J. Jpn. Bot. 92(6): 321–329 (2017)

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

a,d, a,d a,b Tetsuo Ohi-Toma *, Katsuhisa Ichinose , Kana Watanabe-Toma , c a Hiroshi Ikeda and Jin Murata

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: [email protected]

(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 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.

Helwingia Willd., the only in the and Kurosawa 1975, Xiang and Boufford 2005, family Helwingiaceae, is a with an Xiang 2016). epiphyllous on midvein of adaxial Of the species, H. japonica has the widest surface. The genus comprises four species in the range of distribution from the , Sino-Japanese floristic region: two deciduous mainland China through Taiwan to the (H. japonica (Thunb.) F. Dietr. and H. himalaica Japanese archipelago. The species shows large Hook. f. & Thomson ex C. B. Clarke) and morphological variation of shape and size two evergreen (H. chinensis Batalin and H. (Yamanaka 1966), and several intraspecific taxa omeiensis (Fang) H. Hara & S. Kurosawa) (Hara have been described. In the classification of

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Hara and Kurosawa (1975), three subspecies of In the present study, to grasp intraspecific H. japonica were recognized, subsp. japonica variation within Helwingia japonica in Japan, (distributed in Japan, mainland China, and the we investigated the ploidy levels and sequence Himalayas), subsp. liukiuensis (Hatus.) H. Hara variation of the chloroplast psbA-trnH region & S. Kurosawa (in the Ryukyu Islands from through the distributional range. Amami-oshima Island to Okinawa Island), and subsp. formosana (Kaneh. & Sasaki) H. Hara & Materials and Methods S. Kurosawa (in Taiwan). Because the name of Living branches (15–20 cm in length) of the last subspecies is nomenclaturally invalid, Helwingia japonica were collected from a few subsp. taiwaniana Yuen P. Yang & H. Y. Liu plants per a population for cuttings and then has replaced this name. Recently, Ohashi (2017) cultivated in Koishikawa Botanical Gardens, treated subsp. taiwaniana as a synonym of the University of Tokyo (KBGUT). A total subsp. liukiuensis. In addition, several varieties 129 plants of subsp. japonica in Japan and of H. japonica were recognized based on leaf five plants of subsp. liukiuensis (three from morphology in Japan and China (Hara and the Ryukyu Islands and two from Taiwan) that Kurosawa 1975, Xiang and Boufford 2005). were cultivated for more than a decade were In Japan, H. japonica subsp. japonica is used following ploidy and chloroplast DNA widely distributed from southwestern Hokkaido analyses (Appendix 1). We could distinguish through Honshu, Shikoku to Kyushu, and two only typical var. parviflia based on with varieties, var. japonica and var. parvifolia, are flowers but the intermediate forms could not recognized by leaf morphology (Yamanaka be distinguished from typical var. japonica 1966, Hara and Kurosawa 1975, Noshiro 1999). confidently. In the chloroplast analysis, 18 Variety parvifolia, which has smaller leaves (ca. Chinese plants, including H. japonica, H. 2–6 cm long, 1–3 cm wide) with 2–4 pairs of chinensis, and H. omeiensis, were also examined lateral veins and a rather lustrous upper surface, (Appendix 2). is distinguished from var. japonica having larger In the ploidy analysis, firstly, somatic leaves with 4–6 pairs of lateral veins. However, chromosomes of root tip cell from several it is difficult to distinguish them because their plants, which has been kept in KBGUT, were intermediate forms are often observed. observed with optical microscopy according Hara and Kurosawa (1975) also reported that to the method of Yamamoto et al. (2008). A Japanese plants of H. japonica include diploids with known 2n = 38 (diploid; Fig. 1), (2n = 38), tetraploids (2n = 76), and hexaploids whose locality was unknown but morphology (2n = 114). They recognized that var. japonica looks like the intermediate form, was used is a hexaploid distributed from southwestern as a standard in the flow cytometric (FCM) Hokkaido to Kyushu, var. parvifolia is diploid analysis. Nuclei from a piece of fresh leaf (0.5 (and rarely tetraploid) in southwestern Honshu, cm2) were isolated and stained by Cystain UV Shikoku, and Kyushu, and their intermediate precise P Kit (Partec GmbH). After running on forms observed in west Japan are tetraploid. a Ploidy Analyzer PA (Partec GmbH), a peak Noshiro (1999) considered var. parvifolia and position of the fluorescence histogram of relative the intermediate forms as diploid in central genome size was compared with the standard and south Honshu, Shikoku, and Kyushu and by the peak analysis implemented in the Ploidy tetraploid in south-western Honshu, Shikoku, Analyzer. Based on the value of the peak index, and Kyushu, respectively. However, ploidy level ploidy levels were estimated, and samples within the species has been recognized based on indistinguishable from the standard were treated few plants from limited areas. as diploid. December 2017 The Journal of Japanese Botany Vol. 92 No. 6 323

using TCS version 1.21 (Clement et al. 2000) with 99% confidence limits and gap missing.

