Carotenoids in Representatives of the Pseudocyphellaria Genus from South America

Home , Citranaxanthin, Dinoxanthin, Rhodoxanthin, Rubixanthin, Violaxanthin

J Hattori Bot. lab. No. 87: 277- 286 (Nov. 1999)

CAROTENOIDS IN REPRESENTATIVES OF THE PSEUDOCYPHELLARIA GENUS FROM SOUTH AMERICA

1 2 3 BAZYLI CZECZUGA , SUSANA CALVEL0 , LARS ARVIDSSON AND EWA CZECZUGA-SEME !UK 1

ABSTRACT. Column and thin-layer chromatography revealed the presence of the following carotenoids in the thalli of 20 species (39 specimens) of Pseudocyphellaria genus from various habi tats on the South-America: a-carotene, /3-carotene, /3-cryptoxanthin, lute in, 3 '-epilutein, zeaxanthin, lutein epoxide, antheraxanthin, echinenone, hydroxyechinenone, canthaxanthin, astaxanthin, celaxan thin, citranaxanthin, reticulataxanthin, violaxanthin, neoxanthin, dinoxanthin, heteroxanthin, cryptoftavin, mutatoxanthin, chrysanthemaxanthin, auroxanthin, aurochrome, 3,4,3 ',4' -bisdehydro /3-carotene, capsochrome, rhodoxanthin, /3-apo-2 ' -carotenal, /3-citraurin, apo-6'-lycopenal and azafrin. In the thalli of all the 20 species of the Pseudocyphel/aria genus /3-carotene and astaxanthin were found as constant carotenoids. The total content of carotenoids ranged from 16.4 (specimen no. 27 of Pseudocyphellaria faveo lata) to 95.9 µg g- 1 dry mass (specimen no. 8 of Pseudocyphellaria aurata).

INTRODUCTION Initially the lichens belonging to the genus Pseudocyphellaria genus were described as representatives of the Sticta genus (Muller Argoviensis 1879, Zahlbruckner 1905, Gal loway and James 1986). In the forties of the present century the Pseudocyphellaria genus was distinguished from Sticta genus (Magnusson 1940). A great contribution to the knowl edge of the morphological, anatomical and biochemical details of South-American and New Zealand species of Pseudocyphellaria was made by Galloway (1988). The lichen species belonging to Pseudocyphellaria genus occur mainly in the Southern Hemisphere, usually in a cool temperature region (Galloway 1985, 1986, 1989, 1992, 1993, 1994, 1997) though such species as Pseudocyphellaria clathrata or Pseudocyphellaria aurata are to be found in countris with a warm climate like Mexico (Imshang I 956a), the West Indies (lmshang l 956b) or Brazil (Vainio 1890). However, the most numerously represented species are found in Ecuador (Galloway and Arvidsson 1990), on the Galapagos Islands (Weber 1966, 1986) and Patagonia (Guzman et al. 1990, Calvelo 1992). Chemical studies carried out some time ago of thalli of some Pseudocyphel/aria species made it possible to determine a number of chemical compounds not previously noted in lichens (Huneck et al. 1973; Elix et al. 1987, 1990). Ecophysiological adaptations of the lichen genera Pseudo cyphellaria to various conditions egsamined by Snelgar and Green (I 981 ), Renuer and

1 Department of General Biology, Medical University, Kilinskiego I, PL-15-230 Bialystok 8, Poland. 2 Centro Regional Universitario Bariloche, CC-1336, Bariloche, Rio Negro, Argentina. 3 Botanical Museum, University ofGoteborg, Carl Skottsbergs Gata 22, S-413-19 Goteborg, Swe den. 278 J. Hattori Bot. Lab. o. 87 I 9 9 9

Galloway ( 1982), Lange et al. ( 1988), Demmig-Adams et al. ( l 990a, b ), Green and Lange (I 991) and Green et al. (1991 ). Preliminary studies of carotenoids in the thalli of Pseudo cyphellaria aurata and Pseudocyphellaria clathrata from Brazil (Czeczuga and Xavier Filho I 987) prompted us to undertake detailed studies of the carotenoid content in the thalli of different species from places where they are most abundant.

