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Intraspecific Variation of Ploidy Levels and Chloroplast Haplotype within Helwingia japonica (Helwingiaceae) in Japan

Tetsuo Ohi-TOmaa,d,*, Katsuhisa ichinOsea,d, Kana WaTanabe-TOmaa,b, Hiroshi ikedac and Jin muraTaa

aBotanical Gardens, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 112-0001 JAPAN;

bMusashi High School & Junior High School, Nerima-ku, Tokyo, 176-0011 JAPAN;cThe University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 JAPAN;

dEqually contributed*Corresponding author: ooi@ns.bg.s.u-tokyo.ac.jp

(Accepted on June 23, 2017)

We report a high degree of intraspecific variation of ploidy levels and chloroplast haplotype within Helwingia japonica (Thunb.) F. Dietr. (Helwingiaceae) in Japan. In the flow cytometric analysis, diploid, tetraploid, and hexaploid plants were detected. In chloroplast psbA-trnH analysis, 22 haplotypes were distinguished. Based on the combination of ploidy level and haplotype, 32 types of intraspecific variation were distinguished. Helwingia japonica subsp. japonica var. japonica includes a ploidy series of diploid, tetraploid, and hexaploid. It is noteworthy that plants of var. japonica in Hokkaido and Tohoku region are uniformly diploid, while some of var. japonica in west Japan are tetraploid. The distributional ranges of the ploidy levels of subsp. japonica in Japan seem to be roughly segregated, and only three sympatric populations that show two different ploidy levels were found. Considering the distributional range, the haplotype connection in the network, and the haplotype sharing among ploidy levels, one major haplotype has contributed to polyploidization in west Japan. Most polyploids might have been derived from the diversification from polyploids with the major haplotype rather than multiple polyploidzations from different diploids.

Key words: Flow cytometry, Helwingia japonica, Helwingiaceae, Japan, ploidy level, psbA-trnH.

J. Jpn. Bot. 92(6): 321–329 (2017)

Helwingia Willd., the only genus in the family Helwingiaceae, is a shrub with an epiphyllous inflorescence on midvein of adaxial surface. The genus comprises four species in the Sino-Japanese floristic region: two deciduous (H. japonica (Thunb.) F. Dietr. and H. himalaica Hook. f. & Thomson ex C. B. Clarke) and two evergreen (H. chinensis Batalin and H. omeiensis (Fang) H. Hara & S. Kurosawa) (Hara

and Kurosawa 1975, Xiang and Boufford 2005, Xiang 2016).

Of the species, H. japonica has the widest range of distribution from the Himalayas, mainland China through Taiwan to the Japanese archipelago. The species shows large morphological variation of leaf shape and size (Yamanaka 1966), and several intraspecific taxa have been described. In the classification of