Results For all the cultivated plants of Helwingia japonica, diploid (2x), tetraploid (4x, peak index = 1.918–2.082), and hexaploid (6x, peak index = 2.828–3.377) were detected based on relative genome size against the diploid standard in FCM analysis (Appendix 1). For representatives of each ploidy level, the somatic chromosomes were confirmed by optical microscopy. Twelve plants identified as typical var. parvifolia were Fig. 1. Somatic chromosomes of a diploid standard (2n = diploid, and some plants of var. japonica and the 38) of Helwingia japonica used in the flow cytometric intermediate forms were also diploid. Tetraploid analysis. Bar = 10 μm. and hexaploid were observed from var. japonica and the intermediate forms. Five plants of subsp. Total genomic DNA was extracted from liukiuensis were diploid. silica-gel-dried leaf tissue, using the modified In the chloroplast DNA analysis, despite HEPES-CTAB method described by Ohi- the short length of psbA-trnH (342–462 bp), Toma et al. (2010). Nucleotide sequences 22 haplotypes (A–V) were distinguished based of chloroplast psbA-trnH intergenic spacer on nine substitutions, seven tandem repeats by (Hamilton 1999) were determined using slippage, and one inverted repeat for Japanese polymerase chain reaction (PCR) and direct and Taiwanese plants (Appendix 1). All kinds bi-directional sequencing. PCR amplification of haplotypes were detected from H. japonica was conducted using TaKaRa ExTaq (TaKaRa subsp. japonica in Japan. The plants of subsp. Bio), with the following cycling conditions: liukiuensis in the Ryukyu Islands and Taiwan denaturation at 96 °C for 45 sec, followed by have haplotype R and H, respectively. In 30 cycles at 96 °C for 45 sec, annealing at 50 addition, five haplotypes different from Japan °C for 45 sec, extension at 72 °C for 1 min, and Taiwan (X1–X5) were shared among H. and a final extension at 72 °C for 10 min. After japonica, H. chinensis, and H. omeiensis in purifying by using GeneClean III kit (Bio China (Appendix 2). In the haplotype network, 101) or illustra ExoProStar (GE Healthcare), each haplotype was connected by one or two PCR products were reacted with the Big Dye steps, but their relationship was ambiguous as Terminator v3.1 Cycle Sequencing Kit (Applied loops were observed (Fig. 2). Biosystems). Sequencing was conducted using Of the haplotypes of Helwingia japonica Applied Biosystems 373 Genetic Analyzer. subsp. japonica in Japan, eight haplotypes After nucleotide sequences were manually A, B, F, H, R, S, T, and V were detected from aligned, haplotypes were distinguished based on diploids, 14 haplotypes D, G, H, J, K, L, M, nucleotide substitutions and gaps (tandem repeat N, O, Q, R, T, U, and V were from tetraploids, by slippage and inverted repeat). Haplotype and 10 haplotypes C, D, E, H, I, K, L, N, P, and sequences were deposited in the DNA Data R were from hexaploids (Fig. 2). Haplotype Bank of Japan. H was shared among diploids, tetraploids, After gaps were binary coded as a fifth and hexaploids, and was also found from character, the haplotype network was constructed diploid plants of subsp. liukiuensis in Taiwan. 324 植物研究雑誌 第 92 巻 第 6 号 2017 年 12 月

Fig. 2. Statistical parsimony network of chloroplast psbA-trnH haplotypes. A–V (open symbol) were haplotypes detected from Helwingia japonica in Japan and Taiwan, and X1–X5 (gray symbol) were from H. japonica, H. chinensis, and H. omeiensis in China. Small solid circle is a missing haplotype. Each line connecting haplotypes indicates a single mutation. Open circle, open diamond, and open hexagon are diploid, tetraploid, and hexaploid, respectively.

