植物研究雑誌 J. J. Jpn. Bo t. 81: 81: 107-112 (2006)

Karyomorphogical Analysis of Diploid Races of Aucubajaponica Aucubajaponica Thunb. ()

Machiko TSUSAKA ， Hiroshi IKEDA and Tak 吋i HOSHINO

Department Department of Mathematical and Environmental System Science ， Graduate School of Informatics ， Okayama University of Science ，1- 1， Ridai-cho ，Okayama 700-0005 JAPAN E-mail: E-mail: [email protected]

(Received on October 6，2005)

A karyomorphological analysis of diploid races of Aucub α japonica Thunb. ， with special special reference to their sexualities ， was carried ou t. After examining 59 individuals from from 16 localities in the Chugoku and Shikoku Districts ， western Japan ，all individuals proved proved to be diploid with 2n = 16 ， and the smallest pair of chromosomes had a satellite on on each short arm. Among the 59 individuals ， 24 individuals (“ heterotype") possessed a heterogeneous heterogeneous pair of chromosomes composed of median and submedian chromosomes in in the fourth longest pair. The remaining 35 individuals (“ homotype") possessed homoge-

neous neous pair with median chromosomes in the fourth longest pai r. Such cytological varia 副 tion tion did not show a geographical tendency ， but all the “heterotypes" were observed in male male individuals ， and “ homotypes" were in female individuals. Therefore we have con- cluded cluded that the karyomorphological variations in diploid races of A. japonica depend on their their sexualities.

Key words: ，diploid race ，karyomorphology ， sex chromosome.

Aucuba japonica Thunb. (Comaceae) is Chromosome numbers have been reported to distributed distributed throughout Japan ，from southern be 2n = 32 (tetraploid) for v訂 s.japonic αand Hokkaido to Okinawa ，and in Korea and borearis and to be 2n = 16 (diploid) for va r. Taiwan. It is an evergreen dioecious ovoid ω(Meurman 1929 ， Funabiki 1958 ， mainly growing in temperate forests. Aucuba Kurosawa 1971). Karyotype analyses have japonica japonica is usually classified into three va- been made mainly for tetraploid races rieties ， vars. japonica ， borealis Miyabe & (Meurman 1929 ， Sinoto 1929 ， Viinikka Kudo ，and ovoidea Koidz. Variety japonica 1970) ，and such studies of diploid races have is is distributed from the Tohoku District to the not been extensively carried ou t. Kurosawa Chugoku and Shikoku Districts mainly on (1971) compared the karyotypes of diploid the the Pacific Ocean side. Variety bore αlis is and te 佐aploid races. distributed distributed from southem Hokkaido to the Sex chromosomes have been reported in Kinki Kinki District through the Tohoku and several dioecious ，mainly in herbs Hokuriku Districts ，mainly on the Japan Sea such as Rumex (Kihara and Ono 1923 ，Love side. side. Variety ovoidea is distributed from 1943) ，Melandrium (Blackbum 1923 ，Winge eastern eastern part of the Chugoku and Shikoku 1923) ， Humulus (Winge 1923) ， and Districts Districts to the Kyushu District and also in Cannabis (Sinoto 1929) ，and relatively few the the Amami and Okinawa Islands (Hara 1966 ， in woody plants such as 5， αlix (B lackburn 1989 ， Noshiro 1999 ， Ohi et al. 2003). and Harrison 1924) ，and Palmae (Sinoto

-107- 108 108 植物研究雑誌第81 巻第2号 平成 18 年4月

1929). 1929). On the Aucuba ，no sex chromo- Science (OKA Y). some has been reported. Sugiura (1 927) ， Meurman (1 929) and Sinoto (1 929) observed Results and Discussion chromosomes of te 位aploid races of A. japon- After examining chromosomes of Aucub α ica ica and noted that there was no sex chromo- japonica collected from 16 localities in the some among 32 somatic chromosomes. Chugoku and Shikoku Districts ，all individu- Ki hara and Yamamoto (1 935) examined A. als proved to be diploid with 2n = 16 (Table chinensis chinensis (2n = 16) ， and Kurosawa (1971) 1). Figures 1 and 2 show somatic chromo- examined examined A. chinensis and A. himalaica somes and karyotypes of diploid races of A. (2n (2n = 16) cytologically ， but they did not japonica ，respectively. The length of somatic mention mention sex chromosomes. chromosomes 紅 e from 3.6 to 7.1μm ， and This This paper aims to provide an accurate kaηomo 叩hologically ， the complement is report report of karyomorphology of diploid races mono- moda l. Eight chromosome pairs were of of A. japonica ， with special reference to sex recognized by configuration of chromo- or or sex-dependent chromosomes. somes: three large-sized pairs with subterminal subterminal to submedian chromosomes ， one Materials Materials and Methods medium-sized pair with median chromo-