MATERIALS AND M ETHODS The 8 species (21 specimens) from Argentina and 12 species (I 8 specimens) from Ecuador were studied and the name, the site and habitat of these species are presented in Table I. The investigated species of lichens are deposited in the Botanical Museum of Uni versity Goteborg (LA) and in University ofBariloche (SC). Cleaned, air dried thalli were macerated with acetone under nitrogen in dark glass bot tles and the extracts kept in a refrigerator until analysed. Saponification was carried out with I 0% KOH in ethanol at 20°C for 24 ha in the dark under nitrogen. Column and TLC (Czeczuga I 980) were used to separate the carotenoids, which were identified by compari son with standard compounds (Hoffman-La Roche and Sigma Company) by a) the behav iour on column chromatography; b) their UV-VIS spectra (Beckman 2400); c) their parti tion between n-hexane and 95% ethanol; d) their Rf-values on TLC; e) the presence of al

lylic OH-group determined by the acid-CHCl2 test; f) the epoxide test; and g) the mass spectrum ( cf. Vetter et al. I 97 l ). Quantitative determinations were done by UV, VIS spec troscopy (Davies 1976). For the structures of carotenoids see Straub ( 1987).

RESULTS In 39 specimens of 20 species of the Pseudocyphellaria genus from the mountains of South America, the presence of 31 carotenoids was established (Table 2). These were mainly carotenoids commonly occurring in lichens. Celaxanthin, citranaxanthin, dinoxanthin, reticulataxanthin and 3,4,3 ',4' -bisdehydro-/3-carotene were new to lichens and only azafrin, chrysanthemaxanthin and het eroxanthin have to date been found only sporadically in lichens. The total content of carotenoids ranged from 16.4 (specimen no. 27 of Pseudocyphellaria fa veolata) to 95 .9 µ g g- 1 dry mass (specimen no. 8 of Pseudocyphellaria aurata) (Table 3).

DISCUSSION A new carotenoid, celaxanthin, a derivative of rubixanthin, also known as anhydros aproxanthin, was first isolated from red harries, Calastrus scandens (Weedon I 971 ). In the present study it was detected in the thalli of Pseudocyphel/aria hirsuta collected in the Lago Puelo National Park and Pseudocyphellaria subrubella in the Nahuel Huapi National Park in Argentina. Citranaxanthin and reticulataxanthin belong to apocarotenales. They were first detected in citrus fruit (Yokoyama and White I 965, Yokoyama et al. 1965). In lichen of the genus Pseudocyphellaria citranaxanthin was found in the thalli of Pseudo cyphel/aria corrifolia from the Nahuel Huapi ational Park, while reticulataxanthin in the thalli of Pseudocyphellaria granulata collected in the province of Rio Negro in Argentina. Dinoxanthin, a neoxanthin derivative (Goodwin I 980), is characteristic of algae of Pyrro phyta type (Jeffrey et al. 1975, Eugster 1979). In our study it was isolated from the thalli of B. CzECZUGA et al. : Carotenoids in representatives of the Pseudocyphe/laria genus from South America 279

Table I. Investigated species of Pseudocyphellaria genus.

