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The Lichen Genera Anzia and Pannoparmelia in Australia

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The Lichen Genera Anzia and Pannoparmelia in Australia J. Hattori Bot. Lab. No. 74: 287-298 (Nov. 1993) THE LICHEN GENERA ANZIA AND PANNOPARMELIA IN AUSTRALIA 2 ISAO YOSHIMURA1 and JOHN A. Eux ABSTRACT. Two new species of Anzia, A. minor Yoshim. and A. tianjarana Yoshim. et Elix, and two species of Pannoparmelia, P.angustata (Pers. in Gaud.) Zahlbr. and P. wilsonii (Riis.) D. Gall. are recorded from Australia. A key to the species is given and their chemistry, distribution, ecology and taxonomy described. INTRODUCTION In a continuation of a worldwide study (by I. Y.) of the genera Anzia and Pannoparmelia (Yoshimura 1987) we undertook a review of the Australian represen­ tatives of these genera. We now report the discovery of two new species of Anzia from the continent, in addition to the well known Pannoparmelia angustata and P. wilsonii (Bratt, Blackman, and Cashin 1976; Galloway 1978). MATERIAL Material preserved in several herbaria (TNS, US, BM, ANUC, CANB, NICH) including recent collections (mostly by J. A. E.) of the genera Anzia and Pannopar­ me/ia were used for the present study. M ETHODS Cross- and longitudinal-sections of the material were obtained by means of a freezemicrotome (Reichert-Jung; Frigomobile OM and Freezing-microtome 1206). Sections (20-30µm thick) of thalli were stained with lactophenol cotton-blue. Asci were stained with Iodine solution. Sections were observed using a light-microscope and photographed. Methods for the thin-layer chromatography (TLC) of lichen substances essentially followed those of C. Culberson (1972) and Elix et al. (1987, 1988). Fragments of dried lichen specimens were extracted with acetone. Extracts were spotted on to Merck precoated glass plates (Kiesel Gel 60, no. 5721; 100 X200mm) and chromatograms were developed for 20min. at 25 °C in various solvent systems; i.e. benzene, dioxane, acetic acid (90: 25 : 4, v/v/v); hexane, ether, formic acid (5 : 4 : 1, v/v/v); toluene, acetic acid (85: 15, v/v). High-performance liquid chromatography (HPLC) was carried out with a JASCO IC-800 liquid chromatograph at 25 °C. Conditions were as follows: column, JASCO 1 Kochi Gakuen College, 292 Asahitenjinchi, Kochi, 780, Japan. 2 Department of Chemistry, The Faculties, Australian National University, Canberra, ACT 0200, Australia. 288 J. Hattori Bot. Lab. No. 74 I 9 9 3 Finepack SIL C18- 10P 4.6mm X 150mm; solvent system, methanol-water-phosphoric acid (80: 20: 0.9, v/v/v); detector, JASCO UV spectrophotometric detector (250 nm), and Shimazu photodiodearray spectrophotometer (range; 200-400nm, minimum width; 2 nm). Identification of lichen substances by TLC and HPLC was made by comparison with the standard lichen substances isolated by Dr. S. Huneck (protocetraric acid, hypoprotocetraric acid, chloroatranorin, atranorin, usnic acid, divaricatic acid, sekika­ ic acid, and lobaric acid), and Dr. K. Takahashi (sekikaic acid and divaricatic acid). Samples of several species of Anzia were used for comparisons with the substance indicated in parenthesis: A. japonica (anziaic acid), A. gregoriana (anziaic acid), and A. opuntiella ( divaricatic acid). Authentic 4-0-methylhypoprotocetraric acid was isolated from Xanthoparmelia notata (Kurok.) Hale (Cresp, Elix et al. 1972); anziaic