Haplotypes T and V were shared between three populations of central Honshu, two diploids and tetraploids. Five haplotypes D, K, different ploidy levels were distributed together, L, N and R were shared between tetraploids and but did not share the identical haplotypes: one hexaploids. Haplotype R was also found from consisting of diploid (haplotype F) and tetraploid diploid plants of subsp. liukiuensis in the Ryukyu (haplotype D) in Aichi Prefecture, and two Islands. In the haplotype network, haplotypes consisting of diploid (haplotype S) and hexaploid from diploids, tetraploids, and hexaploids were (haplotype D) in Yamanashi Prefecture. scattered in tip and interior positions. Most A few haplotypes have a wide geographical haplotypes of polyploids were connected to range; haplotype H in west Japan, haplotypes A haplotype H at the interior position by one or in Tohoku, and haplotype D in central Honshu. two steps. Most of other haplotypes were localized in The geographical distribution of the ploidy limited areas. levels and haplotypes were plotted on the map (Fig. 3). Diploids were distributed in (1) Discussion Hokkaido and Tohoku region, (2) the pacific side We detected three ploidy levels (diploid, of central Honshu, (3) Shikoku, west Chugoku, tetraploid, and hexaploid) and 22 haplotypes and Kyushu, and (4) the Ryukyu Islands (A–V) from Helwingia japonica in Japan and and Taiwan (subsp. liukiuensis). Tetraploids Taiwan. Based on the combination of ploidy were found from central and western Honshu level and haplotype, 32 types of intraspecific through Shikoku to Kyusyu. Hexaploids were variation were distinguished, indicating a high continuously distributed from south Tohoku degree of intraspecific diversity. through Kanto region to central and western In the previous recognition of Helwingia Honshu (mainly Sea of Japan side). In only japonica subsp. japonica in Japan (Hara December 2017 The Journal of Japanese Botany Vol. 92 No. 6 325 haplotypes. psbA-trnH in Japan and Taiwan. One symbol is one plant and its shape and color indicate a indicate color and shape its and plant one is symbol One Taiwan. and Japan in Helwingia japonica japonica Helwingia Geographical distribution of ploidy levels and haplotypes of of haplotypes and levels ploidy of distribution Geographical

ploidy level: white circle (diploid), gray diamond (tetraploid), and black hexagon (hexaploid). Within each symbol, A–V are chloroplast chloroplast are A–V symbol, each Within (hexaploid). hexagon black and (tetraploid), diamond gray (diploid), circle white level: ploidy Fig. 3. 3. Fig. 326 植物研究雑誌 第 92 巻 第 6 号 2017 年 12 月 and Kurosawa 1975, Noshiro 1999), it was subsp. japonica was subdivided into three considered that typical var. parvifolia with isolated areas each characterized by unique smaller leaves was mostly diploid, var. japonica chloroplast haplotypes: (1) Hokkaido and with larger leaves was hexaploid, and the Tohoku region (one-step connected haplotypes intermediate form was tetraploid. In addition, A and B), (2) the Pacific side of central Honshu subsp. liukiuensis in the Ryukyu Islands and (one-step connected haplotypes F and S, and Taiwan were recognized as diploid. Our distantly related haplotype V, that is connected examination through the distributional range to haplotype B by one-step), and (3) Shikoku, revealed that var. japonica includes a ploidy west Chugoku, and Kyushu region (one-step series of diploid, tetraploid, and hexaploid, connected haplotypes H and T). Haplotypes although it confirmed that typical var. parvifolia A and B in the first area were not shared by and subsp. liukiuensis were diploid. In our polyploids. Haplotype H in the third area was results, it is noteworthy that plants of typical var. majority within tetraploids and hexaploids as japonica in Hokkaido and Tohoku region were well as diploids. In addition, haplotype V in the diploid, while some of typical var. japonica in second area and haplotype T in the third area west Japan were tetraploid. Although polyploids were shared by tetraploids, but their ranges sometimes have larger plant size than diploids were localized in the Pacific side of Kanto and (Lewis 1980), the leaf morphology and ploidy south Kyushu, respectively. In polyploidization levels were not necessarily correlated within H. within subsp. japonica, it is likely that the major japonica subsp. japonica. haplotype H has contributed in west Japan. The distributional ranges of the ploidy levels Considering the connection to haplotype H of H. japonica subsp. japonica in Japan seem in the network and non-sharing of haplotype to be roughly segregated, as is known in other with diploids, other polyploids with rare plants (Stebbins 1971). The different distribution haplotypes in west Japan and most polyploids between diploids and polyploids has been often in central Honshu might have been derived by explained as adaptive selection to environments diversification from tetraploids and hexaploids (Lewis 1980). However, ecological factors with the major haplotype H rather than multiple between ploidy levels and their distribution in polyploidzations from different diploids. subsp. japonica are unknown, because diploids Here, haplotype R was shared by distantly of subsp. japonica were distributed in a more isolated different subspecies and different northern area than polyploids in addition to ploidy levels, a tetraploid and a hexaploid of southwestern area and the Pacific side. subsp. japonica in Kinki region of Honshu and In three sympatric populations that show two diploids of subsp. liukiuensis in the Ryukyu different ploidy levels, diploid and tetraploid Island. The process of distributional pattern and do not share the identical or one step connected polyploidization involved by haplotype R needs haplotype: i.e., diploid (haplotype F) and further investigation. tetraploid (haplotype D) in Aichi Pref., and The forming process of morphological diploid (haplotype S) and hexaploid (haplotype variation, high intraspecific diversity, its D) in two populations of Yamanashi Pref. geographical pattern and polyploidization Thus, the polyploids in these populations within H. japonica seem to be complicated. It is were not derived from the sympatric diploids expected that the investigation by nuclear DNA directly. Reproductive isolation between ploidy at the population level can clarify the process of levels may become one of the factors of their polyploidizations and the reproductive isolation geographical segregation in subsp. japonica. among ploidy levels. The distributional range of diploids of December 2017 The Journal of Japanese Botany Vol. 92 No. 6 327

