STUDIES ON THE LICHEN GENUS USNEA IN EAST FENNOSCANDIA AND PASIFIC NORTH AMERICA

PEKKAHALONEN

Department of Biology

OULU 2000

OULUN YLIOPISTO, OULU 2000

STUDIES ON THE LICHEN GENUS USNEA IN EAST FENNOSCANDIA AND PASIFIC NORTH AMERICA

PEKKA HALONEN

Academic Dissertation to be presented with the assent of the Faculty Science, University of Oulu, for public discussion in Kuusamonsali (Auditorium YB 210), on February 11th, 2000, at 12 noon.

Copyright © 2000Oulu University Library, 2000

OULU UNIVERSITY LIBRARYOULU 2000

ALSO AVAILABLE IN PRINTED FORMAT

Manuscript received 27 December 1999Accepted 10 January 2000

Communicated by Docent Tiina Randlane Docent Soili Stenroos

ISBN 951-42-5524-0

ISBN 951-42-5523-2ISSN 0355-3191

Halonen, Pekka, Studies on the lichen genus Usnea in East Fennoscandia and PacificNorth AmericaBotanical Museum, Department of Biology, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland2000Oulu, Finland(Manuscript received December 27 1999)

Abstract

The shrubby Usnea species of East Fennoscandia and the whole known Usnea flora of BritishColumbia were studied. Furthermore, the status and distribution of Usnea hirta and U. longissima,which have divergent habitat requirements and distribution patterns, were surveyed in EastFennoscandia. The two species also occur in British Columbia and their chemistry, ecology anddistribution were compared in the two study areas. The nomenclature and taxonomy of U. hirtawere also revised.

Nine shrubby Usnea species were documented from East Fennoscandia and 25 species andspecies groups were recorded from British Columbia (when U. fulvoreagens and U. pacificana areincluded as distinct species). U. chaetophora, U. diplotypus and U. nidulans s. lat. were reported asnew to North America, whereas U. esperantiana and U. rigida s. lat. were documented for the firsttime for Canada, and U. ceratina and U. rubicunda are new to British Columbia. U. pacificana wasdescribed as a new species from the Pacific Canada and the United States and U. wasmuthii wasreported from the states of Washington and Oregon as new to North America. Several taxa wererecognized as synonyms and lectotypified in our studies.

In total, 21 secondary medullary substances or compound groups were found in the EastFennoscandian and 24 in the British Columbia Usnea species. Salazinic acid is the most commonsubstance in both areas. New chemotypes were found in three shrubby Usnea species in EastFennoscandia and in six taxa in British Columbia. Differences in the chemistry of some specieswere found when comparing the East Fennoscandian and British Columbia specimens, e.g., in U.hirta and U. longissima.

Al l the East Fennoscandian Usnea species studied have a relatively southern distribution or theyare infrequent in northern regions, while most of the surveyed North American species have moreor less maritime distribution. All these Usneae are primarily epiphytes and the majority of themprefer well-li t and moist sites.

Keywords: chemistry, ecology, nomenclature, taxonomy

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Acknowledgements

My sincerest thanks are due to Teuvo Ahti (University of Helsinki) for his considerasupport and help during my studies, and to Philippe Clerc (Conservatoire et JaBotaniques, Geneva) who has been my teacher for this difficult genus. I am also grato Trevor Goward (Clearwater, B.C., Canada) for his invitation to study the wesCanadian material ofUsnea. I thank Juha Tuomi for his encouragement to write ththesis and for his constructive criticism. The staff members of the herbaria cited arethanked, and I am especially indebted to Orvo Vitikainen (Botanical Museum, Univerof Helsinki) for his kindest help. The official referees of my thesis, Tiina Randla(University of Tartu) and Soili Stenroos (University of Turku), are also acknowledgedare those many persons who have helped me in many ways during my studies and/owhom I have discussed the problems inUsnea; among them are Kristina Articus, LindaGeiser, Maria Angeles Herrera-Campos, Riikka Hälikkä, Bruce McCune and YoshiOhmura. Leena Myllys and Arto Puolasmaa placed their chemical analyses andbasic work on East FennoscandianUsnea at my disposal. Tuuli Timonen, MauriKorhonen and Helvi Mikkola are thanked for photographs and Sirkka-Liisa Peltonenthe map of the biogeographical provinces of East Fennoscandia. James Nimmo rethe English of the summary. I am grateful to Esteri Ohenoja, Henry Väre and TaUlvinen for my good working facilities in the Botanical Museum in Oulu. H. Väre halso been my supervisor. I want to express my warmest thanks to my parents and myVille, Eetu and Henri.

My work was financially supported by the Academy of Finland, through a grant toAhti, and by the Emil Aaltonen Foundation and by the University of Oulu.

Oulu, December 1999 Pekka Halonen

Abbrevations of herbaria

BM Natural History Museum, London

CANL National Herbarium of Canada, Ottawa

H Botanical Museum, University of Helsinki

H-ACH Herbarium Acharius, University of Helsinki

H-NYL Herbarium Nylander, University of Helsinki

KPABG Herbarium, Polar-Alpine Botanical Garden, Kirovsk

KUO Kuopio Museum of Natural History

LBL Herbarium, University of Maria Curie-Sklodowska, Lublin

OULU Botanical Museum, University of Oulu

S Swedish Museum of Natural History, University of Stockholm

TU Herbarium, University of Tartu

TUR Herbarium, University of Turku

TUR-V Herbarium Vainio, University of Turku

UBC Herbarium, University of British Columbia, Vancouver

UPS Botanical Museum, University of Uppsala

WIS Herbarium, University of Wisconsin, Madison

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List of original papers

This thesis is based on the following publications, which are referred to in the textheir Roman numerals:

I Halonen P & Puolasmaa A (1995) The lichen genusUsneain eastern FennoscandiaI. Usnea hirta. Ann Bot Fenn 32: 127–135.

II Halonen P (1997) The lichen genusUsnea in eastern Fennoscandia. II.Usnealongissima. Graphis Scripta 8: 51–56.

