 71–76. [Study of weathering and biodeterioration by several lichen species at this sandstone site.] ———, D. K. Upreti, S. K. Dwivedi & S. Nayaka. 2009. Lichen as quantitative biomonitors of atmospheric heavy metals deposition in Central India. Journal of 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. I have now transferred the Recent Literature on Lichens series into the capable hands of my colleague Brendan Hodkinson (brendan.hodkinson@duke.edu). Please be sure to place him on your mailing list for all future lichen publications. Many thanks. DOI: 10.1639/0007-2745-114.2.408

Lecidea ramulicola (= Lecanora symmicta f. ramulicola) is raised to species rank within Lecanora. It is anatomically similar to Lecanora symmicta, but unlike that species it has atranorin as major secondary product and usnic acid only as an accessory substance, more frequently branched and anastomosing paraphyses, and shorter conidia. The species is known from northeastern North America and central Europe, where it is often found in pine forests, most frequently on dead pine twigs.

Using spectral filters, Cladonia uncialis and Cladina rangiferina were exposed for seven days to visible light supplemented with two intensities of three different bands of ultraviolet in a growth chamber. The concentrations of lichen secondary products separated by thin-layer chromatography were then determined densitometrically. In Cladonia uncialis, higher intensity exposure to visible light enhanced with UV-A, while UV-B and UV-C were excluded, significantly increased usnic acid concentrations over control levels. However, less usnic acid accumulated under high intensity enhancement of both UV-A and UV-B. The effect of supplemental UV-A and UV-B was the same on atranorin concentrations in Cladina rangiferina. This suggested that UV-A caused increased accumulation of both compounds, but that the effect was negated by UV-B. Concentrations of usnic acid in natural populations of Cladina mitis were significantly lower in spring and summer when UV-B flux is greatest.

Lecanora thysanophora R. C. Harris, n. sp. is described. It is a common lichen in eastern North America, most often found as a sterile corticolous crust, but apothecia are not uncommon when the species grows along streams. Besides being recognizable in the field, it is distinctive chemically, containing usnic acid, zeorin, often porphyrilic acid, and several species-specific terpenoids.

Four chemotypes of Dimelaena oreina are reported from the analysis of 140 specimens from the Iberian Peninsula. Chemotypes II (usnic and gyrophoric acids) and V (usnic and stictic acids) were previously known from this area, but chemotype I (usnic and fumarprotocetraric acids) is new to the region, and chemotype VII (usnic, stictic, and gyrophoric acids) is new for the species. Chemical and phytogeographical evidence suggests that chemotypes V and VII are closely related. In addition to geographic distributions, phytogeographic affinities and minor morphological trends, some lichens and lichenicolous fungi growing on Dimelaena oreina chemotypes are reported. Among all the species of Dimelaena hitherto known (e.g., Mayrhofer et al. 1996; Sheard 1974; Sheard & Mayrhofer 1984), Dimelaena oreina (Ach.) Norman is well characterized by its yellowgreen thallus containing usnic acid in the cortex, lecanorine apothecia, and an hyaline hypothecium. It is widespread in Temperate and Boreal regions of the northern hemisphere and is also known in the southern hemisphere (Mayrhofer et al. 1996). In Europe, its distribution is from Scandinavia to the Mediterranean. Dimelaena oreina is one of the best studied lichens from a chemical point of view. It is chemically variable, with six chemotypes recognized worldwide (Culberson et al. 1984; Leuckert & Poelt 1978; Leuckert et al. 1975, 1981, 1987; Mayrhofer et al. 1996; Sheard 1974, 1977; Sheard & Ahti 1975). According to the terminology of Sheard (1974, 1977) and Culberson et al. (1984), each chemotype is designated with a number, and is characterized by the content of the main substances (see Table 1). Except for the chemotype with sphaerophorin, described from California (Culberson et al. 1984) all chemotypes occur in Europe. They are distributed following a north to south gradient, with northern Europe having fewer and less complex chemotypes than southern territories (Leuckert & Poelt 1978). Chemotype III is predominant in northern Scandinavia, chemotype I is the most frequent in some mountainous regions of central Europe (with chemotype IV often associated), and chemotype V becomes the predominant chemotype in other parts of central Europe and in the Mediterranean region. A good correlation between chemistry and elevation has been also demonstrated in a study from South Tyrol (Leuckert et al. 1981). Chemotype V dominated at lower elevations, whereas at higher elevations chemotype I was the most frequent. The overall distribution of the chemotypes is summarized in Table 1. The main aim of this paper is to study the chemical variability of D. oreina in the Iberian Peninsula (Andorra, Portugal and Spain), a particularly interesting territory considering its great climatic and biological diversity, as well as the above mentioned chemical richness and complexity in southern Europe. In the Iberian Peninsula, D. oreina is a widespread lichen, extending from regions close to the coast to the highest mountains, mostly associated with hard siliceous rocks. Sporadic analysis of Spanish and Portuguese specimens by Leuckert et al. (1975), Sheard (1977), and Culberson et al. (1984) reported two chemotypes (II and V) for this territory. A rare occurrence of chemotype III (Sheard 1977) in Spain is here excluded, and needs confirmation because of the low elevation and typical Mediterranean character of the locality. Also, a few samples from the Canary Islands and Morocco have been analyzed; this complementary information is included in "representative specimens studied." MATERIALS AND METHODS The study is based on material kept at BCC, LEB, LISU, MA, MAF, VAB, and Etayo priv. herb. The main morphological and anatomical characters were studied by means of standard light microscopic techniques. A total of 142 samples, 140 from the Iberian Peninsula, one from the Canary Islands and one from Morocco, were analyzed by Thin-Layer Chromatography (TLC) and 12 selected specimens by High-Performance Liquid Chromatography 0007-2745199139-44$O.7510 This content downloaded from 157.55.39.129 on Thu, 28 Jul 2016 04:05:12 UTC All use subject to http://about.jstor.org/terms 40 THE BRYOLOGIST [VOL. 102 TABLE 1. Lichen substances content and distribution of the chemotypes of Dimelaena oreina, excluding rare occurrences FU = fumarprotocetraric acid, GY = gyrophoric acid, ST = stictic acid and SPH = sphaerophorin. Usnic acid present in all the chemotypes. Greenland Eastern Western Southern ChemoRussia FennoscanCentral Southern and North North Hemitype Substances and Asia dia Europe Europe N. Canada America America sphere

15-28. 5 fig. 2 schemes. [Chemical study of Usnea articulata and Parmelia tinctorum.] ABO-KHATWA, A. N., A. A. AL-ROBAI & D. A. AL-JAWHARI. 1996. Lichen acids as uncouplers of oxidative phosphorylation of mouse-liver mitochondria. Natural Toxins 4: 96-102. 5 fig. 1 tab. [Study using usnic acid, vulpinic acid, and atranorin.] AKCAY, H. & G. ARDISSON. 1988. Radioactive pollution of Turkish biotas one year after the Chernobyl accident. Journal of Radioanalytical and Nuclear Chemistry, Letters 128: 273-281. 1 fig. 2 tab.

