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Phycobionts Isolated from Japanese Species of Anzia (Lichenes)
Arch. Protistenkd. 143 (1993): 163-172 Fischer Verlag Jena
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© by Gustav
FOR
PROTISTEN KUNDE
Phycobionts Isolated from Japanese Species of Anzia (Lichenes) 1)
TAKA-AKI IHDA, TAKETO NAKANO, ISAO YOSHIMURA
& ZENNOSKE IWATSUKI
Botanical Institute, Faculty of Science, Hiroshima University, Hiroshima-ken, Japan K6chi Gakuen College, K6chi, Japan
Summary: Eighty-three specimens of six species of Anzia STIZ. (Lichenes) were collected from various areas in Japan. Phycobionts were isolated from their thalli and studied taxonomically: three species of unicellular green algae, Trebouxia gelatinosa AHMADJ. ex ARCHIBALD, T. potteri AHMADJ. ex GARTNER and T. showmanii (HILDR. et AHMADJ.) GARTNER were identified as the phycobionts of Anzia. This is the first report of phycobionts of Anzia. It seems that the lichenization in Anzia is not restricted to a specific algal partner.
Key Words: Lichenes; Anzia Phycobiont; Trebouxia Chlorophyceae.
Introduction
Materials and Methods
Anzia STIz. is a foliose lichen genus which mainly grows on tree bark. 23 species of Anzia have so far been reported, their distribution centres being eastern Asia and tropical America (YOSHIMURA 1987). In Japan, six species have been reported (YOSHIMuRA 1987): Anzia colpota VAIN., A. hypoleucoides MULL. ARG., A. japonica (TUCK) MULL. ARG., A. opuntiella MULL. ARG., A. ornata (ZAHLBR.) ASAH. and A. stenophylla ASAH. Studies of phycobionts of Anzia are practically none with only one suggestion that the phycobionts of Anzia might be Trebouxia (GALLOWAY 1985), and no taxonomical studies. In the present paper, we report some phycobionts isolated from 83 specimens of six species of Japanese Anzia.
Phycobionts were isolated from the following 83 specimens of six species of Anzia in Japan. Anzia colpota VAIN. (13 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 136 (cuI. no. TI 70.), 146 (TI 49), 147 (TI 65). Honshu, Mie-ken, Miyagawa-mura, Mt. Odaiga-hara, May 11, 1990: Ihda 187 (cuI. no. TI 100). Honshu, Hiroshima-ken, Togouchi-cho, Sandan-kyo, May 4, 1989: Ihda 1 (cuI. no. TI 1). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 203 (cuI. no. TI 121), 204 (TI 122), 213 (TI 133), 217 (TI 130), 219 (TI 129). Honshu, Yamaguchi-ken, Hongo-rnura, Mt. Jakuchi, May 4, 1990: Ihda 235 (cuI. no. TI 137), 236 (TI 136). Shikoku, Ehime-ken, Omogo-mura, Mt. Sasaga-mine, May 4, 1990: Matsumoto 202 (cuI. no. TI 132).
J) Contribution from the Phytotaxonomical and Geobotanical Laboratory, Hiroshima University, N. Ser. No. 44. 11*
A. hypoleucoides MULL. ARG. (9 specimens). Hokkaido, Yubari-shi, Mt. Yubari, July 15, 1989, Ihda 107 (cuI. no. TI 44).
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Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 82 (cuI. no. TI 37). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 208 (cuI. no. TI 114), 209 (TI 117), 210 (TI 115), 212 (TI 120), 214 (TI 118), 216 (TI 112), 221 (TI 116).
A. japonica (TUCK) MULL. ARG. (14 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 142 (cuI. no. TI 42-2), 148 (TI 50), 150 (TI 43). Hokkaido, Yubari-shi, Mt. Yfibari, July 15, 1989: Ihda 109 (cuI. no. TI 59), 129 (TI 47), 132 (TI 48). Hokkaido, Yubari-shi, Shupararo river, July 16, 1989: Ihda 134 (cuI. no. TI 56). Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 77 (cuI. no. TI 32), 79 (TI 34), 85 (TI 40). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 220 (cuI. no. TI 138), 222 (TI 124). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, May, 4, 1989: Ihda 19 (cuI. no. TI 18). Shikoku, Ehime-ken, Omogo-mura, Mt. Sasaga-mine, May 5, 1990: Matsumoto 249 (cul. no. TI 131). A. opuntiella MULL. ARG. (39 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 135 (cuI. no. TI 54), 138 (TI 58), 140 (TI 53), 152 (TI 51), 154 (TI 64), 156 (TI 62). Hokkaido, Yubari-shi, Mt. Yfibari, July 15, 1989: Ihda 108 (cuI. no. TI 67), 110 (TI 71), 112 (TI 63). Honshu, Mie-ken, Miyagawa-mura, Mt. Odaiga-hara, May 11, 1990: Ihda 183 (cuI. no. TI 91), 185 (TI 93), 186 (TI 94), 189 (TI 101), 190 (TI 97), 191 (TI 92), 192 (TI 96), 193 (TI 95). Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 78 (cuI. no. TI 33), 80 (TI 35), 81 (TI 36), 86 (TI 41). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 207 (cuI. no. TI 113), 211 (TI 125), 213 (TI 135), 218 (TI 126). Honshu, Hiroshima-ken, Togouchi-cho , Sandan-kyo, May 4, 1989: Ihda 2 (cuI. no. TI 2), 3 (TI 3); April 18, 1990: Matsumoto 172 (TI 109). Shikoku, Ehime-ken, Omogo-mura, Mt. Ishizuchi, August 18, 1990: Matsumoto 446 (cuI. no. TI 211). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, May 4, 1989: Ihda 11 (cuI. no. TI 10), 12 (TI 11), 13 (TI 12), 14 (TI 13), 15 (TI 14), 18 (TI 17); July 5, 1990: Matsumoto 304 (TI 156), 357 (TI 153); October 20, 1990: Matsumoto 534 (TI 219), 564 (TI 220).