328 植物研究雑誌　第 92 巻　第 6 号 2017 年 12 月

T. Ohi 073-2, 6x (2.949), type I; Gifu, Ibigawa, T. Ohi 074, 6x (2.828), type D; Aichi, Shinshiro, T. Ohi 075-1, 4x (1.994), type D & T. Ohi 075-2, 2x, type F; Nagano, Ina, T. Ohi 076-2, 6x (2.914), type D & T. Ohi 076-3, 6x (3.036), type D; Gifu, Nakatsugawa, T. Ohi 077-1, 4x (1.974), type D & T. Ohi 077-3, 4x (1.973), type D; Gifu, Gero, T. Ohi 078, 6x (2.925), type H; Gifu, Takayama, T. Ohi 079, 6x (2.945), type D; Chiba, Minami-Boso, T. Ohi 080, 2x, type S; Shizuoka, Shizuoka, T. Ohi 081-2, 2x, type F & T. 081-3, 2x, type F; Yamanashi, Fujikawa, T. Ohi 082-1, 2x, type S & T. Ohi 082-2, 6x (2.907), type D; Shizuoka, Izu, T. Ohi 083, 2x, type F; Kanagawa, Mastuda, T. Ohi 084, 6x (2.985), type D; Tokyo, Izu-Oshima, T. Ohi 085, 6x (3.377), type D; Ibaraki, Daigo, T. Ohi 086, 6x (3.075), type D; Shimane, Nishinoshima, T. Ohi 089, 6x (3.074), type H; Shimane, Okinoshima, T. Ohi 090, 6x (3.024), type H; Tokyo, Ome, T. Ohi 092, 6x (3.061), type D; Shizuoka, Shimoda, T. Ohi 096-1, 2x, type S & T. Ohi 096-2, 2x, type S; Nagasaki, Tsushima, K. Watanabe 099, 2x, type H; Nagano, Karuizawa, Y. Yamaguchi 100, 6x (2.980), type C; Nagano, Tatsuno, T. Ohi-Toma 101, 6x (2.955), type D; Gifu, Ogaki, T. Ohi-Toma 102-1, 6x (2.984), type D & T. Ohi-Toma 102-2, 6x (3.025), type D; Nara, Uda, T. Ohi-Toma 103-1, 4x (1.966), type H & T. Ohi-Toma 103-2, 4x (2.038), type H; Yamanashi, Ichikawa-Misato, K. Ichinose 104-1, 2x, type S & K. Ichinose 104-3, 6x (3.105), type D; Gifu, Gujo, T. Ohi-Toma 108, 6x (2.993), type H; Miyagi, Ishinomaki, T. Nemoto 109-1, 2x, type A & T. Nemoto 109-3, 2x, type A; Ibaraki, Mt. Tsukuba, T. Ohi-Toma 111-2, 6x (3.021), type D & T. Ohi-Toma 111-2, 6x (3.076), type C; Hokkaido, Hakodate, K. Ichinose 112, 2x, type A; Tochigi, Nasu-Shiobara, K. Ichinose 113, 6x (2.961), type D; Niigata, Aga, T. Ohi-Toma 115, 6x (3.063), type E; Niigata, Joetsu, Y. Iokawa 116, 6x (3.024), type E; Aomori, Mt. Hakamagoshi, T. Nemoto 117, 2x, type A; Ibaraki, Kita-Ibaraki, Y. Endo 119-1, 6x (3.044), type D & Y. Endo 119-2, 6x (2.976), type D; Fukushima, Iwaki, T. Nemoto 120-1, 2x, type A & T. Nemoto 120-2, 2x, type A; Fukushima, Namie, T. Nemoto 121-1, 6x (3.049), type D & T. Nemoto 121-2, 6x (3.152), type C; Okayama, Niimi, T. Ohi-Toma 122-1, 4x (1.994), type Q & T. Ohi-Toma 122-2, 4x (2.003), type Q; Okayama, Kagamino, T. Ohi-Toma

123-1, 6x (3.022), type P & T. Ohi-Toma 123-2, 6x (3.092), type P; Hiroshima, Jinsekikogen, T. Ohi-Toma 124-1, 4x (2.016), type H & T. Ohi-Toma 124-2, 4x (1.956), type H; Iwate, Morioka, T. Nemoto 125, 2x, type A; Saitama, Hanno, K. Ichinose 127, 6x (3.325), type D; Aomori, Towada, K. Sasamura 129, 2x, type A; Akita, Nikaho, K. Sasamura 130, 2x, type A; Ibaraki, Mito, Y. Endo 132, 6x (3.017), type D; Chiba, Shibayama, T. Ohi-Toma 133, 2x, type V; Chiba, Funabashi, T. Ohi-Toma 134, 2x, type V; Shizuoka, Fuji, T. Ohi-Toma & K. Watanabe-Toma 136-1, 4x (1.957), type V & T. Ohi-Toma & K. Watanabe-Toma 136-2, 4x (2.082), type V; Shizuoka, Shizuoka, T. Ohi-Toma & K. Watanabe-Toma 137, 2x, type E.

