Regular Article Cytological Studies on Wild Farfugium Japonicum (Asteraceae)

Chromosome Science 12: 27-33, 2009 Okuno et al. 27

Regular Article

Cytological studies on wild Farfugium japonicum (Asteraceae)

Hajime Okuno, Masashi Nakata and Masahiro Mii

Received: December 16, 2009, Accepted: January 21, 2010 Copyright C 2009 by the Society of Chromosome Research

Abstract on rocky cliffs near seashores in Honshu (mainly in Farfugium japonicum, Asteraceae, Japanese name: Tsu- the southeastern part), Shikoku, Kyushu, Ryukyu and wabuki, is a traditional garden plant of Japan. At pres- Ogasawara Isl. (Koyama 1995). ent more than 100 horticultural cultivars are known, and This species has long been cultivated for ornamental most of them have been selected from wild populations. purpose since the Edo period, and more than 100 In order to obtain cytological information on F. japoni- horticultural cultivars, which have mostly been selected cum as horticultural resource, chromosome numbers, from wild populations, are known at the present time meiotic chromosome behaviors and pollen stainabilities (Kitamura et al. 1994, Ogisu 1997, Okuno et al. 2007). Some were examined on wild plants from 70 collection sites cultivars represent freak morphology such as dwarf form selected from the whole range of distributional areas (e.g., ‘Itohime’), rumpled lamina (e.g., ‘Kimen’), crisped in Japan. Except for one aneuploid (2n=61) plant which margin of lamina (e.g., ‘Yukibeni-botan’), serrated and wavy has a common leaf morphology, all 177 individuals of margin of lamina with short petiole (e.g., ‘Daruma-jishi’), var. japonicum were counted to be 2n=60. Meiotic divi- etc. (cf. Okuno et al. 2007). Generally polyploid plants sion is normal and 30 bivalents were observed at meta- show gigas phenotype, but the opposite feature can be phase I. The frequency of stainable pollen grains was seen in some polyploids (Nishiyama 1952, Stebbins 1971). high (93.9±8.8%, N=60). Other three taxa of this species, High-level polyploidy usually causes malformed leaves and var. giganteum, var. luchuense and var. formosanum, were flowers (Kobayashi et al. 2008, Griesbach and Kamo 1996). also observed to be 2n=60. From the cytogenetical view- Trisomic plants, in which one chromosome is present point, F. japonicum is considered to be a stable diploid, in triplex condition, also differ from a normal diploid in although its chromosome number is relatively high. morphology as exampled in Goodspeed and Avery (1939). Thus, it can be considered that the chromosomal changes in number, such as polyploidy, aneuploidy and mixoploidy Key words: Asteraceae, chromosome number, Farfugi- have caused freak morphology in some cultivars of F. um japonicum, meiosis, Senecioneae japonicum. Chromosome numbers of F. japonicum have been reported to be n=30 (Ishikawa 1916, as Ligularia tussilagi- nea) and 2n=60 for the plants from Japan (Arano 1962, Miyagi 1971, Okuno et al. 2005), Taiwan (Hsu 1970) and Introduction China (Su and Liu 1995, Liu 2001). However, only one or few individuals had been studied in each case and no Farfugium japonicum (L.) Kitam., Asteraceae - Senecio- horticultural cultivars had been examined yet. Thus, we neae, Japanese name: Tsuwabuki, is an evergreen perennial observed mitotic and meiotic chromosomes on both wild herb distributed in Japan, South Korea, Taiwan and the plants and horticultural cultivars in order to clarify the east of China (Kitamura 1981). In Japan it grows mainly origins of horticultural cultivars. In the present study, we will describe chromosome numbers of wild F. japonicum collected in Japan and Taiwan as the first report. Results Hajime Okuno on chromosome numbers of horticultural cultivars will be 3250-201 Fukaisawa-machi, Naka-ku, Sakai, Osaka 599-8236, presented elsewhere. Japan Masashi Nakata (*) Botanic Gardens of Toyama, Materials and Methods 42 Kamikutsuwada, Fuchu-machi, Toyama 939-2713, Japan Plant materials e-mail: [email protected] Masahiro Mii A total of 190 individuals of F. japonicum including Laboratory of Plant Cell Technology, four taxonomic varieties, var. japonicum, var. giganteum Graduate School of Horticulture, Chiba University, (Siebold et Zucc.) Kitam., var. luchuense (Masam.) Kitam. 648 Matsudo, Matsudo, Chiba 271-8510, Japan and var. formosanum (Hayata) Kitam., were collected from 28 Chromosome number of Farfugium japonicum

