Intraspecific Polyploidy of Houttuynia Cordata and Evolution of Chromosome Number in the Saururaceae

Intraspecific polyploidy ofHouttuynia cordata and evolution of chromosome number in the Saururaceae

Kazuo Oginuma1,8, Hisako Sato2, Yoshiko Kono2, Shaotien Chen3, Zuken Zhou3, Ching-I. Peng4, Arata Momohara5, Tomohisa Yukawa6 and Hiroaki Setoguchi7

1Department of Environmental Science, Faculty of Human Life and Environmental Science, Kochi Women’s University, Eikokuji-cho 5-15, Kochi 780-8515, Japan; 2Graduate School of Human Health Science, Kochi Women’s University, Eikokuji-cho 5-15, Kochi 780-8515, Japan; 3Department of Phytotaxonomy and Phytogeography, Kunming Institute of Botany, The Chinese Academy of Sciences, Heilongtan, Kunming 650204, Yunnan, The People’s Republic of China; 4Herbarium (HAST), Research Center for Biodiversity, Academia Sinica, Taipei 115, Taiwan; 5Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan; 6Tsukuba Botanical Garden, National Science Museum,Tsukuba-shi, Ibaraki 305-0005, Japan; 7Department of Biology, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu-cho, Sakyo-ku, Kyoto-shi, Kyoto 606-8501, Japan

8Author for correspondence ([email protected]) Received July 19, 2007; accepted August 25, 2007

ABSTRACT. Intraspecific polyploidy and cytogeography were clarified in the samples of Houttuynia cordata (Saururaceae) studied in the Sino-Japanese region of eastern Asia, ranging from Nepal, China, northern Thailand, Taiwan to Japan. Five chromosome numbers of 2n=72, 80, 96, 112 and 128 were detected, four of which were firstly recorded here. These chromosome numbers suggested that the basic chromosome number of Houttuynia could be x=8. The observed cytotypes were evaluated as 2n=72, 80, 96, 112 and 128, and represented 9x, 10x, 12x, 14x, and 16x, respectively. The continental part of east Asia harbored intraspecific polyploidy ranging from x9 to 16x, and only one cytotype of 12x or 2n=96 was found from the eastern edge of the Asian continent between Taiwan and Japan. The evolution of basic chromosome number was discussed based on a phylogenetic tree of the Saururaceae, suggesting that x=11 was an archaic basic chromosome number in this family. Houttuynia may have experienced a disploid reduction from x=11 to x=8, as an autoapomorphy and subsequent intraspecific polyploidization in continental eastern Asia.

KEYWORDS: Chromosome, Eastern Asia, Houttuynia cordata, Saururaceae, Sino-Japanese region

Houttuynia Thunb. is monotypic and a member of the the wide distribution of this species: n=48 (Japan, Mihara Saururaceae together with three other genera of Anemopsis, 1960), 2n=24 (Taiwan, Hsu 1968), n=52-56 (Japan, Shibata Gymnotheca and Saururas (The Angiosperm Phylogeny and Miyake 1908), 2n=ca. 96 (Japan, Okabe 1934; Nepal, Group [APG II] 2003). Phylogenetic relationships of Kurosawa 1966), 2n=96 (Japan, Okada 1986), and these four genera have been reported based on DNA 2n=100-104 (unknown location, Soderberg 1927). The sequence data; however, the phylogenetic position of long range of H. cordata in the Sino-Japanese region, Houttuynia conflicts in the organelle and nuclear DNA covering over 5000 km, suggests that migration and trees (Meng et al. 2002, 2003). Chloroplast (cp) and intraspecific evolution of H. cordata may have occurred mitochondrial (mt) DNA sequence data suggest that separately in several areas. Intraspecific diversity and Houttuynia is sister to Anemopsis, whereas nuclear (n) geographic structure of chromosome number are expected DNA suggests that Houttuynia is sister to the Saururus- along the corridor in the Sino-Japanese region. Gymnotheca clade. The basic chromosome number of Houttuynia is Houttuynia cordata Thunb., only the species in the controversial, as is the Saururacean chromosome number. genus, is a perennial herb mainly distributed in the Sino- The basic chromosome number of Houttuynia is thought Japanese region of eastern Asia, ranging from Japan to to be x=12 (Okada 1986), while the basic number is x=22 the Himalayas through the Ryukyu Islands, Taiwan, and or 11 (2n=44) in Anemopsis, x=9 (2n=18) in Gymnotheca, China, and extending to southeast Asia. In Japan, the and x=11 (2n=22) in Saururas. Based on variation in species propagates by formation and separation of chromosome number within Houttuynia, the basic chro- underground stems and by parthenogenesis (Shibata and mosome number should be redetermined to understand Miyake 1908; Mihara 1960), whereas reproduction by the evolution of chromosome number in the Saururaceae, sexual reproduction has not been reported. The reproduc- a paleoherb group, i.e., to determine the plesiomorphic tive characteristics of parthenogenetic and microspore characteristic of chromosome number in this paleoherb degeneration are widely accepted (e.g., Mabberley 1998). family. Various chromosome numbers have been reported from Here we present intraspecific polyploidy with new