The common depside atranorin and the depsidones stictic and norstictic acids were found in mycobionts of several crustose lichens cultured on medium with 10% sucrose as well as in natural lichens. Usnic acid and anthraquinone were found in other mycobionts on 0.4 and/or 10% sucrose media.

Two saxicolous species ofLecanora are described as new to science. Lecanora brodoana Lumbsch & Nash from Mexico and southwestern USA contains usnic acid, atranorin, and zeorin and has orbicular, coalescing soralia. Lecanora neosonorensis Lumbsch & Nash contains atranorin and 2'-0-methylperlatolic acid and has pruinose discs. Lecanora helva and L. plumosa are reported as new for North America and L. gangaleoides and L. pseudistera are new for Mexico The genus Lecanora s.str. is widely distributed throughout the Sonoran Desert region and other areas of Mexico. During a continuing revision of extensive collections in ASU and the herbarium of H. T. Lumbsch in Essen we have found two apparently undescribed species belonging to the L. subfusca group. Both are described below. Moreover, four previously described species are recorded for the first time from North America.

Eighteen taxa belonging to the Lecanora subfusca group that contain usnic acid in addition to atranorin are revised on a worldwide basis. The following species are described as new: L. alba Lumbsch from Australia, L. floridula Lumbsch from Florida, L. formosula Lumbsch from Australasia, L. neonashii Lumbsch from Mexico, and L. transvaalensis Lumbsch from South Africa. A xanthone containing population of L. wilsonii is distinguished as L. wilsonii ssp. xanthophora Lumbsch. Lecanora achroella and L. subflavicans are reduced to synonymy with L. achroa, and L. elatinoides var. straminea with L. elatinoides. A key to the taxa treated is included. After consid- eration of the morphological, anatomical, and chemical variation, it is concluded that the usnic acid containing species of Lecanora do not form a natural group and cannot be separated from

We conducted physiological, pharmacological, and chemical studies using tissue cul- tures derived from natural lichens. Cultured tissues of about 200 lichen species were initiated from vegetative thalli collected in various regions of the world. The cultures grew on both complex and defined media. Growth was significantly affected by temperature, as well as by carbon and nitrogen sources. Some tissue cultures produced secondary substances (i.e., depsidones and usnic acid), but most acetone extracts did not show the same patterns of TLC spots as the corresponding natural thalli. Some lichen tissue cultures produced more usnic acid than natural thalli. Several pharma- cological activities were screened for both tissue cultures and natural lichens.

Usnic acid-deficient thalli of the lichen Arctoparmelia centrifuga (L.) Hale, previously known only from Fennoscandia, are reported here from New Brunswick and Newfoundland. Pigment-deficient and pigment-containing thalli growing side by side in New Brunswick show no detectable differences in morphology, reproductive state, or lichenicolous microfungi. The Fennoscandian and eastern Canadian populations of typical A. centrifuga contain different combinations of unidentified fatty acids. Their respective usnic acid-deficient counterparts are similarly distinct, providing strong evidence that the pigment-deficient chemotypes originated separately on the two continents, rather than from a common ancestor. Among a collection of saxicolous lichens which I made in northern New Brunswick, Canada, in 1984 is a specimen identical in morphology to the circumpolar, boreal-arctic species Arctoparmelia centrifuga (L.) Hale, but which lacks the yellow cortical pigment usnic acid. Usnic acid-deficient chemotypes ofA. centrifuga have been reported previously only from Norway, Sweden, and Finland (Hasselrot 1953; Poelt and V6zda 1981; Santesson 1984) and have been recognized at various taxonomic ranks, from forma to species, as follows: Parmelia centrifuga var. dealbata Fr., Summ. Veg. Scand. 103. 1845[1846], nom. nud. Parmelia centrifuga # dealbata Fr. ex Th. Fr., Lichenogr. Scand. 1: 129. 1871. TYPE: Sweden, Nirke, G8tlunda parish, Hasta, 1863, Blomberg, ups, holotype, not seen; CANL, H, topotypes! ("1865 & 1866, Blomberg"). Parmelia centrifuga # dealbata Th. Fr. ex Hellbom, Nerikes lafflora, 16. 1871, not seen. Parmelia centrifuga fo. dealbata (Th. Fr. ex Hellbom) Hillmann, Ann. Mycol. 24: 142. 1926, not seen. Parmelia aleuritica Nyl., Flora 58: 103. 1875. TYPE: Finland, Padasjoki [= North Hime: Pihlajavesi, fide T. Ahti, in litt.], 1874, Norrlin 244, H-NYL 34674, holotype!; H, isotype! (Herb. Lich. Fenn. 204). Parmelia centrifuga fo. aleuritica (Nyl.) Oliv., M6m. Soc. Sci. Nat. Cherbourg 36: 182. 1907. Arctoparmelia aleuritica (Nyl.) Hale, Mycotaxon 25: 252. 1986. Hale (1986) created the genus Arctoparmelia to accommodate the distinctive "Parmelia centrifuga group" and recognized A. aleuritica as "an usnic acid-free mutant of A. centrifuga"--one of five species in the genus and the only one lacking usnic acid in the cortex. As the nomenclatural history of this lichen shows, the epiphet aleuritica also has priority at the rank of forma. Hale did not state explicitly his reasons for recognizing the usnic acid-deficient chemotype at species rank, in preference to a lower rank or not at all. While I was investigating the typification and synonomy of this lichen, Teuvo Ahti brought to my attention another eastern Canadian specimen of usnic acid-deficient A. centrifuga, which he had collected in Newfoundland in 1956. I subsequently carried out an analysis of the secondary product chemistry of a number of Fennoscandian and eastern North American specimens of A. centrifuga, using standardized thin-layer chromatography (TLC) 0007-2745/92/1-4$0.55/0 This content downloaded from 157.55.39.249 on Wed, 03 Aug 2016 06:06:52 UTC All use subject to http://about.jstor.org/terms 2 THE BRYOLOGIST [VOL. 95 (Culberson 1972; Culberson & Johnson 1982). In particular, I cochromatographed the two usnic aciddeficient specimens from Canada with several European collections containing mixtures of usnic acid-containing and usnic acid-deficient thalli. The analyses included an isotype of Parmelia aleuritica Nyl. and a topotype of P. centrifuga (L.) Ach. var. dealbata Fr. ex Th. Fr., as noted above. OBSERVATIONS ON UsNIC ACID-DEFICIENT A. CENTRIFUGA IN NEW BRUNSWICK Usnic acid-deficient thalli were found on a single boulder of rhyolite tuff and breccia during approximately one hour of systematic general collecting on an extensive south-facing scree slope with patches of stunted Picea mariana-Betula papyrifera-Vaccinium-Cladina heath. An exact count of the number of individual gray thalli was not obtained. A patch (single thallus?) of irregular outline with a maximum diameter of ca. 10 cm was present along with several smaller discrete neighboring thalli. In the material collected, the thallus lobes are predominantly tightly attached to the substratum; some are intertwined with or overgrowing adjacent thalli of Cetraria hepatizon and typical yellow-pigmented A. centrifuga. There are no apparent differences in the vigour of the gray and yellow thallus forms. Apothecia are well developed in the gray thalli and contain numerous intact asci with a full complement of ascospores. A more detailed description follows. Thallus lobes 0.5-1.8 mm wide, imbricate. Upper surface smooth, mat, "yellowish white" (no. 92; colors according to Kelly 1965) at lobe ends, grading to "medium gray" (no. 265) in older parts [note: slight yellowish tint not due to usnic acid; adjacent typical thalli of A. centrifuga are a contrasting "pale greenish yellow" (no. 104)]. Lower surface minutely roughened, with a few shallow longitudinal wrinkles per lobe, mostly "pale orange yellow" (no. 73); moderately rhizinate, the rhizines simple, to 0.6 mm in length, concolorous with the lower cortex to brownish black (no. 65). Apothecia common in older parts of thallus, to 2.8 mm diameter, deeply concave; thalline margin entire to slightly crenulate; disc dark reddish brown (no. 44). Ascospores 8-12.5 x 4-6 lim. Pycnidia common, conidia weakly bifusiform (bacilliform), with two, less often one, subapical swelling(s), 4-6 x 0.5-0.8 .m. Both yellow and gray thalli are spottily colonized by two unidentified nonfruiting microfungi. One of these has a coarse dendritic branching pattern, each branch comprised of several dark brown parallel hyphae with quadrate to short rectangular cells. The other consists of tiny black hemispherical structures (to 0.2 mm diameter) superficially resembling emergent perithecia or pycnidia. In anatomical section, however, these show no ostiole or other differentiated tissue beneath a thin layer of necrotic, "carbonized" cells. Specimen examined.--NEw BRUNSWICK. RESTIGOUCHE co.: Mount Carleton Provincial Park, north side of Sagamook Mountain, near summit, ca. 710 m; 47*25'N, 66053'W, Clayden 2909 (CANL, NBM).