III Halonen P, Myllys L, Ahti T & Petrova OV (1999) The lichen genusUsneain EastFennoscandia. III. The shrubby species. Ann Bot Fenn 36: 235–256.

IV Halonen P, Clerc P, Goward T, Brodo IM & Wulff K (1998) Synopsis of the genuUsnea(lichenized Ascomycetes) in British Columbia, Canada. Bryologist 101: 360.

V Halonen P (2000)Usnea pacificana, sp. nov.andU. wasmuthii(Lichenized Asco-mycetes) in Pacific North America. Bryologist, in press.

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Contents

AbstractAcknowledgementsAbbrevations of herbariaList of original papers1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3. Secondary chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.4. Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3. Secondary chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.4. Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.5. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Original papers

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1. Introduction

1.1. Taxonomy

The genusUsneaAdans. (Parmeliaceae, lichenized Ascomycetes) is regarded as onthe taxonomically most difficult genera of macrolichens. Most of the specieswidespread and highly variable in morphology. Many species are also very variabchemistry, and may include several chemotypes. Numerous chemotypes which haveearlier regarded as distinct species have not recently been accorded any taxonomisince they are morphologically indistinguishable. For instance, Asahina (1956) divUsnea longissimaAch. into many ”chemical” taxa, but according to Brodo (1984), theshould not be recognized due to the absence of consistent morphological and geogcriteria to separate them.

For a long timeUsneaused to be placed in the family Usneaceae. Studies onapothecial ontogeny and ascus apical structures have indicated, however, that thecannot be recognized as distinct from Parmeliaceae.Usnea was divided into sixsubgenera by Motyka (1936–1938):ProtousneaMotyka, Neuropogon(Nees & Flot.)Motyka, Lethariella Motyka, Chlorea (Nyl.) Motyka, Eumitria (Stirt.) Motyka andEuusneaJatta. In particular the systematic status ofChloreaandLethariella is not clearand some taxonomists do not include them in the genusUsnea(e.g., Obermayer 1997).Only the subgenusEuusnea(i.e. now subgenusUsnea), which contains the distinctmajority of the species, has been found in East Fennoscandia, i.e. Finland andadjacent areas in Russia, and in British Columbia.

Motyka’s (1936–1938, 1947) world monograph of the genus contains 451 speciesnumerous infraspecific entities, but many of the taxa are now commonly regardeenvironmental modifications (see Clerc 1997) or their delimitation needs revisAccording to Räsänen (1951), the FinnishUsneaflora contains 34 species (plus the eighvarieties mentioned), while Hakulinen (1963) reduced the amount to 25 species and svarieties. My present opinion is that there are about only 12 species in Finland, bunumber is not exact since all the taxonomic problems have not yet been solved.

In Europe and North America the genusUsneahas been under thorough revision inrecent times (e.g., Myllys 1994, Clerc 1997, Clerc & Herrera-Campos 1997), but mwork is still required. Randlane’s (1992) survey of the Estonian Usneae mainly follow

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Motyka’s species concepts. Bystrek (1994) has continued the Motyka tradition anddescribed many new taxa, but both P. Clerc (pers. comm.) and I are convinced thatof his views are not acceptable. Carlin & Swahn (1977) studied theUsnea flora ofSweden, but many of their opinions about the taxonomy are not commonly acceTaxonomic studies onUsneahave also been performed, e.g., in East Africa (SwinscowKrog 1974, 1975, 1976, 1978, 1979) and Australia (Stevens 1999). Walker (19published a world-wide revision of the subgenusNeuropogon.

1.2. Morphology

The genusUsneais characterized by the fruticose habit and especially by the presenca cartilaginous central axis. According to Clerc (1987a,b, 1998), the pigmentation obase, the shape of main branches, the soralia, the brilliancy of the cortex and the ana(i.e. the inner structure) have a special importance in the taxonomy ofUsnea. On theother hand the thickness of branches and the abundance of the papillae and the fibriinstance, are largely controlled by environmental conditions.

All the boreal, northern circumpolar Usneae are secondary species which only raproduce apothecia and fertile primary species do not occur in East Fennoscandia. Prspecies, for example,U. florida (L.) F.H. Wigg. andU. rigida (Ach.) Motykas. lat., arefound in temperate areas of Europe and North America. Secondary species bedispersed almost solely by vegetative diaspores and thallus fragments, and thereformay have apomictic populations.

For detailed descriptions of the morphology, see papers III and IV.

1.3. Secondary chemistry

All Usneaspecies occurring in Europe and North America contain a yellow pigmeusnic acid, in the cortex (I–V). It is the only major pigment present in the EasFennoscandian Usneae, but some other pigments have been found in British Coluspecies (IV):Usnea rubicundaStirt. has a red pigment in the cortex,U. wirthii Clerccontains a red pigment in the cortex and a yellow pigment in the medulla and centralandU. ceratinaAch. produces a rose medullary pigment. The chemical structure of thpigments is not known.

The medulla has a complex chemistry (I–V). In addition to pigments, also present arvarious fatty acids (e.g., bourgeanic and caperatic acids, and the murolic acid comand terpenoids (e.g., zeorin). The majority of the secondary products, howeverclosely relatedβ-orcinol depsidones (e.g., norstictic, protocetraric, psoromic and salazacids) orβ-orcinol depsides (e.g., baeomycesic, barbatic, squamatic and thamnolic ac

15

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1.4. Ecology

All East Fennoscandian and British ColumbiaUsneaare primarily epiphytes, but mostspecies may also occasionally grow on decorticated wood or more rarely on roPrimarily saxicolousUsnea species occur, for instance, in arctic and antarctic are(subgenusNeuropogon), in the southern parts of North America (Clerc & HerreraCampos 1997) and in neotropical mountains. MostUsnea species are more or lesshygrophilous and photophilous, and therefore are found most abundantly in moistopen sites.

On the whole,Usnea species are highly sensitive to atmospheric pollution (e.Hawksworth & Rose 1970) and have become sparse or absent in urban and induareas.Usnea hirta(L.) F.H. Wigg., however, is relatively tolerant to pollutants (Kauppi &Halonen 1992), and occurs to some extent even in urban areas, whileU. longissimahasdrastically declined in Europe partly due to air pollution (Esseenet al.1981).