Fifty-nine Fifty-nine individuals were collected from somes ， and four small 司 sized pairs with 16 16 localities in the Chugoku and Shikoku subterminal to median chromosomes. The Districts ， western Japan (Table 1). Young smallest pair had comparatively large satel- branches branches were propagated hydroponically lites on the short 紅 'ms. Kurosawa (1971) ob- and and wer also arranged as herbarium speci- served several diploid individuals of A. mens. mens. Root tips were pre 佐eated with 0.002 japonica collected from the K yushu District

M 8-hydroxyquinoline solution for an hour and the Okinawa Island. Although the pre 皿 at at 20 0 C and stored 15 hours at 4 0 C ， then they sent study has confirmed that the smallest were were fixed with a 3: 1 mixture of 99.5 % pair has satellites ，it is not clear whether the ethanol ethanol and glacial acetic acid at -20 0 C more large-sized pairs have satellites or no t. Other than than one hou r. The root tips were hydrated methods ， such as FISH ，C-banding ， or silver- and and were soaked in a lN HCl solution for 10 staining methods ， should be applied to con- minutes minutes at 60 oC ， and were transferred to firm the number and position of satellites. Schif f' s reagent at room temperature for an During the analysis of k紅 yomorphology ， hour. hour. Then the materials were macerated we found that 24 individuals out of the 59 II ト with with a solution of a mixture of 2 % cellulase dividuals possessed a heterogeneous karyo- and and 2 % pectinase for 30 minutes at 37 0 C ， type with one median and one submedian and and washed in distilled wate r. The chromosomes in the fourth longest pair meristematic meristematic tissues were placed on slide (“ heterotype"). The remaining 35 individuals glasses glasses and were squashed with an aceto- possessed median chromosomes in the pair

glycerin glycerin (45 % acetic acid with a small (“ homotype") (Figs. 1 ，2). All the “hetero 目 amount of glycerin) and observed by types" were found to be male individuals ， microscope. microscope. Mitotic metaphase chromo- and “ homotypes" to be female individuals somes somes were microphotographed and were (Table 1). As “heterotype" individuals were karyotypically karyotypically analyzed. Chromosomes were distributed throughout the research area and assified c1 assified into several types ， following the found in four populations with “ homotype" nomenclature nomenclature of Levan et al. (1964). individuals (Table 1) ，we have concluded V oucher specimens are deposited in the that the k紅 yomorphological variation in Herbarium Herbarium of Okayama University of diploid races of A. japonica is not geographi- Apri12006 Apri12006 Journal of Japanese Botany Vo l. 81 No. 2 109

Table Table 1. Collection data ，chromosome numbers ，and karyotypes of diploid races of Aucuba japonica

Number of Chromosome Locality Locality (collector ， voucher specimen number ，altitude) Sex individuals number (2n) Karyotype examined

Okayama Prefecture Ukankei ，Shimokamo ， Kibichuo-cho (M. Tsusaka & al. ♂ 16 hetero* TS04100602 ，150 m) Ukankei ，Shimokamo ， Kibichuo-cho (M. Tsusaka & al. ♀ 2 16 homo** TS04100603 ，04100610 ，150m) Ukankei ， Shimokamo ， Kibichuo-cho (M. Tsusaka ♀ 4 16 homo 0505032XF ， 150 m) Ukankei ， Shimokamo ， Kibichuo-cho (M. Tsusaka ♂ 2 16 hetero 0505032XM ，150 m) 。 Iwayakei ， Kawakami-cho ， Takahashi-shi (M. Tsusaka T 16 homo 03052401 ，150 m) Nunose ，Bitchu-cho ， Takahashi-shi (M. Tsusaka & al. ♂ 2 16 hetero 050220A ，150 m) Shimane Prefecture Fukamachi pond ，Nishikawazu-cho ， Matsue-shi (S.-J. Lin ♂ 16 hetero 04041901 ，30 m) oT Naku ，Okinoshima-cho (M. Tsusaka 03102502 ，320 m) 16 homo Yamaguchi Prefecture Kamitama ，Tamagawa-cho (M.Tsusaka 03041303 ， 10 m) ♀ 16 homo Tokushima Prefecture oT Minamikumaso ，Kamiyama-cho (M. Tsusaka 05072201 ，90 16 homo m) Minamikumaso ，Kamiyama-cho (M. Tsusaka 05072201 ，90 ♂ 16 hetero m) Kochi Prefecture 。→切。引 1119H84121咽 ぷ Higashi-ishihara ， Tosa-cho (M. Tsusaka 050314A2 ，560 m) -EA--EurozO/O/O/orOhomo