The effect of environmental factors on the content of usnic acid was examined using the cultivated lichen mycobiont of Ramalina siliquosa. There was no correlation between light intensity and the content of usnic acid or 4-O-demethylbarbatic acid. The content of usnic acid changed with variations in temperature. In our previous studies on the production of lichen substances by an isolated lichen mycobiont (Hamada & Ueno 1987, 1990), we reported that the content of the depside, 4-O-demethylbarbatic acid, in the cultured lichen mycobiont of Ramalina siliquosa was affected by temperature. Similar variations in the content of the depsidone, salazinic acid, were observed in lichens growing under natural conditions (Hamada 1989). Thus, further studies on the effect of environmental factors on the lichen mycobiont would also be valuable. As far as lichen pigments are concerned, Rundel (1969) reported that the concentration of usnic acid in Cladonia subtenuis increased with higher light intensity under natural conditions. He implied that usnic acid was not produced in the absence of sunlight. However, Ahmadjian and Jacobs (1985) reported that crystals of usnic acid were abundantly produced by synthetic lichens growing under low light intensity, and considered that the increased production of usnic acid was due to the favorable growing conditions. Moreover, I too have often found usnic acid in lichen mycobionts cultured in the dark (see Hamada 1989). Indeed earlier studies have shown that usnic acid was produced by cultured lichen mycobionts as well as by lichens grown under natural conditions (Komiya & Shibata 1969). Thus, in the present work the relationship between usnic acid content and light intensity was investigated using the cultured mycobiont from a strain ofR. siliquosa, which produces comparatively large quantities of usnic acid. MATERIALS AND METHODS A fertile thallus ofR. siliquosa was collected in Kasumicho of Hyogo Prefecture (36*N), Japan, 4 May 1988. In this thallus salazinic and usnic acids were detected by TLC (Kieselgel GF254, 0.25 mm thickness) in toluene-acetic acid (20:3), usnic acid (Rf = 0.68), 4-O-demethylbarbatic acid (Rf = 0.47), and salazinic acid (Rf = 0.05). The concentrations of these substances in the sample examined were 0.69% (usnic acid) and 0.82% (salazinic acid) as indicated by HPLC (Hamada 1988). Approximately ten spores discharged from apothecia on the thallus were inoculated into each slant, and the mycelia were cultured for 8 months in 70 test tubes containing a modified malt-yeast extract medium (malt extract 10 g, yeast extract 4 g, sucrose 4 g, agar 15 g, water 1 1, pH 7.0; see Hamada 1989). To examine the effect of light intensity, the mycobiont was cultured at 12"C for 8 months in slant tubes under varied intensities of continuous fluorescent light. The surface of each slant was vertical to the light source. The light intensity was measured by photometer (Minolta T1) and adjusted from 1,100 to 4,400 lux by layers of Victoria lawn (a fine linen fabric) covering the test tubes. The fluorescent lamps (20 W x 6) were changed at intervals of 4 months. The average intensity of these fluorescent lights is shown in Table 2. In a preliminary experiment, the usnic acid content in the lichen mycobiont was found to be affected by the temperature, and is maximal at near 12*C. Therefore, in the experiments investigating the effect of time and light intensity (Tables 1-2), the temperature was maintained at 12*C. For examining the effect of temperature, the mycelia in slant tubes were kept in the phytotron at controlled temperatures of 5, 10, 12, 15, 20, and 25*C, respectively, for 8 months. This experiment was done in darkness to examine the effect of temperature without interference by light. All mycelia cultured at 15, 20, and 25*C were transplanted to fresh medium every 3 months to maintain the cultures on a slightly wet medium; those cultured at 10 and 12?C were transplanted every 4 months; while the mycelia cultured at 5?C were not transplanted at all. The mycobiont colonies coalesced and aggregated compactly on the slant. After 8 months cultivation, the mycelia were harvested, weighed, and used for the chemical determinations. (+)Usnic acid was detected by measurement of optical rotation, [a]D + 507?, in chloroform, and mass spectrometry, EI, 70eV: m/z 344 (M+, 78%), 260 (89%), 233 (100%), 217 (36%), as reported by Komiya and Shibata (1969). In colonies obtained from all test tubes other than those cultured at 25*C, 4-O-demethylbarbatic and usnic acids could be detected by HPLC in H20-methanol-acetic acid (20: 80:1); 4-O-demethylbarbatic acid (Rt = 4.6 min.); usnic acid (Rt = 17.8 min.), with a flow rate of 1.0 ml/min. using a 15 cm x 4.6 mm M & S Pack, C18 column (M & S Co.). The areas of HPLC chromatographic peaks due to usnic and 4-O-demethylbarbatic acids were used for the quantitative measurements. Each value for the acid content was an average of that observed for the colonies 0007-2745/91/57-59$0.45/0 This content downloaded from 157.55.39.78 on Thu, 23 Jun 2016 06:46:55 UTC All use subject to http://about.jstor.org/terms 58 THE BRYOLOGIST [VOL. 94