1.5. Distribution

Usneais a cosmopolitan genus occurring on all continents. Species diversity, howevelow in arid and arctic areas and is highest in humid regions of temperate latitudes.East Fennoscandian and British ColumbiaUsneafloras mostly consist of species that arwidely distributed in the Northern Hemisphere, but also contain some species thanearly cosmopolitan.

Among the East FennoscandianUsneaflora U. hirta andU. longissimarepresent theonly species which also range to the Southern Hemisphere.U. hirta has continentaltendencies, but it has been found on almost every continent (Clerc 1997). The spoccurs nearly in the whole Europe (Poelt 1969) and its North American range extfrom the boreal zone to Mexico (Thomson 1984).U. longissimahas oceanic tendencies(Ahti 1977) and occurs from boreal to tropical regions. The species is extinct or neextinct in most parts of Europe (Sérusiaux 1989). The richest European populations aScandinavia, especially in Norway (Tønsberget al. 1996).U. longissimais still locallycommon in North America occurring along the Pacific coast from northern CaliforniaAlaska, in the eastern parts of Canada and in the north-eastern United States (Brodoet al.2000). The species, however, has also declined in North America (Bennett 1995).

2. Material and methods

The studies were based primarily on herbarium material, including numerous typespecimens, deposited in BM, CANL, H (incl. H-ACH, H-NYL), KPABG , KUO, LBL,OULU (incl. herb. Oulanka), S (incl. S-Motyka), TU, TUR (incl. TUR-V), UBC, UPS,WIS, personal herbarium of Bruce McCune (Corvallis, Oregon), personal herbarium ofBruce Ryan (Tempe, Arizona) and the herbarium of the Siuslaw National Forest(Corvallis, Oregon). I have visited in H, LBL, S, TU, TUR and UPS. In addition toFinland, I have carried out field work in British Columbia, and in the Paanajärvi NationalPark and in the Kutsa Nature Reserve in Russia.

The thickness of the cortex, medulla and central axis were mainly measured by cuttinga short segment (usually 1 mm or less) of a branch, and the layers were measured alongtwo cross-sections from both ends of the segment. Measurements were made with astereomicroscope on the thickest part of the largest branch. For each measured specimen,the thickness is given as an average of all measurements. The ratio of the width of thecortex, the medulla and the central axis, can be given as a percent of the radius for thecortex and the medulla, and as a percent of the diameter for the axis (%C/%M/%A).Because of the sparse material of some taxa their inner structure was studied by themethod of Clerc (1984). In this method the branch is cut to the middle point, and thethickness of the layers is measured from a cross section. Chemistry was examined bymeans of the standardized thin-layer chromatography (TLC), described by Culbersonetal. (1981) and White & James (1985).

I am responsible for the morphology, taxonomy and nomenclature in the first paper,while A. Puolasmaa mostly conducted the herbarium survey and mapping. In the thirdpaper, I am responsible for the taxonomy and for the nomenclature together with T. Ahti,whereas L. Myllys has studied the morphology and the chemistry together with me, andO.V. Petrova has studied and collectedUsneaspecimens from the Murmansk Region. Inthe fourth paper, P. Clerc and I are primarily responsible for the taxonomy, while allauthors have contributed to the chemical studies and field observations.

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3. Results and discussion

3.1. Taxonomy

Nine shrubbyUsneaspecies were documented from East Fennoscandia (Table 1), btreatment of the pendent species, excludingUsnea longissima, is still under preparation.The whole knownUsnea flora of British Columbia was studied and 25 species [U.fulvoreagens(Räsänen) Räsänen included as a separate species] and species grouprecorded from the province (Table 1).U. chaetophoraStirt., U. diplotypusVain. andU.nidulansMotyka s. lat. were new to North America, whereasU. esperantianaClerc andU. rigida s. lat. were documented for the first time for Canada, andU. ceratinaandU.rubicundawere new to British Columbia.U. pacificanaHalonen was described as a newspecies from the Pacific Canada and the United States, andU. wasmuthiiRäsänen wasreported from the states of Washington and Oregon as new to North America (V).

Clerc (1987a) divided the shrubby FennoscandianUsneaspecies into six groups (Table2): U. diplotypusagg.,U. fragilescensagg.,U. glabrescensagg.,U. lapponicaagg.,U.subfloridanaagg. andU. hirta (single species). He did not includeU. glabrata (Ach.)Vain. in any of these aggregates, but in the study of the British ColumbiaUsneawereferred it to theU. fragilescensagg., which also containsU. cornutaKörb. s. lat., U.esperantiana, U. fragilescensLynge var.mollis (Vain.) Clerc andU. wirthii (IV). Thereare also many other different views on the systematics ofUsneapresented in our studieswhen compared to Clerc (1987a) (Table 2). For example, Clerc (1987a) regardeU.fulvoreagensas a member of the ”U. lapponicaagg.”, but we found it to represent a taxonclose to U. glabrescens(Nyl. ex Vain.) Vain. ex Räsänen (III, IV). We includedU.fulvoreagensand U. glabrescensin the U. florida agg. together withU. pacificana, U.subfloridanaStirt. andU. wasmuthii(III, V), while U. lapponicabelongs to theU. rigidaagg. with U. diplotypus, U. rigida s. lat., U. scabrataNyl. s. lat. and U. substerilisMotyka (III, IV). Usnea hirtaandU. longissimacannot be inluded in any of these threaggregates.