The authors thank staff of Koishikawa the index value of peak analysis against the diploid (2x) Botanical Gardens, the University of Tokyo standard was shown in parentheses. Haplotypes are types A–V (accession no. LC311027–LC311048). Their voucher for cultivating plants, and Dr. T. Kurosawa, specimens are kept in herbarium TI. Dr. T. Nemoto, Dr. Y. Endo, Dr. Y. Iokawa, Y. Helwingia japonica subsp. japonica – Ishikawa, Yamaguchi, K. Sasamura, Dr. S. C. Wu, Dr. S. Hakusan, T. Ohi 001, 6x (2.964), type H; Fukui, Echizen, G. Wu, and Dr. C. X. Fu for collecting plants. T. Ohi 002, 6x (2.999), type D; Fukui, Obama, T. Ohi 003, The authors would like to thank Dr. H. Ohashi 6x (2.917), type H; Kyoto, Fukuchiyama, T. Ohi 004, 6x (2.910), type H; Tottori, Iwami, T. Ohi 005, 6x (2.898), type for his encouragement and valuable comments. H; Tottori, Chizu, T. Ohi 006, 6x (2.967), type H; Tottori, Kofu, T. Ohi 008, 6x (3.192), type N; Shimane, Misato, T. 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Ohi 047-1, Wu Z. Y., Raven P. H. and Hong D. Y. (eds.), Flora 4x (2.029), type J & T. Ohi 047-3, 4x (1.972), type H; Mie, of China 14: 227–229. Science Press, Beijing and Odai, T. Ohi 051-1, 4x (2.025), type N & T. Ohi 051-2, 4x Missouri Botanical Garden Press, St. Louis. (2.044), type H; Toyama, Toyama, T. Ohi 052, 6x (2.899), Yamamoto N., Ikeda H. and Hoshino T. 2008. type D; Gunma, Mt. Akagi, T. Ohi 053, 6x (2.959), type D; Cytotaxonomical studies of flowering plants in Niigata, Minami-Uonuma, T. Ohi 054, 6x (3.110), type L; Yakushima Island, Kagoshima Pefecture, Japan. Part I: Fukushima, Aizu-Misato, T. Ohi 057-1, 6x (3.008), type K dwarf taxa. J. Phytogeogr. Taxon. 56: 79–93. & T. Ohi 057-2, 6x (3.001), type D; Yamagata, Shirataka, Yamanaka T. 1966. On Helwingia japonica var. parvifolia. T. Ohi 059, 2x, type A; Yamagata, Tsuruoka, T. Ohi 060, J. Jap. Bot. 41: 375–380 (in Japanese). 2x, type B; Aomori, Aomori, T. Ohi 066, 2x, type A; Iwate, Hirono, T. Ohi 067, 2x, type A; Iwate, Hanamaki, T. Ohi Appendix 1. 068, 2x, type A; Miyagi, Kurihara, T. Ohi 069, 2x, type List of samples of Helwingia japonica in Japan and A; Miyagi, Sendai, T. Ohi 070, 2x, type A; Fukushima, Taiwan with their voucher, ploidy level, and chloroplast Date, T. Ohi 071-1, 6x (2.951), type C & T. Ohi 071-2, 6x psbA-trnH haplotype. Samples of the typical var. parvifolia (3.060), type C; Tochigi, Imaichi, T. Ohi 072, 6x (3.067), were underlined. For tetraploids (4x) and hexaploids (6x), type D; Shiga, Taga, T. Ohi 073-1, 6x (2.891), type H & 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 123-1, 6x (3.022), type P & T. Ohi-Toma 123-2, 6x (3.092), 074, 6x (2.828), type D; Aichi, Shinshiro, T. Ohi 075-1, 4x type P; Hiroshima, Jinsekikogen, T. Ohi-Toma 124-1, 4x (1.994), type D & T. Ohi 075-2, 2x, type F; Nagano, Ina, T. (2.016), type H & T. Ohi-Toma 124-2, 4x (1.956), type Ohi 076-2, 6x (2.914), type D & T. Ohi 076-3, 6x (3.036), H; Iwate, Morioka, T. Nemoto 125, 2x, type A; Saitama, type D; Gifu, Nakatsugawa, T. Ohi 077-1, 4x (1.974), type Hanno, K. Ichinose 127, 6x (3.325), type D; Aomori, D & T. Ohi 077-3, 4x (1.973), type D; Gifu, Gero, T. Ohi Towada, K. Sasamura 129, 2x, type A; Akita, Nikaho, K. 078, 6x (2.925), type H; Gifu, Takayama, T. Ohi 079, 6x Sasamura 130, 2x, type A; Ibaraki, Mito, Y. Endo 132, 6x (2.945), type D; Chiba, Minami-Boso, T. Ohi 080, 2x, (3.017), type