A bioassay was developed to determine the effects of six lichen extracts on food con- sumption and growth of gypsy moth (Lymantria dispar) larvae. The larvae were allowed to feed on leaves of Quercus rubra which had been painted with either acetone or water extracts of Cladina subtenuis, Flavoparmelia caperata, Hypogymnia physodes, Parmelia sulcata, Parmotrema hy- potropum, or Usnea strigosa. Acetone extracts containing depsides, depsidones, and usnic acid did not affect food consumption or larval growth. All water extracts except Flavoparmelia caperata decreased leaf consumption and growth by 30-80%. Although water extracts do not completely deter feeding, they could play an important ecological role in preventing lichenivory by gypsy moths. The gypsy moth, Lymantria dispar, is a common forest pest in eastern United States and can cause serious damage to hardwood forests during out- break years. Gypsy moth larvae are voracious feed- ers and are reported to utilize 485 species of North American plants as food sources (Gerardi & Grimm 1979). Although their preferred foods are leaves of Quercus or Populus species, they will eat a variety of unusual foodstuffs, including plastic tree flagging and styrofoam soaked in sugar (Zielinski 1986). De- spite the fact that larvae of gypsy moths come in contact with corticolous lichens during all stages of their development, our observations suggest that they never utilize lichens as a food source in nature. Secondary compounds in lichens have been shown to play important ecological roles in influencing food selection by lichenivores (Lawrey 1980, 1983, 1984, 1986, 1989; Reutimann & Scheidegger 1987; Run- del 1978). Such compounds include the acetone- soluble depsides, depsidones, usnic and pulvinic acid derivatives, which are exuded as crystals onto the surface of the lichen. Lichen secondary com- pounds-vulpinic acid and a combination of vul- pinic acid and atranorin--have also been shown to deter feeding by an insect which does not normally come into contact with lichens, the yellow-striped armyworm, Spodoptera ornithogalli (Slansky 1979). In order to provide a possible explanation as to why gypsy moth larvae do not feed on lichens, we investigated the effects of water and acetone lichen extracts on larval consumption and growth.

Usnea lecanorica (Ascomycotina, Usneaceae), a new species of the U. florida group from south-central Mexico, differs from all other known species of its genus by producing lecanoric acid as the major medullary constituent. The new Mexican lichen species Usnea lecanorica (Ascomycotina, Usneaceae), the subject of this report, is the first member of its genus known to produce lecanoric acid. Unlike the some 18 0-orcinol products characteristic of many scores of species in this genus, lecanoric acid is a para-depside of the orcinol series, a category previously known to be represented in Usnea by only evernic acid in U. longissima ssp. jesoensis Asah. and U. trichodea Ach. Evernic acid is the 4-O-methyl derivative of lecanoric acid; it probably also occurs in trace amounts in the new species described here. The natural but variable genus Usnea (discussion: Krog 1976) consists of about 500 species. Although thoroughly cosmopolitan, it achieves greatest species diversity in warmtemperate, subtropical and upland-tropical vegetations. The fruticose thallus with its central cord is so distinctive that the chemical data now recorded from approximately 200 species are surely correctly assigned at the generic level. Taxonomy at the species level, however, is confused because in many groups the major evolutionary modifications have been in secondary-product chemistry and amplitude of ecologic tolerance. Usnea lecanorica belongs to the U. florida complex--characteristically tufted lichens with conspicuous apotheciaconstituting in North America a series of 13 chemotypes as determined from a thin-layer chromatographic survey of 358 specimens (Fiscus 1972). No micromorphological analyses have been made, and consequently it is unknown if any of the small morphological variations seen in the U. florida complex consistently accompany major chemical discontinuities. The U. florida group in North America is characterized by certain 0-orcinol depsides (diffractaic, squamatic and thamnolic acids) and depsidones (connorstictic, constictic, 2'O-methylpsoromic (= conpsoromic), fumarprotocetraric, galbinic, norstictic, protocetraric, psoromic, salazinic and stictic acids). Additionally, some members of the U. florida group make fatty acids (including caperatic and bourgeanic acids); they all make the yellow pigment usnic acid and some produce other pigments as well. It was consequently a surprise to find two C+ red Mexican collections that produced the unsuspected orcinol depside lecanoric acid. The two small original collections were made by Robert W. Cruden in 1966 at a mountain locality some 25 km southwest of Toluca in the state of Mexico. We found four more specimens at the same locality ten years later during one of several general collecting trips for lichens of the mountain forests of south-central Mexico. Curiously, these four were the only examples of this chemotype among the many specimens of the U. florida group taken throughout the extensive regions visited. The cool, relatively moist locality near Toluca is above 3000 m elevation and is forested with Abies religiosa and Pinus sp. The new species grows upon both these trees. The most common congeneric companion 007-2745/83/254-256$0.45/0 This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 06:11:51 UTC All use subject to http://about.jstor.org/terms 1983] CULBERSON ET AL.: A NEW USNEA 255