No. Species Coll. No. Collected from Altitude (m)

I. P. arvidssonii D. J. Galloway LA-2070 Ecuador, Napo: Baeza 2000 2. P. arvidssonii D. J. Galloway LA-6711 Ecuador, Napo: road Quito-Baeza 2250 3. P. arvidssonii D. J. Galloway LA-1244 Ecuador, Loja: road Loja-Zamora 2500 4. P. arvidssonii D. J. Galloway LA-839 Ecuador, Pichincha: road Quito-Mindo 2850 5. P. aurata (Ach.) Vain . LA-4074 Ecuador, Tungurahua: Rio Pastaza Valley 1500 6. P. aurora (Ach.) Vai n. LA-6468 Ecuador, Tangurahua: Rio Pastaza Valley 1650 7. P. aurata {Ach.) Vai n. LA-3875 Ecuador, Napo: road Baeza-Teno 1800 8. P. aurata (Ach.) Vain. LA-6627 Ecuador, Napo: road Quito-Baeza 2700 9. P. bartlettii D. J. Gall oway LA-6060 Ecuador, Napo: Cordillera Oriental 3450 10. P. bartlettii D. J. Galloway LA-7150 Ecuador, Azuay: road to Molleturo 3600 11. P. berberiana (Foster) SC-882 Argentina, PNNH'>: on 850 D. J. Galloway et P James Sa:xegothaea conspicua 12. P. berberina (Foster) SC-883 Argentina, PNNH: on 850 D. J. Galloway et P James Sa:xegothaea conspicua 13. P. berberina (Foster) SC-890 Argentina, PNNH: on 850 D. J. Galloway et P James Sa:xegothaea conspicua 14. P. clathrata (L.) Vain . LA-2074 Ecuador, Mapo: Baeza, the village 2000 15. P. clathrata (L.) Vain. LA-6716 Ecuador, Napo: road Quito-Baeza 2250 16. P. corrifolia (Miill. Arg.) Malme SC-67 Argentina, PNNH : Cerro Catedral 1350 17. P. corrifolia (Miill. Arg.) Malme SC-861 Argentina, Prov. Rio Negro: 1000 on Nothofagus antarctica 18. P. corrifo/ia (Miill. Arg.) Malme SC-862 Argentina, Prov. Rio Negro: on Diosteajuncea !OOO 19. P. crocata (L.) Vain . LA-67 10 Ecuador, Napo: road Quto-Baeza 2250 20. P. crocata (L.) Vain. LA- 1243 Ecuador, Loja: road Loja-Zamora 2200 2 1. P. crocata (L.) Vain. LA-7152 Ecuador, Azuay: road to Molleturo 3600 22. P. crocata (L.) Vain . SC-4 Argentina, PNN H: on Nothofagus dombeyii 850 23 . P. dozyana (Mont. Bosch) LA-6465 Ecuador, Tungurahua: Rio Pastaza Valley 1650 D. J. Galloway 24. P. encoensis R. Sant. LA-6565 Ecuador, Chimborazo: Cordillera Oriental 3500 25 . P.faveo/ata (Delise) Malme SC-98 Argentina, TNNH: Lago Frias 850 2 26. P.faveolata (Del ise) Malme SC-866 Argentina, PNL l: on Nothofagus a/pina 1200 27. P.faveolata (Delise) Malme SC-906 Argentina, Prov. Neuquen, on Luma apiculata 110 28. P.flavicans (Hook. Tayloe) Vai n. SC-595 Argentina, PNLP3l : on Nothofagus dombeyii 960 29. P.jfavicans (Hook. Tayloe) Vain . SC-880 Argentina, Prov. Rio Negro, 950 on Nothofagus dombeyii 30. P. gilva (Ach.) Malme SC-301 Argentina, Prov. Rio Negro, between mosses 850 31. P. glabra (Hook. Tayor) Va in. SC-867 Argentina, PNL: on Nothofagus dombeyii 800 32. P. granulata (Chrch., Bab.) Malme SC-59 Argentina, Prov. Rio Negro, Arroyo Casa de Piedra 860 33. P. hirsuta (Mont.) Malme SC-93 Argentina, Prov. Rio Negro, Arroyo Casa de Piedra 810 34. P. hirsuta (Mont.) Malme SC-669 Argentina, PNLP: on rocks 950 35. P. hirsuta (Mont.) Malme SC-873 Argentina, Prov. Rio Negro: Villa Tacul 1000 36. P. intricata (Delise) Vain. LA-6880 Ecuador, Azuay: Paramo de Tinaj illas 3400 37. P. neglecta (Miill. Agr.) H. Magn SC-869 Argentina, PNL: on rocks 1100 38. P. s11brubella Rasanen SC-851 Argentina, PNNH: on Sa:xegothaea conspicua 1120 39. P. vaccinia (Mont.) Malme SC-851 Argentina, P H: Arroyo Goye 1250