Higashi-ishihara ， Tosa-cho (M. Tsusaka 050314A2 ，560 m) 且 hetero 4EEi--EAe-- Shiraiwado ，Kagami ， Kochi-shi (M. Tsusaka 050314 ，285 m) 60T hetero Sakagawa ，Tosayamada-cho (M. Tsusaka 050313Al ，82 m) homo 升 OOT Sakagawa ，Tosayamada-cho (M. Tsusaka 050313Al ，82 m) 且 hetero Nagasawa ， Ino-cho (M. Tsusaka 050314Al ，610 m) 4EaA4a-A4EE homo 判。。十 Nagasawa ， Ino-cho (M. Tsusaka 050314Al ，610 m) hetero O/O/O〆 且唱 Kususe ， Ino-cho (M. Tsusaka TS03060604 ，40 m) OTOT homo Iibo ， Yusuhara-cho (M. Tsusaka 04101301 ，490 m) 'B-A homo

唱・・ Shimokurechi ，Kubokawa-cho (M. Tsusaka 04101303 ，350 A homo m) Ooino ，Kubokawa-cho (M. Tsusaka 04101302 ，320 m) ♀ 16 homo Ehime Prefecture oT Ohkuki ，Uchiko-cho (M. Tsusaka 04101202 ， 160 m) 2 16 homo

*hetero: *hetero: the fourth-1ongest pair with heterogeneous chromosomes.

料 homo: the fourth-longest pair with homogeneous chromosomes.

cal cal nor populational variation ， but co 町es- therefore supposed that the fourth chromo- ponds ponds to the sexuality. Karyomo 中hological some pairs are sex chromosomes. variation variation co 町 esponding to sexuality in A. The reports of sex chromosomes in japonica japonica is reported for the first time. It is dioecious plants have been mainly for herbs 110 植物研究雑誌第81 巻第2号 平成18 年4月

Fig. Fig. 1. Sornatic chrornosornes of diploid races of Aucub α japonica. A-C: Fernale ，D-F: Male. A: Ukankei ‘ Okayarna Okayarna Pre f. B: Nagasawa ， Kochi Pref. C: Ii bo ， Kochi Pre f. D: Nishikawazu-cho ， Shirnane Pref. E: Ukankei ， Okayarna Pref. F: Takahashi-shi ， Okayarna Pref. Arrows indicate the fourth-longest pair with hornogeneous hornogeneous chrornosornes (A-C) and heterogeneous chrornosornes (D-F). Arrowheads indicate satel- lite lite chrornosornes. Bar = 10 flrn.

(Blackbum (Blackbum and Harrison 1924 ，Meurman dioecious evergreen like A. japonica. 1925 ， Lindsay 1930 ，Love 1943 ， Sinoto The heteromorphic chromosomes observed 1929 ，Winge 1923) ， and relatively few for in diploid races of A. japonica are supposed woody plants (Blackbum and Harrison 1924 ， to be sex chromosomes and this is the first Meurman 1925 ， Lindsay 1930 ， Sinoto 1929). report of sex chromosomes for evergreen There There is no report on sex chromosomes for shrub. Apri12006 Apri12006 Journal of Japanese Botany Vo l. 81 No. 2 111

A 、11111 ji l J I I ，.. iも

Fig.2. Fig.2. Kaηotypes of diploid races of Aucuba japonica. A: Nagasawa ， Kochi Pre f. (♀). B: Nishikawazu ，Shimane Pre f. (♂). Arrows indicate the fourth-longest pair with homogeneous chromosomes (A) and heterogeneous chromosomes (B). Arrowheads indicate satellite chromo- somes. somes. Bar = 10μm.