Ramalina intermedia has a North American distribution ranging from New Brunswick south along the eastern seaboard, inland throughout Quebec and Ontario, and has been reported from the Northwest Territories and Alberta. This species is abundant in the Great Lakes region, and has been collected in mountainous areas of Colorado, New Mexico, and Arizona. This species has a single chemical race in North America containing sekikaic, homosekikaic, and usnic acids and variably possessing atranorin and an unknown. Fertile thalli are reported from North America. A new species, Ramalina petrina, is described from the Appalachian Mountains. This small sorediate species is characterized by conspicuous terminal soralia borne on recurved lobe tips. Ramalina petrina is PD+ and contains protocetraric acid, usnic acid, and atranorin. Ramalina intermedia is one of the most widespread, yet most often misidentified small sorediate species of Ramalina in North America. Although it is common and abundant in much of its range and is well represented in herbaria, the species has been poorly understood. It is the purpose of this study to clarify the chemical ecology and distribution of this species in North America. The distribution of R. intermedia is sympatric, in part, with R. farinacea, R. pollinaria, and R. roesleri. All three are species with which it has been confused. Examination of herbarium specimens and field collections has shown that R. intermedia consistently contains sekikaic and homosekikaic acids. The identity of homosekikaic acid in R. intermedia was determined by Chicita Culberson (pers. comm.). Surveys of thallus chemistry were carried out by thin-layer chromatography with Merck Silica Gel F254 pre-coated glass plates using a benzene:dioxane: acetic acid (90/25/4, v/v/v) solvent system as described by Culberson and Kristinsson (1970). Microextraction was effected upon thallus fragments using warm acetone. The plates were developed with 10% H2SO4 and heat. All of the 140 thalli tested by thin layer chromatography contained sekikaic, homosekikaic, and usnic acids. Atranorin and an unknown (Rf class 3 in this solvent system) were found in a few plants. Usnic and homosekikaic acids appeared to vary greatly in concentrations, occasionally present only in trace amounts. Taylor (1968) reported no medullary acids in individuals of R. intermedia from Ohio. We have been unable to confirm the existence of this strain in our studies. Ramalina intermedia has a much broader ecological amplitude and range of substrate This content downloaded from 164.67.185.210 on Fri, 18 Jul 2014 18:06:56 PM All use subject to JSTOR Terms and Conditions 618 THE BRYOLOGIST [Volume 77

Recent literature on patterns of chemical variation in the lichen-forming ascomycetes is reviewed, and correlations of chemical variation patterns with respect to morphology at the generic level are presented. At the subgeneric level, reports can be grouped into: (1) examples showing distinct correlations of lichen chemistry with morphology or geography, and (2) examples showing weak correlations of lichen chemistry with morphology or geography, where chemical patterns follow morphological or geographical trends or tendencies. The recognition of chemical strains, where no correlations of chemistry with morphology or geography exist, is documented in the final section of the paper. During the 1860s William Nylander devised a simple method for identifying chemical differences between lichens by applying spot tests of potassium hydroxide or calcium hypochlorite on lichen thalli to elicit color reactions. These spot tests indicated the presence or absence of various aromatic and aliphatic substances. Today over 500 lichen sub- stances are known to be produced by the lichen- forming ascomycete fungi. Most of these substances have now been named and their structures eluci- dated (C. F. Culberson 1969, 1970; C. F. Culberson et al. 1977c). Unlike vascular plants or other groups of cryp- tograms, chemical characteristics have been em- ployed in lichen taxonomy for over 100 years. The chemical composition of most lichen species ap- pears to be uniform and constant throughout the life of the individual lichen, and the chemistry is uniform throughout the range of many species. Nor- mally one substance (atranorin, usnic acid or a xan- thone) is produced in the cortical layer of the thallus while one or more colorless substances (depsides and depsidones) are deposited on the cell walls of the medullary hyphae. Substances can be even more

One new species of Everniastrum, E. paramense, and four new species of Hypotrachyna, H. cendensis, H. meridensis, H. neoflavida and H. primitiva, are described from the paramo region of Venezuela. Everniastrum paramense Hale & L6pez, sp. nov. (FIG. 1) Thallus foliaceus, corticola vel saxicola, laxe adherens, fere subfruticosus, rigidus, cinereoalbus, 15-30 cm latus, lobis lineari-laciniatis, subdivaricatis, 1-3 mm latis, planis vel convexis atque subcanaliculatis, margine integro, anguste nigro, superne nitidus, emaculatus, sorediis isidiisque destitutus, subtus niger, modice rhizinosus, rhizinis longis, furcatis vel dichotome ramosis. Cortex superior columnaris, 30 Am crassus, stratum gonidiale ca. 25 Am crassum, medulla alba, pallide flava vel nigricascens, 70-90 Am crassa, cortex inferior niger, 26-28 Am crassus. Apothecia numerosa, substipitata, expansa, 4-6 mm lata, latiora quam lobis, disco fisso; hymenium 70-75 Am altum; sporae 8:nae, incolores, simplices, 9-10 x 14-16 Am. Chemistry: Atranorin, secalonic acid A and traces of unidentified substances (determined by Dr. C. F. Culberson). Holotype: VENEZUELA, ESTADO MIRIDA: Paramo de Motumba, Quebrada de El Volcan, 3100-3200 m, on shrubs. M. L6pez 12516, (us; isotype in MERF, Universidad de los Andes). This remarkable species is unusual in several respects. At first we included it in Hypotrachyna because of the plane or more often weakly canaliculate, linear, eciliate lobes and the long dense, furcate to dichotomously branched rhizines. Small specimens in particular show these traits. In addition the chemistry is close to Hypotrachyna (similar unknowns are found in H. palmarum (Lynge) Hale) and not at all like that of Everniastrum (see Hale, 1976). Later examination of larger collections convinced us that it is more probably an Everniastrum, mostly because of the canaliculate lobation, very long divaricate lobes with large apothecia and the columnar cortical structure similar to that in Everniastrum pachydermum (Hue) Hale. It is common on shrubs and on soil in a number of paramos, especially above 3000 m. Additional specimens examined.-VENEZUELA. MERIDA: Same locality as the type, L6pez 12483, 12487, 12521 (us); Pairamo de Aricagua, L6pez 12815, 12818, 12838, 12849, 12861, 12872 (us); Phramo de Cende, L6pez 13061, 13077, 13090, 13111 (us); Piramo de San Jose, L6pez 13921 (us). TACHIRA: Piramo del Rosal, Hale & L6pez 45058; Piramo de Tamai, Hale & L6pez 45209; Phramo de El Zumbador, L6pez & Keogh 9404 (us). TRUJILLO: Phramo de Guaramacal, Ldpez 10381 (us); Paramo de Guirigay, L6pez 10950, 10968, 10982, 10984 (us); Phramo El Jab6n, Ldpez 13369, 13396 (us). Hypotrachyna cendensis Hale & L6pez, sp. nov. (FIG. 2) Thallus foliaceus, fragilis, laxe adnatus, viridi-flavus, usque ad 12 cm latus, lobis brevibus, inaequaliter dilatatis, subimbricatis, 2-5 mm latis, superne planus, nitidus, albo-maculatus, sorediis isidiisque destitutus, subtus niger, sparse vel modice rhizinosus, rhizinis dichotome ramosis. Cortex superior 9-11 Am crassus, stratum gonidiale 24-26 Am crassum, medulla alba, 120-150 sm crassa, cortex inferior niger, 12-14 pm crassus. Apothecia substipitata, usque ad 11 mm diametro, disco piano, fisso, amphithecio rugoso; hymenium ca. 70 sm altum; sporae 8:nae, incolores, simplices, 3-4 x 5-6 Gm. Chemistry: Usnic acid, lecanoric acid and evernic acid. This content downloaded from 157.55.39.157 on Mon, 01 Aug 2016 04:28:36 UTC All use subject to http://about.jstor.org/terms 1978] SHORT ARTICLES 591 ::: ::::i::::::::::::::::I-:::: ::: ::::.:. :::::j:::::::::::::::::::::::::::: :::::::: :-:-::::i-:::-:::::-:::::::_::::i