I) Parque Nacional Nahuel Huapi 2) Parque Nacional Lanin 3) Parque acional Lago Puelo 280 J. Hattori Bot. Lab. No. 87 I 9 9 9

Table 2. Carotenoids found in species of Pseudocyphellaria genus.

Summary Structure No. Carotenoid Semisystematic name formula (see Fig. 1)

I. [3-carotene C40Hs6 B-r-B [3,[3-Carotene 2. a -carotene C40Hs6 A-r-B {3,t:-Carotene 3. [3-cryptoxanthin C40Hs60 B-r-D {3,/3-Caroten-3-ol 4. lutein C40Hs60 2 C-r-D {3,t:-Carotene-3,3 '-diol 5. 3'-epilutein C40Hs602 C-r-D {3,t:-Carotene-3,3 '-diol (stereisomeric) 6. zeaxanthin C40 Hs60 2 D-r-D [3,[3-Carotene-3 ,3-' diol 7. lutein epoxide C40Hs60 2 C-r-0 5,6-Epoxy-5,6-dihydro-[3,£-carotene-3,3 '-diol 8. antheraxanthin C40Hs60 3 D-r-0 5,6-Epoxy-5,6-dihydro-[3,[3-carotene-3,3 ' -diol 9. echinenone C40Hs40 B-r-M. {3,{3-Caroten-4-one 10. hydroxyechinenone C40Hs40 2 B-r-N 3-Hydroxy-/3,/3-caroten-4-one 11. canthaxanthin C40Hs20 2 M-r-M. [3,/3-Carotene-4,4 '-dione 12. astaxanthin C40H s20 4 N-r-N 3,3 '-Dihydroxy-[3,[3-carotene-4,4-dione 13 . celaxanthin C40Hs40 D-r-T 3' ,4'-Didehydro-[3,

17. neoxanthin C40Hs60 4 0-r1-H 5,6-Epoxy-6, 7-didebydro-5,6,5 ',6' -tetrahydro-{3,/3-carotene-3,5,3 '-trio!

18. dinoxanthin C42 HssOs I-r1-0 5' ,6'-Epoxy-6,7-didehydro-5,6,5 ' ,6'- tetrahydro-{3,/3-carotene-3 ,5 ,3 '-triol-3-acetate

19. heteroxanthin C40Hs60 4 F-r1-G 7' .8 ' -Didehydro-5,6-dihydro-[3,[3-carotene- 3,5,6,3 '-tetrol

20. cryptoflavin C40 Hs60 2 B-r1-R 5 ,8-Epoxy-5 ,8-dihydro-[3,[3-caroten-3-ol

21. mutatoxanthin C40Hs60 3 D-r1-R 5,8-Epoxy-5,8-dihydro-[3,[3-carotene-3,3 '-diol 22. chrysanthemaxanthin C40Hs60 3 C-r1-R 5,8-Epoxy-5,8-dihydro-{3,£-carotene-3,3 '-diol 23 . auroxanthin C40Hs604 R-r2-R 5,8,5' ,8' -Diepoxy-5,8,5 ' ,8' -tetrahydro-/3,/3- carotene-3,3 ' -diol 24. aurochrome C40Hs60 2 P-r2-P 5,8,5' ,8' -Diepoxy-5,8,5' ,8' -tetrahydro- [3,[3-carotene 25. 3,4,3' ,4' -bisdehydro- C40Hs20 K-r-K 3,4,3' ,4' -Tetrahydro-{3,/3-carotene />carotene