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Table 1. Main secondary medullary metabolities in the genus Usnea in East Fennoscaand Pacific North America. The North American data is mainly based on the BritColumbia material, except Usnea fulvoreagens and U. wasmuthii (see comments3 and 4).E-FEN = East Fennoscandia, P-AM = Pacific North America,1 = the occurrence in EastFennoscandia is not studied,2 = unpublished data,3 = the material is mainly from theUnited States,4 = all specimens are from the United States, * = the species was not foufrom East Fennoscandia, + = present in all specimens examined,± = present in somespecimens (chemoraces), a = accessory substance, not important for taxonomy andpresent in small amount to faint traces,S = present only in soralia,H = the strain may be ahybrid. ATR = atranorin, BAE = baeomycesic acid, BAR = barbatic a., CNSTIconnorstictic a. (seemingly overlooked in the studied material and it was excluded inCph-1 = convirensic a., Cph-2 = confumarprotocetraric a., CPSO = 2’-O-demethpsoromic a., CRSTI = cryptostictic a., CSTI = constictic a., DBAR = 4-O-demethbarbatic a., DIF = diffractaic a., EVE = evernic a., FAT = fatty acids (bourgeanic acid iU. esperantiana, the murolic acid complex in U. hirta and usually caperatic acid inlapponica), FUM = fumarprotocetraric a., MEN = menegazziaic a., NSTI = norstictic aPRO = protocetraric a., PSO = psoromic a., SAL = salazinic a., SQU = squamatic a., S= stictic a., THA = thamnolic a.

Area ATR EVE DIF BAR DBAR BAE SQU THA Cph-11. Usnea cavernosa E-FEN * * * * * * * * *

P-AM – – – – – – – – –2. Usnea ceratina E-FEN * * * * * * * * *

P-AM – – + + a – – – –3. Usnea chaetophora E-FEN1 * * * * * * * * *

P-AM – – – – – – – – –4. Usnea cornuta s. lat.E-FEN * * * * * * * * *

P-AM – – – – – – – – –5. Usnea diplotypus E-FEN – – – ± a – – – –

P-AM – – – ± a – – – –6. Usnea esperantianaE-FEN * * * * * * * * *

P-AM – – – – – – – – –7. Usnea filipendula s. E-FEN2 – – – – – – – – –

lat. P-AM – – – – – – – – –8. Usnea fragilescens E-FEN * * * * * * * * *

var. mollis P-AM a – – – – – ± – –9. Usnea fulvoreagens E-FEN – – ± – – – – – –

P-AM 2, 3 – – – – – – – – –10.Usnea glabrata E-FEN – – – – – – a – a

P-AM – – – ± a – – – a11.Usnea glabrescensE-FEN – – – – – – – – –

P-AM – – – – – – – – –12.Usnea hesperina E-FEN * * * * * * * * *

P-AM – – – – – – – – –13.Usnea hirta E-FEN – – – – – – – – –

P-AM – – ± – – – – – –14.Usnea lapponica E-FEN – – – – – – – – –

P-AM – – – ± a – – – –15.Usnea longissima E-FEN – – + – – – a – –

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P-AM a ± ± ± a – – – –16.Usnea madeirensisE-FEN * * * * * * * * *

P-AM – – – a a – – – –17.Usnea nidulans s. E-FEN * * * * * * * * *

lat. P-AM – – – – – – – – –18.Usnea pacificana E-FEN * * * * * * * * *

P-AM – – – a a + + – –19.Usnea rigida s. lat. E-FEN * * * * * * * * *

P-AM – – – – – – – – –20.Usnea rubicunda E-FEN * * * * * * * * *

P-AM – – – – – – – – –21.Usnea scabrata s. E-FEN 2 – – – – – – – – –

lat. P-AM – – – – – – – – –22.Usnea subfloridanaE-FEN – – – – – – ± ± –

P-AM – – – – – – ± ± –23.Usnea substerilis E-FEN – – – ± a – – – –

P-AM – – – ± a – – – –24.Usnea trichodea E-FEN * * * * * * * * *

P-AM – – + – – – – – –25.Usnea wasmuthii E-FEN – – – ± a – – – –

P-AM 4 – – – + a – – – –26.Usnea wirthii E-FEN * * * * * * * * *

P-AM – – – – – – – – –

Area Cph-2 FUM PRO NSTI CNSTI STI CSTI CRSTI MEN SAL PSO CPSO FAT1. E-FEN * * * * * * * * * * * * *

P-AM – – – – – – – – – ± – – –2. E-FEN * * * * * * * * * * * * *

P-AM – – – – – – – – – – – – –3. E-FEN * * * * * * * * * * * * *

P-AM – – – – – – – – – ± – – –4. E-FEN * * * * * * * * * * * * *

P-AM – – ± ± a ± a a a ± ± a a5. E-FEN – – a – – – – – – + – – a

P-AM – – – – – – – – – + – – –6. E-FEN * * * * * * * * * * * * *

P-AM – – – – – – a – – + – – +7. E-FEN2 – – a – – – – – – ± – – –

P-AM – – a – – – – – – + – – –8. E-FEN * * * * * * * * * * * * *

P-AM – – – ± a ± a a a ± ± a –9. E-FEN – – – ± a ± a a – ± – – –

P-AM 2, 3 – – – ± a ± a a a – ± a –10. E-FEN a a ± ± a – – – – ± – – a

P-AM a a ± – – – – – – – – – –11. E-FEN – – a ± a ± a a – ± – – –

P-AM – – – ± – ± a a a ± – – –12. E-FEN * * * * * * * * * * * * *

P-AM a – + – – – – – – – – – –

Area ATR EVE DIF BAR DBAR BAE SQU THA Cph-1

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Usnea glaucescensVain., U. pulvinata Motyka ex Räsänenand six infraspecificentities ofU. hirta were found to represent synonyms ofU. hirta (I). Clerc (1992, 1997)regardsU. foveataVain. to be conspecific withU. hirta, but we found that the type ofU.foveatacontains strongly modified thalli which belong to the pendent speciesU. barbata(L.) F.H. Wigg. s. lat. (i.e. U. scabrata s. lat.) (I). Many other Usnea taxa, mainlydescribed by J. Motyka (e.g., Motyka 1936–1938) and V. Räsänen (e.g., Räsänen 1were synonymized as the result of our studies (I, III, IV). For instance,U. stuppea(Räsänen) Motyka, which was described from British Columbia, turned out to bsynonym of U. substerilis (IV). As many as ten taxa were found to representU.glabrescensby us (III), includingU. extensaVain., which has been regarded as a distinspecies by some other taxonomists (e.g., Carlin & Swahn 1977).