High performance liquid chromatography was used to assess the levels of secondary products extractable from thalli in each offour stands of Cladina stellaris (Opiz) Brodo. In each location there were highly significant differences among thalli with respect to perlatolic acid and usnic acid. Homogeneous subsets of thalli within each stand were identified by the use of Duncan's New Multiple Range Test. Since the coefficients of variation were no lower in sites where environmental conditions were apparently uniform than they were in obviously variable sites, it is suspected that the observed intrastand variability in lichen chemistry was a reflection of innate differences among thalli. In three of the four sites there was no indication of a correlation between the levels of usnic and perlatolic acids. Are lichen thalli throughout a single site basically uniform or are there innate differences among thalli? This question is of importance to both systematic or ecological studies, especially when more material may be required for one sample than is available in a single, discrete thallus. For example, some investigations may necessitate the exclusive use of young or actively growing tissue; for analysis of some enzymes, it may be necessary to have samples as large as 450 mg. It is unclear whether or not samples constituted by combining thalli collected even within the same stand would be homogeneous. This would depend to some extent upon the mechanisms of reproduction and dispersal. Although one stand of a given lichen species usually displays little variability in terms of the array of secondary polyphenolics, thin-layer chromatogr phy (TLC) indicates that quantities of compounds may vary significantly from one thallus to another (Fahselt 1979, 1981). More accurate and efficient quantification can be provided by high-performance liquid chromatography (HPLC) where because ofautomation many samples can be processed relatively quickly. The use of HPLC methods for lichen products was pioneered by C. F. Culberson (1972a) and subsequently used to advantage in various studies (e.g. W. L. Culberson & C. F. Culberson 1978; C. F. Culberson, Nash & Johnson 1979; C. F. Culberson & Ahmadjian 1980). The objective in the present paper was to use 007-2745/84/50-56$0.85/0 This content downloaded from 157.55.39.72 on Thu, 15 Sep 2016 06:03:58 UTC All use subject to http://about.jstor.org/terms 1984] FAHSELT: INTERTHALLINE VARIABILITY IN CLADINA STELLARIS 51

A new tridepside, 4-O-methylgyrophoric acid, was extracted from a Jamaican collection of Lobaria cfr. dissecta that also contained gyrophoric acid. The structure of the new product was proven by identification of its methyl ester as tenuiorin by direct comparison with a sample of the latter extracted from L. linita. Microchemical studies on 177 herbarium specimens of 18 taxa of Lobaria showed the presence of 4-O-methylgyrophoric acid, either as a constant component or as an accessory but always accompanying gyrophoric acid in L. crassior, L. dentata, L. cfr. dissecta, L. erosa, and L. quercizans. In addition to the tridepsides gyrophoric acid, 4-O-methylgyrophoric acid, and tenuiorin, the following substances were detected: atranorin, constictic acid, norstictic acid, scrobiculin, stictic acid, usnic acid, thelephoric acid, and several unidentified substances. Species of the genus Lobaria have frequently been studied chemically because they are large and easily collected, but identification of the plants is difficult because no monographic treatment exists for the genus. The data accumulated up to now for Lobaria suggest that, as in other genera of macrolichens, chemistry may be a taxonomically useful adjunct to the usual morphological characters. A number of related species of Lobaria, however, have been reported to contain only gyrophoric acid, and for these species chemistry would seem to be of little taxonomic value. During a recent microchemical survey of herbarium specimens for the occurrence of the meta-depside scrobiculin (Culberson, 1967a), some gyrophoric acid-producing specimens of Lobaria were found to contain an unidentified substance. The present paper gives the previously unreported results from this microchemical survey and the identification and distribution of the new tridepside 4-O-methylgyrophoric acid. Previous workers have identified a number of products in the genus. The most extensively studied species, Lobaria pulmonaria (L.) Hoffm., has an intriguing distribu1 This investigation was supported by U.S. Public Health Service Research Grant GM-08345 from the Division of General Medical Sciences, National Institutes of Health. I thank William Louis Culberson for checking the identification of the lichen specimens used and Carol Watkins for technical assistance. 2 Department of Botany, Duke University, Durham, North Carolina 27706. This content downloaded from 207.46.13.83 on Sat, 22 Oct 2016 06:34:29 UTC All use subject to http://about.jstor.org/terms 20 THE BRYOLOGIST [Volume 72 tion of chemically distinct varieties and strains containing various combinations of p-orcinol depsidones, stictic acid and norstictic acid, accompanied by a new compound, constictic acid (Asahina, 1968), and the tridepside gyrophoric acid (Asahina & Yanagita, 1934; Asahina, Yanagita & Omaki, 1933; Asahina, Yanagita & Yosioka, 1936). The tridepside tenuiorin was originally found in Lobaria (Asahina & Yanagita, 1933). Terpenoid compounds in the genus have been studied (Agarwal et al., 1961; Rao, Sarma & Seshadri, 1965, 1966), but their chemical structures are unknown and no effort to identify these products has been made in the present study. MICROCHEMICAL PROCEDURES3 Fragments of herbarium samples were extracted first with benzene at room temperature and then with hot acetone. All residues from evaporation of the extracts were chromatographed in a benzene-dioxane-acetic acid (90:25:4, v/v) solution on SiO2-G plates prepared in the laboratory. Under these conditions, gyrophoric acid and 4-O-methylgyrophoric acid separate as distinct spots. Although some additional tests (described below) gave satisfactory results with Merck analytical-layer SiO2-Fz, plates, a mixture of 4-O-methylgyrophoric acid and gyrophoric acid showed only a single elongated spot with an Ri value slightly higher than that produced by gyrophoric acid alone. Microcrystal tests did not consistently distinguish samples containing gyrophoric acid alone from those also containing 4-O-methylgyrophoric acid. The pure sample of the latter compound makes needles with parallel extinction in the GAQ solution and beautiful four-sided plates in the GWPy solution. But a series of extracts from the Lobarias tested gave variable crystal forms in the GAQ solution whether gryrophoric acid alone or in combination with 4-O-methylgyrophoric acid had been demonstrated by thin-layer chromatography (TLC). In the GWPy solution, the crystals of 4-O-methylgyrophoric acid, usually highly distorted, were detectable in only some of the samples known to contain this substance. Confirmatory chromatograms were prepared to eliminate the possibility that some Lobaria samples might contain lecanoric acid, since the Rf values of spots for lecanoric acid and 4-0methylgyrophoric acid are very similar by the method described above. The confirmatory chromatograms were prepared on commercial plates developed in ethyl ether-acetic acid (50:1, v/v) solution. This method distinguished the tridepsides from the depsides but did not separate the 4-O-methyl derivatives from their unmethylated analogs. Samples containing gyrophoric acid, with or without 4-O-methylgyrophoric acid, showed Rr values near 0.28 while lecanoric acid and evernic acid have an Ri value near 0.39. Many specimens tested doubtless contain a trace of atranorin that could not be definitely identified in the small samples studied. Except from specimens containing tenuiorin, the benzene extract is very slight. The preparation of a single chromatogram generally exhausts this extract, but the quantity of atranorin is often below the detectable limit of the thinlayer method. Since tenuiorin and atranorin give spots with the same Ri value by the method used here, the chromatographic detection of atranorin required confirmation by another test. It was found better to treat the benzene extracts first with GAo-T. In the present study, specimens were not retested for atranorin if the first test was negative. Substances in Lobaria that gave good spots by TLC are listed with Ri values (Table 1). The spots were visualized by spraying the plates with 10% H2SO, and heating them in an oven at 1100. The spot for constictic acid was identified by chromatographic comparison with the extract from a sample of Usnea trichodeoides Vain. (Almborn's Lichenes Africani 43-DUKE), a collection cited by Asahina (1968) in the original report of this substance. Constictic acid occurs in L. scrobiculata and was referred to as "unknown L.s.-2" in a previous study (Culberson, 1967a). The compound referred to as "unknown 2" in the present study was not detected chromatographically. It was present in the benzene extract of some specimens and gives distinctive deep yellow plates in the GAo-T solution. The compound was most frequently encountered in Lobaria cfr. dissecta, but was never found in sufficiently high concentration to give a good spot on a chromatogram. 3 For the constituents of the microcrystal test solutions and color test reagents, see Hale (1967). This content downloaded from 207.46.13.83 on Sat, 22 Oct 2016 06:34:29 UTC All use subject to http://about.jstor.org/terms 1969] CULBERSON: A NEW TRIDEPSIDE FROM LOBARIA 21 TABLE 1. Typical Ri values for the substances detected in Lobaria by thin-layer chromatography on Merck SiO2-F254 plates and on SiO2-G plates prepared in the laboratory. The chromatograms were developed in a benzene-dioxane-acetic acid (90:25:4) solution to a height of 10 cm.