68 localities in 31 prefectures of Japan and two localities was employed to make chromosome preparation. The in Taiwan (Table 1, Figs. 1, 2). The collection area covers chromosome number of each individual was determined a whole distributional area of F. japonicum in Japan and it by counting three (sometimes two or one) mitotic cells at also includes nearly northernmost places of the both sides metaphase or prometaphase stages. of the Japan Sea and the Pacific Sea, and a southernmost For meiotic observation, pollen mother cells were place of the Ryukyu Archipelago (cf. Horikawa 1972). used. Young flower heads from potted plants were fixed Although the materials have been collected by non- and stored in modified Carnoy’s fixative (99.5% ethanol : intentional selection, some morphological variants were glacial acetic acid : chloroform = 2 : 1: 1) at 5°C for over 20 casually obtained in var. japonicum; a flower-shape variant h. Immature anthers drawn from some florets in a young “tube ligules” (Fig. 2B) from Kagawa Prefecture, a variant heads were placed on a slide glass, stained with 1% acetic with dissected ligules on a zigzag-shaped scape (Fig. 2C) orcein, and squashed under a cover glass. Chromosome from Iriomote-jima Isl. and a tall scape variant (Fig. 2D) behavior was observed at diakinesis, metaphase I, from Tokunoshima Isl. The material plants were planted in metaphase II, anaphase II and tetrad stages. pots individually in Toyama (Botanic Gardens of Toyama) and in Osaka (a private garden of Okuno). Vouchers will be Pollen fertility preserved at the Botanic Gardens of Toyama (TYM). Pollen fertility was estimated by the presence or absence Chromosome observation of cytoplasm. Pollen grains obtained from a head fixed in 70% ethanol were spread on a slide glass and stained with For mitotic chromosome observation, fresh root tips lactophenol cotton blue solution. The percentage of pollen of 5 mm long excised from potted plants were used. They stainability was calculated mostly on more than 1000 were pretreated with 2 mM 8-hydroxyquinoline aqueous pollen grains (the minimum 225 and the maximum 3500). solution at 15°C for 8 h, fixed with Farmer’s fixative (99.5% ethanol : glacial acetic acid = 3 : 1) at 5°C for 20 h, and stored in a freezer (-10°C) until use. The fixed root tips Results and Discussion were macerated in a mixture of 1 mol/L hydrochloric Chromosome number acid and 45% acetic acid (2:1, v/v) at 60 °C for 15 sec, and immediately rinsed with water. Then the meristematic All 177 individuals of wild F. japonicum var. japonicum regions of 1 mm long were cut off from the root tips and had the chromosome number of 2n=60 (Fig. 3A) except for stained with 1% acetic orcein (synthetic; Kanto Kagaku, one individual with the chromosome number of 2n=61 (Fig. Co.) at room temperature for 10 min. Squashing method 3B) collected in Kyoto Prefecture (HO2652). The aneuploid