Helwingia japonica subsp. liukiuensis – Okinawa, Mt. Katsuu, T. Ohi 093, 2x, type R; Kagoshima, Tokunoshima, T. Ohi 094, 2x, type R; Kagoshima, Amami-Oshima, T. Ohi 095, 2x, type R; Taiwan, Hsinchu, Jianshih, S. C. Wu TW02, 2x, type H & S. C. Wu TW03, 2x, type H.

Appendix 2.List of samples of Helwingia in China used in

the chloroplast analysis. Haplotypes are types X1–X5 (accession no. LC311049–LC311057). Their voucher specimens are kept in herbarium TI.

Helwingia japonica – Zhejiang, T. Ohi-Toma & al. 20090805, type X3; Hubei, T. Ohi-Toma & al. 20090810, type X2; Sichuan, T. Ohi-Toma & al. 20090427, type X3; Sichuan, H. Hara A2432, type X3; Sichuan, H. Hara A2433, type X3; Sichuan, H. Hara A2436, type X3; Yunnan, J. Murata & al. 200607, type X1; Yunnan, H. Ohba 93-219, type X2.

Helwingia chinensis – Hubei, H. Hara A2316, type X2; Sichuan, H. Ohba A2543, type X1; Sichuan, H. Ohba 93-220, type X1; cult. Koishikawa Bot. Gard., Univ. Tokyo (KBGUT), s. coll. 88-36, type X5; cult. KBGUT, s. coll. 88-38, type X1.

Helwingia omeiensis – Hubei, T. Ohi-Toma & al. 20090809, type X3; Sichuan, H. Ohba 93-218, type X1; Sichuan, H. Hara A2546, type X3; Yunnan, T. Ohi & al. 200309, type X4; Yunnan, T. Ohi-Toma 20090223, type X3.

大井・東馬哲雄a，一瀬克久a，渡邉・東馬加奈a,b，池田 博c，邑田 仁a：日本産ハナイカダ（ハナイカダ科）における染色体倍数性と葉緑体 DNAの種内多型　日本産ハナイカダ Helwingia japonica の種内多型を把握するために，台湾を含めた分布域を通して，染色体倍数性と葉緑体 DNA の種内多型を調べた．染色体倍数性は，フローサイトメータを用いて相対ゲノムサイズの比較により推定し，二倍体，四倍体，六倍体が区別できた．

葉緑体 DNA psbA-trnH 領域の塩基配列多型からは，22種類のハプロタイプが区別できた．これらを踏まえると，日本産ハナイカダは 32 タイプに区別することができ，高い多様性を持つことが明らかになった．従来の認識とは異なり，ハナイカダ H. japonica subsp. japonica

December 2017 The Journal of Japanese Botany Vol. 92 No. 6 329

var. japonica は二倍体，四倍体および六倍体を含んでおり，東北には二倍体が，西日本には四倍体が分布していることが明らかとなった．また二倍体は地理的に 3 つの地域に分かれて分布していることも明らかになった．典型的なコバノハナイカダ H. japonica subsp. japonica var. parvifolia とリュウキュウハナイカダ H. japonica subsp. liukuiensis は従来の認識通り，二倍体であることを確認した．二倍体，四倍体および六倍体は地理的に分かれて分布しており，3 集団においてのみ，2 つの異なる倍数性が同所的にあることが観察された．倍数レベルと葉緑

体ハプロタイプの地理的分布，ハプロタイプ間の関係，倍数レベル間におけるハプロタイプの共有を考慮すると，1 つの優占ハプロタイプが西日本での倍数化に貢献したと考えられる．その他の倍数体の多くは，異なるハプロタイプをもつ二倍体から生じたというよりは，優占ハプロタイプで生じた倍数体が遺伝的に多様化したであろうと考えられた．

（a東京大学大学院理学系研究科附属植物園，b武蔵高等学校中学校，

c東京大学総合研究博物館）