Cases of human allergy to lichens are reported. The reaction takes the form of contact dermatitis. Two patients showed positive reactions both to lichens containing usnic acid and to the pure substance. Human disease due to invasion of tissues by living fungi or to allergic reactions to fungi has been extensively investigated and documented in the medical literature but the possibility of lichenized fungi as agents in the production of human disease has received little attention until recently. Standard books of medical mycology give no information on the lichens. Liverworts, lichens, and mosses have been implicated in dermatitis occurring in Europe from exposure to 1 Department of Medicine (Dermatology), University of British Columbia, Vancouver 8, B.C., Canada. 2 Addenbrooke's Hospital, Cambridge, England. This content downloaded from 157.55.39.230 on Tue, 07 Jun 2016 06:38:26 UTC All use subject to http://about.jstor.org/terms 19651 MITCHELL & CHAMPION: ALLERGY TO LICHENS 117 wood. One of us (R.H.C.) has observed a housewife who developed allergic dermatitis presumably from exposure to wood dust on her husband's clothing. Allergy tests showed that this was actually due to lichens present on the surface of the wood. In British Columbia "cedar poisoning" is a name commonly given to a skin disease occurring in forest workers. Investigation of these patients has usually given disappointing results. Softwoods have very rarely been observed to produce positive allergy tests and in the majority of forest workers who presented with occupational allergic dermatitis no cause could be found. Recently one of us (J.C.M.) has observed that two forest workers with allergic dermatitis, which occurred only during work in forest areas, showed positive reactions to lichens containing usnic acid and to isolated usnic acid itself. Lichens are plentiful in temperate zone forests and it seems probable that allergy to lichens will be found to account for other cases of skin disease in forest workers and that this may be a problem of considerable economic importance. The allergic reaction of the skin which results from exposure to lichens in a sensitive person takes the form of contact dermatitis, an itching red rash at the sites of contact on the sides and backs of the fingers and on the backs of the hands. The palmar surfaces are less involved because the protective outer horny layer of the skin is thicker at these sites. Spread of the rash to the forearms, face, and other skin areas may occur. The rash persists for several weeks after exposure to the lichen; if no further contact occurs it subsides and the skin returns to normal. Further exposure results in recurrence. The forest workers noted that their skin trouble was worse in the winter and was brought on by handling trees in wet weather. Allergic contact dermatitis tends to be immunologically specific, that is, the allergic patient reacts only to a single chemical or to a group of closely related chemicals. A human subject who is allergic to usnic acid will react to lichens containing usnic acid but not to lichens which do not contain usnic acid. It is planned to find out whether other lichen acids can cause allergic reactions by experimental induction of allergic contact dermatitis in animals and then to study immunological cross reactions within the lichen acid group. It is too early to do more than speculate about other mechanisms whereby lichens may cause human disease. Another form of dermatitis in the human is photosensitivity in which the patient's skin reacts to light. Very often prior exposure to a chemical is required to produce photosensitivity. Furocoumarins which are present in the families Umbelliferae and Rutaceae are well known to dermatologists as photosensitizers. Chemical substances which exhibit fluorescence are liable to be photosensitizers because they can absorb light and release energy which activates biological systems. It is interesting that some lichens exhibit fluorescence and a study of their role as photosensitizers in forest workers requires investigation. Particularly interesting in this respect is the fact that the structure of the molecule This content downloaded from 157.55.39.230 on Tue, 07 Jun 2016 06:38:26 UTC All use subject to http://about.jstor.org/terms 118 THE BRYOLOGIST [Volume 68 of usnic acid (a dibenzofuran derivative) is chemically related to furocoumarins. Respiratory allergy in forest workers is, like skin allergy in forest workers, poorly understood at present. Since some lichens, for example Alectoria, tend to fragment and crumble and to produce an airborne dust in hot weather and since airborne spores are produced by some species, respiratory allergy to lichens may also occur. The frequency of allergic reactions to lichens is unknown and more medical information is required. The purpose of this report, in addition to drawing the attention of lichenologists to allergy to lichens and usnic acid, is to ask for help in discovering further cases. We would welcome information concerning allergic reactions to lichens and would be glad to send relevant medical information to the physician attending the individual concerned. The allergies reported here will be the subject of other more detailed reports (Champion 1965, Mitchell 1965).