87 88 OGINUMA ET AL. chromosome data in H. cordata collected from Japan, 3) 2n=96 One plant each collected in Southeast China Nepal, Thailand, The People’s Republic of China and (Figs. 4 and 9 [collection site 11], Table 1) and Japan Taiwan. We examined the presence or absence of chromo- (Figs. 5 and 9 [collection site 14], Table 1) and four plants some number diversity and discuss its cytogeographical collected from two localities in Taiwan (Figs. 6 and 9 aspects, in addition to discussing chromosome evolution [collection sites 12 and 13], Table 1) showed 2n=96. This in the Saururaceae by combining our original data and chromosome number confirms previous reports (Mihara previously published data. 1960; Okada 1986).

MATERIALS AND METHODS The localities where H. cordata individuals were collected are shown in Table 1. Individual plants were collected in the field and cultivated in the greenhouse, Kochi Women’s University, Japan. The pretreatment, fixation, and chromosome-staining methods were described elsewhere (Oginuma and Nakata 1988). Voucher specimens were deposited in the Herbaria of Kyoto University (KYO) and Academia Sinica, Taipei (HAST). The evolutionary trend of basic chromosome number in the Saururaceae was estimated using the ACCTRAN transformation in MacClade version 3.05 (Maddison and Maddison 1992), based on the Saururacean phylogeny (Meng et al. 2003).

RESULTS AND DISCUSSION Somatic chromosome numbers at metaphase in the plants collected in 14 localities were 2n=72, 80, 96, 112 and 128. Those chromosomes showed gradual variation in length, ranging from 0.6-1.6 µm (Figs. 1-8).

1) 2n=72 The ten plants collected in five localities in China (Figs. 1 and 9 [collection sites 5-8 and 10], Table 1) and two plants collected from Thailand (Figs. 2 and 9 [collection site 3], Table 1) were 2n=72, that was the first Figs. 1-8. Somatic chromosomes at metaphase for record of this chromosome number for this species. intraspecific polyploidy determination of Houttuynia cordata. 1. 2n=72 (Jin Tou, China). 2. 2n=72 (Chiang 2) 2n=80 One plant collected from Teng Chon, Yunnan Mai, Thailand). 3. 2n=80 (Teng Chong, China). 4. 2n=96 (Chong Zuo, China). 5. 2n=96 (Kochi, Japan). 6. 2n=96 County, China, was 2n=80 (Figs. 3 and 9 [collection site (Yangming Shan, Taiwan). 7. 2n=112 (Kakani, Nepal). 8. 4]), that was reported here for the first time. 2n=128 (Teng Chong, China). Scale = 2 µm.

Table 1. Chromosome number of Houttuynia cordata and its collections Chromosome No. of plants Collections number observed 2n=72 China. Yunnan County, ca. 15 km south from Gong Shang. S.Chen & H. Setoguchi 2004073 (KYO) 2 China. Yunnan County, near FuGong, south of Gong Shang. S. Chen & H. Setoguchi 2004074 (KYO) 2 China. Yunnan County, Gong Shang, S. Chen & H. Setoguchi 2004063G (KYO) 2 China. Yunnan County, Xinxian Xiang - Wan Fou Jian. H. Setoguchi et al. 2004378 (KYO) 2 China. Yunnan County, Jin Tou. H. Setoguchi et al. 200611(KYO) 2 Thailand. Chiang Mai, Queen Sirkit Bot. Gard. H. Funakoshi s.n. in 2006 (KYO) 2 2n=80 China. Yunnan County, Tengchong. H. Setoguchi et al. 200410 (KYO) 1 2n=96 Taiwan. Taipei County, Chingshan Waterfall. H. Setoguchi 04T-M1905 (KYO) 2 2n=96 Taiwan. Taipei County, Yangmingshan. Oginuma 0602 (KYO) 2 2n=96 China. Guangxi Zhuangzu Zizhiqu, Chong Zuo Shi. Peng 19746 (HAST) 2 2n=96 Japan. Kochi City. Oginuma 0601 (KYO) 2 2n=112 Nepal. Kathmandu County, Godawari. Oginuma 0602 (KYO) 2 2n=112 Nepal. Nuwakot County, Kakani. Oginuma 0603 (KYO) 2 2n=128 China. Yunnan County, Pin Bian. H. Setoguchi 2004389 (KYO) 2 2n=128 China. Yunnan County, Tengchong. H. Setoguchi et al. 200410 (KYO) 1 INTRASPECIFIC POLYPLOIDY OF HOUTTUYNIA CORDATA 89