acid, subnotatic acid, and nordivaricatic acid were identified by comparison with synthetic material (Djura & Sargent 1977; Elix 1974; Elix & Tearne 1977; Elix & Lajide 1984). RESULTS AND DISCUSSIONS Chemical Substances A diagram of some of the TLC chromatograms is presented in Figure 1, and the UV spectra of six substances are presented in Figures 2- 8. Using HPLC the fo ll owing three substances can be clearly distinguished from one another and identified by their UV spectra and their retention times: protocetraric acid (Fig. 2), hypoprotocetraric acid (Fig. 3), and 4-0-methylhypoprotocetraric acid (Fig. 4). Atranorin (Fig. 5) can be clearly distinguished from chloroatranorin (Fig. 6), although it cannot be separated in currently used solvents (TA = toluene: acetic acid, 85: 15, v/v; HEF = hexane, ether, formic acid, 5: 4 : 1, v/v/v). An unidentified substance (AT2) is the main chemical substance present in one of the chemical races of Anzia tianjarana. A T2 can be distinguished from anziaic acid ( C + red, UV spectrum: Fig. 8) by its color reaction (C-, KC + red), UV spectrum (Fig. 7), the retention time (Rt. 15 .59), and TLC. The main substance (Rt. 12.58; A max 215, 271, 302; A min 244, 298) present in Pannoparme/ia angustata and P. wilsonii was identified as divaricatic acid, while.nordivaricatic acid (Rt. 10.10; A max 215, 271, 304; Amin 242, 294) was identified by TLC and HPLC comparison with authentic material. Although all specimens of P. angustata and P. wilsonii were characteristicall y yellow­ green to dull green in colour, occasionally the usnic acid concentration was so low that it could not be detected chromatographically. THE SPECIES Anzia minor Yoshim., sp. nov. (Figures 9- 12) Thallus laciniatus, laciniis plus minus dichotome divisis, strato medullari axem chondroideum praedito, subtus spongiostrato moniliforme constricto. Species cum thallo ut inAnziajamesii sed ad hac specie thallo saxicolo, parviore, lobis angustioribus, exisidiatis, et acido 4-0-methylhypoprotocetrarico vel acido protocetrarico continente differt. I. YOSHIMURA & J. A. Eux: The lichen genera Anzia and Pannoparmelia in Australia 289 TA(85: J5) Rf. 0.9 0.8 atranorin + chloroatranorin 0.7 0.6 0.5 4-0-methylhypvprotocetraric acid •. ::.:: :.:;, isonotatic acid 0.4 - subnotatic acid 0.3 hypoprotocetraric acid CJ CU> ® 0.2 ., "' . 0.1 4-0-demethylnotatic acid 8 ~ protocetraric acid 0.0 "O <..> V") «: 0 "O <..> ~ '() .c: 00'° ..... «: «: c '°.._ V")°' ("'\ .... .c: 0 °''° <..> v 0 00 .c: <..> c .._ .._ °' t'd °' .._ .... c .... ·~ <:::> 0 c s: ~ .... "':1l . i: .§ .:::; .~ v C:l .s 0. 0 (') -~ ::::: ::: ::: ::: .9 c .... c 0 0. 0 :1l .... ;>., .... --.:: ~ ~ :a "' --.:: 0. ..c:: <..> -;; CD Fig. 1. TLC chromatograms of Anzia minor and comparisons with some authentic substances and some other species of Anzia. Solvents: TA = toluene, acetic acid (85 : 15, v/v). Type. Australia. New South Wales. Sandstone escarpment on the road 8 km east of Nerriga, elevation ea. 750m, Dec. 1, 1965, Syo Kurokawa 6405 (TNS-holotype, ANUC, NICH, US­ isotypes). Thall us foliose, saxicolous, loosely attached, orbicular to spreading, 2-6 cm wide. Lobes sublinear, more or less dichotomously branched, 0.4--0.5 (--0.9) mm wide, margins entire, apices rounded. Upper surface pale grey to bluish grey, becoming brown in the herbarium, smooth, matt or shining, occasionally distinctly maculate, often with tangential cracks in older lobes, lacking soredia and isidia. Medulla white. Lower surface white, shining, smooth, occasionally visible between the spongiostratum cushions and at the lobe tips. Spongiostratum moniliform, forming globose to ellipsoid 290 J. Hattori Bot. Lab. No. 74 I 9 9 3 750 protocetraric acid hypoprotocetraric acid 4-0-mdbylhypoprotocetraric acid m "'80 •oo ff: 4. 