A. ornata (ZAHLBR.) ASAH. (only one specimen). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, July 5, 1990: Matsumoto 316 (cuI. no. TI 188). A. stenophylla ASAH. (7 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 145 (cuI. no. TI 52), 151 (TI 45), 153 (TI 66). Hokkaido, Yfibari-shi, Mt. Yfibari, July 15, 1989: Ihda 106 (cuI. no. TI 46). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 205 (cuI. no. TI 128), 206 (TI 127).
Shikoku, Ehime-ken, Omogo-mura, Mt. Omogo, October 21, 1990: Matsumoto 567 (cuI. no. TI 222). Phycobionts were isolated by the method of NAKANO (1988). Axenic cultures of phycobionts were obtained by the micropipette method (AHMADJIAN 1967). They were cultured on IN BBM (modified by BISCHOFF & BOLD 1963) agar slants under the standard condition (20°C, 2,000 lux, 12 h lightll2 h dark). After about three weeks, observations of the life cycle and morphological features of algal material were made using a light microscope. The position and structure of the pyrenoid were confirmed by Azocarmin G solution, and dilute aqueous methylene blue solution was used to determine the presence of gelatinous matrices. The lichen specimes are deposited in the herbarium of Hiroshima University (HIRO), and unialgal or axenic cultures are deposited in the Botanical Institute, Faculty of Science, Hiroshima University (CCHU).
Results and Discussion Species of the genus Anzia are foliose (Fig. 1, a), and have a spongiostratum with uniseriate filaments, and asci with numerous and crescent-shape spores. The phycobiont forms an algal layer between the upper cortex and the medulla (Fig. 1, b). Vegetative cells of the phycobionts are spherical or subspherical in shape with irregular shaped chloroplasts in which one or a few pyrenoids are observed (Fig. 1, c). Chloroplasts are green in color, but detailed features are indistinct due to the influence of mycobiont hyphae. Autosporangia are rarely observed in some specimens, and zoosporangia and aplanosporangia have not been detected in this study. In log phase cultures of 83 strains of phycobionts, shape of cells and chloroplasts are distinctive, the algae belonging to only one genus, Trebouxia DE PUYMALY (Chlorophyceae). Therefore, these algae were compared with the original descriptions and figures of Trebouxia by ARCHIBALD (1975) and HILDRETH & AHMADJIAN (1981), and with the monographical studies of Trebouxia by GARTNER (1985). Moreover, we used original strains deposited in UTEX for the comparison. We identified these algae as T. gelatinosa AHMADJ. ex ARCHIBALD, T. potteri AHMADJ. ex GARTNER, and T. showmanii (HILDR. et AHMADJ.) GARTNER.
Trebouxia gelatinosa AHMADJ. ex ARCHIBALD, Phycologia 14: 128, fig. 5, 1975. (Fig. 2, 5) Colonies slightly rough, light-green in color. Vegetative cells solitary, spherical or slightly ellipsoidal in log phase of culture, 10-15 (-18) urn in diameter, not
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a
Fig. 1. Anzia colpota VAIN . a thallus. Scale 2 em. b algal layer. Scale = 50 urn. c vegetative cells of the phycobionts in thallus. Scale = 10 urn.
markedly enlarged in stationary phase, with walls about
0.5 urn thick, without gelatinous matrices. Cells uninucleate . Chloroplasts axial, with slightly lobed structure, with a distinctive pyrenoid in the central portion . Pyrenoids spherical, with sieve-like structure and minute fragmental starch sheath . Asexual reproduction by zoospores (16-128 in a spor angium) and aplano spores (16- 128). Zoospores ovoid , pyriform or linear, 2-4 urn wide , 4-8 urn long, without cell wall , with biflag ella of equal length. Stigma obscure . This species was isolated from the following six species (26 specimens) of Japanese Anzia as an algal partner. Anzia colpota : specimen no. Ihda I , 204, 219, 235. A. hypoleucoides : Ihda 212, 221. A. japonica: Ihda 129, 132, 134, 150. A. opuntiella: Ihda 3, II , 86, 110, 140, 154, 192: Matsumoto 172, 304, 446, 534, 564. A. ornata: Matsumoto 316. A . stenophylla : Ihda 205, 206: Matsumoto 567. This species was also isolated from Hypogymnia physodes (MEISCH 1981). ARCHIBALD (1975) and HILDRETH
& AHMADJIAN (1981) described that this species had gelatinous matrices , and that the pyrenoid of this species had a continuous starch sheath. However, GARTNER (1985) reported that cells of this species had no gelatinous matrice s, and pyrenoid had no starch sheath. Our result s support GARTNER'S observation on gelatinou s matrices, but we observed a pyrenoid with many minutel y fragmented starch sheaths . This species is newly found in Japan .