Table 1. Localities, sample numbers, chromosome numbers and vouchers of Farfugium japonicum examined. No. of Prefecture/ Chromosome No.* Locality plants Voucher (Pollen grain stainability (%)) area number (2n) observed var. japonicum Fukushima 1 Tanoami, Hisanohama-machi, Iwaki City 3 60 HO0701, HO0702(95.1), HO0751 Ibaraki 2 Isohara, Isohara‐cho, Kitaibaraki City 3 60 HO0801(97.6), HO0851, HO0852 Chiba 3 Izumi, Misaki-cho, Isumi City 5 60 HO1201(98.3), HO1202(98.7), HO1203, HO1251, HO1252 Kanagawa 4 Kikuna, Minamishitaura-machi, Miura City 4 60 HO1401, HO1402(64.7), HO1451, HO1452 Niigata 5 Aikawakabuse, Sado City 2 60 HO1701(93.3), HO1752 6 Ogikowashimizu, Sado City 1 60 HO1702(94.8) Ishikawa 7 Kurosaki-machi, Kaga City 5 60 HO1901, HO1902, HO1951-1953 8 Togiushioroshi, Shika-machi, Hakui-gun 1 60 MN25245 9 Tsurugiji, Monzen-machi, Wajima City 1 60 MN25246 10 Kitagawa, Monzen-machi, Wajima City 1 60 MN25247 Fukui 11 Akasaki, Tsuruga City 2 60 HO2001, HO2051 12 Ageno, Tsuruga City 4 60 HO2002(97.2), HO2003(93.9), HO2004(96.5), HO2052 Shizuoka 13 Kawana, Ito City 4 60 HO2201(93.5), HO2202, HO2251, HO2252 14 Omaezaki, Omaezaki City 4 60 HO2203, HO2204, HO2253, HO2254 Aichi 15 Onoura, Mihama-cho, Chita-gun 4 60 HO2301(96.5), HO2302(96.1), HO2351, HO2352 Mie 16 E, Futami‐cho, Ise City 3 60 HO2401(99.2), HO2402(94.2), HO2403 Kyoto 17 Kakezu, Amino-cho, Kyotango City 3 60 HO2601, HO2602, HO2651 1 61 HO2652 Osaka 18 Hakotsukuri, Hannan City 4 60 HO2701(85.2), HO2702(94.5), HO2751, HO2752 Hyogo 19 Kiba, Himeji City 4 60 HO2801(97.1), HO2802, HO2851, HO2852 20 Okinoura, Kasumi-ku, Kami-cho, Mikata-gun 4 60 HO2803(99.7), HO2804(96.1), HO2853, HO2854 Okuno et al. 29