D; Chiba, Shibayama, T. Ohi-Toma 133, 2x, type S; Shizuoka, Shizuoka, T. Ohi 081-2, 2x, type F & type V; Chiba, Funabashi, T. Ohi-Toma 134, 2x, type V; T. 081-3, 2x, type F; Yamanashi, Fujikawa, T. Ohi 082-1, Shizuoka, Fuji, T. Ohi-Toma & K. Watanabe-Toma 136-1, 2x, type S & T. Ohi 082-2, 6x (2.907), type D; Shizuoka, 4x (1.957), type V & T. Ohi-Toma & K. Watanabe-Toma Izu, T. Ohi 083, 2x, type F; Kanagawa, Mastuda, T. Ohi 136-2, 4x (2.082), type V; Shizuoka, Shizuoka, T. Ohi- 084, 6x (2.985), type D; Tokyo, Izu-Oshima, T. Ohi 085, Toma & K. Watanabe-Toma 137, 2x, type E. 6x (3.377), type D; Ibaraki, Daigo, T. Ohi 086, 6x (3.075), Helwingia japonica subsp. liukiuensis – Okinawa, Mt. type D; Shimane, Nishinoshima, T. Ohi 089, 6x (3.074), Katsuu, T. Ohi 093, 2x, type R; Kagoshima, Tokunoshima, type H; Shimane, Okinoshima, T. Ohi 090, 6x (3.024), type T. Ohi 094, 2x, type R; Kagoshima, Amami-Oshima, T. H; Tokyo, Ome, T. Ohi 092, 6x (3.061), type D; Shizuoka, Ohi 095, 2x, type R; Taiwan, Hsinchu, Jianshih, S. C. Wu Shimoda, T. Ohi 096-1, 2x, type S & T. Ohi 096-2, 2x, TW02, 2x, type H & S. C. Wu TW03, 2x, type H. type S; Nagasaki, Tsushima, K. Watanabe 099, 2x, type H; Nagano, Karuizawa, Y. Yamaguchi 100, 6x (2.980), type Appendix 2. C; Nagano, Tatsuno, T. Ohi-Toma 101, 6x (2.955), type List of samples of Helwingia in China used in D; Gifu, Ogaki, T. Ohi-Toma 102-1, 6x (2.984), type D the chloroplast analysis. Haplotypes are types X1–X5 & T. Ohi-Toma 102-2, 6x (3.025), type D; Nara, Uda, T. (accession no. LC311049–LC311057). Their voucher Ohi-Toma 103-1, 4x (1.966), type H & T. Ohi-Toma 103- specimens are kept in herbarium TI. 2, 4x (2.038), type H; Yamanashi, Ichikawa-Misato, K. Helwingia japonica – Zhejiang, T. Ohi-Toma & al. Ichinose 104-1, 2x, type S & K. Ichinose 104-3, 6x (3.105), 20090805, type X3; Hubei, T. Ohi-Toma & al. 20090810, type D; Gifu, Gujo, T. Ohi-Toma 108, 6x (2.993), type H; type X2; Sichuan, T. Ohi-Toma & al. 20090427, type Miyagi, Ishinomaki, T. Nemoto 109-1, 2x, type A & T. X3; Sichuan, H. Hara A2432, type X3; Sichuan, H. Hara Nemoto 109-3, 2x, type A; Ibaraki, Mt. Tsukuba, T. Ohi- A2433, type X3; Sichuan, H. Hara A2436, type X3; Toma 111-2, 6x (3.021), type D & T. Ohi-Toma 111-2, 6x Yunnan, J. Murata & al. 200607, type X1; Yunnan, H. (3.076), type C; Hokkaido, Hakodate, K. Ichinose 112, Ohba 93-219, type X2. 2x, type A; Tochigi, Nasu-Shiobara, K. Ichinose 113, 6x – Hubei, H. Hara A2316, type (2.961), type D; Niigata, Aga, T. Ohi-Toma 115, 6x (3.063), X2; Sichuan, H. Ohba A2543, type X1; Sichuan, H. Ohba type E; Niigata, Joetsu, Y. Iokawa 116, 6x (3.024), type E; 93-220, type X1; cult. Koishikawa Bot. Gard., Univ. Tokyo Aomori, Mt. Hakamagoshi, T. Nemoto 117, 2x, type A; (KBGUT), s. coll. 88-36, type X5; cult. KBGUT, s. coll. Ibaraki, Kita-Ibaraki, Y. Endo 119-1, 6x (3.044), type D & 88-38, type X1. Y. Endo 119-2, 6x (2.976), type D; Fukushima, Iwaki, T. Helwingia omeiensis – Hubei, T. Ohi-Toma & al. Nemoto 120-1, 2x, type A & T. Nemoto 120-2, 2x, type A; 20090809, type X3; Sichuan, H. Ohba 93-218, type X1; Fukushima, Namie, T. Nemoto 121-1, 6x (3.049), type D & Sichuan, H. Hara A2546, type X3; Yunnan, T. Ohi & al. T. Nemoto 121-2, 6x (3.152), type C; Okayama, Niimi, T. 200309, type X4; Yunnan, T. Ohi-Toma 20090223, type Ohi-Toma 122-1, 4x (1.994), type Q & T. Ohi-Toma 122- X3. 2, 4x (2.003), type Q; Okayama, Kagamino, T. Ohi-Toma