Fig. 9. Cytogeographical distribution of intraspecific polyploidy in Houttuynia cordata. 1. Kakani, Nepal (2n=112). 2. Godawari, Nepal (2n=112). 3. Chiang Mai, Thailand (2n=72). 4. Teng Chong, China (2n=80, 2n=112). 5. Jin Tou, China (2n=72). 6. Fu Gong, China (2n=72). 7. Southern Fu Gong, China (2n=72). 8. Gong Sahan, China (2n=72). 9. Pin Bian, China (2n=128). 10. Xingxian Xiang, China (2n=72). 11. Chang Zuo, China (2n=96). 12. Yangming Shan, Taiwan (2n=96). 13. Ching Shan waterfall, Taiwan (2n=96). 14. Kochi, Japan (2n=96).

4) 2n=112 Four plants collected from two localities in South China, whereas the cytotype of 2n=112 plant was Nepal showed 2n=112 (Figs. 7 and 9 [collection sites 1 found in Nepal and that of 2n=72 plant was found in and 2], Table 1), that was reported here for the first time. north Thailand. The chromosome number of 2n=ca. 96 was reported earlier in Nepal plants (Kurosawa 1966). 5) 2n=128 Three plants collected in two localities in The individual with the chromosome number of 2n=96 China were 2n=128 (Figs. 8 and 9 [collection sites 4 and may be distributed widely throughout the Sino-Japanese 9], Table 1), that was reported here for the first time. region from the Himalayas to Japan down to South China Houttuynia cordata showed here five different chro- and Taiwan. The continental part of the region harbors mosome numbers, four of which were recorded here for intraspecific polyploidy, ranging from 9x to 16x, and the the first time. However, the species did not verified and 12x (2n=96) cytotype might have migrated and/or prove the previous counts of 2n=24 (Taiwan, Hsu 1968), survived on the eastern edge of the Asian continent, n=52-56 (Shibata and Miyake 1908), and 2n=100-104 between Taiwan and Japan. Recent phylogeographic (Soderberg 1927) during the course of investigation. Our studies in the Sino-Japanese region suggested that some present data and the previous records such as 2n=96 by plant taxa such as Spiraea and Ainsliaea might have Okada (1986), 2n=ca. 96 by Okabe (1934) and Kurosawa allopatrically evolved in the eastern (southeast China – (1966) suggested that the basic chromosome number of Taiwan – Japan) and western (Himalayas – southwest Houttuynia could be x=8. Then, the chromosome number China) sides of the Sino-Japanese region, which is of 2n=24 in the plant from Taiwan (Hsu 1968) would be attributable to the Himalayan uplift in the late Tertiary to either triploid with x=8, or it may represent an erroneous the early Quaternary (Zhang et al. 2006, Mitsui et al. chromosome count. Although some individuals obtained unpublished). The present study showed that the eastern from the locality of the species studied by Hsu (1968) side of the Sino-Japanese region harbored only the 12x were re-investigated in the present work to find the (2n=96) plant, suggesting that eastern China to Japan chromosome number of 2n=24 counted by Hsu (1968), through Taiwan comprises a monotypic 12x cytotype. they showed always the chromosome number of 2n=96. However, our collection sites were insufficient to support Thus, based on the basic chromosome number x=8, the this hypothesis, and further cytological observations are observed cytotypes were evaluated as 9x (2n=72), 10x needed to clarify the cytogeographical distributions of H. (2n=80), 12x (2n=96), 14x (2n=112) and 16x (2n=128). cordata in the Sino-Japanese region. In this study, the chromosome number 2n=72 was Cytogeography of Houttuynia Four of the five cyto- estimated to be 9x, suggesting individuals with this types such as 2n=72, 80, 96 and 128 plants occurred in genome set are infertile. In addition, the Japanese H. 90 OGINUMA ET AL. cordata plants (12x, 2n=96) are reported to be male- the ACCTRAN transformation (Fig. 10). Meng et al. sterile; only parthenogenesis and vegetative reproduction (2003) presented two phylogenetic trees with inconsistent could contribute to propagation of these plants (Okabe topology based on cpDNA and nDNA sequence data. 1934, Mihara 1960). However, the polyploidy found here However, these authors had used only Zipperia (2n=38; suggests the occurrence of sexual reproduction associated Piperaceae) as an outgroup for the Saururaceae, although with male fertility and female meiosis at each ploidy basic chromosome numbers of the Piperaceae are variable level. In our preliminary observation of pollen fertility of (x=11, 13, and 19). Therefore, we discuss the evolutionary 12x (2n=96) plants from Taiwan (population at Ching trend of basic chromosome number on the phylogenetic Shan Waterfall, Table 1), approximately 10% of pollen tree based on parsimonious reconstruction of the character grains were successfully stained with lactophenol state. cottonblue, suggesting they may be fertile. In addition, Based on a parsimonious comparison of the two the stained pollen grains varied in size, which may be evolutionary patterns, chromosome number evolution ascribed to meiosis with unequal chromosome distribution. can be traced with fewer steps on the nDNA-based tree. Formation of the five ploidy levels in H. cordata should In this tree (Fig. 10), Zippelia begoniaefolia with 2n=38 be attributed to the fortuitous formation of fertile game- (Okada 1986) was used as an outgroup. Based on karyo- tophytes with or without meiosis and crossing between individuals of different ploidy levels (e.g., 48 [6x, n] + 80 [10x, 2n]=128). Observations of fertility and polyploidy of gametophytes are needed to clarify the origin of intraspecific polyploidy in association with seed dispersal and range expansion of H. cordata.