13. E-FEN – – – ± – – – – – – – – ±P-AM – – – – – – – – – – – – ±

14. E-FEN – – a – – – – – – ± ± a ±P-AM – – – – – – – – – ± ± a –

15. E-FEN – – – – – – – – – – – – aP-AM – a – – – – a – – ± – – –

16. E-FEN * * * * * * * * * * * * *P-AM – – a – – – a – – + – – –

17. E-FEN * * * * * * * * * * * * *P-AM – – a ± a ± a a a ± ± a –

18. E-FEN * * * * * * * * * * * * *P-AM – – – – – – – – – – – – –

19. E-FEN * * * * * * * * * * * * *P-AM – – + – – – – – – – – – –

20. E-FEN * * * * * * * * * * * * *P-AM – – a + – a a – – + – – –

21. E-FEN2 – – a – – – – – – ± – – –P-AM – – a – – – – – – ± – – a

22. E-FEN – – – – – – – – – – – – –P-AM – – – – – – – – – – – – –

23. E-FEN – – a ±H – – – – – ± – – aP-AM – – – – – – – – – ± – – –

24. E-FEN * * * * * * * * * * * * *P-AM – – – – – – + – – + – – –

25. E-FEN – – a – – – – – – ± – – –P-AM 4 – – – – – – – – – ± – – –

26. E-FEN * * * * * * * * * * * * *P-AM – – – ±S a a a a a – ±S – a

Area Cph-2 FUM PRO NSTI CNSTI STI CSTI CRSTI MEN SAL PSO CPSO FAT

21

andia),a, butlercentlerc

s,iescific

Table 2. The systematic grouping of the shrubby Usnea species occurring in Fennoscby Clerc (1987a) and Halonen et al. (III, IV). 1 = not treated by Halonen et al. (III, IVsince the species has not been found in East Fennoscandia and Pacific North Americit is probably not a member of the Usnea fragilescens agg., 2 = not treated by C(1987a), 3 = a synonym of U. glabrescens (III), 4 = represents an unidentified pendspecies (III), ? = the systematic status of the species is not clearly indicated by C(1987a).

3.2. Morphology

The majority of the East FennoscandianUsneaspecies have a shrubby, divergent thalluwhich may become subpendent (III), while about half of the British Columbia specbecome pendent when mature (IV, V). This is due to the favourable climate on the Pacoast of British Columbia, since many pendentUsneaspecies have a maritime rangethere.

Species

Aggregates Clerc (1987a) Halonen et al. (III, IV)Usnea diplotypusagg. Usnea diplotypus

Usnea substerilis

Usnea fragilescensagg. Usnea cornuta Usnea cornuta

Usnea flammea1 Usnea fragilescens

Usnea fragilescens Usnea glabrata2

Usnea glabrescensagg. Usnea extensa3Usnea glabrescens

Usnea hirta Single species Single species

Usnea lapponicaagg. Usnea fulvoreagens

Usnea lapponica

Usnea mostruosa4

Usnea subfloridanaagg. Usnea florida

Usnea subfloridana

Usnea wasmuthii?

Usnea floridaagg. Usnea florida

Usnea fulvoreagens

Usnea glabrescens

Usnea subfloridana

Usnea wasmuthii

Usnea rigidaagg. Usnea diplotypus

Usnea lapponica

Usnea substerilis

22

dlosepecies

y,

bianlehere

oids)

reas,thermonrand

eratichile,lly in

).

hangical

own

hselt

Taxonomically close taxa such asU. cornuta s. lat./U. fragilescensvar. mollis, U.lapponica/U. substerilis and U. pacificana/U. subfloridana are morphologically mostdistinctly distinguished by their different soralia (III–V). The density of papillae anfibrils or the colour of the cortex, for instance, proved to be less useful in separating cspecies. The inner structure has a remarkable systematic significance, because the swithin theU. florida agg. and theU. rigida agg. are more or less similar in their anatomwhile U. glabrataandU. hirta differ in their anatomy from these two aggregates (III).

Some morphological differences were recognized between some British ColumUsneaand their counterparts in Europe. The most striking difference has been found iU.substerilis, which usually has abundant isidia on soralia in British Columbia (IV), whisoralia are only rarely abundantly isidiate in the East Fennoscandian material (III). Tis often a continuum of different morphotypes ofUsneaspecies, however, and it is noteasy to evaluate the role of genetic and environmental factors.

3.3. Secondary chemistry

In total, 21 secondary medullary substances or compound groups (including terpenwere found in the East Fennoscandian and 25 in the British ColumbiaUsneamaterials(Table 1). Salazinic acid constitutes the most common medullary substance in both aand norstictic and stictic acids are also relatively frequently present. The ocompounds of the stictic acid group and protocetraric acid, for instance, are less comand are often accessory. In most species,β-orcinol depsidones were solely present ooccurred more commonly than depsides. Fatty acids were found in ten speciesterpenoids were relatively common. Some fatty acids such as bourgeanic and capacids and the murolic acid complex have a significant diagnostic importance, wterpenoids were not taxonomically important in theUsneaspecies studied. Furthermoreseveral unknown substances occurred in the material studied, but they were usualow amounts and they had no significance in separating close species.

New chemotypes were found inUsnea glabrescens, U. substerilisandU. wasmuthiiinEast Fennoscandia (III), and inU. cornuta s. str., U. fragilescensvar. mollis, U. hirta, U.madeirensisMotyka, U. nidulans s. lat.and U. rigida s. lat. in British Columbia (IV).Protocetraric acid was not previously known as the main substance inU. rigida s. lat.andthis strain may represent a distinct, undescribed taxon (Goward & Halonen 1999U.pacificanawas included inU. subfloridanain our study of theUsneaof British Columbia(IV), although we predicted that the British Columbia material may represent more tone taxon. Further study revealed that because of its several distinctive morpholocharactersU. pacificana merits a species rank instead of being a chemotype ofU.subfloridana (V). The East Fennoscandian strain ofU. substerilis (one specimen)containing norstictic acid may be a hybrid (Table 1), since the compound was not knfrom the species of theU. rigida agg. before (III). Hybridization in lichens is stillincompletely understood (see Henssen & Jahns 1974, Brodo 1978, Clerc 1984, Fa1991).