26. capsochrome C40Hs60 4 R-r1-S 5.8-Epoxy-3,3 '-dihydroxy-5,8-dibydro- [3,x-caroten-6' -one 27. rhodoxanthin C40Hso0 2 L-r3-L 4' ,5 '-Didehydro-4,5-retro-[3,/3-carotene- 3,3 '-dione

28. /3-apo-2' -carotenal C37H4gO B-r1-V 3 ',4 ' -Didehydro-2 '-apo-{3-caroten-2' -al

29. /3-citraurin C30H4002 D-r1-Y 3-Hydroxy-8' -apo-/3-caroten-8' -al 30. apo-6' -lycopenal C32H420 Y-r-U 6' -Apo-

Table 3. Carotenoid distribution in investigated species of Pseudocyphellaria genus.

Carotenoid Major carotenoids Total content No. Species (see Table 2 and Fig. I) (%) (µg g-1 dry weight)

l. P. arvidssonii D. J. Galloway I, 3, 7, 12, 16, 20, 26 20 (78.5) 94.4 2. P. arvidssonii D. J. Galloway 1 ,3,4,5,~8, 12, 16 7 (31.7) 29.6 3. P. arvidssonii D. J. Galloway I, 6, 7, 12 J2 (64.1) 36.7 4. P. arvidssonii D. J. Galloway I, 4, 5, 7, 8, 12, 20, 26 20 (84.6) 91.4 5. P. aurata (Ach.) Vain. J, 4, 7, 12, 16, 20 20 (87.7) 88.7 6. P. aurata (Ach.) Vain. 1 ,6,~ II, 12, 16,20 12 (80.5) 21.4 7. P. aurata (Ach.) Vain. I, 3, 4, 7, 8, 12, 20, 26 20 (57.2) 66.6 8. P. aurata (Ach.) Vain. I, 7, 12, 16, 20, 26 20 (78.7) 95 .9 9. P. bartlettii D. J. Galloway I, 3, 4, 7, 8, 12, 16, 26 7 (23.8) 38.3 JO. P. bartlettii D. J. Galloway I, 4, 7, 12, 20, 26 26 (34.7) 42.7 I l. P. berberiana (Foster) 1,4,6, 7, 12, 16, 17,26,27 26 (36.9) 31.3 D. J. Galloway et P. James 12. P. berberina (Foster) 1,3,4, 7, II, 12, 17 11 (31.9) 41.2 D. J. Galloway et P. James 13. P berberina (Foster) I, 3, 6, 8, 11, 12 12 (25 .8) 29.6 D. J. Galloway et P. James 14. P. c/athrata (L.) Vain. I, 3, 7, 11, 12, 16, 19, 20, 26 20 (73 .3) 92.0 15 . P. c/athrata (L.) Vain. I, 7, 11, 12, 26 7(41.2) 58.8 16. P. corrifolia (Miill. Arg.) Malme I, 3, 6, 10, 11, 12, 14, 16 12 (30.9) 20.5 J7. P. corrifo/ia (Miill. Arg.) Malme J, 3, 11 , J2, 2J, 26 JI (32.6) 24.4 18. P. corrifolia (Miill. Arg.) Malme I, 3, 8, 9, 12, 16, 17, 18, 26 26 (22.6) 29.9 19. P. crocata (L.) Vain. I, 3, 6, 7, 8, 12, 26 12 (41.\) 49.5 20. P. crocata (L.) Vain. 1,3, 6, 7, II, 12, 26 7 (39.0) 80.9 21. P. crocata (L.) Vain. I, 4, 6, 11, 12, 16, 26 7 (24.6) 72. 1 22. P. crocata (L.) Vain. I , 8, I I, 12, 16, 26 16 (31.6) 21.2 23 . P. dozyana (Mont. Bosch) I, 3, 4, 6, 7, 12, 16, 26 7 (33.0) 45.3 D. J. Galloway 24. P. encoensis R. Sant I, 7, 8, 11, 12, 16, 30 11 (33.5) 33.1 25 . P.faveolata (Delise) Malme 1, 3,8,9, II, 12, 17,26 26 (27. 1) 21.3 26. Pfaveo/ata (Delise) Malme 1,3,6, 10, 11, 12, 16, 17,26,27 26 (26.5) 27.1 27. P.faveo/ata (Delise) Malme I, 3, 11, 12, 17, 22, 28 12 (47.7) 16.4 28. Pjlavicans (Hook. Taylor) Vain. I, 3, 7, 10, 12, 17 12 (29.9) 19.9 29. P.jlavicans (Hook. Taylor) Vain. I, 3, 8, 11, 12, 24, 27 8 (22.8) 17.2 30. P. gilva (Ach.) Malme I, 2, 7, 11, 12, 16, 17 I (38.7) 18.6 31. P. g/abra (Hook. Taylor) Vain. I, 3, 6, 7, 12, 16, 20, 25 16 (32.7) 34.0 32. P. granulata (Chrch., Bab.) Malme I, 3, 7, 11, 12, 15, 16, 17, 26 7 (30.0) 20.5 33. P. hirsuta (Mont.) Malme 1,2, 7, 10, 12, 16, 17 7(50.1) 19.1 34. P. hirsuta (Mont.) Malme I, 3, 7, 11, 12, 13, 16, 17, 23 3 (41.9) 22.4 35. P. hirs.uta (Mont. ) Malme I, 3, 4, 7, 11, 12, 16, 17, 23 11 (32.5) 20.1 36. P. intricata (Delise) Vain. I, 4, 7, 8, 11, 12, 30, 31 7 (33.4) 23. I 37. P. neglecta (Miill. Arg.) H. Magn. I, 3, 7, 10, 11 , 12, 16 10(37.3) 33.6 38. P. subrubella Rasanen I, 3, 9, 12, 13, 16, 17, 26, 29 26 (30.7) 17.4 39. P. vaccinia (Mont.) Malme 1,3, 7, 8, II, 12, 16, 17 7 (32.9) 36.2 282 J. Hattori Bot. Lab. No. 87 I 9 9 9