continued Wakayama 21 Kushima, Miwasaki, Shingu City 2 60 MN25203, MN25204 22 Moriura, Taiji-cho, Higashimuro-gun 1 60 HO3003(98.2) 23 Kitsumoto, Shimotsu-cho, Kainan City 1 60 MN26006 24 Maruda, Shimotsu-cho, Kainan City 2 60 HO3001, HO3002(98.2) Tottori 25 Yurashuku, Hokuei-cho, Tohaku-gun 4 60 HO3101(92.3), HO3102(98.6), HO3151, HO3152 Shimane 26 Chigo-cho, Izumo City 2 60 HO3201(98.4), HO3251 27 Koizu-cho, Izumo City 2 60 HO3202(98.4), HO3203(98.2) 28 Uppurui, Uppurui-cho, Izumo City 1 60 HO3252 Okayama 29 Kashino, Ushimado-cho, Setouchi City 4 60 HO3301(95.5), HO3302, HO3351, HO3352 Hiroshima 30 Ooura, Kamagari-cho, Kure City 4 60 HO3401, HO3402(99.3), HO3451, HO3452 31 Motoujina-machi, Minami-ku, Hiroshima City 5 60 MN25228-25232 Yamaguchi 32 Mukoushima, Houfu City 4 60 HO3501, HO3502(96.9), HO3551, HO3552 33 Chinto, Hagi City 4 60 HO3503, HO3504(95.6), HO3553, HO3554 Tokushima 34 Tosadomariura, Naruto-cho, Naruto City 4 60 HO3601(97.7), HO3602(97.8), HO3651, HO3652 35 Minamibayashi, Minobayashi-cho, Anan City 4 60 HO3603, HO3604(97.1), HO3653, HO3654 Kagawa 36 Koiso, Higashikagawa City. 2 60 HO3701(98.6), HO3751 37 Enohama, Azi-cho, Takamatsu City 2 60 HO3702(96.6), HO3752(95.8) 38 Tarumi-cho, Takamatsu City 2 60 HO3703, HO3753 39 Oomi, Mino-cho, Mitoyo City 2 60 HO3704(98.3), HO3754 40 Yoshino, Syodoshima-cho, Syozu-gun 2 60 HO3705, HO3706(98.9) 41 Shamijima, Sakaide City 1 60 MN26042 Ehime 42 Yoki, Kawanoe-cho, Shikokuchuo City 3 60 MN2506, 25062, 25063 43 Kaminada, Futami-cho, Iyo City 4 60 HO3801(98.1), HO3802(98.9), HO3851, HO3852 Kochi 44 Urado, Kochi City 3 60 MN25055-25057 45 Kaba, Sukumo City 4 60 HO3901(98.1), HO3902(87.8), HO3951, HO3952 Fukuoka 46 Ookura, Yahatahigashi-ku, Kitakyusyu City 3 60 HO4001(96.7), HO4051, HO4052 Saga 47 Nishinohama-machi, Karatsu City 4 60 HO4101(96.5), HO4102(95.3), HO4151, HO4152 Nagasaki 48 Kobago, Saikai-cho, Saikai City 4 60 HO4201(99.2), HO4202(71.5), HO4251(89.3), HO4252 49 Kusubo, Mitsushima-machi, Tsushima City 1 60 MN27068 50 Hirugaura, Mitsushima-machi, Tsushima City 4 60 HO4203, HO4204, HO4205, HO4253 51 Nishizato, Izuhara-machi, Tsushima City 1 60 MN27069 52 Tamanoura, Tamanoura-machi, Goto City 1 60 MN27010 Kumamoto 53 Kawachi, Kawachi-machi, Kumamoto City 4 60 HO4301(56.8), HO4302, HO4351, HO4352 Ooita 54 Usuno, Bungotakada City 2 60 HO4401(98.0), HO4451(93.3) Miyazaki 55 Minamitakanabe, Takanabe-cho, Koyu-gun 4 60 HO4501(97.2), HO4502(98.9), HO4551, HO4552 Kagoshima 56 Miyagahama, Nishikata, Ibusuki City 3 60 HO4602, HO4651(96.4), HO4652(88.3) 57 Kodakara-jima, Toshima-mura, Kagoshima- 1 60 BGT44120 gun 58 Tokunoshima-cho, Ooshima-gun 2 60 MN26001, MN26002 Okinawa 59 Ookawa, Nago City 1 60 BGT30661 60 Kin, Kin-cho, Kunigami-gun 3 60 MN26045-26047 61 Maeda, Onna-son, Kunigami-gun 3 60 MN26048-26050 62 Iriomote-jima, Taketomi-cho, Yaeyama-gun 1 60 HO4702(92.4) 63 Fanan-gawa, Tozato, Ishigaki City 1 60 HO4704(98.0) 64 Uotsuri-jima, Ishigaki City 1 60 HO4701(96.7) (Taiwan) 65 Orchid Island, Taidong, Taiwan 1 60 HO4802(61.4) var. giganteum (Siebold et Zucc.) Kitam. Wakayama 66 Nishiki，Kushimoto-cho，Higashimuro-gun 3 60 MN26038-26040 (63.9) Kagoshima 67 Ibusuki City（ Cultivated in the Makino Bot. 2 60 MN25058, MN25060 (82.9) Gard.） var. luchuense (Masam.) Kitam. Kagoshima 68 Sumiyou-gawa, Sumiyo-cho, Amami City 1 60 MN28027 Okinawa 69 Iriomote-jima, Taketomi-cho, Yaeyama-gun 6 60 MN28021-28026 var. formosanum (Hayata) Kitam. (Taiwan) 70 Manyuehyuan forest park, Taipei, Taiwan 1 60 HO4801(99.3) * Numbers correspond to those in Figure 1. 30 Chromosome number of Farfugium japonicum

Figure 1. Collection sites of Farfugium japonicum in the present study. Uotsuri-jima, Ishigaki-jima, Iriomote-jima and Taiwan are excluded. Numbers correspond to those in Table 1.