Trebouxia potteri AHMADJ. ex GARTNER , Arch . Hydrobiol . Suppl. 71 (Algological Studie s 41): 535, fig. 24, 1985. (Figs. 3, 6) Colonies rough , light-green in color. Vegetati ve cells solitary , spherical or slightly ellipsoidal in log phase of culture, 13-18 (- 22) urn in diameter, markedly enlarged in stationary phase (25 urn), with walls about 0.5 urn thick, without gelatinous matrices. Cells uninucleate. Chloroplasts axial , with deeply lobed structure ,
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a c
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d f
e
9 Fig. 2. Trebouxia gelatinosa
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AHMADJ. ex. ARCHIBALD. a young vegetative cell. b, d, e median optical section of mature vegetative cell. c surface views of the same cell of Fig. 2 b. f first division of protoplast. g zoosporangium. h aplanosporangium. i zoospore . Scale = IO urn.
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c
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Fig. 3. Trebouxia potteri
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AHMAD). ex GARTNER. a young vegetative cell with a pyrenoid. b young vegetative cell with two pyrenoids . c mature vegetative cell. d autosporangium. e ap1anosporangium. f first division of protoplast. g zoosporangium. h, i zoospores. Scale = 10 11m.
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c
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d
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f
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Fig. 4. Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER . a, b young vegetative cells. c median optical section of mature vegetative cell. d surface view of the same cell of Fig. 4c . e autosporangium . f zoosporangium . g aplanosporangium. h, i zoospores. Scale = 10 urn.
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a
d.
Fig. S. Trebouxia gelatinosa AHMADI. ex ARCHIBALD. a, b mature vegetative cells. c first division of protoplast. d, e zoosporangium. f aplanosporangium. Scale 10 urn ,
with distinctive one or more pyrenoids near the central portion. Pyrenoids spherical, with sieve-like structure and minutely fragmental starch sheaths. Asexual reproduction by zoospores (16-128 in sporangium), aplanospores (16-128) and autospores (4-8). Zoospores ovoidal, pyriform or linear, 3-5 urn wide, 4-7 urn long, without cell wall, with biflagella of equal length . Stigma obscure. This species was isolated from the following four species (29 specimens) of Japanese Anzia as an algal partner. Anzia colpota: specimen no. Ihda 136 , 187, 203, 217: Matsumoto 203. A. hypoleucoides : Ihda 107, 208, 210, 216. A. japonica : Ihda 85, 142, 148, 220. A. opuntiella: Ihda 14, 15, 78, 108, 112, 135, 138, 183, 189, 190, 191,193,207,211,218 : Matsumoto 357.
This species was also isolated from Pertusaria sp. and Rhizopla ca chrysoleuca (as Pseudotrebouxia potteri) by HILDRETH & AHMADJIAN (1981). In recent years , this species was isolated from 12 Japanese species of Pertusaria by TAKESHITA et al. (1989). ARCHIBALD (1975) and HILDRETH & AHMADJIAN (1981) described that this species had one pyrenoid with a continuous starch sheath. However, GARTNER (1985) described that this species had many naked pyrenoids. On the other hand , TAKESHITA et al. (1989) reported that this species had many pyrenoids with minutely fragmented starch sheaths. Our results support TAKESHITA et al. (1989).
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Fig. 6. Trebouxia potteri AHMADJ. ex GARTNER. a mature vegetative cell with a pyrenoid. b vegetative cells with a few pyrenoids. c division of protoplast. d zoosporangium. e aplanosporangium (centres). f autosporangium. Scale = lO[.lm.
Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER, Arch. Hydrobiol. Suppl. 71 (A1go1ogica1 Studies 41): 537, fig. 26, 1985. (Figs. 4, 7) Colonies rough, deep-green in color. Vegetative cells solitary, spherical or slightly ellipsoidal in log phase of culture, 10-15 (-18) urn in diameter, not markedly enlarged in stationary phase, with walls about 0.5 urn thick, without gelatinous matrices. Cells uninucleate. Chloroplasts axial, with deep-crinkly structure and a distinctive pyrenoid in central portion. Pyrenoids spherical, with sieve-like structure and minutely fragmented starch sheaths. Asexual reproduction by zoospores (16-128 in sporangium), aplanospores (16-128) and autospores (4-8). Zoospores ovoid, pyriform or linear, 2-5 urn wide, 4-9 urn long, without cell wall, with biflagella of equal length. Stigma obscure.