Clinal variation in the usnic acid content of thalli of Cladonia subtenuis along determined light gradients was demonstrated spectrophotometrically. Integrated measurements of light incidence were made using ozalid paper light sensors. The percentage of usnic acid in the dry podetia is directly related to the percentage of full sunlight in the microhabitat of the plants. Extreme values ranged from 0.13% in plants from a site of low light intensity (3% full sunlight) to 2.82% in plants from a relatively open site (51% full sunlight). The yellowish color in the podetia of sun populations was not markedly altered by extraction of the usnic acid. Cladonia subtenuis (Abb.) Evans is a common lichen in the southeastern United 1 This research was supported in part by U.S. Public Health Research Grant GM-08345 from the Division of General Medical Sciences, National Institutes of Health, to Dr. Chicita F. Culberson. I thank Dr. Culberson for her help in this study. 2 Department of Botany, Duke University, Durham, North Carolina 27706. This content downloaded from 207.46.13.172 on Fri, 07 Oct 2016 06:14:56 UTC All use subject to http://about.jstor.org/terms 1969] RUNDEL: CLINAL VARIATION IN USNIC ACID PRODUCTION 41 States, occurring on soil in openings or at the edge of pine woods and on banks along old roadcuts. This species has long been known for great variation between sun and shade forms. This variation was noted by Evans (1944) when he raised the taxon to specific rank. Evans recognized four forms including f. cinerascens, a grayish-green shade form with little yellow color and little or no usnic acid, which he distinguished from the typical f. subtenuis or sun form, yellowish in color and containing usnic acid. These two forms have been separated solely on the basis of the degree of yellow coloring of the podetia which has been assumed to be related to the content of usnic acid, a yellow lichen substance. A very yellow color of the podetia has been considered to indicate a high content of usnic acid, a gray-green color to indicate little or no usnic acid. In the present study an attempt has been made to relate a quantitative assessment of the usnic acid content in C. subtenuis to the light incidence of the habitat. Usnic acid is one of the most widespread of lichen substances and a considerable body of literature concerning it exists. Few studies, however, have concerned themselves with quantitative determinations of the usnic acid content of lichens. Laaskso and Gustafsson (1952) used a ferric chloride reagent to make quantitative measurements of usnic acid sepctrophotometrically for Cladonia alpestris. Another spectrophotometric method was used by Ramaut et al. (1966) in a study of the chemotaxonomic significance of usnic acid in Cladonia tenuis, C. leucophaea, and C. impexa. In their study the height of the absorption curve at 380 my for a benzene extract of usnic acid from the lichens studied was used to measure usnic acid concentration. A more refined spectrophotometric analysis for usnic acid has been used in the present research. Usnic acid in benzene solution has a very strong ultraviolet absorption peak at 285 m/, and this peak provides a more satisfactory measurement of the usnic acid content than the absorption at 380 my where no peak occurs. MATERIALS AND METHODS Mats of Cladonia subtenuis were collected in Duke Forest (Durham County, North Carolina) from a range of ecological sites in which the percentage of full sunlight reaching the microhabitat of each mat had been measured. Light measurements at these sites were made with deciduous trees in full leaf. From each small mat of C. subtenuis studied, approximately 0.5 g of the terminal 5 mm of the podetia were collected and dried to constant weight in a desiccator over concentrated sulfuric acid. Material in obvious fruiting condition was not used. The dried sample was weighed and immediately extracted. The lichen was extracted in 15 ml of benzene for 10 minutes on a steam table. The filtered solutions from four successive extractions were pooled and diluted to 500 ml with benzene. Three ml of this solution were run on a Bausch & Lomb Spectronic 505 spectrophotometer against a benzene reference to give an absorption curve for the 280-330 my range (Fig. 1). The usnic acid content of the samples was determined from the absorption peak at 285 me. A calibration curve relating concentration of usnic acid in milligrams per 500 ml of benzene was obtained using solutions of known concentration of usnic acid. The relationship between peak height and usnic acid concentration is linear over the range measured. From the calibration chart the usnic acid concentration of each sample was determined; these data and the original dry weight of each sample enabled the calculation of the percentage of usnic acid by weight in each sample. Integrated measurements of light incidence on thallus mats in the field were made with ozalid paper light sensors (Friend, 1961). Packets of light-sensitive ozalid paper were exposed for 24 hours on the surface of the mats to be studied. The number of papers bleached by the sun at a given sample point was compared to that bleached in a totally This content downloaded from 207.46.13.172 on Fri, 07 Oct 2016 06:14:56 UTC All use subject to http://about.jstor.org/terms 42 THE BRYOLOGIST [Volume 72

Scrobiculin, a recently described meta-depside, is a constant com- ponent of Lobaria amplissima and L. scrobiculata, and it is identifiable in fragments of herbarium specimens by microcrystal tests and thin-layer chroma- tography. In L. scrobiculata usnic acid is probably an accessory substance, and two unidentified substances are present in addition to stictic acid and norstictic acid. 4-O-Methylphysodic acid can be identified in Parmelia livida by thin-layer chromatography, but other constituents present interfere with microcrystal tests for this depsidone. Atranorin, lividic acid, and two unidentified substances were also detected as constant components of this species, and two additional, un- known compounds were found in variable concentration. In 1947 Asahina found stictic acid and an unknown substance, recrystallizing as needles in the GE solution, by microcrystal tests on Lobaria verrucosa Hoffm. (=L. scrobiculata (Scop.) DC.). Recently, the unknown substance, named scrobiculin, was isolated and its chemical structure (Fig. 1) determined (C. F. Culberson, 1967). Scrobiculin was the only phenolic compound obtained from Lobaria amplissima, but it occurred with (+)-usnic acid, stictic acid, and norstictic acid in L. scrobiculata. 4-O-Methylphysodic acid is a second new lichen substance which has recently been studied chemically (C. F. Culberson, 1966). The only known natural source of 4-0-methylphysodic acid is Parmelia livida Tayl., from which it was first isolated along with atranorin and a major constituent of unknown chemical structure, which I shall call lividic acid. Earlier, Hale (1958) found atranorin and an unknown substance crystallizing in the GE solution by microchemical tests on the type specimen of Parmelia livida. W. L. Culberson (1961) obtained similar results, described the microchemical test in more detail, and gave a good photograph of the crystals ob- served in the GE solution. These crystals are now known to be formed by the major constituent, lividic acid. The purpose of the present report is to describe microchemical tests which may be useful for the identification of the two new lichen substances scrobiculin and 4-O-methylphysodic acid. These methods were employed in microchemical surveys of the three lichens, Lobaria amplissima, L. scrobiculata, and Parmelia livida, which in- dicate that the new substances are constant components of the species in which they occur. In addition, two unidentified substances were detected microchemically in L. scrobiculata and four in P. livida. Some chromatographic properties of these un- identified compounds are described.