plant, which unfortunately has not yet flowered, showed of the previous report by Peng and Hsu (1978) for a plant no freak morphology of the leaves (Fig. 2A). The flower- from Taipei. shape variant “tube ligules” (HO7886) had the normal chromosome number of 2n=60. Other two variants, the Meiotic chromosomes and pollen fertility zigzag scape with dissected ligules (HO4702) and the tall scape (HO4603) also had the same chromosome number of At diakinesis and meiotic metaphase I of the micro- 2n=60. The chromosome numbers of F. japonicum in Japan sporogenesis in F. japonicum, normal 30 bivalents were have been reported to be n=30 (Ishikawa 1916) and 2n=60 observed (Figs. 4A, B), which confirmed n=30 count (Arano 1962, Miyagi 1971, Okuno et al. 2005). Hsu (1970) by Ishikawa (1916). In most of the materials observed, also reported the chromosome numbers of F. japonicum to abnormal chromosome behaviors, such as lagging be n=30 and 2n=60 for the materials from Taiwan. Su and chromosomes, chromosome bridges and micronuclei Liu (1995) and Liu (2001) reported chromosome numbers formation were not observed in the subsequent stages of and karyotypes of F. japonicum from Wuhan Institute of microsporogenesis (Figs. 4C, D). Thus, normal tetrad (Fig. Botany, Chinese Academy of Sciences (cultivated), and 4E) and pollen grains (Fig. 4F) were formed. from Sichuan Province of China, respectively. Our present Pollen grains of F. japonicum var. japonicum were observations confirmed those previous reports. examined in 62 plants including two exceptional plants The chromosome number of the other three varieties which had no pollen grains. Three plants from Kanagawa, was also confirmed in this study. The var. giganteum was Kumamoto and Taiwan showed low values of stainability, determined to have 2n=60 chromosomes in three plants 64.7, 56.8 and 61.4 %, respectively, although the sizes of from Kushimoto, Wakayama Pref. (MN26038-26040, cf. stained pollen grains were quite similar to those of the Umemoto and Kusanagi 1992) and in two plants from others. The other 57 plants had high stainability of 95.6±4.5 Ibusuki (cultivated in the Makino Botanical Garden, % (ranges: 71.5 – 99.7 %). Pollen grain stainability in var. MN25058, MN25060) (Fig. 3C). This is the first report of japonicum as a whole was 93.9±8.8 % (N=60). The pollen the chromosome number for this variety. The chromosome grain stainabilities of two plants in var. giganteum were number for F. japonicum var. luchuense was also counted to relatively low, 63.9 % and 82.9 %, respectively, while in var. be 2n=60 in one plants from Amami Isl. (MN28027) and formosanum the value of a plant was high, 99.3 %. six plants from Iriomote Isl. (MN28021-28026) (Fig. 3D). The result agreed with that of Miyagi (1971), in which the Cytological remarks locality of the materials had not been described. The same chromosome number (2n=60) was also confirmed in F. Four varieties of Farfugium japonicum examined in the japonicum var. formosanum (Fig. 3E) in a plant collected present study are distinguished from each other mainly by in Taipei, Taiwan (HO4801), which confirmed the result leaf morphology. The var. giganteum is characterized by Okuno et al. 31

Figure 2. Farfugium japonicum as materials used in the present study. A-D: var. japonicum. A: An aneuploid plant collected in Kyoto Pref. (HO2652). B: A head of a flower variant collected in Kagawa Pref. (HO7886). C: Heads of a variant collected in Iriomote Isl., Okinawa Pref. (HO4702). D: A tall scape variant from Tokunoshima Isl., Kagoshima Pref. (HO4603). E: var. giganteum from Ibusuki, Kagoshima Pref. (MN25060). F: var. luchuense from Iriomote Isl., Okinawa Pref. (MN28026). G. var. formosanum from Taiwan (HO4801).