23

strain

fareand

nd in

ot

lowtaic,ost-onedwithapanndiac, 4-oundtoo

amer less,andes isy of

ounds

hytic

from

are

olely

Several differences are found in the chemistry of manyUsneaspecies between BritishColumbia and East Fennoscandia (Table 1). For example, in East Fennoscandia, thewith fatty acids is clearly the most common chemotype ofU. hirta, and the speciesoccasionally produces norstictic acid. In British Columbia, the most common strain oU.hirta contains usnic acid alone, and fatty acids in the murolic acid complexinfrequently present. We also found one British Columbia specimen with usnicdiffractaic acids (IV). This chemotype was not previously known in the species.U. hirtamay also have some further strains in other areas; salazinic acid strain has been fouEast Africa (Clerc 1997). Isousnic acid (Kinoshitaet al. 1993) and atranorin (Yoshimuraet al. 1994) have been identified in culturedU. hirta, although these substances are nknown in natural specimens.

Usnea longissimahas a relatively complex chemistry. Different strains ofU.longissimacontain only usnic acid (probably also other substances are present in veryamounts) or more commonly with various combinations of depsides: evernic, diffracbarbatic, 4-O-demethylbarbatic acids and atranorin (Table 1). Evernic acid is the mcommon medullary substance in British Columbia, and diffractaic, barbatic and 4O-demethylbarbatic acids are also frequently present there (IV). We also foundspecimen with usnic, salazinic, barbatic, 4-O-demethylbarbatic acids and atranorin, anone specimen with usnic and a trace of fumarprotocetraric acids. Strainsfumarprotocetraric or salazinic acid and atranorin have been reported from J(Asahina 1967). However, diffractaic acid is the main compound in East Fennoscatogether with an unknown fatty acid or trace of squamatic acid, and evernic, barbatiO-demethylbarbatic, salazinic and fumarprotocetraric acids and atranorin are not fthere (II). The diffractaic acid strain is the most common chemotype in Norway,(Thøgersen & Høiland 1976).

Many Usneaspecies produce different chemotypes in different areas, but the sspecies may also have several strains in one locality. Chemotypes may be, more oartificial since some compounds may occasionally occur in very low amountstherefore are easily overlooked in TLC studies. The metabolism of lichen substancinsufficiently known and there are controversial opinions about the chemotaxonomlichens. According to Carlin (1986), the chemical composition of any givenUsneaspecies could depend on the metabolic status of the thallus, since many close compare different oxidation states of the same substance.

3.4. Ecology

Usneaspecies present in East Fennoscandia and British Columbia are primarily epip(I–V), but may occasionally occur also on decorticated wood (especiallyUsnea hirta) (I,III, IV). Most of the East Fennoscandian shrubby species have been collected alsorocks (III), but only one saxicolousUsneaspecimen (U. subfloridana) was found in theBritish Columbia material (IV). In East Fennoscandia,Betulaspp.,Picea abiesandAlnusspp. are the most common substrates (III), and in British Columbia most speciesequally common on conifers and deciduous trees (IV).U. rigida s. lat. represents aspecies which displays a distinct phorophyte preference, since it has been found s

24

arkorech

best

as

-arid

astrn andt

uental

d in

a of

he

t

land

orththen

ut

from deciduous trees in British Columbia (IV), whileU. hirta grows mainly on acid barksand lignum, also in East Fennoscandia (I, III, IV). The preference is partly due to bproperties, especially acidity, but climatic conditions have probably much msignificance. For instance,Pinus is the most common tree species in dry habitats whiare most suitable forU. hirta (I, III).

Species occurring in the moist old-growth forests of East Fennoscandia arerepresented byU. glabrescensand U. longissima. The latter has been collected fromPicea abiesand Betula (one site) in East Fennoscandia (II), and mainly fromPiceasitchensis, Tsuga heterophyllaandMalus fuscain British Columbia (IV).U. longissimaisprobably not distinctly host specific since in Norway it does not favour sprucesphorophytes, but it favours old, humid spruce forests (Tønsberget al. 1996).U. hirta hasa divergent ecology being the most xerophytic of theUsneaspecies studied (I, III, IV). Itgrows most frequently in open pine forests and is commonly present even in semiforests in British Columbia.

3.5. Distribution

All the local Usnea species have a more or less southern distribution in EFennoscandia, although most of the species are widespread occurring both in westeeastern regions (III). MerelyU. glabrataandU. longissimarepresent an eastern elemenin the area.U. hirta, U. lapponicaandU. subfloridanaare the only shrubbyUsneathathave also been found rarely from the northernmost regions. The front of the freqoccurrence of the genus roughly follows the northern limit of the Middle Borebioclimatic zone (see Ahtiet al.1968).

Most of the British Columbia species have a coastal range, i.e. they are distributehypermaritime and/or maritime regions (IV, V). The northernmost Pacific range ofU.ceratina, U. esperantiana, U. hesperinaMotyka andU. rubicundain North America is onthe hypermaritime south-west coast of Vancouver Island. Solely hypermaritime taxBritish Columbia are also represented byU. nidulans s. lat.and U. fragilescensvar.mollis. Other coastal species includeU. chaetophora, U. cornuta s. lat. (incl. U.occidentalisMotyka), U. diplotypus, U. longissima, U. madeirensis, U. pacificana, U.rigida s. lat.andU. wirthii , andU. wasmuthiiwas found in three maritime localities fromWashington and Oregon.U. filipendula Stirt. s. lat., U. glabrescens s. lat.(incl. U.fulvoreagens) and U. subfloridana have been collected from both the coast and tintermontane zone being more frequent in coastal areas.U. hirta, U. lapponica, U.trichodeaAch. (only one locality) andU. glabrata (rare in the maritime zone) representhe inland element.U. cavernosaTuck.,U. scabrata s. lat.andU. substerilisare the onlyspecies which occur throughout the entire province, but they are more common in inregions.