We thank M r. Shungo Kariyama ， Kurosawa S. 197 1. Cytotaxonomical studies on the Kurashiki Kurashiki Museum of Natural History ， Mr. genus Aucuba. J. Jpn. Bo t. 46: 231-238. Makoto Ogawa ， Tokushima Prefectural Levan A. ， Fredga K. and Sandberg A. A. 1964. Nomenclature Nomenclature for centromeric position on chromo- Museum ， and Dr. Tetsuo Ohi ， Botanical somes. Hereditas 52: 201-220. Garden Garden of the University of Tokyo ， for their Love A. 1943. Cytogenetic studies on Rumex subgenus valuable valuable information on distribution of Acetosella. Hereditas 30: 1-136. Aucubajaponica. Aucubajaponica. We also appreciate D r. Su- Lindsay R. H. 1930. The chromosomes of some Jang Jang Lin ，Shimane University ， for sending a dioecious angiosperms. Ame r. J. Bo t. 17: 152- 174. 174. materia l. This study is partly supported by Meurman O. 1925. The chromosome behaviour of the the Yakumo Foundation for Environmental some dioecious plants and their relatives with spe- Science Science in 2005 (to M. T.). cial reference to the sex chromosomes. Soc. Sci. Fennica ，Commentat Bio l. 2: 1-105. References References 1929. Chromosome numbers in the family Cornaceae. Cornaceae. Mem. Soc. Fauna Fl ora Fennica 6: 95- Blackburn Blackburn K. B. 1923. Sex chromosomes in plants. 100. 100. Nature Nature 112: 687-688. Noshiro Noshiro S. 1999. Cornaceae. In: Iwatsuki K.， Boufford 一一- and Harrison J. W. H. 1924. A premin 訂 y ac- D. D. E. and Ohba H. (eds.) ，Fl ora of Japan IIc: 254- count count of the chromosomes and chromosome 258. 258. Kodansha ， Tokyo. behaviour behaviour in Salicaceae. Ann. Bo t. 38: 361-378. Ohi T. ，K 司ita T. and Murata J. 2003. Distinct geo- Funabiki Funabiki K. 1958. Distribution and polyploidy of an- graphic graphic structure as evidenced by chloroplast DNA giosperms giosperms 1. Kr omosomo 37-38: 1253-1267. haplotypes haplotypes and ploidy level in Japanese Aucuba Hara H. 1966. Cornaceae. The Flora of Eestern (Aucubaceae). (Aucubaceae). Ame r. J. Bo t. 90: 1645-1652. Himalaya. Himalaya. p. 744. University of Tokyo Press ， Sinoto Sinoto Y. 1929. Chromosome studies in some Tokyo. Tokyo. dioecious dioecious plants ， with special reference to the 一一一 1989. Cornaceae. In: Satake Y. ，Hara H. ， Watari allosomes. allosomes. Cytologia 1: 109-19 1. S. S. and Tominari T. (eds よWild Fl owers of Japan ， Sugiura Sugiura T. 1927. Some observations on the meiosis of Woody Plants 11: 109-112. Heibonsha ，Tokyo (in the the pollen mother cells of Carica papaya ， Myrica Japanese). Japanese). rubra ，Aucuba japonica and Beta vulgaris. Bo t. Ki hara H. and Ono T. 1923. Cytological studies of Mag. Tokyo 41: 219-224. Rumex L. Bo t. Mag. Tokyo 37: 84-90. Viinikka Viinikka Y. 1970. A comparative study of mitotic and 一一and Yamamoto Y. 1935. Chromosomenver- meiotic meiotic chromosomes of Aucub α japonica Thunb. haltnisse haltnisse bei Aucuba chinensis Benth. Agric. Ann. Ann. Bo t. Fennici 7: 203-21 1. Hortic. Hortic. Tokyo 10: 2485-2495. 112 112 植物研究雑誌第81 巻第2号 平成18 年4月

Winge 0. 1923. On sex chromosomes ， sex determina- dioecious plants. Comp t. Rend. Trav. Lab. tion tion and preponderance of females in some Carlsberg 15: 1-26.

津坂真智子，池田 博，星野卓二:アオキ(ミズ キ科)の 2 倍体における核形態学的解析 ミズキ科アオキ (Aucuba japonica Thunb.) の二 ホモ型と，中部動原体染色体と次中部動原体染色 倍体 (2n = 16) について，雌雄性を考慮して核型 体を一本ずつもつヘテロ型の組み合わせをもっ個 分析を行った.中国・四国地方産のアオキ 16 地点 体が見出された.この変異は地理的なものではな 59 個体について解析を行ったところ，すべての個 く，ホモ型の組み合わせをもっ個体はすべて雌で 体で最小の染色体対に付随体がみられ，これまで あり，ヘテロ型の組み合わせをもっ個体はすべて の報告と→致した.しかし これまで指摘されて 雄であることが明らかになった. したがって，ア いた最大の染色体対と三番目に長い染色体対にあ オキの二倍体は，性によって異なる核型を示し， るとされる二次狭窄については，今回の研究では 性染色体をもっていると考えられる. 明瞭に識別することはできなかった.また，四番 (岡山理科大学総合情報研究科 目に長い染色体対に中部動原体染色体を 2 本もつ 数理・環境システム専攻)