Bull. Natl. Mus. Nat. Sci., Ser. B, 33(3 · 4), pp. 133–136, December 21, 2007 Chromosome Numbers of Zannichellia L. (Zannichelliaceae) in Japan Norio Tanaka1, Yu Ito2, Ruriko Matsuyama3 and Koichi Uehara3 1 Tsukuba Botanical Garden, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, 305–0005, Japan E-mail: ntanaka@kahaku.go.jp 2 Botanical Gardens, Koishikawa, Graduate School of Science, The University of Tokyo, Hakusan 3–7–1, 112–0001, Japan 3 Faculty of Horticulture, Chiba University, Matsudo 641, Matsudo, 271–8510, Japan Abstract Chromosome numbers of Zannichellia L. (Zannichelliaceae) found in Japan was studied. Plants collected from six localities had a chromosome number of 2n⫽24. The chromosome numbers of Zannichellia found in Japan were firstly reported with collecting information. According to existing taxonomic keys, Zannichellia plants used in this study were determined to be Z. palustris or Z. pedunculata, a species previously not reported in Japan. Key words : Chromosome number, flow cytometry, Japan, Zannichellia palustris, Zannichellia pedunculata. Introduction Zannichellia L. (Zannichelliaceae) is a genus of annual or perennial submerged aquatic plants, which inhabit brackish and fresh waters. It is widely distributed in tropical and temperate latitudes (Tomlinson, 1982). The taxonomy of Zannichellia has not been studied entirely and has been confused not only in Japan but throughout the world (Cook, 1996; Kadono, 1994). Japanese Zannichellia plants have been identified as Z. palustris L. (Ohwi, 1972; Yamashita, 1982; Kadono, 1994). The chromosome numbers of Zannichellia have been previously reported as 2n⫽12, 14, 24, 28, 32, 34, and 36 (Van Vierssen, 1982; Talavera et al., 1986; Talavera and Garcia Murillo, 1992; Montgomery et al., 1997; Mesicek and Javurkova-Jarolimova, 1992). Zannichellia palustris was also found to have variations as follows: 2n⫽24 from Europe, China and Japan; 28 from Europe; 34 from Europe and China; 36 from Europe (Scheerer, 1940; Tarnavschi, 1948; Harada, 1956; Reese, 1961, 1963, 1967; Hedberg and Hedberg, 1964; van Vierssen, 1982; van Vierssen and van Wijk, 1982; Uotila et al., 1983; Mesicek and Javurkova-Jarolimova, 1992; Sun, 1992; Montgomery et al., 1997). Harada (1956) reported 2n⫽24 as the chromosome number of Z. palustris in Japan. However, the collecting site and voucher specimens are not documented. Therefore, observation of chromosome numbers from locations with collection data is necessary to provide basic information in order to revise the taxonomic treatment. In this paper, we report chromosome numbers of Z. palustris from six localities in Japan. Materials and Methods Six of the eleven localities of Zannichellia plants reported by Tanaka et al. (2006) were used in this study (Table 1). Some individuals from each location were studied. Voucher specimens are deposited in the National Museum of Nature and Science (TNS). Root tips of each plant were cut and soaked in 8 mM aqueous hydroxyquinoline solution at 4°C overnight. After fixation with a 3 : 1 (v/v) mixture of ethanol and acetic acid at room temperature for three hours, they were macerated in 1 N HCl for 10 min at 60°C, stained with 1% aceto-orcein 134 Norio Tanaka et al. Table 1. Localities and voucher specimens of populations investigated in japan. Population Locality Voucher Tofutsu Yudo Takahoko Ogawara Nishiki-Hama Nobeoka Lake Tofutsu, Koshimizu, Hokkaido Pref. Lake Yudo, Toyokoro, Hokkaido Pref. Lake Takahoko, Rokkasyo, Aomori Pref. Lake Ogawara, Kamikita, Aomori Pref. Nishiki-Hama, Higashi-Izumo, Shimane Pref. Nobeoka, Miyazaki Pref. TNS 9528810-9528818 TNS 9528819, 9528820 TNS 9528835, 9528836 TNS 9528778 TNS 9528826-9528831 TNS 9528833, 9528834 Fig. 1. Somatic chromosomes of Zannichellia at metaphase. A: Tofutsu, B: Takahoko, C: Ogawara. Bar indicates 5 m m. for 1 min at room temperature, and squashed on a glass slide. Flow cytometry (FCM) analyses were conducted to indirectly estimate the chromosome number of Zannichellia plants whose roots could not be collected. Samples were prepared using CyStain UV precise P kit (Partec, Germany) for nuclei extraction and staining of nuclear DNA. The genome size was determined by a basic procedure of Ploidy Analyzer PA (Partec, Germany). Oryza sativa L. cv. Nihonbare was used as an internal standard. Results and Discussion The chromosome numbers of Zannichellia plants from Tofutsu, Takahoko, and Ogawara were 2n⫽24 by microscopic observation (Fig. 1). By FCM analyses, the genome size of the plants in Yudo, Takahoko, Nishiki–Hama and Nobeoka, were shown to be nearly equal to each other (Fig. 2, Table 2). Although the peak indices of each samples had minor differences (1.209–1.241, Table 2), these samples were estimated to be 2n⫽24 on the presumption that their chromosome numbers are an even number. These results Table 2. Peak indices of flow cytometry analyses. Peak index* Population Yudo Takahoko Nishiki-Hama Nobeoka 1 2 3 1.239 1.241 1.209 1.221 2.489 2.499 2.408 2.388 — — 4.855 — *Peak index shows the relative location when the peak of internal standard is assumed to be 1.0. showed that the Zannichellia plants from all localities of this study have a chromosome number of 2n⫽24. This is consistent with the only data on chromosome numbers of Japanese Zannichellia (Harada, 1956). Some Zannichellia in this study were not determined to be Z. palustris, which is the only known Zannichellia species in Japan (Ohwi 1972; Yamashita, 1982; Kadono, 1994). According to Talavera et al. (1986), who used features of leaf and fruit as key characters, plants of Tohfutsu, Yudo and Ogawara were determined to be Z. palustris L., while Takahoko, Nishiki-Hama, and Nobeoka, had intermediate characters between Z. palustris and Z. pedunculata Reichenb. Chromosome numbers of Zannichellia 135 Fig. 2. Peaks in fluorescence of Zannichellia plants of Takahoko (Peak 2 and 3), from analyses with flow cytometer. The horizontal scale indicates fluorescent intensity (calibrated by the internal standard, Oryza sativa: Peak 1) and the vertical scale indicates number of cells. (not previously reported in Japan). According to van Vierssen (1982), who used leaf and fruit as key characters, Takahoko, Nishiki-Hama and Nobeoka, were identified as Z. pedunculata. However, this study described the chromosome numbers of Z. palustris and Z. pedunculata to be 2n⫽24 and 2n⫽36, respectively. 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