All the East Fennoscandian Usneae also occur along the Pacific coast of NAmerica, but the relatively low hygric oceanity of East Fennoscandia is indicated byabsence of hypermaritimeUsnea species. Many species have different distributiopatterns, however, when comparing their range in these two areas. For instance,U. hirtais one of the most commonUsneain East Fennoscandia and is distributed througho

25

n abia

t

ingnte isesate.

ality85,bitatsitesriod

. For

g”).

me

Finland (I, III). Thus it is surprising that the species has been found so far only irelatively small area in the southern parts of the intermontane regions in British Colum(IV). The coast of the province is too humid forU. hirta, but the species could be presenin northern intermontane areas and/or in the boreal zone.

Usnea longissimahas a wide range along the coast of British Columbia even belocally common in hypermaritime regions (IV), while it has in total only four knowlocalities in eastern Finland and the nearby areas of Russian Karelia (II). The climaprobably too dry toU. longissimain most parts of East Fennoscandia and the localitifound are situated in areas with relatively high precipitation and a humid local climThe species is regarded as extinct in Finland (Rassiet al. 1992), but it may be found, forinstance, in the Koillismaa province in similar sites such as the nearby Paanajärvi locin Russia.U. longissima, however, has an ineffective long-range dispersal (Esseen 19Gauslaa 1997) and the species often has a patchy occurrence, even in uniform ha(Tønsberget al. 1996). It is also probable that the four known East Fennoscandian sare relicts of a former more frequent occurrence during a warmer post-glacial pewhen the present westernmost areas of Finland were covered by water.

Our studies have also had importance in the classification of threatened speciesexample, based on our investigations (III),U. wasmuthiiwill be added to the list ofthreatened lichens of Finland (status “Nearly Threatened” and “in need of monitorinThe other East Fennoscandian Red ListUsneaspecies areU. barbata s. lat., U. glabrataand U. longissima (Kotiranta et. al. 1998). Only U. rigida s. lat. is reported as athreatenedUsneaspecies in British Columbia (Goward 1996, IV), but there are also soother threatened Usneae in the province, e.g.,U. ceratina and U. rubicundawhich arevery rare there.

4. Conclusions

The genusUsneais probably phylogenetically very old, judging from the fact that manyof the species, including all East Fennoscandian Usneae, have very wide globaldistribution and have adapted to different habitats and climates.Usnea substerilis, forinstance, has a slightly different morphological variation in East Fennoscandia comparedto the British Columbia material, which may partly be due to climatic factors. The speciesdiversity is distinctly higher in British Columbia than in East Fennoscandia, since most ofthe Usneaspecies occurring in these areas have more or less a southern distribution andfavour a hygric maritime climate.

In addition to the considerable morphological variation most of theUsneaspecies aremoderately to very variable in their chemistry, and some species have locally distributedchemotypes. However, without any significant morphologically distinguishablecharacters they cannot be regarded as separate taxa.

There still exist, however, some taxonomical problems which should be solved beforethe whole Usnea floras of East Fennoscandia and Pacific North America are totallyclarified. The most difficult and urgent problem, concerning also the classification ofthreatened species, is the taxonomy and systematics of the pendent speciesU. barbata s.lat., U. chaetophoraandU. filipendula s. lat. Usnea barbata s. lat.andU. filipendula s.lat. may contain many close species and at present we do not know whether these twospecies groups belong to different aggregates although they have a similar chemistry.Furthermore,U. chaetophoramay represent a modification ofU. barbata s. str.Amongthe shrubby species, the taxonomic status ofU. substerilisand the British Columbiamaterial ofU. nidulans s. lat.andU. rigida s. lat.should be cleared up in further studies.The former species could be regarded as a subspecies ofU. lapponicaand the two lattermay be distinct, undescribed taxa. Methods of molecular biology, together withmorphological and chemical investigations, might give considerable help in taxonomicalstudies on the genusUsnea.

gy.

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,

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.

:

5. References

Ahti T (1977) Lichens of the boreal coniferous zone. In: Seaward MRD (ed) Lichen ecoloAcademic Press, London, p 145–181.

Ahti T, Hämet-Ahti L & Jalas J (1968) Vegetation zones and their sections in northwestern EurAnn Bot Fenn 5: 169–211.

Asahina Y (1956) Lichens of Japan. Vol. III. GenusUsnea. Research Institute for Natural ResourcesShinjuku, Tokyo.

Asahina Y (1967) Lichenologische Notizen (§205). J Jap Bot 42: 289–294.Bennett JP (1995) Lichens. In: LaRow ET, Farris GS, Pucket CE, Doran PB & Mac MJ (eds)

Living Resources. National Biological Service, Washington, p 194–196.Brodo IM (1978) Changing concepts regarding chemical diversity in lichens. Lichenologist 10: 1Brodo IM (1984) Lichenes canadenses exsiccati: fascicle III. Bryologist 87: 97–111.Brodo IM, Sharnoff SD & Sharnoff S (2000) Lichens of North America. Yale University Press, N

Haven & London, in press.Bystrek J (1994) Studien über die FlechtengattungenUsneain Europe. Lublin.Carlin G (1986) The chemical strains ofUsnea lapponicaandU. glabrescensin Sweden. Graphis

Scripta 1: 29–32.Carlin G & Swahn U (1977) De svenskaUsnea-arterna. Svensk Bot Tidskr 71: 89–100.Clerc P (1984) Contribution à la revision de la systématique des Usnées (Ascomycotina,Usnea)

d'Europe. I.Usnea florida(L.) Wigg. emend. Clerc. Cryptog Bryol Lichénol 5: 333–360.Clerc P (1987a) Systematics of theUsnea fragilescensaggregate and its distribution in Scandinavia

Norw J Bot 7: 479–495.Clerc P (1987b) On the morphology of soralia in the genusUsnea. Bibl Lichenol 25: 99–102.Clerc P (1992) Some new or interesting species of the genusUsnea(lichenised Ascomycetes) in the

British Isles. Candollea 47: 513–526.Clerc P (1997) Notes on the genusUsneaDill. ex Adanson. Lichenologist 29: 209–215.Clerc P (1998) Species concepts in the genusUsnea(lichenized Ascomycetes). Lichenologist 30

321–340.Clerc P & Herrera-Campos MA (1997) Saxicolous species ofUsneasubgenusUsnea(lichenized

Ascomycetes) in North America. Bryologist 100: 281–301.Culberson CF, Culberson WL & Johnson A (1981) A standardized TLC analysis ofβ-orcinol

depsidones. Bryologist 84: 16–29.Esseen P-A (1985) Litter fall of epiphytic macrolichens in two oldPicea abiesforests in Sweden. Can

J Bot 63: 980–987.

