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 2n24. 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 genusof 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). JapaneseZannichellia plants have been identified as Z.
palustris L. (Ohwi, 1972; Yamashita, 1982;Kadono, 1994).
The chromosome numbers of Zannichellia
have been previously reported as 2n12, 14, 24,28, 32, 34, and 36 (Van Vierssen, 1982; Talaveraet al., 1986; Talavera and Garcia Murillo, 1992;
Montgomery et al., 1997; Mesicek and Javurkova-Jarolimova, 1992). Zannichellia palustris wasalso found to have variations as follows: 2n24
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 vanWijk, 1982; Uotila et al., 1983; Mesicek andJavurkova-Jarolimova, 1992; Sun, 1992; Montgomery et al., 1997).
Harada (1956) reported 2n24 as the chromosome number of Z. palustris in Japan. However,the collecting site and voucher specimens are notdocumented. Therefore, observation of chromosome numbers from locations with collection
data is necessary to provide basic information inorder to revise the taxonomic treatment. In thispaper, 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 usedin this study (Table 1). Some individuals fromeach location were studied. Voucher specimens
are deposited in the National Museum of Natureand 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) mixtureof ethanol and acetic acid at room temperature
for three hours, they were macerated in 1 N HClfor 10 min at 60°C, stained with 1% aceto-orcein

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Table 1.

Localities and voucher specimens of populations investigated in japan.

Population

Locality

Voucher

Tofutsu
Yudo
TakahokoOgawaraNishiki-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 aglass 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 2n24 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 eachsamples had minor differences (1.209–1.241,Table 2), these samples were estimated to be
2n24 on the presumption that their chromosome numbers are an even number. These results

Table 2.

Peak indices of flow cytometry analyses.
Peak index*Population
YudoTakahoko
Nishiki-HamaNobeoka

123

1.239
1.241
1.209
1.2212.489
2.499
2.408
2.388

——
4.855
—

*Peak index shows the relative location when the peakof internal standard is assumed to be 1.0.

showed that the Zannichellia plants from all localities of this study have a chromosome numberof 2n24. This is consistent with the only dataon 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 (Ohwi1972; Yamashita, 1982; Kadono, 1994). According to Talavera et al. (1986), who used featuresof leaf and fruit as key characters, plants of Tohfutsu, Yudo and Ogawara were determined to beZ. palustris L., while Takahoko, Nishiki-Hama,and Nobeoka, had intermediate characters between Z. palustris and Z. pedunculata Reichenb.

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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 tovan Vierssen (1982), who used leaf and fruit as
key characters, Takahoko, Nishiki-Hama andNobeoka, were identified as Z. pedunculata.
However, this study described the chromosome
numbers of Z. palustris and Z. pedunculata to be
2n24 and 2n36, respectively. Van Vierssen’staxonomic description of morphological characters and chromosome numbers are inconsistentwith our results. In the future, a molecular phylogenetic approach would be necessary to identifyand delineate taxa in this genus.
Acknowledgements
We are grateful to Satoru Kinoshita, Tadashi
Minamidani and Heigoro Narusako, Takatomo
Yano, Masahiko Takiguchi, Takashi Mori,Koubei Hosoi, Mami Yamazaki, Hidenobu Kunii,
Kaname Kamiya, Satoru Araki for collecting
plants; Masahiro Mii and Chin Don Poh for giving us the use of ploidy analyzer. This study wassupported by a Grant-in-Aid from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan (No. 17710194).
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