This species was isolated from the following five species (28 specimens) of Japanese Anzia as an algal partner. Anzia colpota: specimen no. Ihda 146, 147,213,236. A. hypoleucoides: Ihda 82,209,214. A. japonica: Ihda 19, 77, 79, 109,222:Matsumoto249.A. opuntiella:Ihda2, 12, 13, 18,80,81, 152, 156, 185, 186,213. A. stenophylla: Ihda 106, 145, 151, 153. This species was first described as Pseudotrebouxia showmanii by HILDRETH & AHMDJIAN (1981) as the phycobiont of Lecanora hagenii. Later, it was reported from five species of Diploschistes (FRIEDL & GARTNER 1988). HILDRETH & AHMADJIAN (1981) described that the pyrenoid of this species had a continuous starch sheath. On the other hand, GARTNER (1985) described that this species had a naked pyrenoid. However, we observed that this species has a pyrenoid with minutely fragmental starch sheaths. This species is newly found in Japan.
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Fig. 7. Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER. a, b mature vegetative cells. c aplanosporangium. d first division of protoplast. e zoosporangium. f autosporangia. Scale = 10 11m.
It is known that some species of lichens have the same algal species as phycobiont. For example, NAKANO (1988) reported that 26 species belonging to four genera of Japanese Graphidaceae had the same algal partner, Trentepohlia lagenifera . It was also reported that the mycobionts of 12 species of Japanese Pertusaria had only one species of alga, Trebouxia potteri (TAKESHITA et al. 1989). From their results, it is considered that lichenization of Japanese Graphidaceae and Pertusaria is restricted to a specific algal partner. On the other hand, it has been reported that lichenization of some lichen species is not restricted to a specific algal partner (FRIEDL & GARTNER 1988). They found that Diploschistes ocellatus and D. scruposus have two algal partners (Trebouxia gigantea and T. showmanii). In this study, we report that four species of Anzia (A. colpota, A. hypoleucoides, A. japonica and A. opuntiella) have
three species of Trebouxia (T. gelatinosa, T. potteri and T. showmanii) . We also report that Anzia stenophylla has two species of Trebouxia (T. potteri and T. showmanii) . Our results are further examples to show that lichenization is not restricted to a specific algal partner. The present paper show s that in 83 specimens of Anzia. 26 have Trebouxia gelatinosa, 29 have T. potteri , and 28 have T. showmanii as phycobionts . Thus, it seems that three species of Trebouxia are favoured physicobionts of
Anzia.
Acknowledgements We would like to express our sincere thanks to Dr. M. SEAWARD for correcting the English text. We also are grateful to Mr. T. MATSUMOTO for collecting Anzia specimens.
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References AHMADJIAN , V. (1967): A guide to the algae occurring as lichen symbionts: isolation, culture, cultural physiology , and identification. Phycologia 6: 127-160. ARCHIBALD, P . A. (1975) : Treboux ia DE PUYMA LY (Chlorophyceae , Chlorococcales) and Pseudotrebouxia gen . nov. (Chlorophyceae , Chlorosarcinales). Phycolog ia
14: 125-137. BISCHOFF, H. W . & BOLD , H. C. (1963): Phycological Studies IV . Some soil algae from Enchanted rock and related algal species . Univ . Te xas Pub!. 6318: 1-95. FRIEDL, T . & GARTNER, G . (1988) : Trebo uxia (Pleurastrales, Chlorophyta) as a ph ycobiont in the lichen genus Dipl oschistes. Arch . Protistenkd. 135 : 147-158. GALLOWAY , D . J . (1985): Flora of New Zealand. Lichens. Wellington , pp . 350. GARTNER , G . (1985): Die Gattung Trebouxia PUYMALY (Chlorellales, Chlorophyceae). Arch. Hydrobio!. , Supp!.
71: 495-548. HILDRETH , K. C. & AHMADJIAN , V. (1981): A study of
Trebo uxia and Pseudotrebouxia isol ates from different lichens . Lichenologist 13: 65-86. NAKANO, T . (1988) : Phycobionts of some Japanese species of Graphidaceae. Lichenologist 20: 353-360. MEISCH, J . P. (1981): Beitrage zur Isolation, Kultur und Systematik von Flechtenalgen. Diss . , Univ. Innsbruck, pp.
160. TAKESHITA, S. , NAKANO , T . & IWATSUKI, Z. (1989) : Phycobionts of some Japanese species of Pertusaria (Pertusariaccae), PI. Syst. Evo!. 165: 49-54. YOSHIMURA, I. (1987): Taxonomy and speciation of An zia and Pannoparmelia. In : Progress and Problems in Lichenology in the Eighties. Bibl. Licheno!. 25: 185-195 . Received: June 16, 1992 Authors' adresses: TAKA-AKI IHDA, TAKETO NAKANO , ZENNOSKE IWATSUKI , Botanical In stitute, Faculty of Science, Hiroshima University , Kag ami -yama , Higashi-hiroshima-shi, Hiroshima-ken 724, Japan ; ISAO YOSHIMURA, Kechi Gakuen College, Asahi-tenjin-cho 292, Kechi 780 , Japan .