28

ingB.C.

,ry of

in

me,

nn

ndiash

3)r

1,

nn

usstern

vien

28.

:

Esseen P-A, Ericson L, Lindström H & Zackrisson O (1981) Occurrence and ecology ofUsnealongissimain central Sweden. Lichenologist 13: 177–190.

Fahselt D (1991) Enzyme similarity as an indicator of evolutionary divergence:Stereocaulon saxatileH. Magn. Symbiosis 11: 119–130.

Gauslaa Y (1997) Population structure of the epiphytic lichenUsnea longissimain a borealPiceaabiescanopy. Lichenologist 29: 455–469.

Goward T (1996) Lichens of British Columbia: rare species and priorities for inventory. WorkPaper 08/1996. Research Branch, B.C. Ministry of Forests and Habitat Protection Branch,Ministry of Environment, Lands and Parks, Victoria.

Goward T & Halonen P (1999)UsneaAdans. In: Goward T The lichens of British Columbiaillustrated keys. Part 2, Fruticose Species. Special Report Series; 9. British Columbia MinistForests, Research Branch, Victoria, p 247–264.

Hakulinen R (1963) Jäkäläkasvio. Werner Söderström, Porvoo & Helsinki.Hawksworth DL & Rose F (1970) Qualitative scale for estimating sulfur dioxide air pollution

England and Wales using epiphytic lichens. Nature (London) 227: 145–148.Henssen A & Jahns HM (1974) Lichenes. Eine Einführung in die Flechtenkunde. Georg Thie

London.Kauppi M & Halonen P (1992) Lichens as indicators of air pollution in Oulu, northern Finland. A

Bot Fenn 29: 1–9.Kotiranta H, Uotila P, Sulkava S & Peltonen, S-L (eds) (1998) Red Data Book of East Fennosca.

Ministry of the Environment, Finnish Environment Institute & Botanical Museum, FinniMuseum of Natural History, Helsinki.

Kinoshita Y, Hayase S, Yamamoto Y, Yoshimura I, Kurokawa T, Ahti T & Yamada Y (199Morphogenetic capacity of the mycobiont inUsnea(lichenized Ascomycete). Proc Jap Acad, SeB(1), 69: 18–21.

Motyka J (1936–1938) Lichenum generisUsneastudium monographicum. Pars systematica. Vol2. Leopoli.

Motyka J (1947) Lichenum generisUsneastudium monographicum. Pars generalis. Leopoli.Myllys L (1994) Usnea glabrata(lichenized Ascomycotina) in East Fennoscandia. Acta Bot Fe

150: 125–130.Obermayer W (1997) Studies onLethariellawith special emphasis on the chemistry of the subgen

Chlorea(Lichenological results of the Sino-German Joint Expedition to south-eastern and eaTibet 1994. IV.). Bibl Lichenol 68: 45–66.

Poelt J (1969) Bestimmungsschlüssel europäischer Flechten. J. Cramer, Lehre.Randlane T (1992) Puuhabemed. Tartu.Rassi P, Kaipiainen H, Mannerkoski I & Ståhls G (eds) (1992) Uhanalaisten eläinten ja kas

seurantatoimikunnan mietintö. Komiteanmietintö 1991:30. Ympäristöministeriö, Helsinki.Räsänen V (1931) Die Flechten Estlands. Ann Acad Sci Fenn Ser A4, 34(4): 1–163.Räsänen V (1951) Suomen jäkäläkasvio. Kuopion Luonnon Yst Yhd Julk A5: 1–158.Sérusiaux E (1989) Liste rouge des macrolichens dans la Communauté Européenne. Liège.Stevens GN (1999) A revision of the lichen family Usneaceae in Australia. Bibl Lichenol 72: 1–1Swinscow TDV & Krog H (1974)UsneasubgenusEumitria in East Africa. Norw J Bot 21: 165–185.Swinscow TDV & Krog H (1975) TheUsnea undulataaggregate in East Africa. Lichenologist 7

121–138.Swinscow TDV & Krog H (1976) TheUsnea bornmuelleriaggregate in East Africa. Norw J Bot 23:

23–31.Swinscow TDV & Krog H (1978) Pendulous species ofUsneain East Africa. Norw J Bot 25: 221–

241.Swinscow TDV & Krog H (1979) The fruticose species ofUsneasubgenusUsneain East Africa.

Lichenologist 11: 207–252.

29

ess,

s of

n of

in

Thomson JW (1984) American Arctic Lichens. 1. The Macrolichens. Columbia University PrNew York.

Thøgersen P-J & Høiland K (1976) Chemical investigation ofUsnea longissimain Norway. Norw JBot 23: 115–116.

Tønsberg T, Gauslaa Y, Haugan R, Holien H & Timdal E (1996) The threatened macrolichenNorway - 1995. Sommerfeltia 23: 1–258.

Walker FJ (1995) The lichen genusUsneasubgenusNeuropogon. Bull Br Mus nat Hist (Bot) 13: 1–130.

White FJ & James PW (1985) A new guide to microchemical techniques for the identificatiolichen substances. Bull Br Lich Soc 57 (suppl.): 1–41.

Yoshimura I, Kurokawa T, Kinoshita Y, Yamamoto Y & Miyawaki H (1994) Lichen substancescultured lichens. J Hattori Bot Lab 76: 249–261.