大井・東馬哲雄a，一瀬克久a，渡邉・東馬加奈a,b，池田 博c， a 邑田 仁 ：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 葉緑体 DNA psbA-trnH 領域の塩基配列多型からは，22 握するために，台湾を含めた分布域を通して，染色体倍 種類のハプロタイプが区別できた．これらを踏まえる 数性と葉緑体 DNA の種内多型を調べた．染色体倍数性 と，日本産ハナイカダは 32 タイプに区別することがで は，フローサイトメータを用いて相対ゲノムサイズの比 き，高い多様性を持つことが明らかになった．従来の認 較により推定し，二倍体，四倍体，六倍体が区別できた． 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 体ハプロタイプの地理的分布，ハプロタイプ間の関係， り，東北には二倍体が，西日本には四倍体が分布してい 倍数レベル間におけるハプロタイプの共有を考慮する ることが明らかとなった．また二倍体は地理的に 3 つの と，1 つの優占ハプロタイプが西日本での倍数化に貢献 地域に分かれて分布していることも明らかになった．典 したと考えられる．その他の倍数体の多くは，異なるハ 型的なコバノハナイカダ H. japonica subsp. japonica var. プロタイプをもつ二倍体から生じたというよりは，優占 parvifolia とリュウキュウハナイカダ H. japonica subsp. ハプロタイプで生じた倍数体が遺伝的に多様化したで liukuiensis は従来の認識通り，二倍体であることを確認 あろうと考えられた． した．二倍体，四倍体および六倍体は地理的に分かれて （a東京大学大学院理学系研究科附属植物園， 分布しており，3 集団においてのみ，2 つの異なる倍数 b武蔵高等学校中学校， 性が同所的にあることが観察された．倍数レベルと葉緑 c東京大学総合研究博物館）