Chromosome evolution in Saururaceae The basic chromosome number of the Saururacean genera has been reported as x=22 for Anemopsis, x=9 for Gymnotheca, x=11 for Saururas, and x=12 for Houttuynia (Table 2). However, the basic chromosome number of Houttuynia is corrected to x=8 here as described above. Fig. 10. Chromosome evolution in the Saururaceae. The phylogenetic tree was drawn following Meng et al. (2003). The evolutionary trend of basic chromosome number was A triangle indicates that polyploidization occurred in that mapped on a phylogenetic tree of the Saururaceae using lineage. See text for further explanation.

Table 2. Chromosome number and basic chromosome number of Saururaceae Total number Number of Genus/Species Chromosome number Base number Refernces of species examined species Anemopsis calfornica 1 1 n=22 x=22 Raven et al. (1965), Pinkava et al. (1976) 2n=44 Okada (1986)

Gymnotheca chinensis 2 2 2n=18 x=9 Liang (1991) G. involucrata 2n=18 Liang, (1991)

H. cordata 1 1 n=52-56 x=8 Shibata & Miyake (1908) 2n=100-104 Soderberg (1927) 2n=ca.96 Okabe (1934) n=48 Mihara (1960) 2n=ca. 96 Kurosawa (1966) 2n=24 Hsu (1968) 2n=96 Okada (1986) 2n=72, 80, Present study 96, 112, 128