Pseudocyphellaria corrifolia (No 18) collected in the province of Rio Negro in Argentina. 3,4,3 ',4' -bisdehydro-/3-carotene, the last new carotenoids found, also called retribisdehy dro-/3-carotene (Straub 1987), was first isolated from a fungus Epicoccum nigrum (Foppen 1971). In lichens it was found in thalli of Pseudocyphellaria glabra (No 31) collected from Nothofagus dombeyii in Argentina in the Lanin National Park. Considering rare carotenoids found in the material examined, azafrin was detected in thalli of Pseudocyphellaria intricata (No 36) from Ecuador. We have already found it in the thalli of Flavoparmelia caperata from Ohio in USA (Czeczuga et al. 1997c). Chrysan themaxanthin was detected in the thalli of Pseudocyphellaria faveolata (Nr 27) collected from Luma apiculata in the province of Neuque'n in Argentina. This carotenoid has al ready been encountered in the thalli of Xanthoria fa/lax collected at an altitude of 1646 m in the State of Mexico, USA (Czeczuga and Worthington 1997). Heteroxanthin was found in the thalli of Pseudocyphellaria clathrata (No 14) from Ecuador, collected at an altitude of 2000 m. It has been already found in the thalli of Parmotrema dilatatum and Usnea japonica from Ogasawara islands on the Pacific (Czeczuga et al. 1997a). In the thalli of all 20 lichen species of Pseudocyphellaria genus, 2 constant carotenoids occurred: /3-carotene and astaxanthin. Our previous studies also included some species of Pseudocyphellaria genus. These species were from such regions as New Zealand (Czeczuga and Taylor 1991), Tasmania (Czeczuga and Kantvilas 1990), Australia (Czeczuga et al. 1992a), Brazil (Czeczuga and Xavier-Filho 1987), South Africa (Czeczuga et al. 1988) and Central Africa (Kenya) (Czeczuga et al. 1992b). Most of the species from these regions were not the same as those Argentina and Ecuador. The only species from Ecuador studied that were the same as those in the some of the above regions were Pseudo cyphellaria aurata from the Amazonas and South Africa, Pseudocyphellaria clathrata from Brazil and Kenya and Pseudocyphellaria crocata from Kenya. The carotenoids found to be common to seven species from New Zealand were found to be /3-carotene, lutein, lutein epoxide and violaxanthin. On the other hand, four species from Tasmania had the following carotenoids in common: a-, /3-carotene, zeaxanthin and its derivative 5-hydroxy-5,6-dihydrozeaxanthin, lutein epoxide, violaxanthin and the pre cursors of astaxanthin-a-doradexanthin. The thalli of Pseudocyphellaria arvidssonii from four different places in Ecuador were found to have in common /3-carotene, lutein epoxide and astaxanthin, whereas those of the Pseudocyphellaria aurata thalli, also from four different places in Ecuador, were /3- carotene, lutein epoxide, astaxanthin and cryptoflavin. These carotenoids, with the excep tion of {3-carotene, occurred, in the thalli of the same species from Brazil. On the other hand, the thalli of Pseudocyphellaria aurata from South Africa contained such carotenoids as /3-cryptoxanthin, zeaxanthin, astaxanthin, lutein epoxide and mutatoxanthin. In the indi viduals from Brazil, the predominant carotenoid was auroxanthin and in those from South Africa - mutatoxanthin. The carotenoids common to the Pseudocyphellaria crocata from four different places in Ecuador and Argentina were /3-carotene, astaxanthin and capsochrome, whereas the Pseudocyphellaria faveolata thalli, from three different places in Argentina, had the fol lowing carotenoids in common: /3-carotene, /3-cryptoxanthin, canthaxanthin, astaxanthin B. CzECZUGA et al.: Carotenoids in representatives of the Pseudocyphe/laria genus from South America 283