ARCHIV
© by Gustav
FOR
PROTISTEN KUNDE
Phycobionts Isolated from Japanese Species of Anzia (Lichenes) 1)
TAKA-AKI IHDA, TAKETO NAKANO, ISAO YOSHIMURA
& ZENNOSKE IWATSUKI
Botanical Institute, Faculty of Science, Hiroshima University, Hiroshima-ken, Japan K6chi Gakuen College, K6chi, Japan
Summary: Eighty-three specimens of six species of Anzia STIZ. (Lichenes) were collected from various areas in Japan. Phycobionts were isolated from their thalli and studied taxonomically: three species of unicellular green algae, Trebouxia gelatinosa AHMADJ. ex ARCHIBALD, T. potteri AHMADJ. ex GARTNER and T. showmanii (HILDR. et AHMADJ.) GARTNER were identified as the phycobionts of Anzia. This is the first report of phycobionts of Anzia. It seems that the lichenization in Anzia is not restricted to a specific algal partner.
Key Words: Lichenes; Anzia Phycobiont; Trebouxia Chlorophyceae.
Introduction
Materials and Methods
Anzia STIz. is a foliose lichen genus which mainly grows on tree bark. 23 species of Anzia have so far been reported, their distribution centres being eastern Asia and tropical America (YOSHIMURA 1987). In Japan, six species have been reported (YOSHIMuRA 1987): Anzia colpota VAIN., A. hypoleucoides MULL. ARG., A. japonica (TUCK) MULL. ARG., A. opuntiella MULL. ARG., A. ornata (ZAHLBR.) ASAH. and A. stenophylla ASAH. Studies of phycobionts of Anzia are practically none with only one suggestion that the phycobionts of Anzia might be Trebouxia (GALLOWAY 1985), and no taxonomical studies. In the present paper, we report some phycobionts isolated from 83 specimens of six species of Japanese Anzia.
Phycobionts were isolated from the following 83 specimens of six species of Anzia in Japan. Anzia colpota VAIN. (13 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 136 (cuI. no. TI 70.), 146 (TI 49), 147 (TI 65). Honshu, Mie-ken, Miyagawa-mura, Mt. Odaiga-hara, May 11, 1990: Ihda 187 (cuI. no. TI 100). Honshu, Hiroshima-ken, Togouchi-cho, Sandan-kyo, May 4, 1989: Ihda 1 (cuI. no. TI 1). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 203 (cuI. no. TI 121), 204 (TI 122), 213 (TI 133), 217 (TI 130), 219 (TI 129). Honshu, Yamaguchi-ken, Hongo-rnura, Mt. Jakuchi, May 4, 1990: Ihda 235 (cuI. no. TI 137), 236 (TI 136). Shikoku, Ehime-ken, Omogo-mura, Mt. Sasaga-mine, May 4, 1990: Matsumoto 202 (cuI. no. TI 132).
J) Contribution from the Phytotaxonomical and Geobotanical Laboratory, Hiroshima University, N. Ser. No. 44. 11*
A. hypoleucoides MULL. ARG. (9 specimens). Hokkaido, Yubari-shi, Mt. Yubari, July 15, 1989, Ihda 107 (cuI. no. TI 44).
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Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 82 (cuI. no. TI 37). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 208 (cuI. no. TI 114), 209 (TI 117), 210 (TI 115), 212 (TI 120), 214 (TI 118), 216 (TI 112), 221 (TI 116).
A. japonica (TUCK) MULL. ARG. (14 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 142 (cuI. no. TI 42-2), 148 (TI 50), 150 (TI 43). Hokkaido, Yubari-shi, Mt. Yfibari, July 15, 1989: Ihda 109 (cuI. no. TI 59), 129 (TI 47), 132 (TI 48). Hokkaido, Yubari-shi, Shupararo river, July 16, 1989: Ihda 134 (cuI. no. TI 56). Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 77 (cuI. no. TI 32), 79 (TI 34), 85 (TI 40). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 220 (cuI. no. TI 138), 222 (TI 124). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, May, 4, 1989: Ihda 19 (cuI. no. TI 18). Shikoku, Ehime-ken, Omogo-mura, Mt. Sasaga-mine, May 5, 1990: Matsumoto 249 (cul. no. TI 131). A. opuntiella MULL. ARG. (39 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 135 (cuI. no. TI 54), 138 (TI 58), 140 (TI 53), 152 (TI 51), 154 (TI 64), 156 (TI 62). Hokkaido, Yubari-shi, Mt. Yfibari, July 15, 1989: Ihda 108 (cuI. no. TI 67), 110 (TI 71), 112 (TI 63). Honshu, Mie-ken, Miyagawa-mura, Mt. Odaiga-hara, May 11, 1990: Ihda 183 (cuI. no. TI 91), 185 (TI 93), 186 (TI 94), 189 (TI 101), 190 (TI 97), 191 (TI 92), 192 (TI 96), 193 (TI 95). Honshu, Hiroshima-ken, Togouchi-cho, Mt. Osorakan, June 11, 1989: Ihda 78 (cuI. no. TI 33), 80 (TI 35), 81 (TI 36), 86 (TI 41). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 207 (cuI. no. TI 113), 211 (TI 125), 213 (TI 135), 218 (TI 126). Honshu, Hiroshima-ken, Togouchi-cho , Sandan-kyo, May 4, 1989: Ihda 2 (cuI. no. TI 2), 3 (TI 3); April 18, 1990: Matsumoto 172 (TI 109). Shikoku, Ehime-ken, Omogo-mura, Mt. Ishizuchi, August 18, 1990: Matsumoto 446 (cuI. no. TI 211). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, May 4, 1989: Ihda 11 (cuI. no. TI 10), 12 (TI 11), 13 (TI 12), 14 (TI 13), 15 (TI 14), 18 (TI 17); July 5, 1990: Matsumoto 304 (TI 156), 357 (TI 153); October 20, 1990: Matsumoto 534 (TI 219), 564 (TI 220).