Saurus cernuus 2 2 2n=22 x=11 Baldwin & Speese (1949), Okada (1986) S. chinensis 2n=22 Suzuka (1950), Chuang, et al. (1963), Okada (1986), Ge et al. (1989) INTRASPECIFIC POLYPLOIDY OF HOUTTUYNIA CORDATA 91 morphology, Okada (1986) suggested that the 2n=38 of vascular plants of Taiwan (II). Taiwaniana 14: 11-27. Zippelia might originate from x=22, which is a doubling Kurosaw, S. 1966. Cytological studies on some Eastern Himalayan plants, pp. 658-670. In: H. Hara ed., The flora of x=11, and a subsequent disploid reduction from 2n=44 of Eastern Himalaya. Univ. Tokyo Press. to 2n=38. Thus, the original basic chromosome number Liang, H. X. 1991. Karyomorphology of Gymnotheca and of x=19 of Zippelia would be b=11. Thus, karyomorphology phylogeny of four genera in Saururaceae. Acta Bot. suggests that x=11 is an archaic basic chromosome Yunnan. 13: 303-307. Mabberley, D. J. 1998. The Plant Book. A portable dictionary number in the Saururaceae. Okada (1986) also suggested of the vascular plants, 2nd ed. Cambridge University that the basic chromosome number of the Piperaceae, a Press, Cambridge. sister family of the Saururaceae, is x=11. Therefore, x=11 Maddison, W. P. and Maddion, D. R. 1992. MacClade: Analysis of Phylogeny and Character Evolution, ver. 3.0. should be a symplesiomorphic character in the Piperaceae- Sinauer, Sunderland. Saururaceae clade. Meng, S. W., Chen. Z. D., Li, D. Z. and Liang, H. Z. 2002. Polyploidization might have occurred from the basic Phylogeny of Saururaceae based on mitochondrial matR chromosome number of x=11 to x=22, in the ancestor of gene sequence data. J. Plant Res. 115: 71-76. Meng, S. W., Douglas, A. W., Li, D. Z., Chen, Z. D. and the genus Anemopsis and its sister group. In the Houttuynia Yang, J. B. 2003. Phylogeny of Saururaceae based on – Saururus – Gymnotheca clade, Houttuynia may have morphology and five regions from three plant genomes. experienced a disploid reduction from the basic chromo- Ann. Missouri Bot. Gard. 90: 592-602. Mihara, T. 1960. On the reduction division of Houttuynia some number of x=11 to x=8. The basic chromosome cordata Thunb. Bot. Mag. Tokyo 73: 498 number of x=11 is still retained in Saururus, but a similar Oginuma, K. and Nakata, M. 1988. Cytological studies on dispolid reduction seems to have occurred from x=11 to phanerogams in southern Peru I. Karyotype of Acaena x=9 in Gymnotheca. Thus, Houttuynia might have a ovalifolia. Bull. Natl. Sci. Mus. Ser. B 14: 53-56 Okabe, S. 1934. Uber die Parthenogenesis bei einigen reduced basic chromosome number from x=11 to x=8, as japanischen Pflanzen. Bot. Mag. Tokyo 48: 6-7. an autoapomorphy, with subsequent intraspecific Okada, H. 1986. Karyomorphology and relationships in some polyploidization in continental parts of eastern Asia. genera of Saururaceae and Piperaceae. Bot. Mag. Tokyo 99: 289-299. Pinkava, D. J., Keil, D. J. and Mcgill, L. A. 1976. IOPB ACKNOWLEDGMENTS. We thank Dr. Hidenobu Funakoshi, chromosome number reports LIII. Taxon 25: 483-500. Shinshu University, and Ms. Min Deng and Mr. Yongsheng Yi, The Raven, P. H., Kyhos, D. W. and Hill, A. J. 1965. Chromosome Kunming Institute of Botany, The People’s Republic of China for numbers of spermatophytes, mostly Californian. Aliso 6: collecting plants in the field. This study was supported by Grants- 105-113. in-Aid for Scientific Research (13575011 and 15405014) from Shibata, K. and Miyake, H. 1908. Ueber Parthenogenesis bei Japan Society for the Promotion of Science. Houttuynia cordata. Bot. Mag. Tokyo 22: 141-144 Sodenberg, E. 1927. Uber die Chromosomenzahl von Houttuynia cordata. Svensk Bot. Tidskr. 21: 247-250. LITERATURE CITED Suzuka, O. 1950. Chromosome numbers in pharmaceutical Baldwin, J. T. and Speese, B. M. 1949. Cytogeography of plants. I. Seiken Ziho (Rept. Kihara Inst. Biol. Res.) 4: Saururus cernuus. Bull. Torrey Bot. Club 76: 213-216. 57-58. Chuang, T. I., Chao, Y. C., Hu, W. W. L. and Kwan, S. C. The Angiosperm Phylogeny Group (APG II). 2003. An 1963. Chromosome numbers of the vascular plants of update of the angiosperm phylogeny group classification Taiwan. I. Taiwaniana 1: 51-66. for the orders and families of flowering plants. Bot. J. Ge, C. J., Li, Y. K., Wan, P. and Hsu, P. S. 1989. Chromosome Linn Soc. 141: 399-436. numbers of 31 medicinal plants from Shandong Province. Zhang, Z., Fan, L., Yang, J., Hao, X., Gu, Z. 2006. Alkaloid In: Plant Chromosome Research (1987) (Hong, D., ed.). polymorphism and ITS sequence variation in the Spiraea Organizing Committee Sino-Japanese Symposium on japonica complex (Rosaceae) in China: traces of the Plant Chromosome, Beijing, pp. 267-272. biological effects of the Himalaya-Tibet Plateau uplift. Hsu, C. C. 1968. Preliminary chromosome studies on the Amer. Jour. Bot. 93: 762-769.