A . s. c. D. E. F.

G. H. J. K. L. 0 .. ~ 0: HM HO

M. N. o. P. R.

s. T. u. V. 0 w. II p ;(CHO /(\ z. ;t\/COOH

Fig. 1. Structural features of carotenoids from investigated materials. 284 J. Hattori Bot. Lab. No. 87 I 9 9 9 and neoxanthin. In the thalli of the same species from the Amazonas 13-carotene, a-, 13- cryptoxanthin, neoxanthin, aurochrome, auroxanthin and mutatoxanthin. Auroxanthin oc curred in the largest amounts in the thalli, while the predominant carotenoid in this of this species in Kenya was found to be astaxanthin. Furthermore the thalli from Kenya contained two apocarotenals. In the individulas of Pseudocyphellaria crocata from USA the common carotenoid was 13-cryptoxanthin, lutein epoxide, astaxanthin and violaxanthin (Czeczuga et al. 1997c). In the thalli of Pseudocyphe/laria hirsuta collected from three pleaces in Ar gentina, the common carotenoids were 13-carotene, lutein epoxide, astaxanthin, violaxan thin and neoxanthin. The studies on carotenoids in lichen thalli of other genera, such as Cladina and Cladonia (Czeczuga 1985, Czeczuga and Doll 1991, Czeczuga et al. 1991 , Czeczuga & Hammer 1994 ), Hypotrachyna and Parmotrema (Czeczuga et al. 1997b), have shown that, like in Pseudocyphellaria, the li chen thalli of these genera growing in various environ ments have a larger number of different rather than common carotenoids. This indicates that environmental factors largely modify the carotenoid set of lichen species belonging to these genera, including the genus Pseudocyphellaria.

A CKNOWLEDGEM ENTS The authors would like to thank Professor M.R.D. Seaward for their penetrating re marks and offering valuable suggestions.

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