© 2010 The Japan Mendel Society Cytologia 75(4): 335–339, 2010 Chromosome Counts of Some Wetland Cyperaceous Species from the Mekong Delta, Vietnam Hiroshi Uchiyama1*, Hideyuki Matoba1, Tomoko Aizawa1, Hiroaki Sumida2 and Do Minh Nhut3 1 Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan 2 Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–0880, Japan 3 Kien Giang Agriculture Extension Center, 1224 Nguyen Trung Truc, Rach Gia, Kien Giang, Vietnam Received March 15, 2010; accepted July 15, 2010 Summary Chromosome numbers were counted in root tip cells of 6 cyperaceous species growing in wetlands in the Mekong Delta, Vietnam. The results were as follows: Cyperus haspan, 2nϭ26; Eleocharis dulcis, 2nϭca. 212; Eleocharis spiralis, 2nϭ42; Fimbristylis ferruginea, 2nϭ20; Fimbristylis polytrichoides, 2nϭ10; Lepironia articulata, 2nϭ34. They were reported here for the first time using plant materials from Vietnam. Furthermore, the results of 2 species, Eleocharis spiralis and Lepironia articulata, were the first to be reported for these species. In addition, the dimorphism of Eleocharis dulcis shoots representing emerged and submerged culms was recorded. Key words Aquatic plants, Chromosome number, Cyperaceae, Diffuse centromere, Mapanioideae, Vietnam. The Cyperaceae is a large cosmopolitan family comprising 104 genera and over 5,000 species (Goetghebeur 1998) and shows intriguing cytological characteristics such as diffuse centromeres, post-reductinal meiosis and agmatoploidy (Greilhuber 1995, Hipp et al. 2009). According to the inventory by Roalson (2008), chromosome numbers have been counted in only approximately 16% of the species in the family, and there is a particular lack of chromosome counts in tropical genera. Indeed, there seems to be few chromosomal reports on cyperaceous species from Southeast Asia in genaral. The Mekong Delta, most of which lies in southern Vietnam, is a flat wide plain that floods annually during the rainy season. A large part of the delta wetlands is utilized for agriculture, mainly rice cropping, and fishery. However, acid sulfate soils (ASSs) and/or saline soils are causing problems for agricultural production in many areas. Under the research title, “Development of new bioremediation systems for the destroyed environment”, launched as part of the 21st Century Center of Excellence (COE) Program (Sasaki 2008), we previously performed research activities aimed at understanding the characteristic features of wild plants and other organisms, especially those growing in ASS fields. During this research, we revealed the chromosome numbers of some aquatic and wetland plants growing in ASS fields from central Thailand (Matoba and Uchiyama 2009). In this paper, we report the chromosome numbers of 6 cyperaceous species collected from ASS and saline soil fields in the Mekong Delta, Vietnam. Materials and methods Materials for the present study are listed in Table 1. Plant specimens were collected in wetland * Corresponding author, e-mail: [email protected] 336 H. Uchiyama et al. Cytologia 75(4) areas such as backswamps of mangroves and paddy fields in Kien Giang Province, southern Vietnam, then cultivated in a greenhouse of the College of Bioresource Sciences, Nihon University, Japan. Voucher specimens were deposited in the herbarium of Nihon University, the College Museum of Bioresource Sciences, Fujisawa, Japan. Taxonomic treatment followed Simpson and Koyama (1998). Root tips were cut into 2–3 mm sections then pretreated with 2 mM 8-hydroxyquinoline at 20°C for 3 h before being fixed in ethanol–acetic acid (3 : 1) at 4°C for 24 h. Fixed root tips were washed 3 times in distilled water, then 10 to 30 samples were placed in a microcentrifuge tube containing 500 ml of enzyme solution (4% Cellulase Onozuka-RS (Yakult Honsha) and 2% Pectolyase Y-23 (Kikkoman), pH 4.2) and incubated at 37°C for 1 h. After washing 3 times with distilled water, some of the washed root tips were placed on a glass slide onto which 10–20 ml of fixative was dropped; the root tips were immediately spread apart. Air-dried slides were then Table 1. Chromosome numbers of the Cyperaceae species investigated in this study Species and Present Previous report Locality count (Voucher) (2n) n 2n Reference Cyperus haspan L. Hon Dat, paddy field 26 8 India (Sarkar et al. 1978) (Uchiyama 07056-58) 16 India (Sanyal 1972) 13 India (Rath and Patnaik 1978) 26 India (Nijalingappa et al. 1978, Tejavathi and Nijalingappa 1990) Thailand (Matoba and Uchiyama 2009) 30 Nigeria (Baquar 1978) 16 32 Japan: (Tanaka 1942) 18 India (Mehra and Sachdeva 1975) Eleocharis dulcis (Burm. f.) Trin. ex Hensch. Hon Dat, paddy field ca.212 38 India (Rath & Patnaik 1978) (Uchiyama 05087-88) c.172 Australia (Briggs 1970)* Kien Luong, paddy field ca.212 c.196 Japan (Yano et al. 2004) (Uchiyama 06102-103) Ͼ100 India (Rath and Patnaik 1974) c.212 Thailand (Matoba and Uchiyama 2009) c.108 India (Patnaik and Guru 1968; cited in Rath and Patnaik 1974) Eleocharis spiralis (Rottb.) Roem. & Schlt Kien Luong, backswamp 42 (Uchiyama 07079) Fimbristylis ferruginea (L.) Vahl Kien Luong, backswamp 20 5 India (Mehra and Sachdeva 1976, Rath and Patnaik (Uchiyama 07062-64) 1978, Bir et al. 1986) 10 India (Sanyal and Sharma 1972, Nijalingappa 1975) 10 India (Bir et al. 1992) 20 India (Sarker et al. 1978) Australia (Briggs 2002) 32 India (Subramanian 1988) Fimbristylis polytrichoides (Retz.) R. Br. Kien Luong, backswamp 10 5 India (Bir et al. 1992) (Uchiyama 07059-61) 10 India (Nijalingappa and Tejavathi 1984) 20 India (Sanyal and Sharma 1972) Lepironia articulata (Retz.) Domin Kien Luong, paddy field 34 (Uchiyama 05032-33) *: as E. equiseina. 