91 (• u ) lfl ·l. 46( .. n) 2 3 tT : ll.!6(1in) 4 636 2ZI "' atranorin "' cbloroatranorin unidentified substance (A T2) anziaic acid JO ... , m 2!9 tT : IS. St<1in) 7 Figs. 2- 8. UV spectra of 7 chemical substances found in Anzia minor and A. tianjarana. Fig. 2. Protocetraric acid. Fig. 3. Hypoprotocetraric acid. Fig. 4. 4- 0-Methylhypoprotocetraric acid. Fig. 5. Atranorin. Fig. 6. Chloroatranorin. Fig. 7. Unidentified substance (AT2). Fig. 8. Anziaic acid. cushions, up to 1.0 mm thick, brown-black to black. Rhizines scattered, simple, up to 1.2 mm long, with a tuft at the apex, born singly at the borders of the spongiostratum cushions. Upper cortex ea. 65 µm thick, arranged more or less vertical to the surface of the lobes (palisade); a chondroidal axis of parallel, longitudinally arranged hyphae present in the middle of the lobes, axis 220 X 350µm, originating from the apical meristematic portion of the lobes; medulla up to 50µm thick, composed of loosely interwoven hyphae, 7-IOµm thick; hyphae of spongiostratum derived from the apical meristematic portion of the lobes, 12- 17 µm wide; hyphae of spongiostratum pale brown to black-brown, composed of one row of lumina; rhizines derived from the apical meristematic portion of the lobes, connected to the chondoidal band. Apothecia common, 1.5- 2.0mm diam., pedicellate, weakly convex to more or less flat; disc dark brown to black; thalline exciple concolorous with the thallus and smooth on the upper part; cortex prosoplectenchymatous, ea. 200µm thick; lumina of cortical cells lµm wide, often branched to form net-like spongiostratum; hymenium ea. lOOµm high; paraphyses branched to form nets, septate; apices swollen; asci 55 X 20µm, multi­ spored; spores curved, worm-shaped, 12 X 2- 3 µm. Pycnidia globose, immersed in the I. YosJ-llM URA & J. A. Eux: The lichen genera Anzia and Pannoparmelia in Australia 291 Figs. 9- 12. Anzia minor Yoshim. Fig: 9. Thallus, dorsal view. Fig. 10. Thallus, ventral view. Fig. 11. Longitudinal section of the apical portion of a lobe. Fig. 12. Cross-section of a lobe. Figs. 9, 10. Scale = mm. Figs. 11, 12. Scale bar= 10 µm. lobes; ostiole black-brown, punctiform; conidia cylindrical, 4-5 X 0.5 µm. Chemistry. Chemical race 1: Cortex K + yellow; medulla K - , C - , KC - , P - . Containing atranorin, chloroatranorin, 4-0-methylhypoprotocetraric acid (major) and traces of isonotatic acid, subnotatic acid, hypoprotocetraric acid, 4-0-demethylnotatic acid (by TLC and HPLC). Chemical race 2: Cortex K + yellow; medulla K - , C - , KC - , P + orange-red. Containing atranorin, chloroatranorin, protocetraric acid (major). Specimens Examined: Chemical race 1. AUSTRALLA. New South Wales. On sandstone rocks in open Eucalyptus woodland, Morton National Park, 8 km north-east of Nerriga, 35 °07 'S, 150°08 'E, 750 m, J. A. Elix 5081, 31. X. 1978, J. A. Elix 9159, 18. X. 1981 , J. A. Elix 10171, I. V. 1982, J. A. Elix 11386 and J. Johnston, 6. X. 1983 (ANUC). Chemical race 2. AusTRALLA. New South Wales.
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