A. ornata (ZAHLBR.) ASAH. (only one specimen). Shikoku, Ehime-ken, Omogo-mura, Omogo-kei, July 5, 1990: Matsumoto 316 (cuI. no. TI 188). A. stenophylla ASAH. (7 specimens). Hokkaido, Hurano-shi, Mt. Ashibetsu, July 18, 1989: Ihda 145 (cuI. no. TI 52), 151 (TI 45), 153 (TI 66). Hokkaido, Yfibari-shi, Mt. Yfibari, July 15, 1989: Ihda 106 (cuI. no. TI 46). Honshu, Hiroshima-ken, Tsutsuga-mura, Mt. Garyu, April 20, 1990: Ihda 205 (cuI. no. TI 128), 206 (TI 127).
Shikoku, Ehime-ken, Omogo-mura, Mt. Omogo, October 21, 1990: Matsumoto 567 (cuI. no. TI 222). Phycobionts were isolated by the method of NAKANO (1988). Axenic cultures of phycobionts were obtained by the micropipette method (AHMADJIAN 1967). They were cultured on IN BBM (modified by BISCHOFF & BOLD 1963) agar slants under the standard condition (20°C, 2,000 lux, 12 h lightll2 h dark). After about three weeks, observations of the life cycle and morphological features of algal material were made using a light microscope. The position and structure of the pyrenoid were confirmed by Azocarmin G solution, and dilute aqueous methylene blue solution was used to determine the presence of gelatinous matrices. The lichen specimes are deposited in the herbarium of Hiroshima University (HIRO), and unialgal or axenic cultures are deposited in the Botanical Institute, Faculty of Science, Hiroshima University (CCHU).
Results and Discussion Species of the genus Anzia are foliose (Fig. 1, a), and have a spongiostratum with uniseriate filaments, and asci with numerous and crescent-shape spores. The phycobiont forms an algal layer between the upper cortex and the medulla (Fig. 1, b). Vegetative cells of the phycobionts are spherical or subspherical in shape with irregular shaped chloroplasts in which one or a few pyrenoids are observed (Fig. 1, c). Chloroplasts are green in color, but detailed features are indistinct due to the influence of mycobiont hyphae. Autosporangia are rarely observed in some specimens, and zoosporangia and aplanosporangia have not been detected in this study. In log phase cultures of 83 strains of phycobionts, shape of cells and chloroplasts are distinctive, the algae belonging to only one genus, Trebouxia DE PUYMALY (Chlorophyceae). Therefore, these algae were compared with the original descriptions and figures of Trebouxia by ARCHIBALD (1975) and HILDRETH & AHMADJIAN (1981), and with the monographical studies of Trebouxia by GARTNER (1985). Moreover, we used original strains deposited in UTEX for the comparison. We identified these algae as T. gelatinosa AHMADJ. ex ARCHIBALD, T. potteri AHMADJ. ex GARTNER, and T. showmanii (HILDR. et AHMADJ.) GARTNER.
Trebouxia gelatinosa AHMADJ. ex ARCHIBALD, Phycologia 14: 128, fig. 5, 1975. (Fig. 2, 5) Colonies slightly rough, light-green in color. Vegetative cells solitary, spherical or slightly ellipsoidal in log phase of culture, 10-15 (-18) urn in diameter, not
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Fig. 1. Anzia colpota VAIN . a thallus. Scale 2 em. b algal layer. Scale = 50 urn. c vegetative cells of the phycobionts in thallus. Scale = 10 urn.
markedly enlarged in stationary phase, with walls about
0.5 urn thick, without gelatinous matrices. Cells uninucleate . Chloroplasts axial, with slightly lobed structure, with a distinctive pyrenoid in the central portion . Pyrenoids spherical, with sieve-like structure and minute fragmental starch sheath . Asexual reproduction by zoospores (16-128 in a spor angium) and aplano spores (16- 128). Zoospores ovoid , pyriform or linear, 2-4 urn wide , 4-8 urn long, without cell wall , with biflag ella of equal length. Stigma obscure . This species was isolated from the following six species (26 specimens) of Japanese Anzia as an algal partner. Anzia colpota : specimen no. Ihda I , 204, 219, 235. A. hypoleucoides : Ihda 212, 221. A. japonica: Ihda 129, 132, 134, 150. A. opuntiella: Ihda 3, II , 86, 110, 140, 154, 192: Matsumoto 172, 304, 446, 534, 564. A. ornata: Matsumoto 316. A . stenophylla : Ihda 205, 206: Matsumoto 567. This species was also isolated from Hypogymnia physodes (MEISCH 1981). ARCHIBALD (1975) and HILDRETH
& AHMADJIAN (1981) described that this species had gelatinous matrices , and that the pyrenoid of this species had a continuous starch sheath. However, GARTNER (1985) reported that cells of this species had no gelatinous matrice s, and pyrenoid had no starch sheath. Our result s support GARTNER'S observation on gelatinou s matrices, but we observed a pyrenoid with many minutel y fragmented starch sheaths . This species is newly found in Japan .