2010 Chromosome numbers of some Vietnamese Cyperaceae 337 mounted in Vectashield mounting medium (Vector Lab.) containing 500 ng/ml 4Ј, 6-diamidino-2- phenylinodole (DAPI). At least 10 metaphase plates in the cytoplasm were observed and fluorescent photomicrographs were taken with an Olympus microscope BX60 using an Olympus DP50 CCD camera. Results and discussion Chromosome numbers counted in this study and those reported previously are listed in Table 1. Localities of the materials used for previous chromosomal observations are also shown. Photomicrographs of somatic chromosomes investigated in this study are shown in Fig. 1A–F. The chromosome number of Cyperus haspan L. was counted as 2nϭ26 (Fig. 1A), which is in agreement with reports from India and Thailand. The following chromosome numbers have also been reported for this species: nϭ8, 2nϭ16 and nϭ18 from India, 2nϭ30 from Nigeria, and nϭ16 and 2nϭ32 from Japan. The plants used in this study did not have creeping rhizomes as found in Thai materials (Matoba and Uchiyama 2009). In Eleocharis dulcis (Burm. f.) Trin. ex Hensch., approximately 200 chromosomes have been observed from India, Thailand and Japan; nϭ38 and 2nϭc.172 have also been reported from India and Australia, respectively. Although in the present study we were unable to fix the chromosome number due to the small size of the chromosomes (Ͻ1 mm), approximately 212 were counted in many cell plates observed (Fig. 1B). Interestingly, plants collected from 2 localities, both of which dry up during the dry season, had dimorphic shoots; one robust emerged culm, which is normally described in this species, and another tender submerged culm (Fig. 2). The submerged culm has been reported in this taxon from India (Joseph and Sivarajan 1984) but is rare, and to the best of our knowledge is recorded here for only the second time. The somatic chromosome number of Eleocharis spiralis (Rottb.) Roem. & Schult. was counted as 2nϭ42 (Fig. 1C), and is reported here for the first time for this species. This chromosome number is smaller than that of the species in the subgenus Limnochloa reported previously; E. acutangula (ϭfistulosa) (2nϭ54), E. ochrostachys (2nϭ74), E. sphacelata (2nϭ94–100, c.140, c.157, 176– Fig. 1. Fluorescent photomicrographs (color-reversed images) of the somatic chromosomes of 6 Cyperaceae species (A–F) investigated in this study. A, Cyperus haspan, 2nϭ26. B, Eleocharis dulcis, 2nϭca. 212. C, Eleocharis spiralis, 2nϭ42. D, Fimbristylis ferruginea, 2nϭ20. E, Fimbristylis polytrichoides, 2nϭ10. F, Lepironia articulata, 2nϭ34. Scale bar indicates 10 mm. 338 H. Uchiyama et al. Cytologia 75(4) 184, c.188) and E. dulcis (Briggs 1970, Rath and Patnaik 1978, Yano et al. 2004). Intraspecific polyploidy with xϭ5 has been investigated in the genus Fimbristylis. Chromosome numbers of F. ferruginea (L.) Vahl and F. polytrichoides (Retz.) R. Br. used in this study were tetraploid with 2nϭ20 (Fig. 1D) and diploid with 2nϭ10 (Fig. 1E), respectively. In both species, diploids and tetraploids have been reported from India. In F. ferruginea, tetraploids (2nϭ20) and hetero- ploids (2nϭ32) have also been reported from Australia and India, respectively. The somatic chromosome number of Lepironia articulata (Retz.) Domin (Fig. 1F) was counted as 2nϭ34, and is reported here for the first time. Furthermore, this is the first report for the species in the subfamily Mapanioideae (Goetghebeur 1998, Roalson 2008), which has been considered primitive in the family Cyperaceae. Diffuse centromeres are one of the distinct chromosomal features in the family Fig. 2. Photograph of the dimorphic shoots of Cyperaceae, and are considered as a Eleocharis dulcis growing in a rice field in Hon synapomorphy for the Cyperaceae-Juncaceae Dat, Kien Giang Province. lineage (Greilhuber 1995). Although a localized centromere has occasionally been described in some genera including Cyperus haspan, Fimbristylis ferruginea and F. polytrichoides (Sanyal 1972, Sanyal and Sharma 1972, Nijalingappa 1975, Bir et al. 1986, Subramanian 1988, Tejavathi and Nijalingappa 1990), obvious primary constrictions were hardly observed in these species used in this investigation (Fig. 1). Acknowledgements This work was supported by a Grant-in-Aid for the 21st COE Program from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. References Baquar, S. R. 1978.