gigas phenotype, i. e., having tall scapes, long and wide leaf trisomic plants sometimes show differed phenotypes from blades, and long petioles (Koyama 1995). In the present the corresponding diploids as shown in Nicotiana sylvestris study, one plant with the same phenotype was also found in (Goodspeed and Avery 1939) and Datura stramonium var. japonicum. In general, plants with low-level polyploidy (Avery 1959) of the Solanaceae, the aneuploid plant of F. such as triploid and tetraploid show gigas phenotype. japonicum had no abnormality at least on leaf morphology. However, no such polyploids were found in F. japonicum Concerning the karyotype, F. japonicum var. japonicum var. giganteum and the tall scape variant of var. japonicum is characterized by four telocentric chromosomes as shown in the present study. These results suggest that gigas previously (Okuno et al. 2005). Some of the telocentric phenotype of F. japonicum is not caused by the gene dosage chromosomes sometimes had a satellite (Fig. 3A, marked but is controlled by the specific gene(s). with an asterisk) or a minute chromatin segment on the In Asteraceae, the frequency of spontaneous occurrence centromeric side, thus, they sometimes looked acrocentric. of aneuploid with 2n=2x+1 has previously been reported In the present study, the four telocentric or acrocentric as 0.25% (10/4000) in Crepis tectorum (2n=8) (Nawaschin chromosomes were also verified in the aneuploid individual 1926) and 0.9% (4/439) in Brachycome lineariloba cytodeme (HO2652, Fig. 3B) as well as in other three varieties (Figs C (2n=16) (Watanabe et al. 1985). The ratio (0.5 %) of 3C-E). the spontaneous aneuploid of 2n=61 found in the present In conclusion, wild F. japonicum is considered to be observation is comparable with these results. Although cytogenetically stable diploid species based on the results 32 Chromosome number of Farfugium japonicum

Figure 3. Mitotic metaphase chromosomes of Farfugium japonicum. A & B: var. japonicum. A: A plant from Okinawa Pref. (MN26046), 2n=60. B: An aneuploid plant from Kyoto Pref. (HO2652), 2n=61. C: var. giganteum from Ibusuki. (MN25060), 2n=60. D: var. luchuense from Amami Isl. (MN28027), 2n=60. E: var. formosanum from Taiwan. (HO4801), 2n=60. Arrows indicate four telocentric or acrocentric chromosomes, which sometimes have a satellite as designated by an asterisk in Fig. A, or have a minute chromatin segment. Scale bars indicate 10μm.

Figure 4. Meiotic chromosomes and pollen grains in wild Farfugium japonicum. A: Diakinesis. 2n=30II. B: Metaphase I, polar view. 2n=30II. C: Metaphase II. D: Telophase II. E. Tetrad. F: Mature pollen grains including a sterile one stained with cotton blue. Scale bars indicate10μm (A-E) and 100μm (F), respectively. Okuno et al. 33 of stable chromosome number, meiotic chromosome as a traditional garden plant in Japan – a historical review and the configuration of 2n=60=30II followed by the normal present cultivars–. Bull Bot Gard Toyama 12: 19-35. (in Japanese chromosome behavior, and high values of pollen with English summary) stainability. Peng CI, Hsu CC (1978) Chromosome numbers in Taiwan Composi- tae. Bot Bull Acad Sin 19: 53-66. Stebbins L (1971) Chromosomal Evolution in Higher Plants. Edward Arnold Ltd, London. Acknowledgements Su YJ, Liu QH (1995) The karyotype analysis of Farfugium japonicum. J Wuhan Bot Res 13: 18-20. (in Chinese with English summary) The authors are grateful to Ms. Yasu Nakamura, Mr. Umemoto S, Kusanagi T (1992) Farfugium japonicum (L. fil.) Kitamu- Osamu Kume, Mr. Kaoru Hashimoto, Mr. Toshiaki Shiuchi, ra var. giganteum (S. et Z.) Kitamura extending to the Kii Penin- Mr. Tadashi Kanemoto and the Makino Botanical Garden sula, Japan. The Nanki Seibutsu 34: 20-21. (in Japanese) for their kind supply of the materials, and to Dr. Kuniaki Watanabe K, Carter CR, Smith-White S (1985) The cytology ofBrachy - Watanabe for valuable information on literature. come lineariloba. 9. Chromosome heterogeneity in natural populations of cytodeme C (2n=16). Can J Genet Cytol 27: 410-420.

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