Trebouxia potteri AHMADJ. ex GARTNER , Arch . Hydrobiol . Suppl. 71 (Algological Studie s 41): 535, fig. 24, 1985. (Figs. 3, 6) Colonies rough , light-green in color. Vegetati ve cells solitary , spherical or slightly ellipsoidal in log phase of culture, 13-18 (- 22) urn in diameter, markedly enlarged in stationary phase (25 urn), with walls about 0.5 urn thick, without gelatinous matrices. Cells uninucleate. Chloroplasts axial , with deeply lobed structure ,
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d f
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9 Fig. 2. Trebouxia gelatinosa
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AHMADJ. ex. ARCHIBALD. a young vegetative cell. b, d, e median optical section of mature vegetative cell. c surface views of the same cell of Fig. 2 b. f first division of protoplast. g zoosporangium. h aplanosporangium. i zoospore . Scale = IO urn.
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c
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Fig. 3. Trebouxia potteri
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AHMAD). ex GARTNER. a young vegetative cell with a pyrenoid. b young vegetative cell with two pyrenoids . c mature vegetative cell. d autosporangium. e ap1anosporangium. f first division of protoplast. g zoosporangium. h, i zoospores. Scale = 10 11m.
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Fig. 4. Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER . a, b young vegetative cells. c median optical section of mature vegetative cell. d surface view of the same cell of Fig. 4c . e autosporangium . f zoosporangium . g aplanosporangium. h, i zoospores. Scale = 10 urn.
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a
d.
Fig. S. Trebouxia gelatinosa AHMADI. ex ARCHIBALD. a, b mature vegetative cells. c first division of protoplast. d, e zoosporangium. f aplanosporangium. Scale 10 urn ,
with distinctive one or more pyrenoids near the central portion. Pyrenoids spherical, with sieve-like structure and minutely fragmental starch sheaths. Asexual reproduction by zoospores (16-128 in sporangium), aplanospores (16-128) and autospores (4-8). Zoospores ovoidal, pyriform or linear, 3-5 urn wide, 4-7 urn long, without cell wall, with biflagella of equal length . Stigma obscure. This species was isolated from the following four species (29 specimens) of Japanese Anzia as an algal partner. Anzia colpota: specimen no. Ihda 136 , 187, 203, 217: Matsumoto 203. A. hypoleucoides : Ihda 107, 208, 210, 216. A. japonica : Ihda 85, 142, 148, 220. A. opuntiella: Ihda 14, 15, 78, 108, 112, 135, 138, 183, 189, 190, 191,193,207,211,218 : Matsumoto 357.
This species was also isolated from Pertusaria sp. and Rhizopla ca chrysoleuca (as Pseudotrebouxia potteri) by HILDRETH & AHMADJIAN (1981). In recent years , this species was isolated from 12 Japanese species of Pertusaria by TAKESHITA et al. (1989). ARCHIBALD (1975) and HILDRETH & AHMADJIAN (1981) described that this species had one pyrenoid with a continuous starch sheath. However, GARTNER (1985) described that this species had many naked pyrenoids. On the other hand , TAKESHITA et al. (1989) reported that this species had many pyrenoids with minutely fragmented starch sheaths. Our results support TAKESHITA et al. (1989).
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Fig. 6. Trebouxia potteri AHMADJ. ex GARTNER. a mature vegetative cell with a pyrenoid. b vegetative cells with a few pyrenoids. c division of protoplast. d zoosporangium. e aplanosporangium (centres). f autosporangium. Scale = lO[.lm.
Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER, Arch. Hydrobiol. Suppl. 71 (A1go1ogica1 Studies 41): 537, fig. 26, 1985. (Figs. 4, 7) Colonies rough, deep-green in color. Vegetative cells solitary, spherical or slightly ellipsoidal in log phase of culture, 10-15 (-18) urn in diameter, not markedly enlarged in stationary phase, with walls about 0.5 urn thick, without gelatinous matrices. Cells uninucleate. Chloroplasts axial, with deep-crinkly structure and a distinctive pyrenoid in central portion. Pyrenoids spherical, with sieve-like structure and minutely fragmented starch sheaths. Asexual reproduction by zoospores (16-128 in sporangium), aplanospores (16-128) and autospores (4-8). Zoospores ovoid, pyriform or linear, 2-5 urn wide, 4-9 urn long, without cell wall, with biflagella of equal length. Stigma obscure.
This species was isolated from the following five species (28 specimens) of Japanese Anzia as an algal partner. Anzia colpota: specimen no. Ihda 146, 147,213,236. A. hypoleucoides: Ihda 82,209,214. A. japonica: Ihda 19, 77, 79, 109,222:Matsumoto249.A. opuntiella:Ihda2, 12, 13, 18,80,81, 152, 156, 185, 186,213. A. stenophylla: Ihda 106, 145, 151, 153. This species was first described as Pseudotrebouxia showmanii by HILDRETH & AHMDJIAN (1981) as the phycobiont of Lecanora hagenii. Later, it was reported from five species of Diploschistes (FRIEDL & GARTNER 1988). HILDRETH & AHMADJIAN (1981) described that the pyrenoid of this species had a continuous starch sheath. On the other hand, GARTNER (1985) described that this species had a naked pyrenoid. However, we observed that this species has a pyrenoid with minutely fragmental starch sheaths. This species is newly found in Japan.
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Fig. 7. Trebouxia showmanii (HILDR. et AHMADJ.) GARTNER. a, b mature vegetative cells. c aplanosporangium. d first division of protoplast. e zoosporangium. f autosporangia. Scale = 10 11m.
It is known that some species of lichens have the same algal species as phycobiont. For example, NAKANO (1988) reported that 26 species belonging to four genera of Japanese Graphidaceae had the same algal partner, Trentepohlia lagenifera . It was also reported that the mycobionts of 12 species of Japanese Pertusaria had only one species of alga, Trebouxia potteri (TAKESHITA et al. 1989). From their results, it is considered that lichenization of Japanese Graphidaceae and Pertusaria is restricted to a specific algal partner. On the other hand, it has been reported that lichenization of some lichen species is not restricted to a specific algal partner (FRIEDL & GARTNER 1988). They found that Diploschistes ocellatus and D. scruposus have two algal partners (Trebouxia gigantea and T. showmanii). In this study, we report that four species of Anzia (A. colpota, A. hypoleucoides, A. japonica and A. opuntiella) have
three species of Trebouxia (T. gelatinosa, T. potteri and T. showmanii) . We also report that Anzia stenophylla has two species of Trebouxia (T. potteri and T. showmanii) . Our results are further examples to show that lichenization is not restricted to a specific algal partner. The present paper show s that in 83 specimens of Anzia. 26 have Trebouxia gelatinosa, 29 have T. potteri , and 28 have T. showmanii as phycobionts . Thus, it seems that three species of Trebouxia are favoured physicobionts of
Anzia.
Acknowledgements We would like to express our sincere thanks to Dr. M. SEAWARD for correcting the English text. We also are grateful to Mr. T. MATSUMOTO for collecting Anzia specimens.
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References AHMADJIAN , V. (1967): A guide to the algae occurring as lichen symbionts: isolation, culture, cultural physiology , and identification. Phycologia 6: 127-160. ARCHIBALD, P . A. (1975) : Treboux ia DE PUYMA LY (Chlorophyceae , Chlorococcales) and Pseudotrebouxia gen . nov. (Chlorophyceae , Chlorosarcinales). Phycolog ia
14: 125-137. BISCHOFF, H. W . & BOLD , H. C. (1963): Phycological Studies IV . Some soil algae from Enchanted rock and related algal species . Univ . Te xas Pub!. 6318: 1-95. FRIEDL, T . & GARTNER, G . (1988) : Trebo uxia (Pleurastrales, Chlorophyta) as a ph ycobiont in the lichen genus Dipl oschistes. Arch . Protistenkd. 135 : 147-158. GALLOWAY , D . J . (1985): Flora of New Zealand. Lichens. Wellington , pp . 350. GARTNER , G . (1985): Die Gattung Trebouxia PUYMALY (Chlorellales, Chlorophyceae). Arch. Hydrobio!. , Supp!.
71: 495-548. HILDRETH , K. C. & AHMADJIAN , V. (1981): A study of
Trebo uxia and Pseudotrebouxia isol ates from different lichens . Lichenologist 13: 65-86. NAKANO, T . (1988) : Phycobionts of some Japanese species of Graphidaceae. Lichenologist 20: 353-360. MEISCH, J . P. (1981): Beitrage zur Isolation, Kultur und Systematik von Flechtenalgen. Diss . , Univ. Innsbruck, pp.
160. TAKESHITA, S. , NAKANO , T . & IWATSUKI, Z. (1989) : Phycobionts of some Japanese species of Pertusaria (Pertusariaccae), PI. Syst. Evo!. 165: 49-54. YOSHIMURA, I. (1987): Taxonomy and speciation of An zia and Pannoparmelia. In : Progress and Problems in Lichenology in the Eighties. Bibl. Licheno!. 25: 185-195 . Received: June 16, 1992 Authors' adresses: TAKA-AKI IHDA, TAKETO NAKANO , ZENNOSKE IWATSUKI , Botanical In stitute, Faculty of Science, Hiroshima University , Kag ami -yama , Higashi-hiroshima-shi, Hiroshima-ken 724, Japan ; ISAO YOSHIMURA, Kechi Gakuen College, Asahi-tenjin-cho 292, Kechi 780 , Japan .